Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 29 Detection of Cholesterol in Suaeda Baccata (Chenopodiaceae) Suhad S. Al-Mohammadi *1 *De pa rtme nt o f Pharm cognac y,Co lleg e of Pharmacy, Unive rsity o f Bagh dad, Baghda d-Iraq Abstract This s tudy de tects the presence of choleste rol in a n Iraqi pla nt named Suaeda ba cca ta Forsk of the family Che nopodiacae , wildly and widely grown in Ira q. The abs ence of any publica tion c oncerning the sterol content of this Suaeda s pec ie , and the indus tria l importa nce of choles te rol depending on its role as a precursor in the synthes is of some hormones , like progesterone , acquired this s tudy its value . The inves tiga tions revealed the prese nce of cholesterol tha t was proved by TLC together with the s ta nda rd compound cholesterol, and anisaldehyde spray reagent using three different solvent sys te ms, the n authe ntic ated by HPLC, in whic h the retention time of both the s tandard c holes te rol and the pla nt extrac t cholesterol were identical. Key Words: Cholesterol, Suae da Bacca t and Chenopodiaceae الخالصة مى بـ ول في نبات عراقي مس ستير ول سة تختص بالكشف عن مادة الك را bacca هذه الد ta Suaeda (Chenopodia ceae) ق را ريا و بشكل واسع في الع وب ورية تتناول المحتوى .الذي ينم ود اي مجلة علمية او د ان عدم وج ول للصناعة الد ستير ول مية الك عض الهرمونات الستيرويدي لهاذا الصنف من النبات وأه ائية لتصنيع ب ونة المادة االبتد وائية بك ستيرون مة هذة الدراسة,مثل البروج ود مادة الكولستيرول والذي اكد بواسطة استخدام . يبين قي بينت التجارب البحثية وج ماتوغرافيا الطبقة الرقيقة واسطة ثالثة أنواع من المحاليل الناقلة TLCتقنية كرو كيدي لمادة كما تم إجراء .ب ص التأ الفح ول القياسي HPLCالكولستيرول في المستخلص النباتي بواسطة ستير ول ق الك .والذي طاب In troduction The Chenopodiac eae (go osefoot fa mily) is a la rge fa mily includ ing about 1 02 ge ne ra , and 1400 sp ec ies of low growing plants. (1) Me mb ers o f this fa mily inc luding Sua eda baccata mos tly grow naturally in soils co ntaining much s alt (halop hyte s) (2). Sua eda bacca ta specie is distrib uted in Iraq , in so uth of Ja zira Distric t, So uthern De se rt District, We stern Des ert Ce ntra l Alluvial Plain District a nd Eas te rn Alluvial Distric t (3).the photo of this p la nt is demonstrated bellow Lite ra ture survey indicated tha t d ifferent spec ies of the ge nus Suae da co ntain several different comp ounds , including s te ro ls (4, 5), th us it was d eeme d desirable to find out the sterol conte nt o f this p lant. S te ro ids constitu te a natural p roduc t class of c omp ounds tha t is wid ely distributed thro ughout nature. (6) The che mical s tructure of steroids is ba se on (a) perhydrocyclopenta p henanthre ne nucleus. The steroida l nuc le us is deriv ed from is ope ntyl pyrop hosphate as fo llows :- Aceta te me valonate isope ntenyl p yrophosphate sq ualene cho lesterol pa thway .(7,8) 1corresp ond in g author ema ilsu hadsami04@yahoo.comsu hadsami04@yahoo.com Rece ived 28-1-2006 Accepted 4 -11-2006 mailto:suhadsami04@yahoo.com Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 30 accata] Suae da) [pho togra phy o f Figure (1 The firs t steroids isolated fro m nature were a se ries of C2 7 –C2 9 alco hols tha t were found in lipid fractio n of ma ny tis sues . These co mpounds were solids and therefore name d sterols fro m the Gree k stereos, me aning so lid. The most widely occurring sterol is c holesterol, (Greek Word, c ho le , me aning b ile ). It was first isolated from huma n ga llstones . Until rece ntly choles te rol was thought to be res tric ted to the anima l kingdom; however, it has now bee n identifie d in p la nts.(7, 9, 10). The re is a wide sprea d b elie f among the public a nd ev en a mong the chemists tha t plants do not c ontain choles te ro l and this is a n erro r, further mo re e ven some refe rence s re fe r to this mistake (11, 12). The fa ct is that the cho le sterol wid es pread in the plant kingdom (a ltho ugh othe r related sterols, s uch as β-sitosterol ge nerally o ccur in highe r qua ntity) (13, 14). Cho le sterol o cc urs in b oth free a nd e sterified. It occ urs as a c ompo ne nt o f plant memb ra ne s and a s part of the s urfa ce lipids of lea ve s whe re it is s ome time s the ma jo r sterol. The qua ntity o f choles te ro l is ge nerally sma ll when exp re ss ed a pe rc ent o f to ta l lipid. While cho le sterol a verages p erhap s 50 mg/kg total lipid in p la nts, it can b e as high as 5 g/kg (or more) in animals. (15). The qua ntities o f choleste rol in a numbe r of vegetab le (plant) oils are giv en in the fo llowing tab le . Ta ble (1) Cho le sterol conte nt o f so me plant oils Source Cholste rol (mg/kg ) Re fe re nces 1 Pa lm oil 20 16 2 Pa lm ke rnel 17 17 3 Coconut o il 14 17 4 Cotto n se ed oil 45 17 5 Soybean o il 29 17 6 Corn oil 55 17 7 P eanut oil 24 17 8 S un flo wer oil 14 17 9 Cano la oil 53 17 1 0 Av ogadro oil < 30 18 1 1 Olive oil 0.5-2 19 ,2 0 1 2 .S esame o il Ca.1 19 ,2 0 Chole sterol and its e sters a re imp orta nt constituents o f plant me mbrane s. The follo wing ta ble re prese nts so me da ta on the sterol frac tion o f s ome plant o rgane lles . Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 31 Table (2) S ub-ce llu la r distribution of choles te ro l in plant While c hole sterol is us ua lly a minor co nstituent of the sterol fraction in plants , it is the major constituent of s ome p la nt s urfa ce a s shown in table (3) Table (3) Ste ro l co nte nt of Rape (Canola) (23) So urce Choles te rol % Sitos te rol % 1. Leaves a. S urfa ce 71.5 0.6 b. Intra ce llular 15 30 2 . Se e ds a. S urfa ce 7 .2 62 b. Intra ce llular 0 .7 67 3 . Se e dpods a. S urfa ce 35 21 The propo rtion of choles te ro l in the sterol frac tion o f Lilia cea e, Solanac eae a nd Scrop hulariace ae familie s is e sp ecially la rge. (24, 25) Chole sterol (C27 H46 O), 3β-chole …..s ta-5-e n- 3ol, mol.wt. 3 86.66 , is practica lly inso luble in water, s lightly s oluble in alco hol, more solub le in hot a lc ohol, one gram diss olves in 2.8 ml of ether, in 4 .5 ml of chlo ro fo rm, In 1.5ml of pyridine, also it is solub le in pe trole um ethe r, benze ne(26) . Choles te ro l s truc ture is rep re se nted b ello w. (26) Figure (2) c hole ste rol s truc ture Altho ugh tota l synthes is o f so me med icinal steroids is e mployed co mmercially, there is also a great d ema nd for natural p ro ducts which will se rve as a s tarting ma te rial for the ir p artial synthe sis (2) Acc ordingly, c holes terol has s erve a s a precurs or for the synthes is of p roges te ro ne (27) as repres ente d in the follo wing diagram, and this a cq uire d this study its impo rtance. So urce Free Cho le s - tero l% Cho le ste ro l - ester% Re fer ence 1. Gre en bean leav es a . Whole 1 1 2 1 b. Chloroplasts 2 4 33 c. Mito chondria --- --- d. Mic ro so mes 1 28 2 .Etio la ted be an leav es a . Whole 6 23 2 1 b. Chloroplasts 2 7 26 c. Mito chondria --- --- d. Mic ro so mes 6 34 3 . Orga nelles o f day maiz e -21 shoo ts a . N ucle i 2 2 76 2 2 b. Chloroplasts 2 52 c. Mito chondria 1 32 d. Mic ro so mes 1 32 CH3 CH3H3C CH3 CH3 HO A B C D Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 32 Figure (3 ) :conve rsion o f choles te ro l to pro ge ste rone (27) Material a nd Meth ods The p la nt materia l (a eria l pa rt) wa s collec te d during mo nths o f July, Augus t, and S epte mber 200 5. F ro m the high wa ys of Baghdad c ity. The p la nt was identified by the De pa rtment of Pha rmaco gno sy , c olle ge of Pha rmacy/University of Baghda d ; and authentica te d b y the Na tional He rb arium of Iraq . Bo ta ny Directorate a t Abu-Ghra ib , Ira q. Forty grams o f the dried a eria l pa rts were first mac erated with 50 0 ml o f n-hexane for 2 4 ho urs. The res id ual plant pa rt then wa s drie d at ro om te mperature, s oa ked in wa te r fo r 2 4 ho urs, d ried , and then refluxe d with 2 NHCl solution fo r two hours . Afte r filtra tio n by Buc hne r funnel, 5% ammonia so lution was add ed to the re sidual plant part, and then washed b y distilled wate r se veral times , until neutral. This plant pa rt a fte r drying ov er night will be e xtracted again with 25 0 ml of petroleum e ther (60 0C -800C ) for ten hours by the use o f soxhlet a ppa ra tus. The pe troleum ether filtrate will then b e ev apo ra te d to dryness to b e re ady for the id entifica tion o f s te ro id by TLC. (28) The following d ia gram re pres ents the extraction proc ed ure o f ste roids from Sua eda bac ca ta . HO HO OH OH HO O O O HO O O O O2 NA DPH O2 NA DPH NAD + keto-e nol t automerism enol-keto tautomerism P rogesteron e Cholesterol Preg nen olo ne Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 33 40 g m. Of the d ried aerial p lant part Ma ce ra tion with 50 0 ml n-he xa ne Fo r 24 hr. Res id ual plant pa rt Filtrat 1 . Drie d at room te mp. 2. S oak in wa te r for 2 4hr. 3. Reflux with 2NHCl fo r 2hr. F iltra te residual plant part Add 5% NH3 Wash several times with D.W Plant p art 1 - Dried ov er night at roo m te mp 2 - reflux with p etro le um ethe r (60-80 0)Fo r 10 hr. (s oxhlet). Marc filtra te Ev apo ra te to d ryness TLC de te ctio n for s te ro ids bacc ata Suae da Sche matic proce dure for the e xtrac tion o f ste roids from)Figure (4 Id entification of th e Stero id (Ch ole ster ol) Id entific atio n wa s p erfo rme d firs t by TLC, us ing silic a gel G, anisalde hyde sp ra y re agent (29,30)[0.5ml Anisa ld ehyde is mixe d with 10 ml glac ia l ac etic acid, fo llowe d by 8 5ml metha nol and 5 ml c oncentrate d s ulphuric ac id in that order. the TLC plates a re s pra ye d with 10 ml, he ated at 1 100 for 5-10 minute s.], s tand ard choleste rol, a nd diffe re nt s olve nt s ys te ms that were: (31,32) Solve nt (1 ): Toluene: ethyl ac etate (9 0:10) Solve nt (2 ): Benzen: ac etone (9 0:10) Solve nt (3): Pe troleum e ther (600-800): e thyl ac etate (7 5:25) The n this ide ntifica tion wa s authe nticated b y HPLC Result a nd Disc ussions Cho le sterol can b e fo und in p la nt eithe r in the free state or c onjugate d as s imp le glyco side(33) therefore the e xtra ctio n proce dure inc luded the us e o f wa ter and ac id , ne ces sa ry fo r the clea vage of the glyc osidic linkage and the re le as e of the a glyc one p art (cholesterol), and the pos sible suga r site atta chment is C3- a to m in its structure. As the c holes terol is so luble in petroleum ethe r, the re fo re this s olve nt was use d in its e xtra ctio n. The identific ation of c holes terol was performed b y TLC using thre e diffe re nt solve nt sys tems S1 , S2 , S3 . As rep re sente d in the fo llowing diagrams :- Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 34 A B B A B=ae rial plant e xtract B A The Rf. va lue of e ach solvent system of the standard c holesterol and the plant e xtra ct cho lesterol, a re represente d in the fo llowing ta ble. Ta ble (4) [Rf va lues o f bo th, the ae rial plant part e xtrac t and standard cho leste rol] Solve nt s ys te m Rf. of standard c holes te ro l Rf. o f the plant extra ct S1 (Toluene: ethyl ace ta te (9 0:10) 0.73 9 0.730 S2(Benze n:acetone (90 :10) 0.19 2 0.192 S3 (Petro le um ether (6 00-800): ethyl ac etate (75 :25) 0.51 3 0.521 Figure (5 ): TLC plate of the aeria l plant extra ct, a nd standard, using S 1 mobile pha se A=Standard cho le sterol B=ae rial plant e xtract Figure (6) :TLC plate o f the ae rial pla nt extra ct, and standard, using S2 mo bile pha se ) A=S tandard choles te rol B=ae rial plant e xtract Figure (7 ): TLC plate of the aeria l pla nt e x tract, a nd standard, us ing S3 mo bile phase A=Sta nda rd c holes tero l B=ae rial plant e xtract Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 35 Furthe r id entifica tio n to the choles te ro l in the plant extract was perfo rmed b y HPLC. In which the retention ti me of bo th the s ta nd ard choles te rol and the plant extrac t c holesterol were identical a s rep rese nted in the cha rt bellow. Table (5) :Ta ble of the HLPC conditions Conditions of c hole ste ro l HPLC (19) Mob ile phase Ace to nitrile:methanol 50 :50 containing 3% D.W Column C18 Detec to r 2 10 Flow rate 1 ml/mi n Inje ction Volume Fig ure (8) :HPLCchart of pla nt e xtrac t Figure (9) :HPLC c hart o f standard cho le ste rol Conclusion Sua eda bac ca ta F orsk (Che nop odia cae ), a wild Iraq i plant, c onta ins cho le sterol a s one of its constituents . Refe ren ces 1. Rowley GD, Ja co bse n H. (Ed ):”A Hand Book Of S ucc ulent Plants .” Vol.1, Lo ndo n: Bla nd ford Pres s. 1 960 , P.1 . 2. Tre as e and Eva ns ;” Pha rma cogno sy” Unive rs ity of Nottingha m, Nottingham, UK, 15thEd. 200 2, p .2 2, 2 90 . 3. Ali-Alrawi; “ Wild Plant of Iraq ” Minis try Of Agric ulture , Direc to ra te General of Agric ulture Res earch and Pro je cts. 196 4, p17 6 4. Rizk, A.M.; Heiba , H.I; Ma’ayergi, H, A; Batanouny, K.H.;”Co ns tituents Of Plants Gro wing In Qa ta r.” S ci. Appl. Res .c ent. Unive rs ity. Qatar, Doha, Argent. 1 986 , 57 (1 ), 3-9(Eng). 5. S ubrah mangam, C.; Rao , K.Bha skara; Ra o, C.Venkates ara;”Chemica l Examina tion Of The Mangro ve S pec ie s S uae da maritima And Sua eda monic a.”(Dep . Org. Chem. F ood s, Drugs Water, Andhra Univ ersity Vis ha kha pa tnam, 53 000 3 Ind ia ). Ac ta ciene. Indica Chem. 199 2, 1 8(1), 7-8(Eng). 6. Varro E.Tyler, Lynn R. Bra dy and James E. Robb ers:”P harmac ognos y” Philade lp hia, U.S .A. 9thEd. 1 99 7, p .1 56. 7. ibid pp . 1 58,16 0. Ir aq i J.Pha rm.Sc i., Vol.15 (2 ) ,2006 Detec tion o f choles tero l in su aed a bacca ta 36 8. Erich H.”Bios ynthes is of P la nt Steroids ” Jo urnal. Na tura l Prod uct (Lloyd ia ) 19 68, vol.31, 1-4 p.29 3, 1 71. 9. Koji N, To shio G., ShoI, Shinsa ku N., and Shigeo N.” Na tura l P ro duc ts Che mis try” Kod ans ha LTD. Aca demic Pres s, INC. New York and London, 19 74, p.422 . 10. J . B. Ha rb ornc” Phyto chemica l Me thod ” London Cha pman a nd Ha ll 197 3, pp.11 0, 1 13. 11. Horto n, H.R.; Moran, L. A., Ochs, R.S .; Rawn, J .D., S crimgeo ur, K.G." Princ iple s of Bio che mis try" 3rd ed , P re ntic e Hall; up per Sad dle River, 2 002 ; p p 275 . 12. Zuba y, G.L.; Parso n, W.W.; Vance , D.E." Principles o f Biochemistry" 199 5 pp3 85. 13. S hukla VKS et al., "J AOCS " 200 2,Camelina oil, 79, 965 . 14. Noda M et al., "Lipids " 19 88, 23, 439 . 15. Be hrama n E.J . and Venkat Gopa la n"J. of Chemica l Educa tion", Ohio S ta te Unive rs ity, Columbus , 20 05,Vo l.82 , No .1 2, . 16. Gurr, M.I., "Role Of Fats In F ood and Nutrition" 2nd Edn.; Els ev ie r,Londo n, 19 92, pp.3 6. 17. Ross ell, J.B." Analys is o f oils ee ds, fa t, and fatty food s", Els evier, London 19 91, chapter7, Tab le 7 .1 1. 18. Itoh, T.;Ta murat T.;Mas umoto, T., Dup agine P."Fruits"1 97 5, 3 0, P P.687 - 69 8. 19. Casta ng J . Ana . F alsif. Exp ert Chim. 19 81, 74, pp 6 97-700 . 20. Kochha r, S.P."Prog. Lipid. Res ." 198 3, 22, pp 1 61-188 . 21. Bra ndt R.D.; Benve niste, P." Biochemistry and Bio physics " 19 72, 282 , 85 -92 . 22. Ke mp, R.J; Me rc er E.I." Bio chemistry J."1 968 , 11 0,119 -1 25. 23. No da , M.; Ta na ka, M. ,Se to Y., Aib a, T."Lipids ", 199 8, 2 3, 2 39-44 4. 24. Akihisa T.; Kokke, W.C.; Tamura , T." Physiology a nd Bio che mistry of Sterol" American oil chemist's So ciety, Cha mpaign, 1 991 ; chap te r 7 . 25. Ha rtma nn, M.A."Lipid Metabo lism and Membrane Biogene sis" Dann G., ed n, Springe r, Berlin 20 04, chapte r 5. 26. Me rc k and CO., Inc. Ra hwa y, N.J .:” The Merc k Inde x” 8 th Ed. U.S .A. 19 66, pp. 369 . 27. De wik, P .M. "Med ic inal Natural Products, A Bios ynthetic Ap proac h" J ohn Wile y and sons Ltd, Chi Che ster, reprinted 19 97 . 28. Ab dul-Wahe d M. “ Inves tiga tio n o f the Steroidal S ap oge nins of Tribulus sp p. wild ly Gro wn in Iraq” B.S c 200 2 p.38 . 29. Wa gne r, W.; Bla dt, S. and Zga inski, E.M: “Plant Drug Ana lysis” s pringe r-ve rlag Be rlin Heidelberg New Yo rk-To kyo 1 98 4 p.299 -3 04. 30. S ta hl E. “ Thin Layer Chromatograp hy” Springe r- Verla g Berlin P . 19 99 PP .3 23, 325 . 31. Tagree d MH. ” Plant Tis sue Culture of Trigo ne lla foe num-gra ec um for Dios genin Pro ductio n” 19 97 p .4 8. 32. P ushpa K. and Sa tish C.J.”Dios ge nin, Gitogenin and Tigogenin from Trigonella fo enum-gra ce um Tis sue Cultures .” Lloyd ia 197 3,vol.36 p.36 , 33. J .B. Ha rb ornc”Phytochemical Metho d” London Chap man a nd Ha ll 1973, p.110 , 1 13.