Study of Iraqi Spinach Leaves ( Phytochemical and Protective Effects Against methotrexate-Induced hepatotoxicity in rats) Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 8 Study of Iraqi Spinach Leaves (Phytochemical and Protective Effects Against methotrexate-Induced hepatotoxicity in rats) Farah K. Abdul-Wahab * ,1 and Thukaa Z. Abdul Jalil ** * Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad, Baghdad, Iraq ** Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract Spinach, Spinacia oleracea L is a popular vegetable belonging to the family Chenopodiaceae. This study was concerned with extraction of compounds in Iraqi spinach leaves, preliminary phytochemical evaluation, identification of two biological important flavonols, quercetin and kaempferol in spinach leaves and evaluation of the protective effect of aqueous spinach extract on methotrexate (MTX) induced hepatotoxicity in rats. The percentage yield of extraction procedure, identification of spinach by chemical tests and identification of flavonols by thin layer chromatography (TLC) and High performance liquid chromatography (HPLC) were fully described in this study. The results indicate that the percentage of quarcetin in spinach leaves is more than the percentage of kaempferol in the same plant. The rats were divided into three groups as control, MTX group following a single dose of MTX (20 mg/kg, i.p) saline was administered for 5 days and the MTX+aqueous spinach extract group were rats received 200mg/kg orally of aqueous spinach extract 7days before and 5 days after MTX treatment. MTX administration increased the MDA and decreased GSH, ALP while these changes were reversed in aqueous spinach extract treated group. Histological changes observed in MTX treated group was improved by aqueous spinach extract treatment. The protective effect of aqueous spinach extract against MTX- induced hepatotoxicity could be attributed to the combined effects of its constituents. Key words: Spinach, Kaempherol , Quercetin , Methotrexate, Oxidative stress. انىاجج عه انكبذ جسمم ضذ انىاقيةوجاثيراجها كيمياء انىباجيةان) انعراقي انسباوخ أوراق دراسة عه )في انجرران انميثىجريكسيث *فرح قيس عبذانىهاب ،1 **ركاء زهير عبذ انجهيمو . ، بغذاد ، انعشاقجبيعّ بغذاد ، نتانظٛذ تكهٛ ،فشع االدّٔٚ ٔانسًٕو * . ، بغذاد ، انعشاقجبيعّ بغذاد ،نتانظٛذ تكهٛ ،فشع انعمبلٛش ** انخالصة بسخخالصب حٓخى ْزِ انذساست Chenopodiaceaeعبئهت ُخًٙ إنٗح شعبٛت خضبس عببسة عٍْٕ Spinacia oleracea L انسببَخ فٙ كٕٚشسٛخٍٛ، كبًٚبفٛشٔل ٔ ببٕٚنٕجٛب انٓبيخٍٛ انفالفٌٕ، ٔححذٚذ األٔنٛت انكًٛٛبئٛت انُببحٛت، ٔحمٛٛى انعشالٙ انسببَخ أٔساق انًشكببث انفُٕٛنّٛ يٍ ٔطف انُسبّ حى( فٙ انجشراٌ . MTX) انخأثٛش انٕلبئٙ نًسخخهض انسببَغ انًبئٙ ضذ حسًى انكبذ انز٘ ٚسببّ انًٛثٕحشكسٛج ٔحمٕٚى انسببَخ أٔساق انفظم انهَٕٙ ٔ نهفظم انهَٕٙ ّٔححذٚذ انفالفٌٕ بٕاسطّ انطبمّ انشلٛم كبيم بشكم هسببَغناالخخببساث انكًٛٛبئّٛ ٚجانًئّٕٚ نهًسخخهض ٔاجش .َفس انُببث فٙ كبًٚبفٛشٔل َسبّ ْٕ أكثش يٍ انسببَخ أٔساق فٙ كٕٚشسٛخٍٛ َسبّحشٛش انُخبئج انٗ اٌ فٙ ْزِ انذساسّ. انسبئم راث انكفبءِ انعبنّٛ ( انبشٚخٌٕ يهغى / كغى فٙ 02) MTXبعذ جشعّ ٔاحذِ يٍ MTX يجًٕعتانُحٕ انخبنٙ انسٛطشِ , يجًٕعبث عهٗ إنٗ ثالد انجشراٌ حى حمسٛى يهغى / كغى( يسخخهض انسببَغ انًبئٙ 022+ يسخخهض انسببَغ انًبئٙ حهمج انجشراٌ ) MTXفٙ يجًٕعّ أٚبو 5نًذة انًحهٕل انًهحٙ حى إعطبء فٙ حٍٛ GSH ٔALPٔاَخفبع فٙ MDAصٚبدِ فٙ يسخٕٖ MTXءاعطب سبب .MTXاٚبو بعذ اعطبء 5اٚبو لبم ٔ 7عٍ طشٚك انفى MTXيجًٕعت انخٙ نٕحظج فٙ انُسٛجٛت حغٛٛش حى ححسٍٛ يعبيهت .انخغٛٛشاث فٙ انًجًٕعّ انخٙ حهمج يسخخهض انسببَغ انًبئٙ اَعكسج ْزِ انخبثٛش انًشخشن انٗ MTXضذ حسًى انكبذ انخٙ ٚسببٓب انسببَخ انًبئٙ نًسخخهض ًٔٚكٍ أٌ ٚعضٖ انخأثٛش انٕلبئٙ ببعطبء يسخخهض انسببَغ انًبئٙ .ًكَٕبث أساق انسببَغ ن .انحاكسذي االجهاد ,ميثىجريكسيث , كىيرسيحيه , ، كايمبفيرول انسباوخ :انكهمات انمفحاحية Introduction Spinach is a leafy green vegetable that came originally from south western Asia and is now grown in most parts of the world. Scientifically it is known as Spinacia oleracea L. belonging to the family Chenopodiaceae (Figure1), locally known as ispanakh. (1) Spinach is packed with vitamins, minerals and a number of anti- oxidants components like polyphenols, flavonoids and carotinoids which are shown to possess anti- inflammatory effects, anti-mutagenic potential, anti-neoplastic effects, as well as chemo-preventive activities. (2, 3) 1 Corresponding author E- mail : farah77kais@yahoo.com Received : 26/2/2012 Accepted :15/5/2012 Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 9 Figure 1: Spinacia oleracea At least 13 different flavonoid compounds had been discovered by researchers in Spinach leaves (4) among these flavonoids, quercetin and kaempferol are the most important and wid'ely spread flavonol class (containing the typical C6-C3-C6 structure) (5) these flavonols provide the body with anti-oxidant protection, so they have significant influence against kidney damage, toxic liver damage, neurovascular complications and diabetic complications (6, 7) . Methotrexate (MTX), a folic acid antagonist is widely used as a cytotoxic chemotherapeutic agent; however its associated with hepatotoxicity which is considered to be a major clinical side-effect. (8) The toxicity of MTX in the liver seems to relate to the generation of reactive oxygen species (ROS) (9,10) .The present study deals with the investigation of phytochemicals found in the plant by ethanolic and aqueous spinach leaves extract and the possible protective effects of aqueous spinach extract against hepatic damage caused by methotrexate (MTX ) in rats. Materials and Methods Plant materials Fresh leaves of Spinach were purchased from vegetable markets of Baghdad; the plant was identified by the department of pharmacognacy, college of pharmacy/ university of Baghdad; and authenticated by the Herbarium of Baghdad University.100gram of fresh spinach leaves collected and dried under shade, powdered and divided into two parts: the first part, 50 grams of powdered leaves were packed in a thimble of soxhlet extractor. 100 ml of petroleum ether (40-60 ºC) was used in soxhlet for 3 hours to get rid of lipids and fats to give fraction 1 (F1). The defatted powder after drying over-night were extracted with 100 ml of 96% ethanol for 72 hours by soxhlet extractor to give fraction two (F2) as shown in (Figure 2) (11) . In the second part, 50 grams of powdered plant material was extracted with 100 ml of distilled water by cold maceration method, this step was repeated for three times to give fraction three (F3) as shown in (Figure 2) (12) .After that, the extracts (F1, F2 and F3) were filtrated and then concentrated under reduced pressure at a temperature not exceeding 40 0 C to give greenish colored residues. Figure 2: Schematic procedure for first and second parts of spinach leaves extraction (11, 12) Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 10 Phytochemical evaluation Phytochemical evaluation of spinach leaves were done by making general tests for fixed oils, glycosides, alkaloids, phenolic compound, tannins and flavonoids. 1. Test for fixed oils (13) Small quantities of various extracts (F1, F2 and F3) were separately pressed between two filter papers; the appearance of permanent spot oil on the paper indicates the presence of fixed oils. 2. Test for glycosides (14) a -Baljet’s test: 1ml from each extracts (F1, F2 and F3) + 1ml of picric acid make it alkaline with sodium hydroxide →orange color. b- Keller-Kiallian’s test: 1ml from extracts (F1,F2 and F3)+ 2ml of glacial acetic acid+ 1 drop of 0.1% ferric chloride solution → then add 1ml of H2SO4 (drop by drop) → the junction between two liquid layers indicates the presence of the sugar part of glycosides. c- Brontrager’s test: 1ml of F1 and F2 extract+1ml of diluted ammonia solution → pink color. While F3 was treated with chloroform and then chloroform layer was separated. To this equal amount of dilute ammonia solution added → pink color, showing the presence of glycosides. 3. Test for saponins (15) Each extract (F1, F2 and F3) was diluted with 10 ml of distilled water and it was agitated in test tube for 15seconds and standing for 15 minutes, the formation of not less than 1cm layer of foam shows the presence of saponins. 4. Test for alkaloids (16) A small portion of extracts (F1, F2 and F3) were tested with various reagents for the presence of alkaloids. Mayer’s reagent →cream precipitation. Dargendroff’s reagent→ orange brown precipitation. Wagner’s reagent →reddish brown precipitation. 5. Test for phenolic compounds and tannins (14) The tests for phenolic compounds and tannins were carried out with following reagents: a. 5% ferric chloride solution→ deep green or deep blue. b. 10% lead acetate solution →white precipitation. c. 1% potassium dichromate solution → orange precipitate. 6. Test for flavonoids (11) a. With aqueous sodium hydroxide solution Anthrocyanins → blue to violet color. Flavones and flavonol→ yellow color. Flavonones → yellow to orange color . b. With concentrated sulphuric acid Anthrocyanins → yellow orange color. Flavones and flavonol→ yellow to orange color. Flavonones → orange to crimson color. Identification of quercetin and kaempferol as example of flavonoids Spinach is loaded with flavonoids which act as antioxidants; identification of these flavonoids was performed by: 1. Identification of flavonoids (quercetin and kaempferol) by TLC: - (6) Using ready made aluminum plates of silica gel GF254, ultraviolet light detector at 254 nm wave length as detection method, standard flavonoids in comparison with three different solvent systems S1, S2 and S3. Standard flavonoids are: quercetin (Fluka-Austia) and kaempferol (Sigma- Aldrich, USA). Different developing solvent systems were:- S1=Chloroform: Acetone: Formic acid (75:16.5: 8.5) S2 = Chloroform: Methanol (90:10) S3 = Toluene: Chloroform: Acetone (40:25:35). 2- Identification of flavonoids in HPLC Further identification to the flavonoids in plant extracts (F2 and F3) were performed by HPLC. For qualitative estimation comparison of retention time obtained at identical chromatographic conditions of plant extract with authentic standards was done. Table 1: HPLC conditions (6) Sample Mobile phase Column Flow rate Detection Quercetin Acetonitrile: methanol : glacial acetic acid (70:30: 0.1) C18 5 mm x 150 mm 0.5 ml / min UV. Detector at 306 ג nm Kaempherol methanol : water ( 7.5 : 92.5 ) C18 ODS 1.5 ml / min UV. detector at 308 ג nm For quantitative estimation the following equation was used to calculate the percentage of the compounds in the plant: - (17) Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 11 Percentage of compound in the plant {( )} The weight of the plant used in the extraction was 50 gm AUC = Area under the curve. DF = Dilution factor. Conc. St. = Concentration of the standard used in HPLC. Pharmacological studies Preparation of aqueous spinach extract The roots of spinach were removed by sharp knife then the remaining portion was thoroughly washed several times with tap water and finally with distilled water and the water was removed by keeping on drying cabinet for 2 hours at room temperature. These were kept on a rectangular netted plastic basket for 2 hours to remove washing water. Then, the leaves and soft stalks were separated and chopped into small pieces and kept under an electric fan at room temperature then these were dried fully. The dried spinach was grinded with an electric grinder and then the grinded mass was sieved with 2mm wire sieve to get fine powder. Fifteen gram of spinach powder was weighed and thoroughly mixed with 1000 ml deionized distilled water and kept for 24 hours at room temperature. After that the solution was filtered and used. (18) Experimental protocol Eighteen white Albino rats of both sexes, weighing 200-250g were used in this study; the rats were obtained from and maintained in the animal house of the College of Pharmacy, University of Baghdad under conditions of controlled temperature. The animals were fed commercial pellets. The rats were divided into three groups: Group 1 - normal control rats. Group 2- MTX (20 mg/kg i.p) following a single dose of MTX saline will be administered for 5 days (19) . Group 3 – MTX(20 mg/kg i.p) + spinach extract 200 mg/kg body weight orally in drinking water daily 7 days prior to and 5 days after MTX administration. Biochemical estimations Determination of the serum concentrations of the liver enzymes AST, ALT, ALP and Bilirubin were measured in serum samples obtained from all groups of rats. Liver tissue homogenate was prepared by standard procedure (20) and the contents of malondialdehyde (MDA) (21) and reduced glutathione (GSH) (22) were analyzed in liver tissue homogenate. Microscopic evaluation For the light microscopic investigations, tissue specimens from the liver were fixed with 10% formaldehyde and processed routinely for embedding in paraffin. Tissue sections of 5 μm were stained with hematoxylin and eosin (H and E) (23) . Statistical analysis Results were expressed as mean ± SD (standard deviation). Using Student’s t-test, the level of statistical significance was set at P<0.05. Results The dried leaves of Spinacia oleraceae L. was divided into two parts, the first one defatted with petroleum ether and extracted with 96% ethanol by soxhlet apparatus while the second one extracted with distilled water by cold maceration method. The percentage yield of plant extracts was as following: Petroleum ether → 1.2% Ethanol→ 5.5% Aqueous → 6.25% Preliminary phytochemical evaluation The various extract of dried leaves were subjected for phytochemical screening which shows the presence of different compounds in plant extracts as shown in (Table 2). Table 2 : Preliminary phytochemical evaluation of dried leaves of Spinacia oleraceae. Active constituents Tests F1 F2 F3 Fixed oil Spot test + - - Glycosides Baljet’s test Keller kiallan’s test Brontrager’s test - - - + + + + + + Saponins Foam test - + + Alkaloids Mayer’s test Dragendroff’s test Wagner’s test - - - - - - - - - Phenolic compounds and tannins FeCl3 test Lead acetate test Potassium dichromate test - - - + + + + + + Flavonoids Dil. NaOH solution Conc. H2SO4 - - + + + + Where + =present ; - =absent Identification of flavonoids by TLC TLC of the extracts (F1, F2 and F3) obtained from dried leaves of Iraqi spinach Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 12 confirms the presence of quercetin and kaempferol in F2 and F3 only but not in F1 when comparison was made with standards, as represented in (Table 3) and (Figures 3, 4 and 5). Table 3 : Rf value of plant extracts (F2 and F3), standard quercetin and kaempferol. S1 S2 S3 Solvent system 0.5 0.42 0.72 Rf value of quercetin in F2 0.49 0.41 0.71 Rf value of quercetin in F3 0.48 0.4 0.7 Rfvalue of kaempferol standard 0.7 0.6 0.82 Rf value of kaempferol F2 0.72 0.61 0.84 Rf value of kaempferol F3 0.71 0.59 0.83 S1=Chloroform: Acetone: Formic acid (75:16.5:8.5) S2 = Chloroform: Methanol (90:10) S3 = Toluene: Chloroform: Acetone (40:25:35). Q Aq Alc Pet K Figure 3 : TLC plate of spinach leaves extracts and standard, using S1 mobile phase K Pet Alc Aq Q Figure 4: TLC plate of spinach leaves extracts and standard, using S2 mobile phase Aq K Q Pet Alc Figure 5: TLC plate of spinach leaves extracts and standard, using S3 mobile phase Where Q=quercetin standard, K= kaempferol standard, Pet=petroleum ether extract, Alc=alcoholic extract and Aq=aqueous extract. Identification of flavonoids by HPLC Further identification to the quercetin and kaempferol in plant extracts (F2 and F3) were performed by HPLC in which the retention time of both standards (quercetine and kaempferol) and the plant extracts (F2 and F3) were identical as represented in the figures bellow: Figure 6: HPLC analysis of kaempferol standard Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 13 Figure 7: HPLC analysis of aqueous extract of Iraqi spinach leaves Figure 8: HPLC analysis of alcoholic extract of Iraqi spinach leaves Figure 9 : HPLC analysis of quercetin standard Figure 10 : HPLC analysis of alcoholic extract of Iraqi spinach leaves Figure 11: HPLC analysis of aqueous extract of Iraqi spinach leaves For quantitative estimation, the percentage of quercetin in both F2 and F3 were higher than the percentage of kaempferol in the same extract, as shown in table (4). Table 4: data showing extractive values (perecentage w/w ) of quercetin and kaempferol in Iraqi spinach leaves Extraction solvent quercetin kampferol Ethanolic extract 0.268 % 0.12 % Aqueous extract 0.149 % 0.096 % Biochemical parameters As shown in table 5, the level of MDA, a major degradation product of lipid peroxidation, was found to be significantly higher and level of GSH was significantly decreased in the MTX- Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 14 treated group when compared with control group (p < 0.05).Methotrexate cause no significant change in AST, ALT and bilirubin level (p > 0.05) and decrease in ALP level compared to control group (p < 0.05).While Rats treated with an oral concentrations (200 mg / kg) of Spinach extract 7 days prior to and 5 days after single i.p of methotrexate (20 mg/kg) resulted in significant decrease in MDA and increase GSH and ALP (p < 0.05) and produce no significant change in bilirubin, ALT and AST compared to methotrexate group (p > 0.05).In the livers of rats from the control-treated rats, hepatocytes showed a normal histological appearance (Figure12). In the MTX- treated rats, showed rare scattered dysplastic hepatocytic changes, periportal inflammatory cell infiltration and sinusoidal dilatation were observed (Figure 13 and 14) , while in the MTX+ aqueous spinach extract treated rats affected to a significantly less degree than those in the group given MTX alone showed minimal periportal lymphocytic infiltration and lesser sinusoidal dilatation(Figure 15). Table 5: Effect of methotrexate and aqueous spinach extract on liver biochemical parameters, MDA and GSH of rats Control MTX MTX+ spinach AST(U/L) 105.7 ± 19.05 121.67 ±17.45 118.67 ± 40.1 ALT(U/L) 64.65 ± 24.32 45.83 ± 6.31 73.5 ± 23.25 ** Alp (U/L) 519 ± 93.92 185.33 ± 68.85 * 339.5 ±179.82 ** Bilirubin (U.Mol/L) 0.54 ± 0.11 0.61 ± 0.02 0.61 ± 0.12 MDA (nmol/gtissue) 1522.22 ± 277.08 4386.96 ±508.72 * 3069.01 ± 244.7 ** GSH (µmol/g tissue) 38.87 ± 4.28 29.21 ±1.93 * 35 ± 2.23 ** Data are presented as mean ± SD. n= number of animals. *P<0.05 with respect to control group. **P<0.05 with respect to methotrexate group. Figure 12: Normal histological appearance of liver in control group (H and E X 200) Figure 13 : Rare scattered dysplastic hepatocytic changes in methotrexate treated group (H&E x 400) Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 15 Figure 14: Periportal inflammatory cell infiltration (→)and sinusoidal dilatation (→)in methotrexate treated group (H&E x 200) Figure 15: minimal peiportal lymphocytic infiltration and sinusoidal dilatation (→)in MTX + aqueous spinach extract treated group (H&E x 200) Discussion Preliminary phytochemical studies showed the presence of flavonoids, glycosides, tannins, saponins and polyphenolic compounds on both ethanolic and aqueous extracts. Identification of quercetin and kaempferol as examples to flavonoids by TLC and HPLC indicates the presence of these flavonoids and give the results that the percentage of quercetin in Iraqi spinach leaves is more than the percentage of kaempferol in the same plant.The study examined the role of oxidative stress in an attempt to suggest a possible mechanism of MTX-inducd hepatotoxicity in a rodent model.The results of the studies indicate that MTX causes oxidative tissue damage by increasing lipid peroxidation in the liver tissue and decreasing the level of GSH. These results were consistent with other studied. (24, 25) The increased MDA levels as observed in our study can be attributed to the lipid peroxidation can be induced by MTX itself or as a result of a possible increase in ROS induced by MTX . (.26) The ROS thus formed may lead to cellular damage by peroxidation of membrane lipids, sulfhydryl enzyme inactivation, protein cross-linking and DNA breakdown . (27,25 ) It has been shown that treatment with MTX leads to a reduction in the effectiveness of antioxidant defense systems and that cellular levels of glutathione are reduced by MTX . (26) Although statistically no significant differences in AST was observed, ALT and bilirubin level in methotrexate treated group and this result are consistent with other studies. (25, 28) this may be due to administration of methotrexate for short duration or may need high dose to produce change in the level of these enzymes but the level of ALP decreased. This reduction could be explained by the noticeable damage that affected the endothelial cells lining the blood sinusoids and blood vessels in the portal tracts, which are considered to be the main sites at which the ALP acts. (29) Previous studies have shown that many antioxidants, including N- acetylcysteine (30) and grape seed extract (31) have protective effects in the MTX injury of the liver in rat. At microscopic level, a single dose of MTX resulted in significant liver injury. The mechanism by which MTX causes hepatotoxicity due to its binding to the enzyme dihydrofolic reductase, thus preventing conversion of folic acid to its active form, folinic acid. This in turn blocks the synthesis of nucleic acids, certain amino acids and indirectly proteins which lead to damage of organelles and plasma membranes of hepatic parenchymal cells interfering with their function (32) . Aqueous spinach extract supplementation reduced MTX-induced oxidative liver damage these data are in accordance with the findings of a recent study which demonstrated that spinach leaves contain natural antioxidant system which provide free radical scavenger properties and hepatic protection against CCL 4 (33) and radiation (34) induced by oxidative stress . Conclusion Phytochemical investigation of the aqueous and ethanolic extracts of leaves of Spinacia oleraceae showed the presence of flavanoids, glycosides, saponins, tannins, phenolic compounds and treatment of rats with aqueous spinach extract before and after MTX application provided significant protection from the hepatotoxicity of MTX could be attributed to the combined effects of its constituents as the leaves are rich in flavonoids and p-coumaric acid. References Iraqi J Pharm Sci, Vol.21(2) 2012 Study of Iraqi spinach leaves 16 1. Subhash G p, Virbhadrapp SR. and Vasant O K. Spinacia oleracea Linn: A pharmacognostic and pharmacological overview. IJRAP. 2010; 1:78-84. 2. Boivin D., Lamy S., Lord-Dufour S., Jackson J., Beaulieu E. and Cote M., Moghrabi A, Barrette S, Ginras D and Beliveau R Antiproliferative and antioxidant activities of common vegetables: Acomparative study. Food Chem. 2009; 112: 374–380. 3. Hait-Darshan R., Grossman S., Bergman M., Deutsch M., and Zurgil, N. 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