Iraqi Journal Of Pharmaceutical Sciences Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 16 Hepatoprotective Effect of Echinops tenuisectus (Compositae) on CCl4 Induced Hepatic Damage in Rats Munaf H. Abdulrazzaq* , Enas J. Khadeem** , Suhad S. Al- Muhammadi** *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 Flavonoids are known to play a vital role in the management of various liver disorders.They are a large family of compounds synthesized by plants; they belong to a group of natural substances with variable phenolic structures. In this study we aim to scan the types of flavonoids in a newly studied, wild Iraqi plant named Echinops tenuisectus of Compositae family. The medicinal importance of flavonoids on one hand, and the absence of any phytochemical investigation on tenuisectus species of Echinops genus on the other hand, acquired this study itۥs importance. Three flavonoids were identified in the seed ,s extract of this plant (Silymarin, Rutin, Quercetin ) by two chromatographic methods, first Thin layer chromatography (TLC) using TLC ready made GF254 plates, UV detector at 254 nm, and two different solvent systems in which the Rf value of the standards (Silymarine, Rutin, Quercetin) matched with the Rf value of the Silymarin, Rutin and Quercetin found in the plant seed ٫s extract. High pressure liquid chromatography (HPLC) was the other chromatographic method that used to identify the presence of these flavonoids in the plant seed. The plant seed sۥ aqueous extract was evaluated for its efficacy in rats by inducing hepatotoxicity with CCl4.Single oral dose of 250mg/kg of Seeds Extract was given to ratۥs for 7 days. Serum activities of transaminases (ALT and AST) were used as the biochemical marker of hepatotoxicity. Histopathological changes in ratُُs liver section were also examined. The results of the study indicated that, the pretreatment of rats with Echinops extract before the hepatotoxins agent (CCl4) offered a hepato- protective action. Key words: Echinops, Flavonoids ةصالخلا ريلافهمعنمرنا راثال واد ا ا هعلافمادا يولا ا مهه اريفبهىادواردىرياريفلم .ري مفادوابهواريمورولابعاتفتاديوايفاىفلا را يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدعرراريفلورللادواريلافهمعنمرنافما يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدلولاتىرفهلاعمنمفاي ا موعاو أل . يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدهىراييبفهلاريبلهلايرلافهمعنمرنادواع لفا تملا ععلاألا دمكعورناترفهلا وم اريففع يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةد ناريفهفه هلاي هواريملولفاألهنابهواريمورولاأبفهو . اأموك فاتاناأ يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدعررادواريلافهمعنمرنافما ديولرلاريله وايرمل نا)رييرهف ونوفاو هوفامعروووهو( أعروبلاوىنلوهوادواوى اريفى د عاىرفه فارا يذابما لمهلا اأ يبع اU.Vا م نارا الافع اريلمليوهلاGF254انرناريمعتهلاTLCاأئوولمرلاوف با(TLC)مى د عاىرفه اريبلللاريىفهلل ايرلافهمعنمرناريفعععلفافماRf ايرلافهمعنمرناريله وهلاو ألاافهفلRfا تاتلادث يهتا يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةد فرلادلورللفا هحاأةافهفلا254nmريفععما اريوماأممنا ععلاريلافهمعنمرنافماريفيولرلاريمل ما(HPLC)ريفيولرلاريمل م .ت اوىنللامى د عاىرفه ا ثااريلا اريا يما ايفتادواريلافهمعنمرناريله وهلا اريلافهمعنمرنافماريفيولرلاريمل م . ا لهه اريلا يهلاريااعهلاي هواريملولاretention timesأوب أبا ادرل /مرا ا250ا هحا ارتب اريفيولرلاريف ماتواوىنباريل اأوىتلافموب اCCl4ترذاملماريلاىرةاريوما ارووثمرت اأعروبلا اريهلانلهواريلا يهلاريعف هلا ريااعهلاي هواريملولافماردىرياريفلم .ASTا ALT يفمفاولالاأن لا افه عاديوععارا يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةددن ا Introduction The Echinops tenuisectus belong to the Family Compositae (Fig1) is a wild, Iraqi plant first studied in Iraq. The Echinops genus consist of 100 spp.(1) which are widely distributed in Sharaban, Diyalah, Badrah ((Upper Tigris Plain))(2) . Preliminary investigation indicated that, this plant contain different types of flavonoids in accepted amount. Among these flavonoids: Silymarin (figure 2) which is a flavonolignan that has been introduced fairly recently as a hepatoprotective agent(3,4,5,6,7). Silymarin has been found to provide hepatoprotection through its antioxidants properties (scavengers and regulators of the intracellular content of glutathione)(8,9,10) , as cell membrane stabilizers and permeability regulators that prevent hepatotoxic agents from entering hepatocytes(11,12). Also as inhibitors of the transformation of satellite hepatocytes in to myofibroblasts, the process responsible for the deposition of collagen fibers leading to cirrhosis (13, 14, 15). 1 Corresponding author : E-mail : enassara@yahoo.com Received : 21/11/2007 Accepted : 24 /5 /2008 Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 17 Figure 1: Photography of Echinops tenuisectus Figure 2: Structure of silybin (commercially called silymarin) The other flavonoids found in this plant are the Quercetin and Rutin (Figure 3,4), both of them possess a powerful antioxidant activity which help to prevent free radical oxidative damage to cells, also help in the treatment and prevention of alcohol and chemical - induced hepatotoxicity by increase glutathione in the liver(16). Glutathione responsible for detoxifying a wide range of hormones, drugs, and chemicals. High level of glutathione in the liver increases its capacity for detoxification. Quercetin and Rutin increase the level of the important antioxidant enzyme superoxide dismutase in the cell cultures (17). In addition they stimulate protein synthesis in the liver, which results in an increase in the production of new liver cells to replace the damaged one (18). Shoskes 1999 demonstrate that Quercetin and Rutin also inhibit the synthesis of leukotrienes (mediators of inflammation, which can result in psoriasis) (19).Recently, flavonoids can help in prevention of cancer through several pathway: inhibiting proliferation and inducing apotosis(20,21)or through inhibiting tumor invasion and angiogenesis(22,23). This wide variety of beneficial health effects of these flavonoids acquired this study its importance in finding a new uninvestigated source of these important flavonoids , contained within Echinops tenuisectus of the Family Compositae and evaluate their possible protective effect against the experimentally- induced liver damage in rats by CCl4 . Liver, the largest organ in vertebrate body, is the major site of intense metabolic activities. Liver injury caused by toxic chemicals and certain drugs has been recognized as a toxicological problem. Herbal drugs are playing an important role in health care programs world wide, and there is a resurgence of interest in herbal medicines for treatment of various ailments including hepatopathy(24). CCl4 is reported to produce free radicals which affect the cellular permeability of hepatocytes and it causes massive histopathological changes like necrosis, congested vessel and fatty changes (steatosis). So, the reverse of these phenomenon can be considered as the index of hepatoprotective(25). Figure 3: Quercetin; R = H Figure 4: Rutin ; R= rhamno-glucosyl Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 18 Materials and Methods A. Plant materials: The plant material was collected during July 2005 From Sharaban,Dyala city. The plant was identified by the Department of Pharmacognosy, College of Pharmacy /University of Baghdad; and authenticated by the Herbarium of Baghdad University. Fifty grams of the powdered plant material (seed part) were first defatted by reflux with 100 ml of petroleum ether (60º-80ºC) for one hour and filtered. The defatted dried plant material was then extracted by reflux using 100 ml of 70% ethanol for three hours.This step was repeated for four times, then the combined filtrates were evaporated under reduced pressure using Buchi rotatory evaporator attached to vacuum pump at 40ºC, to a thick residue of ethanol extract (F1). This residue was then hydrolyzed with 2N HCl in aqueous methanol (1:1) under reflux for three hours; the resultant solution was then partitioned with 100 ml of ethyl acetate (F2). This fraction was evaporated under reduced pressure to dryness, as shown in the following diagram (Figure 5). Echinops plant (50gm.) seed part Defatted with Petroleum ether (60ºC -80ºC) Using reflux for 1hr. Filtrate Residual plant part Extract with 70% ethanol Using reflux for 3 hr. Marc Ethanolic Filtrate Evaporate to thick liquid F1 1. Hydrolyzed with 2N HCL In aqueous methanol (1:1) 2. Partition with 100ml Ethyl acetate F2 (27) gm contain Flavonoids Figure-5 [Schematic representation of flavonoids extraction From Echinops tenuisectus ] F2 (ethyl acetate fraction)ا→اevaporationاtoاdrynessاunderاreduceاpressureا→اblack- greenish oily residue, TLC and HPLC indicated that this fraction contain three compounds which are silymarin, rutin and quercetin and by preparative thin layer chromatography and HPLC we can separate each one and calculate the percentage of each one by weighting. F2ا(oilyاresidueاfraction)ا→اdissolvedاinاwaterا→اsuspentionا(readyاforاhepato- protective study) Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 19 B. Identification of Silymarin,Quercetin and Rutin in the plant seed extract. The Identification of these flavonoids in the seed extract was performed by: 1. Identification of Flavonoids by TLC: Using TLC ready made Gf254 plates, UV detector at 254 nm, Standard flavonoids and two different solvent systems that were (26) : Solvent (1): chloroform: acetone: formic acid (75:16.5:8.5) (Figure 6) Solvent (2): n.butanol: glacial acetic acid: water (40: 10:50) (Figure 7) (Table-1) showed the Rf values of the standards Silymarin, Quercetin and Rutin, and the Rf value of plant seed part extract. B D C A Figure 6: TLC Gf254 plate of the seed extract and standards using S1 mobile phase. A Plant seed extract C Quercetin standB Silymarin standard D Rutin standard A C B D Figure 7: TLC Gf254 plate of the seed extract and standards using S2 mobile Phase A Plant seed extract C Quercetin standard B Silymarin standard D Rutin standard Table 1: Rf values of standard silymarin, rutin and quercetin and seed extract. Solvent system Standard silymarin Standard Quercetin Standard Rutin Seed extract S1 0.43 0.35 0.28 0.4,0.33,0.2 S2 0.2 0.81 0.56 0.21,0.8,0.5 Figure 8: HPLC of plant seed extract of Echinops tenuisectus. Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 20 Figure 9: HPLC of standard Silymarin. Figure 10: HPLC of standard Quercetin. Figure 11: HPLC of standard Rutin. Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 21 2. Identification of Flavonoids by HPLC: Silymarin, Quercetin and Rutin were authenticated by HPLC . (Figures 8-11) The HPLC conditions are listed in the following table. (Table-2) Table 2: HPLC conditions. HPLC Conditions Mobile phase Methanol:water (50:50) Column C18 25cm Flow rate 1ml/min Detector 288 nm C. Hepatoprotective studies: 1. Experimental animals: Eighteen – Albino rats of both sexes weighting 150-200 gm (both sex) were used in this study. Animals were kept in the animal house of the College of Pharmacy/ University of Baghdad, under standardized condition (12 hr light dark cycle at room temperature). The animals were fed standard chow and given water ad libitum. 2. Experimental design: The animals were divided in to three groups (each group have 6 animals) and treated as follows: Group (1): Six rats received normal saline for 7 day orally and secreted at along 7, saved as control Group (2): Six rats received single oral dose of CCl4 (1mg/kg) diluted by corn oil in ratio of 1:1 v/v for the induction of liver damage and animals were sacrificed after 24 hr of CCl4 administration. Group (3): Six rats received oral dose of the seed extract of Echinops tenuisectus Plant in amount equivalent to 250mg/kg by gavages tube for 7 days, befor CCl4 (1mg/Kg diluted by corn oil in a ratio of 1:1 v/v), then the rats were sacrificed after 24 hr, after CCl4 administration. 3. Biochemical estimation: Serum was prepared from the collected blood and subjected to biochemical estimation of ALT and AST. 4. Histopathology: Portion of liver tissue in each group was fixed in 10% formalin (Formalin diluted to 10% with normal saline) and proceeded for histopathology. After paraffin embedding and block making, serial section of 5µ thickness were made, stained with Haematoxylin and Eosin and examined under microscope. 5. Statistical analysis: The significance of difference between the mean values was calculated using unpaired student's t-test. P-value less than 0.05 were considered significant for all data showed in our results. 6. Results: A) Biochemical parameters: Table-3 showed a significant elevation in the activities of both ALT and AST in CCl4- treated rats compared to control group. Pre- treatment rats with seed extract of Echinops tenuisectus (250mg/kg) showed a significant decline in the activities of ALT and AST compared with CCl4 treated rats (Table 3 , Figure 12 and 13). Table 3: Effects of seed extract of Echinops tenuisectus on the activities of serum ALT and AST in rats treated with CCl4. - Each value represents Mean ± standard deviation. - Values with non=identical superscripts (a,b) within each parameter are significantly different ( P< 0.05) - N= Number of animals. Control CCl4 Seed extract +CCl4 (a) Figure 12: Bar chart comparing the effects of seed extract of Echinops tenuisectus pre- treated with CCl4 on serum ALT activity. Treatment Serum ALT U/L Serum AST U/L Control N=6 10.24±1.21 45±3.8 CCl4-treated N=6 64.4±7.53 a 68.6±1.67a Seed extract + CCl4 N=6 13.6±1.34 54.4±3.28b 0 10 20 30 40 50 60 70 80 Control CCl4 Seed extract +CCL4 A L T a c ti v it y U /L Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 22 Control CCl4 Seed extract +CCl4 (a) (b) Figure 13: Bar chart comparing the effects of seed extract of Echinops tenuisectus pre- treated with CCl4 on serum AST activity. B) Histological examination: Histological examination of ratُs liver treated with CCl4 showed that, there was centrilobular hemorrhage, with heavy inflammation and necrosis. In addition to steatosis and individual necrosis were observed compared with control (Figure 14 and 15). Pre-treatment of rats with seed extract of Echinops tenuisectus before CCl4, exhibit variable degrees of recovery with slight centrilobular congestion, marked reduction in inflammatory reaction. Furthermore, neither necrosis nor steatosis was observed in ratۥs liver section (Figure 16). Figure 14: Section showing normal rat's liver. Magnification: 40X, staining: haematoxylline and eosin. Figure 15: Section showing morphological alteration of liver from CCl4-treated rats. Black arrow represents fatty changes (steatosis), white arrow represent haemorrhage. Magnification: 40X, staining: haematoxylin and eosin. Figure 16: Section showing the administration of seed extract of Echinops tenuisectus improved CCl4-induced hepatic damage. Magnification: 40X, staining haematoxylin and eosin. Discussion: Many compounds exhibit hepatoprotective activity, demonstrated either by decreasing the harmful effect of hepatotoxic compound or by maintaining the normal hepatic physiology. The present study showed that, the seed extract of Echinops tenuisectus have good hepatoprotective effect against CCl4-induced hepatotoxicity in rat manifested by attenuating the increases in the serum activities of ALT 0 10 20 30 40 50 60 70 80 Control CCl4 Seed extract +CCL4 A S T a c ti v it y U /L Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Hepatoprotective effect of Echinops flavonoids 23 and AST (Table 3, Figure 12and13) and by reversing the histological damage induced by CCl4, this was attributed to the presence of flavonoids, especially the silymarin, rutin and quercetine which possess antioxidant properties (8,16) which can improve the normal physiology of hepatocyte (17,18,19). Conclusion The present study showed that, seed extract of Echinops tenuisectus improve the hepatic damage and steatosis induced by CCl4 toxicity. Acknowledgment The authors gratefully thank Prof. Dr. Ali- Al- Mussawi for supporting this project. References: 1. 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