HEPATOPROTECTIVE EFFECTS OF GINKGO BILOBA AGAINST CARBON TETRACHLORIDE INDUCED HEPATIC INJURY IN RATS

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1 Indin Journl of Phrmcology 21; 33: RESEARCH PAPER K. ASHOK SHENOY et l. HEPATOPROTECTIVE EFFECTS OF GINKGO BILOBA AGAINST CARBON TETRACHLORIDE INDUCED HEPATIC INJURY IN RATS K. ASHOK SHENOY*, S. N. SOMAYAJI**, K. L. BAIRY* * Deprtment of Phrmcology, Kstur Medicl College, Mnipl, Krntk. ** Deprtment of Antomy, Interntionl Centre for Helth Sciences, Mnipl, Krntk. Mnuscript Received: Revised: Accepted: SUMMARY Ojectives: To ssess the protective ctivity of Ginkgo ilo (GB) ginst CCl4 induced heptotoxicity in rts nd proe into its mechnism of ction. Methods: Liver dmge ws induced in Wistr rts y dministering (15-25 g) CCl4 (.5 ml/kg, i.p.) once dily for 7 dys. GB (5 mg/kg, i.p.) ws given for one week. Silymrin (2 mg/kg, p.o.) ws given s reference drug. Levels of mrker enzymes (AST, ALT, ALP) nd totl proteins (TP), lumin (Al) were estimted in serum. A proe into the mechnism of ction ws ttempted y estimting thiorituric cid rective sustnces (TBARS) nd glutthione (GSH) levels in liver homogentes in order to evlute the degree of lipid peroxidtion. Histopthologicl studies were lso done to confirm the iochemicl chnges. Results: The men + SEM serum AST, ALT, ALP levels in control nimls were , nd IU/L respectively wheres in CCl 4 treted rts, the level rose to , nd IU/L respectively. GB reduced the AST, ALT nd ALP levels to , nd IU/L respectively. Silymrin reduced AST, ALT nd ALP levels to , nd IU/L respectively. There ws significnt decrese in serum TP nd Al levels fter CCl 4, which ws reversed y GB nd silymrin. The tissue men + SEM vlues of TBARS nd GSH in control nimls were nmol of mlondildehyde/g of wet tissue nd mg/g of wet tissue respectively. In CCl 4 treted nimls, the TBARS nd GSH levels were nd respectively. GB reduced TBARS to nmol of MDA/g of wet tissue nd incresed GSH level to mg/g of wet tissue. Silymrin reduced TBARS to nmol of MDA/g of wet tissue nd incresed GSH level to mg/g of wet tissue. Conclusion: GB hs protected the liver from CCl 4 dmge. Prole mechnism of ction is y protection ginst oxidtive dmge produced y CCl 4. KEY WORDS Heptoprotective effect Ginkgo ilo cron tetrchloride silymrin mlondildehyde glutthione INTRODUCTION The Ginkgo ilo (GB) tree, lso known s midenhir tree is the sole representtive of once flourishing otnicl division, tht of the so clled Ginkgophytes. The leves re recommended s eing eneficil to the hert nd lungs; inhltion of decoction of leves is used in ronchil sthm 1. Extrcts of the leves of GB hve een used for cererl insufficiency due to degenertive or vsculr cuses, to improve lerning nd memory, peripherl vsculr diseses, s crdioprotective nd mny other diseses 2-4. GB exhiits vriety of interesting phrmcologicl properties such s oxygen free rdicl scvenging ctivity, cyclonucleotide phosphodiesterse inhiition, memrne stilising effect, increse in lood fluidity nd improvement in cognitive function 2,3,5,6. Among the vrious mechnisms involved in heptotoxic effect of cron tetrchloride, one is oxidtive dmge through free rdicl genertion 7,8 nd ntioxidnt property is climed to e one of the mechnisms of heptoprotective effect of indigenous Correspondence: K.L. Biry e-mil: office-mhe@mnipl.edu

2 HEPATOPROTECTIVE EFFECTS OF GINKGO BILOBA 261 sustnces. GB hs ntioxidnt properties 4,5,9-12. Hence the ojective of the study ws to evlute the effect of GB on CCl 4 induced heptotoxicity. MATERIALS AND METHODS Drugs nd chemicls: Ginkgo ilo dry extrct (contining.24 mg of ginkgoflvonglycosides/g of dry extrct) ws kind gift from Rnxy Lortories, Delhi. Cron tetrchloride ws otined from S.D. Fine-Chem Ltd., Boisr. Silymrin ws otined from Inden s.p.., Miln, Itly. Thiorituric cid (TBA), 5-5'- Dithio-is 2-Nitroenzoic cid (DTNB) nd Glutthione (GSH) were otined from Sigm Chemicl Compny, St. Louis, MO, USA. All other chemicls were otined from locl sources nd were of nlyticl grde. Animls: Twenty-four loclly red dult mle lino rts (15-25 g) of Wistr strin were used. They were housed in clen polypropylene cges nd fed with commercil pelleted rt chow (M/S Hindustn Lever Ltd., Mumi) nd wter d liitum. Before strting the experiment, permission from Institutionl Animl Ethics Committee ws otined. Experimentl procedure: A totl of 24 nimls were eqully divided into 4 groups (n=6 in ech group). Group I, which served s norml control, received distilled wter intrperitonelly (i.p.). Group II received CCl 4.5 ml/kg, i.p. once dily for 7 dys 13. Group III received CCl4.5 ml/kg, i.p. nd GB 5 mg/kg, i.p. 14 simultneously for 7 dys. Group IV received CCl4.5 ml/kg, i.p. nd silymrin 2 mg/kg, p.o. 15 simultneously for 7 dys. At the end of the tretment, lood smples were collected y direct crdic puncture nd the serum ws used for the ssy of mrker enzymes viz., sprtte minotrnferse (AST), lnine minotrnferse (ALT), lkline phosphtse (ALP) nd totl proteins (TP) nd lumin (Al). The rts were scrificed y cervicl disloction, the livers were removed immeditely, wshed with ice-cold sline nd 5% homogente prepred in.5m sodium phosphte uffer (ph 7.). The homogente ws centrifuged t 7 x g for 1 minutes t 4 o C nd the superntnt ws used for the estimtion of mlondildehyde (MDA), the end product of lipid peroxidtion nd glutthione (GSH). Enzyme ssys: The ctivities of serum heptic mrker enzymes nmely sprtte minotrnsferse (AST), lnine minotrnsferse (ALT) nd lkline phosphtse (ALP) were ssyed in serum using stndrd kits from Lupin Lortories nd Pointe Scientific respectively. The results were expressed s units/litre (IU/L). Protein estimtion: The levels of TP nd Al were estimted in serum of experimentl nimls y iuret method nd romcresol green method respectively 16. Stndrd kits from Rnxy Lortories, Delhi were used for these estimtions. Lipid Peroxidtion: The quntittive mesurement of lipid peroxidtion ws done y mesuring the concentrtion of TBARS in liver using the method of Ygi 17. The mount of mlondildehyde (MDA) formed ws quntitted y rection with Thiorituric cid (TBA) nd used s n index of lipid peroxidtion. The results were expressed s nmol of MDA/g of wet tissue using molr extinction coefficient of the chromophore (1.56 x 1-5 M -1 cm -1 ). Glutthione estimtion: GSH ws estimted in the liver homogente using DTNB y the method of Buetler 18. The sornce ws red t 412 nm nd the results were expressed s mg GSH/g of wet tissue. Histopthologic exmintion: Animls were scrificed on the dy of withdrwl of lood nd liver ws removed, sliced nd wshed in sline. Liver pieces were preserved in 1% formldehyde solution for histopthologic study. The pieces of liver were processed nd emedded in prffin wx. Sections mde were out 4-6 µm in thickness. They were stined with hemtoxylin nd eosin nd photogrphed. Sttisticl nlysis: The sttisticl nlysis were crried out y One-wy Anlysis of Vrince (ANOVA). P vlues <.5 were considered significnt. RESULTS Biochemicl prmeters: There ws significnt (p<.5) increse in the serum heptic enzyme levels nd significnt (p<.5) decrese in the TP nd Al levels fter CCl 4, which ws reversed with GB (Figures 1 nd 2). These effects were comprle to silymrin. In order to proe the possile mechnism y which GB prevents heptic dmge cused y CCl 4,

3 262 K. ASHOK SHENOY et l. Figure 1. Effect of Ginkgo ilo (GB) nd silymrin on serum mrker enzymes in CCl 4 induced heptic dmge (men + SEM). n = 6 in ech group. IU/L I (Control) II (CCl4) III (CCl4 + GB) IV (CCl4 + Silymrin) p<.5 Vs Group I p<.5 Vs Group II n=6 P <.5 Vs Group I P <.5 Vs Group II AST ALT ALP Figure 2. Effect of Ginkgo ilo nd silymrin on serum totl proteins (TP) nd lumin (Al) in CCl 4 induced liver dmge (men + SEM). n = 6 in ech group. g/dl I (Control) II (CCl4) III (CCl4 + GB) IV (CCl4 + Silymrin) p<.5 Vs Group I p<.5 Vs Group II n=6 P <.5 Vs Group I P <.5 Vs Group II TP Al

4 HEPATOPROTECTIVE EFFECTS OF GINKGO BILOBA 263 Figure 3. Effect of Ginkgo ilo on thiorituric cid rective sustnces (TBARS) levels in liver in CCl 4 induced liver dmge (men + SEM). n = 6 in ech group. 5 nmol of MDA/g of wet tissue TBARS I (Control) II (CCl4) III (CCl4 + GB) IV (CCl4 + Silymrin) p<.5 Vs Group II n =6 P <.5 Vs Group II Figure 4. Effect of Ginkgo ilo on glutthione (GSH) levels in liver in CCl 4 induced liver dmge (men + SEM). n = 6 in ech group. 3 GSH (mg/g of wet tissue GSH I (Control) II (CCl4) III (CCl4 + GB) IV (CCl4 + Silymrin) p<.5 P <.5 Vs Group Vs II Group II n=6

5 264 K. ASHOK SHENOY et l. Figure 5. Liver tissue of control rts showing norml histology. Figure 6. Liver tissue of CCl 4 treted nimls showing hydropic chnges nd stetosis. Congestion of centrl vein nd sinusoids nd mild portl chronic inflmmtory cell infiltrte is seen. Figure 7. Liver tissue of rts treted with CCl 4 nd GB showing lmost norml histology. There is no evidence of prenchyml injury. Figure 8. Liver tissue of rts treted with CCl 4 nd silymrin showing lmost norml histology. There is no evidence of prenchyml injury. investigtion on levels of TBARS nd glutthione were crried out. TBARS were found to e elevted fter the dministrtion of CCl 4, which ws significntly (p<.5) reversed y GB (Figure 3). There ws significnt (p<.5) rise in GSH content of liver fter tretment with GB (Figure 4). The effects of GB were comprle to tht of silymrin. Histopthologic exmintion: In control nimls, liver sections showed norml heptic cells with wellpreserved cytoplsm, prominent nucleus nd nucleo- lus nd centrl vein (Figure 5). In CCl 4 treted nimls the sections showed hydropic chnges in centriloulr heptocytes with single cell necrosis surrounded y neutrophils. Congestion of centrl vein nd sinusoids were seen with cute nd chronic inflmmtory cells infiltrting sinusoids minly in the centrl zone. The midzonl nd periportl heptocytes showed mild to moderte degree of ftty chnge (Figure 6). Pretretment with GB showed mild ftty chnge nd mild sinusoidl congestion

6 HEPATOPROTECTIVE EFFECTS OF GINKGO BILOBA 265 (Figure 7). Pretretment of the nimls with silymrin showed mild centrl venous congestion nd mild ftty vcuoltion (Figure 8). DISCUSSION Cron tetrchloride is one of the most commonly used heptotoxins in the experimentl study of liver diseses 19. The heptotoxic effects of CCl 4 re lrgely due to its ctive metolite, trichloromethyl rdicl 19,2. These ctivted rdicls ind covlently to the mcromolecules nd induce peroxidtive degrdtion of memrne lipids of endoplsmic reticulum rich in polyunsturted ftty cids. This leds to the formtion of lipid peroxides, which in turn give products like mlondildehyde (MDA) tht cuse dmge to the memrne. This lipid peroxidtive degrdtion of iomemrnes is one of the principl cuses of heptotoxicity of CCl 4 21,22. This is evidenced y n elevtion in the serum mrker enzymes nmely AST, ALT nd ALP. GB hs significntly reduced these liver enzyme levels. Further, GB hs incresed the level of totl proteins nd lumin in the serum, which indictes heptoprotective ctivity. Stimultion of protein synthesis hs een dvnced s contriutory heptoprotective mechnism, which ccelertes the regenertion process nd the production of liver cells 23. Since GB hs incresed the glutthione content of liver, it my lso e useful in heptotoxicity induced y other gents. However, glutthione levels were not ffected in CCl 4 treted groups (Figure 4). In our study, elevtion in the levels of end products of lipid peroxidtion in liver of rts treted with CCl 4 were oserved. The increse in MDA levels in liver suggests enhnced lipid peroxidtion leding to tissue dmge nd filure of ntioxidnt defense mechnisms to prevent formtion of excessive free rdicls. Pretretment with GB significntly reversed these chnges. Hence it is possile tht the mechnism of heptoprotection of GB is due to its ntioxidnt effect. Histopthologicl studies showed tht CCl 4 cused stetosis nd hydropic degenertion of the liver tissue. GB pre-tretment exhiited protection, which confirmed the results of iochemicl studies. All the effects of GB were comprle with those of silymrin, proven heptoprotective. The results of our study indicte tht simultneous tretment with GB protects the liver ginst CCl 4 induced heptotoxicity. REFERENCES 1. Newll CA, Aderson LA, Phillipson JD. Ginkgo. In, Herl Medicines: A Guide for Helth-Cre Professionls. The Phrmceuticl Press, London, 1996, Kleijnen J, Knipschild P. Ginkgo ilo. Lncet 1992; 34: Pul C. Ginkgo ilo extrct (EG 761) in perspective, 2nd Ed. Adis Interntionl Ltd., New Zelnd, Pietri S, Murelli E, Drieu K nd Culcsi M. Crdioproective nd ntioxidnt effects of the terpenoid constituents of Ginkgo ilo extrct (EG 761). J Mol Cell Crdiol 1997; 29: Pincemil J, Dupuis M, Nsr C, Hns P, Hg-Berrurier M, Anton R et l. Superoxide nion scvenging effect nd superoxide dismutse ctivity of Ginkgo ilo extrct. Experienti 1989;45: Clostre F. From the ody to the cell memrne: the different levels of phrmcologicl ction of Ginkgo ilo extrct. Presse Med 1986;15: DeLeve LD, Kplowitz N. Mechnisms of drug-induced liver disese. Gstroenterol. Clin N Am 1995;24: Frrel GC. Liver disese cused y drugs, nesthetics nd toxins. In, Sleisenger & Fordtrn s Gsrointestinl nd Liver Disese: Pthophysiology /Dignosis / Mngement. 2nd Ed. (Feldmn M, Schrschmidt BF nd Sleisenger MH, editors), WB Sunders Co., Phildelphi, 1998, Mitr I, Mrcocci L, Droy-Lefix MT, Pcker L. Peroxyl rdicl scvenging ctivity of Ginkgo ilo extrct EG 761. Biochem Phrmcol 1995;49: Mrcocci L, Mguire JJ, Droy-Lefix MT, Pcker L. The nitric oxide scvenging properties of Ginkgo ilo extrct EG 761. Biochem Biophys Res Commun 1994;21: Mrcocci L, Pcker L, Droy-Lefix MT, Sekki A, Grdes- Alert M. Antioxidnt ction of Ginkgo ilo extrct EG 761. Methods Enzymol 1994;234: Kose K, Dogn P. Lipoperoxidtion induced y hydrogen peroxide in humn erythrocyte memrnes. 2. Comprison of the ntioxidnt effect of Ginkgo ilo extrct (EG 761) with those of wter-solule nd lipid-solule ntioxidnts. J Int Med Res 1995;23: Ro PGM, Ro SG, Kumr V. Effects of Heptogrd ginst

7 266 K. ASHOK SHENOY et l. cron tetrchloride induced liver dmge in rts. Fitoterpi 1993;LXIV: Toski A, Engelmn DT, Pli T, Engelmn RM, Droy-Lefix MT. Ginkgo ilo extrct (EG 761) improves postischemic function in isolted preconditioned working rt herts. Coron Artery Dis 1994;5: Morzzoni P, Bomrdelli E. Silyum mrinum (Crduus mrinus). Fitoterpi 1995;LXIV: Gowenlock AH, McMurry JR, McLuchln DM. Plsm Proteins. In, Vrley s Prcticl Clinicl Biochemistry, 6th Ed, CBS Pulishers, New Delhi, 1988, Ygi K, Rstogi R. Assy for lipid peroxides in niml tissues y thiorituric cid rection. Ann Biochem 1979; 95: Buetler E, Duron O, Kelly BM. Improved method for determintion of lood glutthione. J L Clin Med 1963;61: Johnston DE, Kroening C. Mechnism of erly cron tetrchloride toxicity in cultured rt heptocytes. Phrmcol Toxicol 1998; 83: Srivstv SP, Chen NO, Holtzmn JL. The in vitro NADPHdependent inhiition y CCl 4 of the ATP-dependent clcium uptke of heptic microsomes from mle rts. Studies on the mechnism of inctivtion of the heptic microsoml clcium pump y the CCl 3 rdicl. J Biol Chem 199;265: Cotrn RS, Kumr V, Roins SL. Cell injury nd cellulr deth. In, Roin s Pthologic Bsis of Disese, 5th Edition, Prism Book Pvt. Ltd., 1994, Kplowitz N, Aw TY, Simon FR, Stolz A. Drug-induced heptotoxicity. Ann Int Med 1986;14: Awng D. Milk Thistle. Cn Phrm J 1993;23: ISOLATED TISSUE If you stretch it or let it dry der little tissue is sure to die For if the temperture is too high then my e it will fly Wht to do to mke it ply tht I'll discover y & y It will respond, or it won't ut give up, I don't Though it hs mind of its own this much I too hve sworn Frustrted, I shll not e for, in few yers I'll e free PG Students of Phrmcology PGIMER, Chndigrh, (Sent in y Dr. Dinesh Kumr Bdyl, Deprtment of Phrmcology, Christin Medicl College, Ludhin)