Hepatoprotective Effects of Liv.52 on Ethanol-induced Liver Damage in Rats
Rajat Sandhir* and Gill, K.D. Department of Biochemistry, Postgraduate Institute of Medical Education & Research Chandigarh, India
SUMMARY
The mechanism of protective effects of Liv.52, a multiherbal hepatoprotective drug, on ethanol induced hepatic damage has been investigated. The results indicate that Liv.52 treatment prevents ethanol induced increase in the activity of the enzyme ϒ-glutamyl transpeptidase. Concomitantly there was also a decrease in ethanol accentuated lipid peroxidation in liver following Liv.52 treatment. The activity of antioxidant enzymes; superoxide dismutase, glutathione peroxidase and the levels of glutathione were decreased following ethanol ingestion. Liv.52 treatment was found to have protective effects on the activity of superoxide dismutase and the levels of glutathione. The results obtained from the study indicate hepatoprotective nature of Liv.52, which might be attributed to its ability to inhibit lipid peroxidation.
Ethanol is currently recognized as the most prevalent known cause of abnormal human development. Alcohol abuse and alcoholism represents one of the major health, social and economic issues facing the world. Liver is among the organs most susceptible to the toxic effects of ethanol1. It is now generally accepted that alcohol can induce in vivo changes in membrane lipid composition and fluidity2, which may eventually, effect cellular functions. Among the mechanisms responsible for the effects of alcohol, lipid peroxidation appears to be a likely candidate, since this process can account for alterations in membrane phospholipid composition observed after ethanol intoxication3,4. Aykae et al.5, have observed an increase in hepatic lipid peroxidation following chronic ethanol ingestion.
Traditional medicines are effective in certain disorders and are based on experience in the use of plant products in amelioration of common diseases. Liv.52, an Ayurvedic multiherbal formulation is widely used in various hepatic disorders6-8. Liv.52 has recently been reported to have protective effects in carbon tetrachloride9,10, paracetamol11 and ethanol12 toxicity. However, very less scientific data regarding the identification and effectiveness of these herbs is available. Therefore, this study has been designed with an aim to understand the mechanism by which Liv.52 may exert its hepatoprotective effects following ethanol exposure.
MATERIALS AND METHODS
Ethanol was obtained from E. Merck, Munich, Germany and Liv.52 was a kind gift from The Himalaya Drug Co., Bangalore, India. Every 2.5 ml of Liv.52 syrup contains an extract of the following: Capparis spinosa (17 mg); Cichorium intybus (17 mg); Solanum nigrum (8 mg); Cassia occidentalis (4 mg); Terminalia arjuna (8 mg); Achillea millefolium (4 mg); and Tamarix gallica (4 mg). Glutathione, NADPH, DTNB, thiobarbituric acid, BSA. Tris were obtained from Sigma Chemical Co., USA. All other chemicals were obtained from local sources and were of analytical grade.
Animals and treatment - Male albino rats (Wistar strain) of 8-10 weeks of age weighing between 100 and 120 g were used for the study. The animals were housed in polypropylene cages, fed on pellet diet (Hindustan Lever Ltd., India) and water ad libitum. Animals were divided into three groups of 6 animals each. Group I received normal saline, intragastrically. Group II received ethanol 3 g/kg body weight, intragastrically, for a period of 4 weeks. Group III received ethanol (3g/kg body weight) and Liv.52 (1.0 ml/kg body weight) for 4 weeks intragastrically.
At the end of treatment, animals were anaesthetized with ether and sacrificed by decapitation. Blood was drawn from the supraorbital sinus, and serum separated for ϒ-glutamyl transpeptidase assay. Livers were removed, washed with ice cold saline (0.15 M) and a 10% (w/v) homogenate prepared in 0.1 M Tris HCl, pH 7.4 for lipid peroxidation and glutathione estimation. The postnuclear fraction for catalase was obtained by centrifugation of homogenated at 1000 g for 20 min at 4°C and for other enzyme assays, the post nuclear fraction was centrifuged at 12,000 g for 60 min at 4°C.
Lipid peroxidation - The quantitative measurement of lipid peroxidation was performed according to the method of Wills13. The amount of malondialdehyde (MDA) formed was quantitated by reaction with thiobarbituric acid and used as an index of lipid peroxidation. The results were expressed as nmol MDA/mg protein using molar extinction co-efficient of the comophore (1.56 x 10-5 M-1 cm-1).
Enzyme assays – The activity of antioxidant enzymes, viz. superoxide dismutase, catalase and glutathione peroxidase was assayed in livers of experimental animals and the activity of γ-glutamyl transpeptidase was assayed in serum of rats.
Superoxide dismutase was assayed according to the method of Martin et al.14, wherein the autooxidation of hematoxylin to hematin is inhibited by the enzyme. The results were expressed as units/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 50%.
Catalase was assayed by the method of Luck15 wherein breakdown of H2O2 by the enzyme is measured at 240nm. Enzyme activity was calculated using the millimolar extinction coefficient of H2O2 (0.07) and the results were expressed as µmol H2O2 decomposed/min/mg protein.
Glutathione peroxidase was assayed by the method of Lawrence and Burk16, wherein oxidation of NADPH by H2O2 was followed at 340nm. Enzyme activity was calculated using the molar extinction coefficient of NADPH (6.22 x 10-6) and the results expressed as nmol NADPH oxidized/min/mg protein.
ϒ-glutamyl transpeptidase activity was ascertained in serum by the method of Szasz17, wherein the transfer the ϒ-glutamyl group of ϒ-glutamyl-4-nitroanilide to glycyl-glycine is measured. The results were expressed as IU/L.
The control activities of various enzymes studied are in accordance to those previously reported18.
Glutathione estimation – Glutathione (GSH) was estimated in the samples by the method of Ellman19 and the results were expressed as µmol GSH/mg protein.
Protein estimation - Protein in the samples was quantitated by the method of Lowy et al20. using bovine serum albumin as standard.
Statistical analysis was carried out using the Student’s t-test. Values having p<0.05 were considered significant.
Refference: http://www.himalayahealthcare.com/pdf_files/liv197.pdf
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