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1 Journal of Global Pharma Technology ISSN: Available Online at RESEARCH ARTICLE Hepatoprotective Effect of Vitamin C and E on Antitubercular Drugs Induced Hepatotoxicity in Albino Rats Babul Kumari *, Tirath Kumar Department of Pharmaceutical Sciences, Kumaun University, Bhimtal Campus, Bhimtal (Utarakhand). *Corresponding Author babulpharm@gmail.com Abstract Objective- The hepatoprotective effects of Vitamin C and E were investigated in anti tubercular drugs induced hepatotoxicity in albino rats. Methods- Liver necrosis was induced by a combination of three anti tubercular drugs [isoniazid (I) 7.5mg/kg, Rifampin (R) 10 mg/kg and Pyrazinamide (P) 35mg/kg] given orally as a suspension for 30 days in rats. The liver damage was evidenced by the histological architectures of the liver tissues and the elevated levels of the liver enzymes like SGOT, SGPT, ALP, Total bilirubin, and unconjugated bilirubin. Results- Treatment with Vitamin C (250 mg/kg) and E (250 mg/kg,) significantly (P<0.05-P<0.001) reduces the anti tubercular drugs induced elevations of the liver enzymes like SGOT, SGPT, ALP, Total bilirubin, and unconjugated bilirubin. Histological architectures of the liver tissues become normal in vitamin -treated group a comparison to the hepatotoxic group. Conclusion-The results of this study suggest that Vitamin C and E have a hepatoprotective effect against anti tubercular drugs induced hepatotoxicity in albino rats. Keywords: Vitamin C, Vitamin E, Isoniazid, Rifampicin, Pyrazinamide, liver enzymes. Introduction Tuberculosis is a dreadful disease from ancient times and still remains a major public health challenge in the 21 st. centuries. India has a 21% of the world s TB population and is the country with the highest TB burden.1 The incidence of TB in India is 1.96 million cases annually, contributing to >300,000 deaths annually, including 1000 deaths every day. 1 However the Ist line anti tubercular drugs like isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin prove successful but their combination produces hepatotoxicity as a major side effect. 2 Patients complain vomiting, nausea, abdominal pain, and patients are unable to take normal food and drinks. Therefore Scientists are more concentrating nowadays to provide better treatment for anti tubercular combination therapy induced hepatotoxicity. From the literature search, it has been found that vitamins including vitamin C and vitamin E can produce hepatoprotection action against many druginduced hepatotoxicities. Vitamin C is chemically ascorbic acid comprising of six carbon organic acids. It is a potent reducing agent and levo-isomer is pharmacologically active. It is watersoluble vitamin in which human is unable to synthesize in the body. Deficiency of vitamin C leads to produce scurvy. It has shown many biological activities such as anticancer, prevention of cardiovascular syndrome (CVS) and hepatoprotection because of its antioxidant properties. 3 Vitamin C is an important free radical scavenger in extracellular fluids trapping radicals and protecting bio membrane of hepatocyte cells due to peroxide free ions attack. 4 Vitamin E, isolated from wheat germ oil by Evans in 1936, was named as α-tocopherol. All green plants contain some Tocopherol and there is evidence that some green vegetables such as lettuce and rose hips contain more than wheat germ. α-, β-and γ-tocopherol has the greatest biological activities. For long decades investigation has been carried out to find out the biochemical mechanism of vitamin E. The biochemical functions of vitamin E are to prevent the oxidation of lipids , JGPT. All Rights Reserved 12
2 The α-tocopherol donates its electron to the free radicals to neutralize then thus protecting the cellular oxidative damage. 3 The present study was made to investigate the protective actions of vitamin C and vitamin E against hepatotoxicity caused by anti tubercular drugs. Material and Methods The Present study was conducted in the Department of Pharmaceutical Sciences, Kumaun university Bhimtal, Nainital (U.K.) India from September 2017 to October Animals Healthy albino rats of either sex weighing 150 g -250g were procured from the departmental animal house and were housed in polypropylene cages at room temperature (25 ± 20C) with 12/12 hours light and dark cycle. Animals were fed with standard pellet diet and water ad libitum. Animals were allowed to acclimatize for a period of 2 weeks before starting the experiment. The study protocol was approved by the Institutional Animal Ethical Committee of the Department (KUDOPS/47). Drugs All the drugs(isoniazid, rifampicin, pyrazinamide, Ascorbic acid (Vit C) (powder dosage form) α-tocopherol (Vit E) and Corn oil (liquid form) used were purchased from Yarrow Chem. Products, Mumbai. Doses and Route of Administration INH 7.5mg/Kg RIF 15mg/Kg, PZN 35mg/Kg and Vitamin C 250mg/kg were dissolved in distilled water separately. 5,6 Vitamin E 250 mg/kg was dissolved in corn oil. Route of administration was oral. 7 Study Protocol The rats were divided into four groups with six animals in each group. Group I Normal control [n=6, the animals were given normal saline only for 30 Days]. Group II Hepatotoxic control [n=6, the animals were given isoniazid (7.5mg/kg) + rifampicin (10mg/kg) + pyrazinamide (35mg/kg) for 30 Days] Group III Vitamin group [n=6, the animals were given isoniazid (7.5mg/ kg) + rifampicin (10mg/ kg) +pyrazinamide (35mg/ kg) + Vitamins {Vitamin C(250mg/ kg) + Vitamin E (250 mg/kg) for 30 days]. Group IV Standard group [n=6, the animals were given Silymarin only for 30 Days.] Collection of Sample The 30th day of drug administration, the animals were anaesthetized under light ketamine anaesthesia and the blood samples were collected from retro-orbital plexus for estimation of biochemical parameters such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin, unconjugated bilirubin and Total Protein. Serum was separated by centrifuging blood at 6000 rpm for 15 minutes and the levels of SGOT, SGPT, ALP, bilirubin, unconjugated bilirubin total protein were analyzed by using diagnostic kits ERBA and semi auto analyser (Transasia-Model ERBA, CHEM 5V2). The two animals from each group were then sacrificed; livers were removed and preserved in 10% formalin for histopathological studies. Histopathological Analysis Small sections of liver tissues were fixed in 10% formalin solution were processed and embedding in paraffin. Sections of 5 microns were cut and stained with hematoxylin and eosin and examined for histopathological changes under the microscope. Statistical Analysis The statistical analysis was done by using Graph pad version The results were recorded as mean± SEM (standard error of the mean). Statistical significance p<0.05(*),p<0.01(**), or p<0.001(***) was determined by using Turkey t-test or ANOVA. Result Biochemical Result SGOT, SGPT, ALP, total bilirubin, unconjugated bilirubin and total protein are indicators of hepatic function , JGPT. All Rights Reserved 13
3 The anti tubercular drugs (isoniazid, rifampicin, and pyrazinamide) treated group significantly elevated the levels of SGOT, SGPT, ALP, total bilirubin and unconjugated bilirubin whereas reduced the levels of total protein (p<0.001) as compared to the control group. Administration of anti tubercular drugs(isoniazid, rifampicin, and pyrazinamide) along with vitamins (Vit C, Vit E) showed a significant reduction in the levels of SGOT, SGPT, ALP, total bilirubin and unconjugated bilirubin and increased the levels of total protein(p<.0001).(table 1 and Fig 1 to 6). Table 1: Effect of treatment of anti tubercular drugs (isoniazid, rifampicin, and pyrazinamide) and anti tubercular drugs (isoniazid, rifampicin, and pyrazinamide) + vitamins (Vit C and Vit E) on liver enzymes, bilirubin, and total protein Parameters Control group Hepatotoxic group Treatment (Vitamin) group Standard (Silymarin) group SGOT 33.7± ± ± ±1.580 SGPT 79.75± ± ± ±11.56 ALP ± ± ± ±3.204 Bilirubin ± ± ± ± Unconjugated ± ± ± ± bilirubin Total protein ± ± ± ± In Fig 1: Hepatotoxic group (SGOT) vs Vitamin group (SGOT) ***p<0.001, Control group (SGOT) vs Vitamin group (SGOT) (***p<0.01). Fig 2: Hepatotoxic group (SGPT) vs Vitamin group (SGPT) ***p< Control group (SGPT) vs Vitamin group (SGPT) ns p> , JGPT. All Rights Reserved 14
4 Fig 3: Hepatotoxic group (ALP) vs Vitamin group (ALP) ***P<0.001.Control group (ALP) vs Vitamin group (ALP) ns P>0.05. Fig 4: Hepatotoxic group (Total bilirubin) vs Vitamin group (Total bilirubin) **P<0.01, Control group (Total bilirubin) vs Vitamin group (Total bilirubin) ***P< Fig 5: Hepatotoxic group (unconjugated bilirubin) vs Vitamin group (unconjugated bilirubin) *P<0.05, Control group (unconjugated bilirubin) vs Vitamin group (unconjugated bilirubin) ***P< , JGPT. All Rights Reserved 15
5 Fig 6: Hepatotoxic group (Total protein) vs Vitamin group (Total protein) ***P<0.001, Control group (Total protein) vs Vitamin group (Total protein) ns P>0.05. Histopathological Results The histopathological results are as follows In Fig 1:.Control group [Central vein (Normal); 2-Sinosoides (Normal) [Fig 2: Hepatotoxic group [1- Central vein (enlarged), 2-Sinosoides (Enlargement] Fig 3: Vitamin group [1-Central vein (Normal), 2-Sinosoides (Normal), 3- Hepatocytes. (Normal)]. Fig 4: Standard (Silymarin) group [Sinosoides (Normal)]. Discussion In this study, we investigated the hepatoprotective effect of vitamins against anti tubercular drugs induced hepatotoxicity in rats. The presently used Ist line anti tubercular drugs like isoniazid, rifampicin, and pyrazinamide produces drug induced , JGPT. All Rights Reserved 16
6 hepatotoxicity as a major adverse effect. Each drug Isoniazid, Rifampicin, and Pyrazinamide is potentially hepatotoxic in itself, their toxicity is increased in a synergistic manner, when given in combination. 8 Isoniazid- induced hepatotoxicity is mainly due to acetyl hydrazine, a metabolite of Isoniazid. 9 An active metabolite, 25 -desacetyl rifampicin, the bio transformed product of rifampicin is responsible for hepatotoxicity. It reduces the enzymes of drug metabolism and inhibits the nucleic acid and protein synthesis by specially binds. 10 Rifampicin is a powerful inducer of CYT P450 and it increases the metabolism of isoniazid, therefore, acetyl hydrazine production increases. Acetylhydrazine converted into toxic metabolites that lead to hepatotoxicity. 11 When there is hepatic injury the enzymes like AST, ALT, ALP, Total Bilirubin and Unconjugated Bilirubin leak into the bloodstream and its elevated level indicate hepatotoxicity. In the present study after anti tubercular drugs (isoniazid + rifampicin+ pyrazinamide) exposure, the serum marker enzymes like AST, ALT, ALP, Total Bilirubin and Unconjugated Bilirubin levels were elevated. Total protein level was decreased. The values were reversed to normal after combined treatment of vitamin C and vitamin E. This is because of stabilization of plasma membrane as well as repairmen of hepatic tissue injury caused by anti tubercular drugs like isoniazid, rifampicin, References 1. accessed 23: (2011). 2. Aithal PG, Day CP (1999) The natural history of a histologically proved drug - induced liver disease. Gut, 44: Beale Jr, John M (2004) Block John H Wilson and Griswold s Textbook of Organic Medicinal and Pharmaceutical Chemistry, 12: Adikwu Elias, Nelson Brambaifa (2012) Hepatoprotective effect of vitamin E. American Journal of Pharmacology and Toxicology, 7 (4): Pari L, Kumar NA (2002) Hepatoprotective activity of Moringa and pyrazinamide. Ascorbic acid is a potent antioxidant it inhibited the chain reactions of chemical agent generated free radicals or scavenged the reactive free radicals before they reached their hepatic targets. 12 Vitamin E acts in the 2 ways in the membrane: It is fat soluble and located primarily within the lipid bilayer of the cell membrane and protecting polyunsaturated fats and other components of the cell membrane from oxidation by free radicals. 13 Also, Vitamin E breaks the antioxidant chain and prevents cell membrane damage from Reactive oxygen species. 14 On treatment with Vitamins[Vitamin C (250 mg/kg) + Vitamin E(250 mg/kg)], 1 hr prior to anti tubercular drugs the hepatic toxicities were near to normal indicating a reversal of anti tubercular drugs induced hepatotoxicity and confirming the free radical scavenging property of Vitamins. Conclusion The results of the present study suggest the ameliorating effect of vitamin C and E against anti tubercular drugs induced hepatotoxicity in rats. This may be due to the inhibition of free radical generation and free radical scavenging activity. Acknowledgment The author is very thankful to staffs especially Dr. L.S. Rautela of Department of Pharmaceutical Sciences, Kumaun University, Bhimtal campus, Bhimtal, Nainital for good support during the research work. oleifera on antitubercular drugs-induced liver damage in rats. J. Med. Food 5(3): Tasduq SA, Kaiser P, Gupta DK, et al (2005) Protective effect of a 50% hydroalcoholic fruit extract of Emblica officinalis against anti-tuberculosis drugs induced liver toxicity. Phytother Res,. 19: Hamden Khaled, Boujbiha Mohamed Ali, et al (2009) Combined vitamins (C and E) and insulin improve oxidative stress and pancreatic and hepatic injury in alloxan diabetic rats. Biomedicine & Pharmacotherapy, 63: , JGPT. All Rights Reserved 17
7 8. Ramappa VIdyasagar, Aithal Guruprasad P (2013) Hepatotoxicity related to anti tuberculosis drugs mechanisms and management: J. Clin. exp. hepatol., 3: Jiang Y, Ren-Xiu P, Jing Y, Rui KG, Juan L (2004) CYP 2E1mediated isoniazidinduced hepatotoxicity in rats.acta Pharmacologica Sinica, 25: Vishal BJ, Vishnu N, Thakare, Anupama AS, Avinash DD, Suresh RN (2010) Hepatoprotective activity of Luffa acutangula. Indian journal of experimental biology 48: Bhupinder SK, Sarita A, Nita k, Usha G (2007) Effect of cimetidine on hepatotoxicity induced by isoniazid rifampicin combination in rabbits. Indian journal of gastroenterology 26: Odigie IP, Okpoko FB, Ojobor PD (2007) Antioxidant effects of vitamin C and E on phenylhydrazine-induced hemolysis in Sprague Dawley rats: evidence for a better protection by vitamin E. Nig Post grad Med J., 14: Bradford A, Atkinson J, Fuller N et al (2003) The effect of vitamin E on the structure of membrane lipid assemblies. J. Lipid Res 44: Brigelius R, Traber M (1999) Vitamin E: function and metabolism. Faseb. J., 13: , JGPT. All Rights Reserved 18
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