International Journal of Pharma and Bio Sciences

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1 Research Article Pharmacology International Journal of Pharma and Bio Sciences ISSN HEPATOPROTECTIVE ACTIVITY OF COMBINATION OF PHYLLANTHUS NIRURI AND CURCUMA LONGA EXTRACTS AGAINST ETHANOL INDUCED TOXICITY IN WISTAR RATS VINAY BS 1, SHALINI ADIGA 1*, SHOBHA KAMATH 2, MOHANDAS RAO KG 3 AND AVIN S 1 1 Department of Pharmacology, Kasturba Medical College, Manipal University 2 Department of Biochemistry, Kasturba Medical College, Manipal University 3 Department of Anatomy, Melaka Manipal Medical College, Manipal University ABSTRACT Alcohol is an important substance with addictive potential. Alcohol is metabolised in the liver. Free radicals generated during the metabolism of alcohol is responsible for hepatic damage. Options available for treatment of alcohol induced liver disease was inadequate in modern medicine. Phyllanthus niruri and Curcuma longa have been shown to have hepatoprotective activity. Hence this study was undertaken to evaluate the effects of Phyllanthus niruri and Curcuma longa in combination to prevent alcohol induced hepatotoxicity. Evaluation was done by comapring the levels of liver enzymes, bilirubin, antioxidants, thiopentone induced sleeping time and liver hisotpathology in various groups of albino rats after administration of ethanol. Treatment with extracts of Phyllanthus niruri and Curcuma longa caused a significant reduction in liver enzymes, bilirubin levels and increase in antioxidants. The results of our study indicate that combination of Phyllanthus niruri and Curcuma longa possess significant hepatoprotective activity against alcohol induced liver damage. KEYWORDS: Curcuma longa, hepatoprotection, antioxidants, liver enzymes SHALINI ADIGA Department of Pharmacology, Kasturba Medical College, Manipal University P - 12

2 INTRODUCTION Alcoholism is a broad term for any drinking of alcohol that results in health problems. The per captia consumption of alcohol has increased in the Asian subcontinent by over 50% between 1980 and The pattern of drinking in India has changed from occasional and ritualistic use to social use. So, there is concerns about the health and social consequences of excessive drinking 2. Alcohol is metabolised primarily in the liver 3. Metabolism of alcohol generates reactive oxygen species (ROS) like superoxide, hydroxyl radical and hydrogen peroxide in the hepatic cells, which oxidize glutathione. Glutathione depletion leads to a build up of ROS which leads to lipid peroxidation of protein and DNA resulting in hepatic damage 4. Alcoholic liver disease is a global health issue, which manifests mainly in the form of fatty liver, alcoholic hepatitis and liver cirrhosis. About 80% of chronic alcoholics develop steatosis, 10-35% of them develop alcholic hepatitis and approximately 10% develop liver cirrhosis 4. The options available for the treatment of liver diseases like cirrhosis, fatty liver and hepatitis were inadequate in the modern medicine. Corticosteroids, antiviral and immunosuppressants are the mainstay of drugs used to treat hepatic diseaseas. These drugs have serious adverse effects, they may even lead to hepatic damage on prolonged use. Alternative drugs, in the form of herbal medicines are used now, instead of currently used drugs of doubtful efficacy and safety 5. Phyllanthus niruri (family Euphorbiaceae) is a plant which possesses several pharmacological properties. It is used in ayurveda to treat gastric lesions 6, and urolithiasis 7. It is shown to have effective hepatoprotective activity against paracetamol 8, carbon tetrachloride 9, and thioacetamide 10 induced heaptotoxicity. It is also shown to have an effective anti-oxidant property 9,11. Curcuma longa is a perennial herb and member of Zingiberaceae family. Studies done earlier have demonstrated its hepatoprotective activity against Carbon tetrachloride 12, paracetamol 13, induced hepatotoxicity. There is no report on the hepatoprotective activity using combination of phytoconstituents of Phyllanthus niruri (PN) and Curcuma longa (CL) extract on alcohol induced hepatotoxicity, hence this study was undertaken and the effects were compared with Liv 52. MATERIALS AND METHODS Preparation of combination of Phyllanthus niruri and Curcuma longa extract. Dry extract of combination of Phyllanthus niruri and Curucma longa was gifted by Arjuna Natural Extracts Limited, Kerala. The extract mixture contained Phyllanthus niruri and Curcuma longa in 1:1 ratio. Phyllanthus niruri contains active phytoconstituents like phyllanthin, hypophyllanthin and flavinoids. Curcumin, demethoxy curcumin, bisdemethoy curcumin and essential oils were present in Curcuma longa as active constituents. Dimethyl sulfoxide (DMS) was used as a solvent. Animals Male adult albino rats of wistar strain weighing grams were used for the study. The animals were housed in clean polypropylene cages under standard environmental conditions. The animals were provided with a standard rat feed (pellets procured from VRK nutritional solutions,sangli) and water ad libitum. This study was approved by Institutional Animal Ethics Committee, KMC No.IAEC/KMC/78/ Materials Absolute ethanol was obtained from Hayman Limited, Essex parx, England. Liv 52 was obtained from Himalaya Drug Company, Bangalore. DMSO were obtained from Merck (India) Limited, Mumbai. Kits used for the estimation of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP) and Total Bilirubin (TB) were obtained from Agappe Diagnostics Limited, Ernakulam, Kerala. All other buffers and reagents used were of analytical grade. Experimental Procedure Rats were divided into 4 groups, each group contained 6 rats. Group I (Control) received DMS 3ml/day, orally. Group II, III and IV received ethanol 3.7g/kg orally 14. Along with ethanol, Group III and IV also received PN and CL extract (400 mg/kg, p.o.) 15,16. and Liv 52 (100 mg/kg, p.o.) 17 respectively. All rats were treated for 28 days. At the end of treatment that is on 29 th day, rats in all groups were given thiopentone sodium (40 mg/kg, i.p) 18 and time taken for onset of sleep and total duration of sleep was recorded. After the rats recovered from thiopentone induced sleep, blood was collected from retro-orbital puncture using heparinised capillary tubes to estimate serum enzyme levels. Serum Enzyme Assays After blood collection, the samples were centrifuged at 3000rpm for 10 minutes in a centrifuge. The serum obtained was used for the estimation of AST, ALT, ALP and TB. Liver Antioxidant Assays Rats were sacrificed under excess urethane on the day of blood withdrawal. Liver was removed and washed with saline. The liver was then homogenized (1:20 w/v) in potassium phosphate buffer (0.1 molar, ph 7.4, containing 0.25M Sucrose). The homogenate was centrifuged at 1500rpm for 10 minutes in a high speed refrigerated centrifuge and the supernatant was used for assay of Glutathione-S-Transferase (GST) and Malondialdehyde (MDA). Histopathological Studies Paraffin sections were prepared using liver tissue and cut into 5µ thick sections. The sections were then stained with hematoxylin and eosin (H&E) dye and studied using light microscope for histopathological changes and photmicrographs were taken. Statistical Analysis The results were analysed using One-way Analysis of Variance (ANOVA) followed by post hoc analysis done using Tukey method. P value of <0.05 was considered P - 13

3 as statistically significant. The analysis was carried out using SPSS (Statistical Software for Social Sciences) version 14 for windows. RESULTS Serum Enzyme Levels Ethanol administration for 28 days caused significant (p<0.05) increase in serum levels of ALT, AST, ALP and TB when compared to Control group indicating liver cell damage. Coadministration of PN and CL along with ethanol significantly (p<0.05) prevented the rise in ALT, AST, ALP and TB values. The effect of PN and CL in reducing ALT, AST and ALT levels was comparable to Liv.52. TB values were better in the Liv 52 group compared to PN and CL group, but this was not statistically significant.(table No.I) Liver Antioxidants Assay Ethanol administration significantly reduced (p<0.05) GST levels in comparison to normal control values which indicates reduction in enzyme antioxidant activity in liver. Ethanol administered group showed significant (p<0.05) increase in MDA levels when compared to normal control values indicated an increase in process of lipid peroxidation. This was significantly prevented (p<0.05) and comparable to Liv52. Reduction in MDA levels was significantly (p<0.05) better in Liv.52 group compared to PN and CL group.(fig I and Fig II) Thiopentone Induced Sleeping Time Ethanol administration caused significant (p<0.05) decrease in the time taken for onset of sleep and it also significantly (p<0.05) increased the total duration of sleep when compared to normal control values indicating an impairement of hepatic metabolizing enzymes. Coadministration of PN and CL extracts along with ethanol significantly prevented the alterations in the time taken for onset of sleep and total duration of sleep. This effect was comparable to Liv.52. (Table No.II). Table I Effect of combination of PN and CL extracts on serum enzyme and Total Bilirubin levels Groups ALT (U/L) AST (U/L) ALP (U/L) TB (mg/dl) Group I ± ± ± ± 0.04 Group II ± 0.6 a 9.81 ± 0.30 a ± 1.16 a 2.03 ± 0.14 a Group III ± 1.07 b 6.88 ± 0.34 b ± 1.17 b 1.29 ± 0.1 b Group IV ± 0.34 b 6.78 ± 0.34 b ± 1.33 b 1.06 ± 0.06 b Values are expressed as Mean ± SEM (n=6). a = p<0.05 vs Group 1, b = p<0.05 vs Group II. One way Analysis of Variance (ANOVA) followed by Tukey post-hoc test. Table II Effect of combination of PN and CL extract on Thiopentone induced sleeping time Groups Onset (in seconds) Duration (in minutes) Group I 178 ± ± 2.92 Group II ± 3.17 a ± 5.06 a Group III ± 2.47 ab ± 1.96 ab Group IV ± 2.96 b ±2.94 ab Values are expressed as Mean ± SEM (n=6). a = p<0.05 vs Group 1, b = p<0.05 vs Group II. One way Analysis of Variance (ANOVA) followed by Tukey post-hoc test. Values expressed as Mean ± SEM (n=6). a = p<0.05 vs Group 1, b = p<0.05 vs Group II. One way Analysis of Variance (ANOVA) followed by Tukey post-hoc test. P - 14

4 Values expressed as Mean ± SEM (n=6). a = p<0.05 vs Group 1, b = p<0.05 vs Group II. c = p<0.05 compared to Group II. One way Analysis of Variance (ANOVA) followed by Tukey post-hoc test. Histopathological Studies Control rats (Group I) showed normal hepatic parenchyma, with lobular arrangement of cells (Fig III). Histological section of liver from Ethanol treated (Group II) rats showed vacoules (Fatty changes), congestion of central vein & sinusoids with lymphocytic infiltration and derangement of hepatic parenchyma (Fig IV). Sections from PN and CL extract (Group III) treated rats, showed almost normal hepatic parenchyma, with normal lobular architecture with minimal lyphocytic infiltration (Fig V). Sections from Liv.52 (Group IV) treated rats, showed normal lobular architecture, without any evidence of parenchymal injury (Fig VI). Figure III Control Group P - 15

5 Figure IV Ethanol treated group Figure V (PN and CL) extracts + Ethanol group Figure VI Liv 52 + Ethanol group DISCUSSION In our study, chronic administration of ethanol at a dose of 3.7g/kg significantly increased the values of AST, ALT, ALP and TB levels when compared to control group, indicating hepatic damage. Administration of PN and CL at a dose of 400mg/kg significantly prevented the elevation in AST, ALT, ALP and TB values. Oxidative stress is one of the main factors in ethanol induced liver damage. The excessive Reactive Oxygen Species (ROS), generated during metabolism of alcohol to acetaldehyde, rapidly react with cellular proteins, altering their function leading to leakage of the liver enzymes 19. By maintaining the integrity of cell membranes (antioxodant property) PN and CL protects against ethanol induced hepatotoxicity. Studies in the past have shown similar hepatoprotective effects of PN 20 and CL 21 against alcohol induced liver damage P - 16

6 when given individually. The excessive ROS generated during ethanol metabolism also rapidly react with lipid membranes. This initiates the lipid peroxidation chain reaction producing reactive aldehydes like MDA 22. In our study, ethanol administration significantly increased liver MDA levels reflecting the causal role of lipid peroxidation in ethanol induced liver damage. Coadministration of PN and CL with ethanol significantly reduced the MDA levels in liver when compared to ethanol administered group. This can be attributed to antioxidant actitivties of PN and CL. Glutathione-S-Transferase is an important enzyme required to maintain normal cellular glutathione pool, an important antioxidant, which prevents accumulation of toxic metabolites 23. In the ethanol group, a significant decrease in GST level was observed, indicating the reduction in enzyme antioxidant levels. Coadministration of PN and CL with ethanol showed tendency for restoration of altered GST level towards near normal, which depicts antioxidant potential of PN and CL. Thiopentone induced sleep time was significantly increased in ethanol treated group. This indicates derangement of hepatic metabolizing enzymes which results in delayed clearance of thiopentone sodium, which prolongs the hypnotic effect. Similar results were seen in previously done studies 24. Coadministration of PN and CL along with ethanol significantly decreased thiopentone induced sleep time, an indirect evidence of their hepatoprotective activity. Histopathological findings in liver correlate with biochemical estimations. Histopathological changes which were seen in the Ethanol group was prevented when PN and CL was given with ethanol. Various active constituents like phyllanthin, hypophyllanthin, of PN and curcumin, demethoxy curcumin of CL, along with various flavanoids of both PL and CL were postulated to be responsible for hepatoprotective activity. Mechanisms by which these constituents of PN and CL work may be different and hence when used in combination they may act synergitically to each other. Based on the observed results, combination of PN and CL, may open up new possibilities in the treatment of liver disorders. However, the findings will have to be confirmed by well planned clinical studies. CONCLUSION Combination of PN and CL extract showed significant hepatoprotection against ethanol induced liver injury. The observed hepatoprotective activity can be attributed to various antioxidant principles present in the extract. Combination of PN and CL may open up new possibilities in the treatment of various liver disorders. However, the observed findings needs to be confimed by well planned clinical studies. 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