HEPATO-PROTECTIVE ACTIVITY OF STEM BARK EXTRACTS OF FICUS RELIGIOSA LINN IN RATS

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INTRODUCTION
Liver is a vital organ play a major role in metabolism and excretion of xenobiotics from the body. Liver injury or liver dysfunction is a major worldwide health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. Liver disease is an important cause of morbidity and mortality in the United States, affecting persons of all ages, but most frequently individuals in the productive years of life, between the ages of 40 and 60 years 1 . They are mainly caused by chemicals and some drugs when taken in very high doses. Despite advances in modern medicine, there is no effective drug available that stimulates liver function, offer protection to the liver from damage or help to regenerate hepatic cells. The available synthetic drugs to treat liver disorders in this condition also cause further damage to the liver. There is urgent need, therefore, for effective drugs to replace/supplement those in current use. The plant kingdom is undoubtedly IJBR2[8] [2011]466-475 www.ijbr.ssjournals.com valuable as a source of new medicinal agents 2 . Paracetamol (PCM) also known as Acetaminophen, taken in overdose can cause severe hepatotoxicity and nephrotoxicity. PCM is activated and converted by cytochrome P450 enzymes to toxic metabolite NAPQI (N-acetyl-pbenzoquinoneimine) that causes oxidative stress and liver damage 3 . Ficus religiosa (moraceae) commonly known as peepal tree is distributed throughout India. It is popular indigenous system of medicine like Ayurveda, Siddha, Unani and Homeopathy and used treditionaly as, analgesic 4 , antiinflammatory 4,5 , wound healing 5 , antioxidant 5,6 , anticonvulsant 7 , asthma 8 , antidiabetic, and ulcer 9 , liver and kidney diseases 10 . Preliminary phytochemical screening of F. religiosa barks, showed the presence tannins, saponins, flavonoids, steroids, terpenoids and cardiac glycosides. The barks of F. religiosa also showed the presence of bergapten, bergaptol, lanosterol, βsitosterol, stigmasterol, lupen-3-one, βsitosterol-d-glucoside (phytosterolin), vitamin k1, β-, leucocyanidin-3-0-β-Dglucopyrancoside, lupeol, lupeol acetate, α-amyrin acetate. 11 Literature revealed that plant containing lupeol (triterpenoids) 12,13 , flavonoids 14 , phenolic compounds were responsible for hepatoprotective activity and the F. religiosa also having these constituent so it was hypothesized that it may also be able to protect the liver. Keeping this view the present study scientifically validated the traditional use of Ficus religiosa for liver disorders.

Acute Toxicity Study:
A safe oral dose of Ficus religiosa extract was determined through the acute oral toxic test in rats as described by the Organization of Economic Co-Operation and Development (OECD) as per 420 guidelines (OECD Guidelines for the Testing of Chemicals, 2010). The Ficus religiosa extracts, at different doses up to 2000 mg/kg, was prepared and then administered (p.o.). Animals were observed for behavioral changes, any toxicity and mortality up to 48 h. Onetenth of this dose were selected as the therapeutic dose for the evaluation 9, 16 .

Evaluation of Hepatoprotective activity:
Determination of hepatoprotective activity (curative aspect) in Paracetamol induced hepatotoxicity 17 . Wistar albino rats weighing between 150-200g, divided into 7 groups, and each group containing 6 animals. Group 1-Normal control (vehicle treated, p.o for 10 days) The animals were sacrificed by cervical dislocation after 24 hours after the last dose administration. The blood samples were collected by cardiac puncture in heparinized microfuge tubes. The blood samples thus collected were immediately centrifuged at 2200rpm for 15 minutes. When serum clearly separated out, the serum was analyzed for alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin were estimated using commercially available kits by the method proposed by Reitman and Frankel 18,19 .

Histopathology study:
Liver is dissected out and the liver samples were excised from the experimental animals of each group and washed with the normal saline. Initially the materials were fixed in 10% buffered neutral formalin and then with bovine solution. They were processed for paraffin embedding following the microtome technique. The sections were taken at 5µ thickness processed in alcohol-xylene series and were stained with alum-haematoxylin and

RESULTS
All the Extracts thus obtained were concentrated under vacuum, dried, weighed and stored in desiccators. The yields were found to be 3.2g (1.28%w/w of crude drug) of petroleum ether extract with semisolid mass of Blackish green color, 5.3g (2.12 %w/w of crude drug) of ethyl acetate extract with Blackish green color semisolid mass, 38.4g (15.36 %w/w of crude drug) of methanol extract with Reddish orange color semisolid mass, 20.5g (8.2%w/w of crude drug) of water extract with dark green color semisolid mass for Ficus religiosa was recorded. In qualitative analysis, various chemical tests were performed for the identification of common phytoconstituents in Ficus religiosa extracts. Preliminary phytochemical screening was performed for each extract. (Table 1) The Ficus religiosa extracts did not cause any mortality up to 2000 mg/kg and were considered as safe. The effect of methanol and aqueous extracts of Ficus religiosa on normal liver function, it was found to be non toxic at the selected dose (200 mg/kg) since the parameters SGPT, SGOT, ALP and total bilirubin were within like that of control. Paracetamol intoxication in normal rats elevated the levels of SGPT, SGOT, ALP, and total bilirubin significantly, indicating acute centrilobular necrosis. The rat treated with alcoholic extract and aqueous extract showed a significant reduction in all the biochemical parameter elevated by paracetamol. Ethyl acetate and pet ether extract showed moderate reduction of all three biochemical parameters. This reduction in biochemical parameter exhibited by alcoholic and aqueous extracts is similar when compared with that of silymarin. Reduction of all biochemical parameters against the hepatotoxin is given in Table 2, Figure  1, 2, 3 & 4.
The histopathological profile of the rat treated with alcohol and aqueous extracts showed no visible changes confirming the safety of the extract at selected dose. Histopathological examination of liver section of control group showed normal cellular architecture with distinct hepatic cells, sinusoidal space. In the liver section of the rats intoxicated with paracetamol, there is disarrangement and degeneration of normal hepatic cells with intense centrilobular necrosis. The liver section of the rats treated with pet. ether and ethyl acetate extracts and intoxicated with paracetamol showed moderate hepatoprotective activity while rats treated with silymarin and intoxicated with paracetamol showed less disarrangement and degeneration of hepatocytes, indicating marked regeneration activity. (Figure 5)

DISCUSSION
Most of the hepatotoxic chemicals damage liver cells either by lipid peroxidation or by other oxidative stress mechanisms induced cellular damage. Activation of some enzymes in the Cytochrome P-450 system such as CYP-2E1 also leads to oxidative stress and injury to hepatocytes, bile duct cells

IJBR2[8][2011]466-475
www.ijbr.ssjournals.com causing accumulation of bile acid inside liver and which in turn promotes further liver damage. Acetaminophen (APAP) is a safe, effective and widely used antipyreticanalgesic drug however; An overdose can induce severe hepatotoxicity in experimental animals and humans. Acetaminophen dose is metabolized in the liver via glucuronidation and sulfate conjugation. The remaining 10 -15% undergoes oxidative metabolism via cytochrome P450 isoenzymes (CYP) 2E1 and 1A2, which produces the hepatotoxic metabolite, N-acetyl-p-benzoquinonimine (NAPQI). Excessive administration of APAP can cause over production of ROS during formation of N-acetylpbenzoquinoneimin(NAPQI) by cytochrome P450. This mechanism has been suggested to participate in the development of oxidative stress and injury in APAP-induced hepatotoxicity 21 . Chronic administration of drug (paracetamol) to rats increased the levels of marker enzymes like ALT, AST and ALP as these are stored in the liver cells and increase in the levels of these marker enzymes in serum indicate damage to the liver cells 22 . The silymarine and the methanolic extract of Ficus religiosa significantly decreased the paracetamol induced elevated levels of the enzymes in the treatment group, indicating the enhancement of structural integrity of hepatocytic cell membrane or regeneration of damaged liver cells by the extract. Decrease in the bilirubin after treatment with Ficus religiosa indicated the effectiveness of the extract in the normal functional status of the liver. Histopathological analyses were good in agreement with the biochemical changes. Many research findings. Therefore, there is a possibility that the extract of Ficus religiosa may possess hepatoprotective activity.

CONCLUSION
The methanolic extract has shown the ability to maintain the normal functional status of the liver. From the above preliminary study, we conclude that the methanolic extract of Ficus religiosa, is proved to be one of the herbal remedies for liver ailment.