Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 37, Num. 3, 2005, pp. 165-168

Indian Journal of Pharmacology, Vol. 37, No. 3, May-June, 2005, pp. 165-168

Research Paper

Evaluation of hepatoprotective activity of stem bark of Pterocarpus marsupium Roxb.

Mankani KL, Krishna V, Manjunatha BK, Vidya SM, Jagadeesh Singh SD, Manohara YN, Raheman Anees-Ur, Avinash KR

Department of PG Studies and Research in Biotechnology, Kuvempu University, Shankaraghatta, Karnataka
Correspondence Address:Department of PG Studies and Research in Biotechnology, Kuvempu University, Shankaraghatta, Karnataka Email:krishnabiotech2003@yahoo.co.in

Date of Submission: 03-Jun-2004
Date of Decision: 16-Oct-2004
Date of Acceptance: 17-Oct-2004

Code Number: ph05041

Abstract

Keywords: Red Kino; serum marker enzymes

Introduction

Materials and methods

The stem bark of P. marsupium was collected from Joldhal range forest, Davanagere, Karnataka. The plant was authenticated by comparing with the herbarium voucher specimen deposited at Kuvempu University Herbaria[7] (FDD-147) Shankaraghatta. The material was air dried under shade, powdered mechanically and stored in airtight containers. About 1 kg of the powdered material was boiled with 5 l of distilled water for 30 min and filtered to obtain the aqueous extract.[8],[9] The extract was concentrated under reduced pressure and finally dried using the water bath. Another 1 kg of the powdered material was subjected for soxhlation. It was refluxed with methanol for 48 h in batches of 250 g each. The extracts were pooled together and concentrated in vacuum using rotary flash evaporator (Buchi, Flawil, Switzerland).

Drug formulations
Oral suspensions containing 25 mg/ml of the methanol and aqueous extracts were prepared in 1% w/v gum tragacanth.

Animals
Male Wistar albino rats weighing 150-200 g were obtained from the National College of Pharmacy, Shimoga, Karnataka. They were maintained at standard housing conditions and fed with commercial diet (Hindustan Lever Ltd., Bangalore) and provided with water ad libitum during the experiment. The institutional animal ethical committee (Reg. No. 144/1999/CPCSEA/SMG) permitted the study.

Evaluation of hepatoprotective activity
The acute toxicity studies were carried out as per stair case method.[10] Fifty male rats were divided into five groups of 10 each and were administered with aliquot doses of the extracts orally (100, 150, 200, 250 and 300 mg/kg). Mortality was not noticed up to 200 mg/kg, whereas, 100% mortality was noticed in the dose of 300 mg/kg. The LD₅₀ of the extracts was found to be 250 mg/kg body weight. One-tenth of this dose was selected as the therapeutic dose for the evaluation.[11]

Five groups of animals containing six each were used for the study. The animals from Group I served as the control and received the vehicle 1% w/v gum tragacanth at a dose of 1 ml/kg/day of p.o. for 14 days. Groups II-V received 0.1 ml/kg/day i.p. of CCl₄ (E-Merck, Mumbai, India) for 10 days.[12] The standard drug Silymarin (Ranbaxy Lab. Dewas) was administered to Group III animals in the dose of 100 mg/kg/day p.o. for 14 days. While, Groups IV and V were treated with methanol extract and aqueous extract of P. marsupium in the dose of 25 mg/kg/day, p.o. (as per acute toxicity studies) for 14 days, respectively. The CCl₄, silymarin and the extracts were administered concomitantly to the respective groups of animals.

Assessment of hepatoprotective activity
All the animals were killed on day 14 under light ether anaesthesia. The blood samples were collected separately by carotid bleeding into sterilized dry centrifuge tubes and allowed to coagulate for 30 min at 37°C. The clear serum was separated at 2500 rpm for 10 min and biochemical investigations were carried out to assess liver function viz ., total bilirubin,[13] total protein,[14] serum transaminases[15] and serum alkaline phosphatase[16] (Raichem, San Diego, CA 92111-1203).

The results are expressed as mean±SEM of six animals from each group. The data were evaluated by one-way ANOVA followed by Tukey′s multiple comparison test. P values < 0.01 were considered statistically significant.

Histopathology
After draining the blood, liver samples were excised, washed with normal saline and processed separately for histological observations. Initially, the materials were fixed in 10% buffered neutral formalin for 48 h and then with bovine solution for 6 h. Paraffin sections were taken at 5 mm thickness, processed in alcohol-xylene series and were stained with alum hematoxylin and eosin.[17] The sections were examined microscopically for histopathological changes.

Results

The administration of CCl₄ to the animals resulted in a marked increase in total bilirubin, serum amino transaminases (AST and ALT) and serum alkaline phosphatase activities. However, the serum total protein level was decreased. The toxic effect of CCl₄ was controlled in the animals treated with the methanol and aqueous extracts by way of restoration of the levels of the liver function biochemistry similar to that of the standard drug silymarin [Table - 1]. Among the extract-treated groups, significant hepatoprotective activity, was observed in those treated with methanol extract.

Histological profile of the control animals showed normal hepatocytes [Figure - 1]. Group II animals exhibited intense centrilobular necrosis (N), vacuolization (F) and macrovesicular fatty change [Figure - 2]. The sections of liver taken from the animals treated with standard drug silymarin showed the hepatic architecture, which was similar to that of control [Figure - 3]. The animals treated with methanol extract exhibited significant liver protection against the toxicant as evident by the presence of normal hepatic cords, absence of necrosis and lesser fatty infiltration [Figure - 4]. However, moderate accumulation of fatty lobules [Figure - 5] was noticed in the sections of animals treated with the aqueous extract.

Discussion

The CCl₄ has been used as a tool to induce hepatotoxicity in experimental animals.[18],[19] This toxic chemical caused peroxidative degradation in the adipose tissue resulting in fatty infiltration of the hepatocytes. The increase in the levels of serum bilirubin reflected the depth of jaundice and the increase in transaminases and alkaline phosphatase was the clear indication of cellular leakage and loss of functional integrity of the cell membrane.[20]

Administration of methanol extract and aqueous extract of P. marsupium stem bark showed significant hepatoprotective activity, which was comparable with the standard drug silymarin. The effect was more pronounced with methanol extract. Many phytochemical reports revealed that the methanolic extract of the plant was found to contain higher concentrations of flavonoids and glycosides.[21] The qualitative phytochemical investigations on the methanolic extracts of P. marsupium also showed positive for flavonoids by ferric chloride, alkaline reagent and Shinoda tests. Further, it has been reported that the flavonoid constituents of the plant possess antioxidant properties[22] and was found to be useful in the treatment of liver damage.[23] The administration of hepatoprotective drugs may induce the hepatocytes to resist the toxic effect of CCl₄. The results indicate that the methanolic extract of P. marsupium has significant hepatoprotective activity. This may be probably due to the higher content of flavonoids. The earlier investigators[24] have screened the hepatoprotective activity of the flavonoid compound, rutin, isolated from Artemisia scoparia , which is also claimed to have free radical scavenging and antilipid peroxidant activities against CCl₄-induced hepatic toxicity. The isolation and characterization of the flavonoids from the stem bark of P. marsupium and screening of the pharmacological action against the liver damage is being investigated. In most of the developed and developing countries, the incidence of viral hepatitis is more so, the investigation for an efficient hepatoprotective drug from the natural resource is an urgent necessity.

Acknowledgments

References

Copyright 2005 - Indian Journal of Pharmacology

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