Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 36, Num. 5, 2004, pp. 284-287

Indian Journal of Pharmacology, Vol. 36, No. 5, October, 2004, pp. 284-287

Research Paper

Protective activity of Glycyrrhiza glabra Linn. on carbon tetrachloride-induced peroxidative damage

Rajesh MG, Latha MS

School of Biosciences, Mahatma Gandhi University, P. D. Hills, P. O., Kottayam - 686 560
Correspondence Address:School of Biosciences, Mahatma Gandhi University, P. D. Hills, P. O., Kottayam - 686 560 mslathas@eudoramail.com

Code Number: ph04098

ABSTRACT

INTRODUCTION

The liver is an organ of paramount importance, which plays an essential role in the metabolism of foreign compounds entering the body. Human beings are exposed to these compounds through environmental exposure, consumption of contaminated food or during exposure to chemical substances in the occupational environment. In addition, human beings consume a lot of synthetic drugs during diseased conditions which are alien to body organs. All these compounds produce a variety of toxic manifestations.[1] Conventional drugs used in the treatment of liver diseases are often inadequate. It is therefore necessary to search for alternative drugs for the treatment of liver diseases to replace the currently used drugs of doubtful efficacy and safety.

India is well known for a plethora of medicinal plants. The medicinal use of many plants (as hepatoprotectants) like Andrographis paniculata, Azadirachta indica, Cassia fistula, Elephantopus scaber, Hibiscus rosasinensis, Phyllanthus debilis, Picrorrhiza kurroa has been reported in the literature.[2],[3]

Glycyrrhiza glabra Linn. of the family Fabaceae is a tall perennial undershrub. Its underground stems and roots are used medicinally. Its hypocholesterolaemic and hypoglycemic activities have been reported.[4] It is known in the traditional system of medicine for its use in liver diseases. It is a major component of many antihepatotoxic polyherbal formulations.[5] Isoflavan derivatives glabridin, hisplaglabridin A, hisplaglabridin B and 4′O-methyl glabridin have been isolated from G. glabra. These chemicals were reported to provide protection against oxidative stress.[6] The biochemical damage produced by active oxygen species and free radicals has emerged as a fundamental pathway of liver injury. Despite the use of G. glabra in liver disorders, no systematic studies on its active oxygen scavenging properties have been reported. In this communication, we present the antiperoxidative effect of G. glabra on CCl₄-induced oxidative damage in rats, supported by histopathological evidence.

MATERIAL AND METHODS

Male albino rats of Sprague-Dawley strain, weighing between 120 g to 150 g were used for the experiment. They were housed in polypropylene cages under standard conditions (23±2^oC, humidity 60-70%, 12 h light/dark cycles) and given standard pellet diet (M/s Hindustan Lever Ltd, Mumbai, India). Water was given ad libitum. The animals were divided into 3 groups of 6 rats each. Group I served as pair-fed control which received the normal feed. Groups II and III received a dose of 0.3 ml CCl₄ in liquid paraffin (3:1, v/v) per 100 g body weight subcutaneously twice a week for a period of two months. Group III rats, in addition to CCl₄ received a dose of 1000 mg/kg body weight/ day of G. glabra root powder mixed with the feed for two months. The concentration of the powder in the feed was adjusted to the amount of food consumed. The dose of the medicinal plant was ascertained by a pilot study over a range of dosages varying from 500 mg/kg, body weight to 1500 mg/kg, body weight/day. Over the range of dosages studied, the plant did not show any toxicity.

Glycyrrhiza glabra roots were collected from the crude drug market, Pala, Kottayam district, Kerala. They were dried overnight at 45^oC and powdered. This preparation was used for the experiment.

At the end of the experimental period, the animals were killed by decapitation. The liver and kidney were dissected out immediately and transferred into ice-cold physiological saline for various biochemical estimations.

Sections of liver tissues were collected in 10% formol saline for proper fixation. Slices of fixed tissues were processed, embedded in paraffin, sectioned to a thickness of 5 mm, mounted on glass slides, and stained with hematoxylin and eosin for histopathological evaluation.

A 10% tissue (liver and kidney) homogenate was prepared using tris-HCl buffer (0.1M; pH 7.5) and used for the analysis. Lipid peroxidation was assessed in terms of thiobarbituric acid reactive substances (TBARS) and conjugated dienes (CD).[7],[8] Changes in the antioxidant status were determined by estimating the activities of catalase, superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione (GSH) and glutathione peroxidase (GSH-Px) in the liver and kidney.[9],[10],[11],[12],[13] The protein content of the tissues was estimated by the method of Lowry et al.[14] The results expressed as percent hepatoprotective activity (H) were calculated by the formula,

H= [l - (HC-N) / (C-N)] x 100 where HC, C and N are the parameters measured in herbal preparation + CCl₄ treated rats, CCl4 treated rats and normal (pair-fed) control rats, respectively.

Statistical analysis
The results are presented as the mean ± SEM. One-way analysis of variance (ANOVA) followed by the Bonferroni test were applied for statistical analysis with the level of significance set at P<0.05.

RESULTS

Feeding CCl₄ to rats for two months resulted in significant loss of body weight. Treatment with the medicinal plants along with CCl₄ prevented the loss in body weight.

There was a significant increase in the concentrations of TBARS and CD during CCl₄treatment as compared with the pair-fed control. Administration of G. glabra together with CCl₄ resulted in significant decrease of TBARS and CD in the liver and kidney compared with the corresponding CCl₄-intoxicated group [Table - 1].

The activities of SOD, catalase, GST and GSH-Px in the tissues studied were significantly decreased in the CCl₄-treated rats compared with pair-fed control. There was also a decrease in the content of GSH in the tissues of Group II rats. Administration of G. glabra along with CCl₄ restored the activities of the above antioxidant enzymes and the level of glutathione to near normal compared to the corresponding CCl₄ administered rats.

Histopathological studies (compared to controls) demonstrated fatty change and ballooning degeneration of hepatocytes induced by CCl₄-liquid paraffin. The liver also showed distorted architecture with nodule formation, distorted central vein and the portal triad showed fibrous portal expansion with moderate fibrosis and moderate inflammation [Figure - 1]a-c. Administration of the root powder of G. glabra at a dose of 1000 mg/kg, body weight exhibited significant improvement [Figure - 1]d.

As a part of the pilot study, we also evaluated the effect of G. glabra on normal rats. Over the range of dosages studied, the plant did not show any alteration of the antioxidant defense and on liver function tests.

DISCUSSION

The reactive metabolites such as trichloromethyl (CCl₃·) and trichloromethyl peroxy (CCl₃OO·) radicals emanated from CCl₄ initiate peroxidation of membrane unsaturated fatty acids. This lipid peroxidation of membrane seriously impairs its function and produces liver injury.

The antioxidant enzymes SOD, catalase and peroxidases constitute a mutually supportive team of defense against reactive oxygen species (ROS).[15],[16] The decrease in the activity of SOD in the liver and kidney of CCl₄-treated rats may be due to the increased lipid peroxidation or inactivation of the enzyme by cross-linking with malondialdehyde. This will cause an increased accumulation of superoxide radicals, which could further stimulate lipid peroxidation. GST binds to lipophilic compounds and acts as an enzyme for GSH conjugation reactions.[3] The decrease in the activity of GST during CCl₄ toxicity may be due to the decreased availability of GSH and suggests a total inhibition of drug metabolism during CCl₄-intoxication.

Depletion of GSH results in enhanced lipid peroxidation, which in turn causes increased GSH consumption.[3] The medicinal herb-treated rats restored the changes in the activity of the antioxidant enzymes and the level of glutathione.

Hepatotoxins develop hypoxic conditions which can damage the perivenular zone of the hepatic acinus. The highest expression of Cytochrome P450 2E1 (CYP2E1) in the perivenular region produces oxy-radicals that contribute to the injury. Moreover, hepatocytes in the perivenular area contain less antioxidant factors and antioxidant enzymes.[17] Thus, while the lipid peroxidation mediated by oxy radicals is likely to be the highest in the perivenular area, the detoxifying capacity of the hepatocytes here is reduced, therefore, the production may exceed the detoxification in the perivenular area.

In short, CCl₄-induced damage produces alteration in the antioxidant status of the tissues, which is manifested as an abnormal histopathology. G. glabra restored all these changes. So, it can be concluded that the herb is a potential antioxidant and attenuates the hepatotoxic effect of CCl₄ by acting as an in vivo antioxidant and thereby inhibiting the initiation and promotion of lipid peroxidation or by an accelerated scavenging of free radicals and their products by conjugation with GSH aided by GST.

ACKNOWLEDGEMENTS

REFERENCES

Copyright 2004 - Indian Journal of Pharmacology

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