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In This Article
 »  Abstract
 »  Introduction
 »  Material and Methods
 »  Results
 »  Discussion
 »  Acknowledgement
 »  References

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RESEARCH PAPER
Year : 2004  |  Volume : 36  |  Issue : 5  |  Page : 284-287
 

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


School of Biosciences, Mahatma Gandhi University, P. D. Hills, P. O., Kottayam - 686 560, India

Date of Submission23-May-2003
Date of Decision10-Feb-2004
Date of Acceptance15-Feb-2004

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

 » Abstract 

OBJECTIVE: To evaluate the potential efficacy of Glycyrrhiza glabra Linn. (Fabaceae) in protecting tissues from peroxidative damage in CCl4-intoxicated rats. MATERIAL AND METHODS: Peroxidative hepatic damage in rats was studied by assessing parameters such as thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), superoxidedismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px) and glutathione (GSH) in liver and kidneys. The effect of co-administration of G. glabra on the above parameters and histopathological findings of the liver in experimental animals was studied. RESULTS: The increased lipid peroxide formation in the tissues of CCl4-treated rats was significantly inhibited by G. glabra. The observed decreased antioxidant enzyme activities of SOD, CAT, GSH-Px, GST, and antioxidant concentration of glutathione were nearly normalized by G. glabra treatment. Carbon tetrachloride-induced damage produces alteration in the antioxidant status of the tissues, which is manifested by abnormal histopathology. G. glabra restored all these changes. CONCLUSION: Glycyrrhiza glabra is a potential antioxidant and attenuates the hepatotoxic effect of CCl4.

How to cite this article:
Rajesh M G, Latha M S. Protective activity of Glycyrrhiza glabra Linn. on carbon tetrachloride-induced peroxidative damage. Indian J Pharmacol 2004;36:284-7


How to cite this URL:
Rajesh M G, Latha M S. Protective activity of Glycyrrhiza glabra Linn. on carbon tetrachloride-induced peroxidative damage. Indian J Pharmacol [serial online] 2004 [cited 2020 Jul 9];36:284-7. Available from: http://www.ijp-online.com/text.asp?2004/36/5/284/12646



 » Introduction Top

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 Top

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±2oC, 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 45oC 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 Top

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 CCl4 treatment 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 Top

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.

 » Acknowledgement Top

The first author is extremely grateful to M.G. University, Kottayam, Kerala, India for providing financial assistance in the form of JRF. 

 » References Top

1.Athar M, Zakir Hussain S, Hassan N. Drug metabolizing enzymes in the liver. In: Rana SVS, Taketa K, editors. Liver and Environmental Xenobiotics. New Delhi: Narosa Publishing House. 1997.  Back to cited text no. 1    
2.Rajesh MG, Latha MS. Hepatoprotection by Elephantopus scaber Linn in CCl4-induced liver injury. Indian J Physiol Pharmacol 2001;45:481-6.  Back to cited text no. 2  [PUBMED]  
3.Anandan R, Deepa Rekha R, Devaki T. Protective effect of Picrorrhiza kurroa on mitochondrial glutathione antioxidant system in D-galactosamine- induced hepatitis in rats. Curr Sci 1999;76:1543-5.  Back to cited text no. 3    
4.Sitohy MZ, el Massry RA, el- Saadany SS, Labib SM. Metabolic effect of licorice roots (Glycyrrhiza Glabra) on lipid distribution pattern, liver and renal functions of albino rats. Nahrung 1991;35:799-806.  Back to cited text no. 4    
5.Rajesh MG, Beena Paul, Latha MS. Efficacy of Kamilari in alcoholic liver cirrhosis. Antiseptic 2000;97:320-1.  Back to cited text no. 5    
6.Haraguchi H, Yoshida N, Ishikawa H, Tamura Y, Mizutani K, Kinoshita T. Protection of mitochondrial functions against oxidative stresses by isoflavans from Glycyrrhiza glabra. J Pharm Pharmacol 2000;52:219-23.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Nichans WG, Samuelson B. Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-30.  Back to cited text no. 7    
8.Klein RA. The detection of oxidation in liposome preparations. Biochim Biophys Acta 1983;210:486-9.  Back to cited text no. 8    
9.Abei H. Catalase. In Bergmeyer HU, editor. Methods in Enzymatic analysis. New York: Academic press 1983.  Back to cited text no. 9    
10.Marklund S, Marklund G. Involvement of superoxide anion radical in auto oxidation of pyrogallol and a convenient assay of super oxide dismutase. Eur J Biochem 1974;47:469-74.  Back to cited text no. 10  [PUBMED]  
11.Habig WH, Pabst MJ, Jackpoby WB. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130-9.  Back to cited text no. 11    
12.Rotruck JT, Pope AL, Gantter HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical roles as a component of glutathione peroxidase. Science 1973;179:588-90.  Back to cited text no. 12    
13.Beutler E, Kelley BM. The effects of sodium nitrate on red cell glutathione. Experientia 1963;19:96-7.  Back to cited text no. 13    
14.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin-phenol regent. J Biol Chem 1951;193:265-75.  Back to cited text no. 14  [PUBMED]  
15.Bandhopadhay U, Das D, Banerjee KR. Reactive oxygen species: Oxidative damage and pathogenesis. Curr Sci 1999;77:658-65.  Back to cited text no. 15    
16.Tabatabaie T. Floyd RA. Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents. Arch Biochem Biophys 1994;314:112-9.  Back to cited text no. 16    
17.Kera Y, Sippel HW, Penttila KE, Lindros KO. Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes. Biochem Pharmacol 1987;36:2003-6.  Back to cited text no. 17  [PUBMED]  
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