International Journal of Pharma and Bio Sciences

RESEARCH ARTICLE
PHARMACOLOGY

ANTIOXIDANT ACTIVITY OF PEDALIUM MUREX FRUITS IN CARBON TETRA CHLORIDE- INDUCED HEPATOPATHY IN RATS

Corresponding Author

MADHU BABU A
Department of Pharmacology, Vikas college of Pharmacy, Jangaon, Andhra Pradesh. India.

Co Authors

SRINIVAS P , VENKATESHWARULU L1 AND ANIL KUMAR CH2

Department of Pharmacology, Vikas college of Pharmacy, Jangaon, Andhra Pradesh. India. Department of Biochemistry, Vikas College of Pharmacy, Jangaon, Andhra Pradesh, India.

ABSTRACT
In this study antioxidant activity of methanol extract of fruits of pedalium murex (MEC) was investigated using carbon tetrachloride (CCl4)- intoxicated rat liver as the experimental model. The hepatotoxic rats were administered MEC for 90 days (daily, orally at the dose of 70 mg per kg body weight). Lipid peroxidation (LPO) in CCl4 - intoxicated rats was evidenced by a marked increment in the levels of thiobarbituric acid reactive substances (TBARS) and diene conjugates (CD), and also a distinct diminution in glutathione (GSH) content in the liver. In CCl4 + MEC treated rats these biochemical parameters attained an almost normal level. The decreased activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GRD) in CCl4 intoxicatedrats, and its retrieval towards near normalcy in CCl4 + MEC- administered rats revealed the efficacy of MEC in combating oxidative stress due to hepatic damage. Elevated level of glutathione transferase(GTS) observed in hepatotoxic rats too showed signs of returning towards normalcy in MEC coadministered animals, thus corroborating the antioxidant efficacy of MEC. The findings provide a rationale for further studies on isolation of active principles and its pharmacological evaluation.

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KEY WORDS
Antioxidant enzymes, Carbon tetrachloride, Pedalium murex Linn, Lipid peroxidation.

INTRODUCTION
Pedalium murex L. (Pedaliaceae) is a diffuse, more or less succulent herb found near the sea coast of south India,1. The fruits as well as the leaves and stems produced milk mucilage when agitated, and

it is recommended as a treatment for gonorrhea2. An infusion or extract prepared from leaves is diuretic and demulcent, useful in treating disorders of the urinary system such as ardor urine, dysuria, spermatorrhoea, and incontinence of urine. As an emmenagogue, the juice is used in puerperal diseases and also to promote lochial discharge 3.The mucilage from leaves and young shoots is used as an aphrodisiac in seminal debility 4. The petroleum ether extract of P. murex is effective against Japanese encephalitis vector culex 5. The aqueous extract of the whole plant has been found to possess analgesic and antiExtensive inflammatory properties6. phytochemical investigations on the plant have revealed the presence of Pedalitin and Pedalin (major flavanoids) along with Diosmetin, Dinatin,Dinatin-7-glucoronide, Quercetin, Quercimeritin, and Quercetin-7glucorhamnoside7.Triterpenoids such as amyrin acetate, Rubusic acid, ursolic acid, and lupeol acetate are reported8. Steroids such as sitosterol9, Sapogenins10 and Diosgenin11 have also been reported. Lipids 12, phenolic acids such as caffeic acid, ferulic acid, protocathechic acid, and vanillic acid 9, and amino acids such as aspartic acid, glutamic acid, and histidine are other phytoconstituents present in P. murex 13. Although the plant contains several phytoconstituents, they have not been evaluated for their pharmacological activities in detail. Since no scientific data are available on the plant. Therefore in the present work attempt has been made to study the antioxidant effect of

fruits of Pedalium murex in CCl4-intoxicated experimental rats. It has been hypothesized that one of the principal causes of CCl4 induced liver injury is LPO by free radical derivatives of CCl4. Thus the antioxidant activity or the inhibition of the generation of free radicals is important in the protection against CCl4 induced Hepatopathy14 Antioxidant action has been reported to play a crucial role in the hepatoprotective capacity of many plants, In Ayurveda, an indigenous system of medicine in India, has a long tradition of treating liver disorders, with plant drugs. Thus search for crude drugs of plant origin with antioxidant activity has become a central focus of study of hepatoprotection. This may prove effective in alleviating tissue damages prevalent in organisms as a consequence of exposure to toxins of extrinsicor intrinsic origin.

MATERIALS AND METHODS

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for further use. LD 50 of MEC was found to be 180 mg/kg body weight of animals. Experimental Animals Albino rats either sex, weighing 230-250 were obtained from the animal house of Lalitha College of pharmacy. The animals were divided into 3 groups eight animals each. The Study was conducted after clearance from the Animal ethical committee. CCl4 induced liver damage Hepatopathy was induced in animals by subcutaneous (sc) administration of CCl4 at lower Abdomen twice a week at the dose of 1 ml per kg. Body weight in double the volume of liquid paraffin (lp) which served as a vehicle. CCl4 was administered on the first and fourth day of every week. Experimental Procedure Body weight of animals was recorded and then they were divided into 3 groups of 8 rats each. Group I animals served as control, which received sc administration of lp only twice a week at the dose of 3ml per kg body weight of each animal. Group II constituted the hepatotoxic group which received sc administration of CCl4 + lp twice a week as mentioned elsewhere. Group III were the herbtreated ones which received sc administration of CCl4 + lp twice a week as mentioned above. They also received MEC daily at the dose of 70 mg/kg body weight (effective dose) of each rat in a suspension of 1 ml water, orally by intubation. A pilot study revealed that MEC evoked hepatoprotection at doses ranging 40-120 mg/kg body weight of animals. Animals were maintained at laboratory conditions for a period of 90 days. Animals were fasted overnight on the

89th day. On the next day, after recording body weight, the animals were sacrificed by decapitation and blood was collected by the incision of jugular vein. The liver was dissected out, blotted off blood, rinsed in phosphate buffered saline (pH 7.4) and immediately proceeded for biochemical estimations. Serum was prepared from the collected blood. Biochemical Estimations The measurement of thiobarbituric acid reactive substances (TBARS) was done as an index of LPO.15 CD content was found out by the method of Klein 16. Activities of SOD and CAT were determined by the methods of Marklund and Marklund, 17 and Aebi.H, 18, respectively. GSH content was determined after deproteinisation by the method of Beautler and Kelly, 19. GPX was assayed by the method of 20. Glutathione transferase (GTS) and GRD were assayed by the methods of 21, and 22 respectively. Statistical Analysis The results were presented as the mean SEM. Studentst test was used to analyse statistical Significance.

RESULTS
The concentration of TBARS and CD was significantly higher in liver of CCl4- treated rats, as compared to normal control animals. (Table 1). These constituents were found to attain a near Conversely, GSH content in liver of Group II animals showed a significant decline when compared with controls. But in Group III animals GSH content was found to attain near normalcy.

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Table: 1 Effect of Pedalium murex fruit on the antioxidant status of liver in rats

Parameters

Group I

Group II

Group III

Thiobarbituric acid reactive substances TBARS ( mol / mg protein) Diene conjugates CD ( mol / 100 g tissue) Reduced glutathione GSH ( mol / 100 g tissue) 378.6 17.6 206.4 10.8* 349.2 11.9** 0.81 0.05 1.08 0.04* 0.86 0.04**

0.24 0.03

0.72 0.06*

0.28 0.05**

Values are mean SEM of 8 animals in each group. * P < 0.01 as compared to Group I. ** P < 0.01 as compared to Group II.

Activities of antioxidant enzymes are presented in Table 2. The levels of SOD, CAT, GPX, and GRD recorded a significant decline in CCl4- administered rats, when compared with normal controls. In CCl4 +MEC- treated rats, the activities of these enzymes attained a near-normalcy. However, the activity of GTS was significantly higher in CCl4 treated animals, which was brought down towards normalcy in herb-treated rats. Table: 2 Effect of Pedalium murex Linn on activity of antioxidant enzymes

Parameters Superoxide dismutase - SOD (U / mg protein) Catalase - CAT (U / mg protein) Glutathione peroxidase GPX (U / mg protein) - GRD (U / mg protein) 0.44** Glutathione transferase GTS ( mol / mg protein)

Group I

Group II

Group III

12.63 0.31 8.38 0.39 0.82 0.09 6.94 0.52

7.69 0.38* 4.86 0.32* 0.59 0.07 * 2.99 0.32*

11.93 0.59** 7.93 0.41** 0.80 0.09** 5.99

8.28 0.82

15.61 0.94*

8.19 0.61**

Values are mean SEM of 8 animals in each group * p < 0.01 as compared to Group I. ** p < 0.01 as compared to Group II.

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DISCUSSION
Ample experimental and epidemiological studies support the involvement of oxidative stress in the pathogenesis and progression of several chronic diseases.23 it is now known that oxygen, Indispensable for maintaining life, sometimes becomes toxic and results in the generation of most aggressive agents such as reactive oxygen species (ROS). The high reactivity of ROS may trigger a host of disorders in body resulting in tissue damage and necrosis in many instances.24 CCl4 mediated hepatotoxicity was taken here as the experimental model for liver injury. It has been established that CCl4 is accumulated in hepatic parenchymal cells and metabolically activated by cytochrome P-450 dependent monoxygenases to form a trichloromethyl free radical (CCl3) which alkylates cellular proteins and other macromolecules with a simultaneous attack on polyunsaturated fatty acids in the presence of oxygen to produce lipid peroxides leading to liver damage.25A study using methanol extract of fruits of pedalium murex having doses ranging 40 - 120 mg/ kgbody weight revealed the extract with dose 70 mg/ kg body weight offering the maximum hepatoprotection with respect to different liver marker enzymes, such as aspartate aminotransferase (AST ), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT). The body has an effective mechanism to prevent and neutralize the free radical induced damage. This is accomplished by a set of endogenous antioxidant enzymes, such as SOD, CAT, GPX and GRD etc. When the balance between ROS production and antioxidant defenses is lost, oxidative stress results, which through a series of events deregulates the cellular functions leading to various pathological conditions26. Any compound, natural or synthetic, with antioxidant properties might contribute towards the partial or total alleviation of this type of damage.

In the present study, elevated level of TBARS and CD observed in CCl4- treated rats indicates excessive formation of free radicals and activation of LPO system resulting in hepatic damage. TBARS produced as byproducts of LPO that occurs in hydrophobic core of bio-membranes.27 The significant decline in the concentration of these constituents in the liver tissue of CCl4 +MEC administered rats indicates anti-lipid peroxidative effect of Pedalium murex. GSH is a major non-protein thiol in living organisms which plays a central role in coordinating the bodys antioxidant defence processes. Perturbation of GSH status of a biological system has been reported to lead to serious consequences.26 Decline in GSH content in the liver of CCl4- intoxicated rats, and its subsequent return towards nearnormalcy in CCl4 +MEC- treated rats reveal antioxidant effect of Pedalium murex fruits. Explanations of the possible mechanism underlying the hepatoprotective properties of drugs include the prevention of GSH depletion and destruction of free radicals28. These two factors are believed to attribute to the hepatoprotective properties of fruits of pedalium murex SOD, CAT and GPX constitute a mutually supportive team of defence against ROS. SOD is a metalloprotein and is the first enzyme involved in the antioxidant defence by lowering the steadystatelevel of O2- . CAT is a hemeprotein, localized in the peroxisomes or the microperoxisomes. This enzyme catalyses the decomposition of H2O2 to water and oxygen and thus protecting the cell from oxidative damage by H2O2 and OH. GPX is a selenoenzyme two third of which (in liver) is present in the cytosol and one third in the mitochondria. It catalyses the reaction of hydroperoxides with reduced glutathione to form glutathione disulphide (GSSG) and the reduction product of the hydroperoxide. In our study, decline in the activities of these enzymes in CCl4-

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administered rats revealed that LPO and oxidative stress elicited by CCl4 intoxication have been nullified due to the effect of fruits of pedalium murex. This observation perfectly agrees with those of 29 who investigated hepatoprotective and antioxidant activity of Bohemia nivea. GTS plays an essential role in liver by eliminating toxic compounds by conjugating them with glutathione. GRD is concerned with the maintenance of cellular level of GSH (especially in the reduced state) by effecting fast reduction of oxidized glutathione to reduced form. The activities of these enzymes were found to be in the reverse order. In liver tissues of CCl4- administered rats, level of GTS registered a significant increment, whereas that of GRD recorded a decline. However, these enzymes restored an almost normal activity in CCl4 + MEC administered rats, thus unearthing the antioxidant effect of pedalium murex.

Natural antioxidants strengthen the endogenous antioxidant defences from ROS ravage and restore the optimal balance by neutralizing the reactive species. They are gaining immense importance by virtue of their critical role in disease prevention. In conclusion, it can be said that methanol extract of Fruits of pedalium murex exhibit a liver protective effect against CCl4- induced hepatotoxicity and possessed anti-lipid peroxidative and antioxidant activities. Efforts are in progress here to isolate and purify the active principle involved in the hepatoprotective efficacy of this medicinal plant.

ACKNOWLEDGEMENT
Authors are thankful to L.Venkateshwarulu, Head Department of Pharmaceutical chemistry. Vikas college of Pharmacy, Jangaon, for providing necessary laboratory facilities and the encouragement.

REFERENCES
1. 2. Nadkarni KM. Indian Materia Medica. Popular Prakashan, Bombay; 1982. Mhaskar KS, Blatter E, Caiur JF. In: Kritikar and Basus illustrated Indian Medicinal Plants, their usage in Ayurveda and Unani medicines, Vol-8, Indian Medicinal Science Series No 93,PID; New Delhi, 2000, pp. 2555-2559. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants, National Institute of cience Communication (CSIR), New Delhi; 1996. Shukla VN, Khanuja SPS. Chemical, Pharmacological and Botanical studies on Pedalium murex. Journal of Medicinal and Aromatic Plant Sciences 26: 64-69, 2004. Venkatarathina KT, Muthusamy VA, Ramanathan S et al. Evaluation of the petroleum ether extracts of Pedalium murex against Japanese encephalitis vector culex Tritaenlorhynchus.Antiseptic 102: 335336, 2005. 6. Muralidharan P, Balamurugan G. Analgesic and anti-inflammatory activities of aqueous extract of Pedalium murex Linn. Biomedicine 28: 84-87, 2008. 7. Subramanian SS, Nair AGR. Flavonoids of the leaves of Pedalium murex Linn. Phytochemistry 11: 464-465, 1972. 8. Prasad TNV, Sastry KV. A note on the chemical examination of Pedalium murex Linn. leaves. Indian Drugs 25: 84-85, 1998. 9. ShuklaYN, Thakur RS. Heptatriacontan-4one, Tetratriacontanyl octacosanoate and other constituents from Pedalium murex Linn. Phytochemistry 22: 973-974, 1983. 10. Harvey SK. A brief comparative pharmacognostic study of certain

3.

4.

5.

This article can be downloaded from www.ijpbs.net

P - 627

indigenous drugs. Natural Medicinal Journal 9: 519. 11. Mangle MS, Jolley CI. HPTLC studies on Tribulus terrestris (Chota ghokru) and Pedalium murex (Bada ghokru). Indian Drugs 35: 189-194, 1998. 12. Bhakuni RS, Shukla YN, Thakur RS. Flavonoids and other constituents from Pedalium murex Linn. Phytochemistry 31:2917-2918, 1992. 13. Rastogi JN, Sharma OD, Loiwal SD. Amino acids in certain medicinal plants. Bull Pure Appl Sci 1: 11-12, 1982. 14. Castro, J.A., Ferrya, G.C, Castro, C.R., Sasame, H, Fenos, O.M. and Gillette, J.R. Prevention of Carbon tetrachloride-induced necrosis by inhibitors of drug metabolism. Further studies on the mechanism of their action. Biochem. Pharmacol 23, 295-302, (1974). 16. Yagi, K. Lipid peroxides and Human disease. Chem. Phys. Lipids. 45, 337-351, (1987). 17. Habig, W.H., Pabst, M.J. and Jakpoby, W.B. Glutathione transferase : A first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139, (1974). 18. Aebi, H. Catalase In : Methods in Enzymatic Analysis, Ed Bergmeyer. H.U. Academic Press, New York, Vol 3, p. 276- 286, (1983). 19. Beautler, E. and Kelley, B.M. The Effects of Sodium nitrate on red cell glutathione. Experientia 19, 96-97, (1963). 20. Rotruck, J.T., Pope, A.L. and Gantter, H.E. Selenium: Biochemical roles as a component of glutathione peroxidase. Sci. 179, 588-590, (1973). 21. Habig, W.H., Pabst, M.J. and Jakpoby, W.B. Glutathione transferase : A first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139, (1974).

22. Racker, E Glutathione reductase ( liver and yeast ) In : Methods in Enzymology, Eds Colowick, S.P. and Kaplan, N.O. Academic Press, New York, Vol VII, p 722-725, .(1955 ) 23. Sunita Tewari, Vani Gupta and Sandeep Bhattacharya. Comparative study of antioxidant potential of tea with and without additives. Ind. J. Physiol. Pharmacol. 44 (2), 215-219, (2000). 24. Prasad Varier, S., Venkatachalam, S.R., Ramesh Chander. and Paul Thomas. Dietary antioxidantsnatural defence against disease. Aryavaidyan 12(3), 149158, (1999). 25. Recknagel, R.O. A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sci. 33, 401-408, (1983). 26. Uday Bandyopadhyay, Dipak Das. and Ranajit Banerjee, K. Reactive oxygen species: oxidative damage and pathogenesis. Curr. Sci. 77 (5), 658-665, (1999). 27. Fraga, C., Leibovitz, B. and Tappel, A Halogenated compounds as inducers of lipid peroxidation in tissue slices. Free Rad. Biol. Med. 3, 119-123, (1987). 28. Valenzuela, A., Lagos, C., Schmidt, K. and Videla, K, Silymarin protection against hepatic lipid peroxidation induced by acute ethanol intoxication in the rat. Biochem. Pharmacol. 3, 2209-2212, (1985). 29. Chun-Chun Lin., Ming-Hong Yen, TsaeShiuan Lo. and Jer-Min Lin. Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea var. nivea and B. nivea var, tenacissma. J. Ethnopharmacol 60, 9-17, (1998).

This article can be downloaded from www.ijpbs.net

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