Antilipid Peroxidative Property of Shilajit

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Antilipid Peroxidative Property of Shilajit
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  PHYTOTEERAPY RESEARCH, VOL. 10,269-270 1996) SHORT CO MM NlCATlO N Antilipid Peroxidative Property of Shilajit Yamini B. Tripathi,* Savita Shukla, Savita Chaurasia and Shashikant Chaturvedi Biochemistry Section, Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India We have investigated the effect of shilajit on lipid peroxidation and glutathione content in rat liver homogenate. It inhibited lipid peroxidation induced by cumene hydroperoxide and ADP/Fe++ complex in a dose dependent manner. It also reduced the rate of oxidation of reduced glutathione content and inhibited ongoing lipid peroxidation, induced by these agents immediately after its addition to the incubation system. Keywords: Shilajit; lipid peroxidation; antioxidant. INTRODUCTION Shilajit is a blackish brown exudation, consisting of organic substances, metal ions and minerals from steep rocks of different formations commonly found in the Himalayan belt (1OOO-3000 m) from Nepal to Kashmir. Chemically it possesses oxygenated biophenyls, oxygenated benzocu- marins, several phenolic compounds, amino acids and triterpenes. It appears that latex and debris of several plants growing in those areas are responsible for the formation of shilajit. Shilajit is in use for the management of bronchial asthma, diabetes, genitourinary infections, wound healing and stomach ulcer. It is also used in cold stress and as a general tonic. Shilajit shows a positive response on memory and anxiety. In the Ayurveda, it is also recommended as antiaging, as medhya rasayana, adaptogenic and mast cell stabilizing property (Ghosal et al. 1989). We report, for the first time, the antioxidant property of shilajit on a liver homogenate system, induced by cumene hydroperoxide and ADP-complexed iron. The results indi- cate its protective response on both enzymatic and non-enzymatic lipid peroxidation and glutathione content. Besides the low molecular weight compounds, there are also several high molecular weight compounds that are present; such as humic acid (HA), fulvic acid (FA) and its derivatives, consisting of H-rich aromatic moieties. This suggests the involvement of humification in the formation of shilajit (Ghosal, 1988). MATERIALS AND METHODS Shilajit, collected from Nepal, was powdered and dissolved in water. The soluble fraction was saved and dried to recover the pure shilajit. This process was repeated three times. Pure shilajit showed peaks in the ultraviolet range at 371 nm and 270 nm. n vitro experiments were canied out on 5 rat liver homogenate in phosphate buffer saline (pH 7.4) as described earlier (Pandey et al. 1994). In brief, the homogenate was preincubated for different time intervals Author to whom correspondence should be addressed with different concentrations of shilajit and then cumene hydroperoxide (CHP, 1.5 mM) was added. At different time intervals, samples were collected and the concentration of malondialdehyde (MDA) and reduced glutathione was estimated by standard methods of Ohkawa et al. (1979) and Ellman (1959) respectively as described earlier (Tripathi et al. 1995; Sharrna et al. 1995). Similarly ADP-complexed iron (Hogeberg et al. 1975), an example of enzymatic lipid peroxidation, was also added to the system and the above parameters were evaluated. RESULTS AND DISCUSSION Protective effect of shilajit on induced lipid peroxidation The homogenate was preincubated with different concentra- tions of shilajit for 20 min and then 1.5 mMcumene hydroperoxide and ADP-complexed iron were added in 1 4 10 20 30 40 5 TIME (in min.) Figure 1. Effect of shilajit on CHP induced glutathione level in rat liver hornogenate. Control V), hilajit V), CHP (01, shilajit+CHP W. CCC 095141 8)</96/030269-02 0 1996 by John Wiley Sons, Ltd. Accepted revised) 6 April 1995  270 Y . TRIPATHI ETAL. 6 u .c 400 5 A 3 i? z C - 200 cn 2 I I.. 20 31 4 0 [, 10 TIME (in min.) Figure2. Curative effect of shilajit on CHP induced lipid perox- idation. No drug V), 5 min V), 0 min 0). different plates to induce lipid peroxidation. Shilajit inhib- ited the lipid peroxidation in the dose dependent manner Table 1) in both conditions. Effect on reduced glutathione GSH) content In normal conditions, the glutathione content declined sharply and reached to the basal level in 50 min as a result of autooxidation, but in the presence of shilajit 1 mg/mL), lsble 1. Protective effect of shilajit on cumene hydroperoxide and ADP-complexed iron induced lipid peroxidation. Values are mean SD f six animals in each group) Shilajit TEARS nW100 rng protein) lrng/rnL) Curnene hydroperoxide ADP<ornplexed iron 0.00 480.682 11.50 477.82 .98 0.30 420.43 f 10.93 438.78k9.79 0.60 365.302 11.25' 393.82k6.59' 0.90 298.40 10.23 301.642 8.79' 1.20 216.302 10.83 225.30k9.32' 2.40 140.60+ 11.23 186.25+ 8.75' Level of significance 'p<0.001. Cumene hydroperoxide= 1.5 mM. ADP-complexed iron=1.6 mM ADP+62 *M FeCI,. the rate of decline was significantly reduced Fig. 1). Its normal level was maintained up to 60 min. This effect was seen even in the presence of 1.5 mMcumene hydro- peroxide. Curative response of shilajit In a separate experiment the curative response of shilajit was investigated. Here, it was added to the incubation mixture after 4 min and 8 min of CHP addition. The results Fig. 2) indicate a significant inhibition in the production of malondialdehyde. It is now well established that free radicals are involved in several diseases Cheeseman and Slater, 1993). The therapeutic use of shilajit in the cure of asthma, diabetes, wound healing, cold stress, ulcers Tewari et al. 1993; Jaiswal and Bhattacharya, 1992) and also the presence of several triterpenes, and phenolic compounds Ghasal, 1988) led us to investigate the effect of shilajit on lipid peroxidation, because in these diseases, free radicals are involved and compounds of this chemical structure, have a free radical scavenging property. Interestingly, the water extract of shilajit also exhibits significant antioxidant property on both enzymatic and nonenzymatic lipid perox- idation. This property is not common with other antioxidants which are in common use and raises the therapeutic value of shilahjit. On comparison with an established antioxidant of plant srcin, i.e. Rubia cordifolia, we found that the activity of shilajit was more than the alcohol extract of R. cordifoliu and comparatively less than parabenzoquinone, a synthetic antioxidant Tripathi et al., 1995). The ED,, ratio indicates that 1.2 mg of shilajit is equivalent to 2.3 mg of R. cordiofolia extract and 0 06 mg parabenzoquinone. This study opens a ray of hope that although many plants described in ayuwedic literature are now extinct, they may be utilized therapeutically by using their exudate in the form of humous of that locality that has been formed over the centuries. Acknowledgements The authors are thankful to Mukta Sharma for her help in experiments and Dr. C. B. Jha for providing the sample of shilajit. Y.B.T. s the recipient of a UGC Career Award. REFERENCES Cheeseman, K. H. and Slater, T. F (eds) (1993). n Free Radicals in Medicine The British Council, Churchill Livingstone, UK. Ghosal, S. (1988). The facets and facts of Shilajit. Proc. National Symp. on Development of lndigenous Drugs in lndia during Past 5 years pp. 3-7. Institute of History of Medicine Medical Research, Hamdard Nagar, New Delhi. Ghosal, S. Lal, J., Srivastava, R. S. et a/., (1989). Immunomodu- latory and CNS effects of sitoindosides IX and X. Phytother. Res. 3,201-206. Hogeberg, J., Moldeus, P Arborgh, 6.. Obrion, Pand Orrenius, S. (1975). The consequences of lipid peroxidation in isolated hepatocytes. Eur. J. Biochem. 59,457-462. Jaiswal, A. K. and Bhattacharya, S. K. (1992). Effects of Shilajit on memory, anxiety and brain rnonoamines in rats. lndian J. Pharmacol. 24,12-17. pp. 481-493. Ohkawa, H., Ohishi, N. and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95,351. Pandey, S. Sharma, M., Chaturvedi, I and Tripathi, Y. 6. (1994). Protective effect of R. cordifolia on lipid peroxide formation in isolated rat liver homogenate. lndian J. Expo. Biol 32, 180-1 83. Sharma, M., Tripathi, F? Singh, V. F? and Tripathi, Y. 6. (1995). Hepato-protective and toxicological evaluation of a com- pound herbal medicine. lndian J. Exp. Biol. 33,34-37. Tewari, P V. Tewari, K. C., and Joshi, P (1973). An interpretation of Ayarvedic findings on Shilajit. J. Res. lnd. Med. 8 53-55. Tripathi, Y. 6.. Shukla, S Sharma, M. and Shukla, V. K. (1995). Antioxidant property of Rubia cordifolia extract and its comparison with vitamin E and parabenzoquinone. Phyt- other. Res. 9 (6). 440-443.
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