Title of Invention

A PHARMACEUTICAL COMPOSITION USEFUL AS AN ANTIOXIDANT

Abstract

This invention relates to a pharmaceutical composition useful as antioxident with free radical scavenging activity . This invention particularly relates to berginin as an antioxident with free radical scevenging activity obtained from a new source namely Tinospora cripsa. This invention also relates to a process for the isolation of bergenin from Tinospora crispa, a new source, with new antioxidant and free radical scavenging activity. Particularly this invention relates to a process of isolation of bioactive bergenin, a new antioxidant and free radical scavenger from Tinospora crispa, having the formula 1 accompanying this specification, by extracting powdered stems in a polar solvent like rectified spirit, methanol, in glass percolator or in Soxhlet extractor, removing fatty nonpolar constituents by triturating with hexane, dichloromethane, chloroform or ethyl acetate, to get rich bioactive fraction, on crystallisation with polar solvents furnished bergenin (1).

Full Text

This invention relates to a pharmaceutical composition useful as antioxident with free radical scavenging activity . This invention particularly relates to berginin as an antioxident with free radical scevenging activity obtained from a new source namely Tinospora cripsa. This invention also relates to a process for the isolation of bergenin from Tinospora crispa, a new source, with new antioxidant and free radical scavenging activity. Particularly this invention relates to a process of isolation of bioactive bergenin, a new antioxidant and free radical scavenger from Tinospora crispa, having the formula 1 accompanying this specification, by extracting powdered stems in a polar solvent like rectified spirit, methanol, in glass percolator or in Soxhlet extractor, removing fatty nonpolar constituents by triturating with hexane, dichloromethane, chloroform or ethyl acetate, to get rich bioactive fraction, on crystallisation with polar solvents furnished bergenin (1). (Figure Removed) Tinospora crispa Mier. (Synonyms Tinospora tuberculata Beumee and Tinospora rumphii Boerl. (Family Minispermaceae). It is found in Assam, Burma, cultivated in Malaya Peninsula and Ceylon [Chadha, Y. R., The Wealth of India, Raw materials, Publication and Information Directorate, CSIR, New Delhi, Vol. X, Page 252, 1976]. The whole plant is exceedingly bitter, and is employed by Malays in the cure of intermittent fever, in India and China the stem is considered a febrifuge of great importance, widespread opinion that it is an excellent tonic during convalescence from exhausting diseases. In the Philippine Islands it is considered to be panacea to be applied to all bodily afflictions. It is given in general debility in chronic rheumatism, in malarial fevers [Kiritikar, K. R., Basu, B. D. and An I. C. S., Indian Medicinal Plants, Second Edition, International Book Distributors, Dehradun, India, Vol. I, Page 76, 1987]. So far very little chemistry have been done on Tinospora crispa. Tinocrisposide, a bitter furanoditerpene glycoside has been isolated from Tinospora crispa stem [Pachaly, P. and Adnan, A. Z., Arch. Pharm. (Weinheim) 325, 705-708 (1992). NFormylannonaine, N-formylnornuciferine and N-acetylnomuciferin have been reported to be present in stems. Many furano diterpenoid glycosides borapetoside A-G have been isolated from Tinospora tuberculata [Fukuda, N., Yonemitsu, M., and Kimura, T., Liebig. Ann. Chem., 491-494 (1993)]. Furano diterpenes tinotufolin A and B were isolated from Tinospora tuberculata [Fukuda, N., Nakamura, M., Yonemitsu, M., Kimura, T., Isobe, R. and Komori, Tetsuya [Liebig. Ann. Chem. 325-327 (1993)]. Several furano diterpene glucosides have been isolated from Tinospora rumphii stems [Martin, T. S., Ohtani, K., Kasai, R. and Yamasaki, K., Phytochemistry, 40, 1729- 1736 (1995) and Martin, T. S., Ohtani, K., Kasai, R. and Yamasaki, K., Phytochemistry, 42,153-158(1996)]. Till today there is no report of the isolation of bergenin from Tinospora crispa. However, it has been isolated earlier from Bergenia crassifolia roots [Hay, J. E. and Haynes L. J., Journal of Chemical Society, 2231-2238(1958)] and from Bergenia ligulata roots [Jain, M. K. and Gupta, R., Journal of Indian Chemical Society, 39, 559-560 (1962). The isolation procedure described by Hay et al was as follow: The roots and rhizomes of Bergenia crassifolia (1 kg. wet weight) were thoroughly dried in vacuumoven at 60° and ground to powder (446 g) which was poured into acetone (1 1) contained in a glass column (24 x 2.5 in.). After 16 hr. the column was eluted with acetone (total volume, 2.5 1) and three fractions were removed. Paper chromatography of each fraction in butanol-acetic acid-water (4:1:5) and butanol-ethanol-water (4:1:5) showed the same phenolic component to be present in each fraction, detected as an orange spot on spraying of the air-dried paper with diazotised sulphanilic acid in 10% aqueous sodium carbonate; no other phenolic products were detected. The fractions were concentrated to brown syrups, dissolved in acetone, and set aside. Crystalline bergenin separated from each fraction, in a total yield of 15 g. Recrystallisation from water yielded the hydrate, m.p. 133°. Several recrystallisation from methanol followed by thorough drying (6 hr. at 150°) gave anhydrous bergenin, m.p. 238°. However, there has been no report on the isolation of bergenin, which is useful as a free radical scavenging agent from Tinospora crispa. Word "antioxidants" and "oxidative stress" are widely used but surprisingly difficult to define precisely. For example, the term antioxidant as used in the literature is often implicitly restricted to chain-breaking antioxidant inhibtors of lipid peroxidation. In particular, food scientists frequently equate antioxidants to inhibitors of lipid peroxidation because they use antioxidants largely to prevent rancidity. However, free radicals generated in vivo damage proteins, lipids, DNA and other molecules in addition to lipids. For that reason an antioxidant is any substance, when present at low concentrations compared to these of an oxidizable substance, significantly delays or prevents oxidation of that substance [Halliwell, B. and Gutteridge, J. M. C, Mehods Enzymol., 186, 1-85 (1990)]. The term oxidizable substrate includes almost everything found in living cells, including proteins, lipids, carbohydrates and DNA. Oxidative stress, denoting a disturbance in prooxidant/antioxidant balance in favour of prooxidants, leading to potential damage. However, the novel role of oxidants and antioxidants as part of signalling cascade, as mediators of adaptive responses has opened new area of active research. Although it is too early to speak of "free radical diseases", but evidences are there for the crucial roles of oxidants and antioxidants in disease states. As most diseases ultimately lead to cell injury or cell death. It is postulated that several of the stress-induced diseases may be due to oxidative stress brought about by either increased generation of reactive oxygen species (ROS) or decreased free radical scavenging activity.[ Richard, R. T. and Sharma, M. M., Ind. J. Clin. Pract., 2, 15-18 (1991)]. It has been proposed that neutrophils could contribute to the ulceration process through the release of tissue damaging proteases and reactive oxygen metabolites. Role of oxygen radical have been implicated in causation of several diseases such as liver cirrhosis, athlerosclerosis, cancer, diabetes, ageing etc. and the drugs that can scavenge oxygen radical have great potential in ameliorating these disease processes. Although the body has several mechanisms to protect the action of oxygen radicals at times these protective mechanisms are not sufficient when compared to the insult produced to the body. Supplementation of non-toxic antioxidants may have achemoprotective role in the body in these conditions [Joy, K. L., Kuttan, G. and Kuttan, R., Ind. J. Expt. Biol., 37, 1177-1181 (1999)]. The superoxide anion (O2"), H202 and hydroxyl radical ('OH) are the major ROS which induce cell degeneration by increasing lipid peroxidation of cell membrane lipids. The toxic end products of the structural and functional integrity of cell membranes, break DNA stand and denaturate cellular proteins [Bhattacharya, A. and Ghosal, S., Ind. J. Expt. Biol., 38, 877-880 (2000)]. The natural cellular antioxidant enzymes include superoxide dismutase (SOD), which scavenges superoxide radicals by speeding up their dismutation, Catalase (CAT) a haeme enzyme which removes H2O2 and glutathione peroxidase (GPX), a seleniumcontaining enzyme which scavenges H2O2 and other peroxidases [Halliwell, B. and Gutteridge, J. M. C., "Free radicals in Biology and Medicine", 2nd Ed. Clarendon, Press, Oxford, 1989]. Detoxification of the superoxide anion is not a terminating step in free radical scavenging, since the enzyme catalysed dismutation results in the production of H2O2 which accumulates in the mitochondria and cytosol. Unless the peroxide is scavenged by CAT and GPX, it in the presence of iron, may also lead to production of HO'. These ROS together with singlet molecular oxygen, may attack lipid, protein and DNA of cells following increased lipid peroxidation chain reaction resulting in wide spread cellular injury and ultimately pathophysiological conditions. In the recent years, ROS and lipid peroxides have been implicated in the pathogenesis of a large number of diseases and environmental toxicities, these include rheumatoid, arthritis, dermatitis, hepatitis, ahterosclerotic cardiovascular disease, diabetes melitus, chronic bronchitis, crohn's disease, ulceration, side-effects of radiation and carcinogenesis [Niwa, Y., Ind. J. Clin. Prac., 1(8), 23-27 (1991)]. Agents that degenerate ROS and/or lipid peroxidation might therefore, be beneficial in the treatment of many of these disorders by removal of O2, scavenging reactive oxygen/nitrogen species or their precursors, inhibiting ROS/RNS formation, binding metal ions needed for catalysis of ROS generation and upregulation of endogenous antioxidant defences. Thus the main objective of the present invention is to provide berginin as an antioxident with free radical scavenging activity obtained from a new source namely Tinospora cripsa Another object is to provide a process for the isolation of bioactive bergenin from Tinospora crispa. Another object of this invention is to provide antioxidant and free radical scavenging activity of bergenin, isolated from Tinospora crispa, having the formula 1 accompanying this specification. Accordingly,the presently invention relates to a pharmaceutical composition useful as antioxidant with free radical scavenging activity comprising an effective amount of bergenin and pharmaceutically acceptable additives. hi an embodiment of the present invention wherein, the additive may be selected from nutrients, which are pharmaceutically acceptable carriers. hi yet another embodiment of the invention in the form of tablet, syrup, powder, capsule.. hi yet another embodiment of the invention bergenin shows inhibition of free radical in the range of 10 to 57 % at adose level of 25 to 200 micro gram per ml. .hi still another embodiment of the invention a method for the treatment of radical scavenging and antioxidant comprising the steps of administration of an effective amount of bergenin to a subject in need thereof. Accordingly, the present invention provides a process for the isolation of bioactive bergenin from Tinospora crispa, having the formula 1 which comprises: (a) powdering of the stems of Tinospora crispa, (b) extracting the powder plant material, with protic organic solvent, (c) concentrating the extract by conventional methods, (d) triturating the above said extract successively with organic solvent in order of increasing polarity to give residue, (e) crystallisating the residue with alcohol, by known method furnished bergenin. In an embodiment of the present invention the protic solvent used for extention in step (b) is selected from ractified spirit, methanol and mixture thereof. In another embodiment of the invention the organic solvent used in step (d) is selected from hexane, chloroform, ethylacetate and methanol. hi yet another embodiment of the invention the alcohol used for crystallisation may be methanol. In our copending patent application no nf 260/01, we have claimed and disclosed a process for the isolation of bioactive bergenin from Tinospora crispa. Characterisation of bergenin (1). Bergenin (1) obtained as crystalline compound, m.p. 237°C, [a]o20 -36.9° (c 1.85, methanol), FABMS m/z 351 [M+Na]+, Molecular formula Ci4H16O9. UV spectrum recorded in methanol showed absorption maxima at 224, 273, 310 nm. IR (KBr) spectrum showed absorption's at 3300, 1710, 1620, 1600, and 1470 cm'1. 1H NMR (200.13 MHz, Pyridine-ds) showed signals at δ 7.67 (1H, s), 5.16 (1H, d, J=10.2Hz), 4.00-4.70 (6H, m, sugar protons) and 3.94 (3H, s, Ar-OCHO. The I3C NMR spectrum also showed signals at δ 165.7, 152.0, 149.0, 142.0, 119.0, 117.1, 110.5, 82.0, 80.5, 75.0, 73.5,71.1,62.0,60.1. Further the structure of bergenin (1) was confirmed by converting it to pentacetate. 1 (100 mg) was dissolved in pyridine (2ml), to this was added acetic anhydride (2ml); the reaction mixture was kept at room temperature over night. The solvents were removed under high vacuum. The residue obtained was crystallised with mixture of hexane-ethyl acetate, MP. 201°C, !H NMR (200.13 MHz, CDC13) showed signals at δ 7.80 (IH, s), 5.52 (IH, t, J=9.3Hz), 5.16 (IH, t, J=9.4Hz), 4.84 (IH, d, J=10.6Hz), 4.39 (IH, t, J=10.4Hz), 4.35 (IH, m), 4.18 (IH, dd, J=12.6, 3.7Hz), 3.95 (3H, s), 3.85 (IH, m), 2.38 (6H, s), 2.14 (6H, s), 2.10 (3H, s). This is the first reported isolation of bergenin from genus Tinospora. Thus the location of collection of the raw material (Tinospora crispd), extraction and process of isolation of 1 has been standardised for maximum yield of the compound. The process being simple and novel is particularly useful for quick isolation of the compound from the plant for all purposes. Bergenin is a potent antioxidant and free radical scavenging molecule. On testing it against sodium nitroprusside induced nitric oxide production (for direct free radical scavenging) and DPPH interaction (a stable free radical) showed highly significant and dose related inhibition of free radical production and scavenging. The invention is described in detail by the examples given below which should not be construed to the limit of scope of the present invention. Example 1: The shade dried, powdered Tinospora crispa stems (0.5 kg) were extracted with rectified spirit (2.0 1) by hot continuous extraction in a Soxhlet apparatus for 32 hours. The rectified spirit was evaporated under reduced pressure to obtain a brown mass, and this extract was triturated successively with hexane (200 ml x 4), chloroform (200 ml x 4), and ethyl acetate (200 ml x 4). The residue left was dissolved in methanol (35 ml) and left in refrigerator overnight, filtered the solid separated, crystallised with methanol, furnished bergenin (1.5 g). Example 2: The shade dried, powdered Tinospora crispa stems (0.5 kg) were extracted with methanol by hot continuous extraction in a Soxhlet apparatus for 32 hours. The methanol (2.0 1) was evaporated under reduced pressure to obtain a brown mass, and this extract was triturated successively with hexane (200 ml x 4), chloroform (200 ml x 4), and ethyl acetate (200 ml x 4). The residue left was dissolved in methanol (35 ml) and left in refrigerator overnight, filtered the solid separated, crystallised with methanol, furnished bergenin (1.4 g). Example 3: The shade dried, powdered Tinospora crispa stems (0.5 kg) were percolated with rectified spirit (4x1.5 1) for a period 64 hours. The rectified spirit was evaporated under reduced pressure to obtain a dark green mass, and this extract was triturated successively with hexane (200 ml x 4), chloroform (200 ml x 4), and ethyl acetate (200 ml x 4). The residue left was dissolved in methanol (35 ml) and left in refrigerator overnight, filtered, the solid separated, crystallised with methanol, furnished bergenin (1.0 g). Example 4: The shade dried, powdered Tinospora crispa stems (0.5 kg) was percolated with methanol (4x 1.5 1) for a period 64 hours. The solvent was evaporated under reduced pressure to obtain a dark green mass, and this extract was triturated successively with hexane (200 ml x 4), chloroform (200 ml x 4), and ethyl acetate (200 ml x 4). The residue left was dissolved in methanol (35 ml) and left in refrigerator overnight, filtered, the solid separated, crystallised with methanol, furnished bergenin (0.9g). The activity testing illustrated in the following examples, which should not be construed to the scope of invention: Example 5: Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be estimated by use of Griess reagent. 0.5 ml of sodium nitroprusside (5mM) in phosphatebuffered saline was mixed with compound 1 at different concentrations dissolved in 30% DMSO and incubated at 25°C for 150 min. A similar control was also run in which an equal amount of DMSO was added in place of test drug. After 150 minutes of incubation the samples were diluted with 0.5 ml of Griess reagent. The absorbency of the chromophore formed during diazotization of sulphanilamide with nitrite and subsequent coupling with napthylethylenediamine was read at 546nm. Example 6: 0.5 ml of sodium nitroprusside (5mM) in phosphate-buffered saline was mixed with compound 1 at different concentrations dissolved in 30% DMSO and incubated at 25°C. Samples were taken after every 30 minute, starting from 0 to 210 minutes and diluted with 0.5 ml of Griess reagent. The absorbance of the chromophore formed during diazotization of sulphanilamide with nitrite and subsequent coupling with napthylethylenediamine was read at 546nm. The time dependent oxygen scavenging was calculated as compared to the control group. Example 7: Interaction of compound 1 with DPPH. To a solution of DPPH (final concentration 100 umol/L) in absolute ethanol, an equal volume of the compound dissolved in ethanol was added at various concentrations (25-200 µg/ml). A similar control was also run where only ethanol was added in place of test drug. After 20 min. at room temperature, absorbance was recorded at 517 nm. Each experiment was performed in triplicate and the standard deviation in absorbance values was less than ±5%. The percent inhibition of DPPH interaction was calculated as compared to the ethanol treated control as shown in table 1. Antioxidant and free radical scavenging activity. It is postulated that several of the stress induced diseases may be due to oxidative stress brought about by either increased generation of Reactive Oxygen Species (ROS) or decreased free radical scavenging activity [Richard, R. T. and Sharma, M. M, Ind. J. Clin. Pract., 2, 15-18 (1991)]. It has been proposed that neutrophils could contribute to the ulceration process through the release of tissue damaging proteases and reactive oxygen metabolites. The superoxide anion (CV), H2O2 and hydroxyl radical (HO) are the major ROS which induce cell degeneration by increasing lipid peroxidation of cell membrane lipids. The toxic end products of peroxidation induce damage of the structural and functional integrity of cell membranes, break DNA strands and denaturate cellular proteins [Bhattacharya, A. and Ghosal, S., Ind. J. Expt. Biol., 38, 877-880 (2000)]. Our study with bergenin demonstrates it to be a potent antioxidant and free radical scavenging molecule. On testing it against sodium nitroprusside induced nitric oxide production (for direct free radical scavenging) and DPPH interaction (a stable free radical) showed highly significant and dose related inhibition of free radical production and scavenging. Bergenin when tested against nitric oxide production, a free radical generation from sodium nitroprusside, it showed a dose related and significant inhibition, at higher doses, of NO production. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide [Green, L. C., Wishnok, J. and Tannenbaum, S. R., Anal. Biochem. 126, 131-136 (1982) and Marcocli, L., Maguire, J. J., Droy-Lefaiz, M. T. and Packer, L., Biochem. Biophys. Res. Commun., 201, 748-755 (1994] which interacts with oxygen to produce nitrite ions and can be estimated by use of Griess reagent [Marcocli, L., Packer, L., Droy-Lefaiz, M. T., Sekaki, A. and Gardes-Albrt, M., Methods Enzymol., 234, 462-475 (1994)]. Scavengers of nitric oxide compete with oxygen leading to reduced production of NO. NO generated from sodium nitroprusside reacts with oxygen to form nitrite. Bergenin inhibits nitric oxide formation by competing with oxygen to react with nitric oxide. The inhibition in the NO formation might be a result of direct scavenging of presence of free radicals. It produced an inhibition of 10 to 57% at a dose level of 25 to 200 µg/ml where as the standard drug used silymarin showed 68% inhibition at a dose of 50 µg/ml (Table 1). The results of bergenin against the interaction with the nitrogen-centred stable free radical 1, l-diphenyl-2-picrylhydrazyl (DPPH) showed slightly less inhibition as compared to the sodium nitroprusside induced NO production. The effect was significant at higher dose level (200 µg/ml) Table-1. The interaction of the compounds against DPPH expresses the reducing activity of the compound and indicates the ability of scavenging free radicals [Ratly, A. K., Sunammoto, J. and Das, N. P., Biochem. Pharmacol. 37, 989-991 (1988)]. The interaction of bergenin with DPPH attributes its free radical scavenging activity where as the nitric oxide scavenging ability speaks its role as potent antioxidant [Sreejayan and Rao, M. N. A., J. Pharm. Pharmacol. 46, 1013- 1016(1994). Table-1: Effect of bergenin on direct oxygen scavenging by NO produced from sodium nitroprusside and interaction with DPPH. Treatment (Table Removed) Bergenin has shown a significant antioxidant and free radical scavenging activity against sodium nitroprusside induced nitric oxide production for direct free radical scavenging and DPPH interaction (a stable nitrogen centred free radical). Since the molecule has shown free radical scavenging and antioxidant activity, it can be a useful remedy from natural source for the reduction of reactive oxygen species mediated damage to the cells and ultimately for the amelioration of diseases caused by imbalance between oxidative stress and antioxidant defence. We claim: 1 . A pharmaceutical composition useful as antioxidant with free radical scavenging activity comprising an effective amount of bergenin along with a pharmaceutically acceptable additives. 2. A composition as claimed in claim 1 wherein, the additive is selected from nutrients, which are pharmaceutically acceptable carriers. 3. A composition as claimed in claim 1 wherein, the composition is in the form of tablet, syrup, powder, capsule.. 4. A composition as claimed in claim 1 wherein bergenin is used as an oxident at a dose level of at least 200 µg / ml 5. A method for the treatment of radical scavenging and antioxidant comprising the steps of administration of an effective amount of bergenin to a subject in need thereof. 6. .A method as claimed in claim 5 wherein percent inhibition achieved interaction with DPPH is 38.54. 7. A method as claimed in claimed in claim 5 wherein percent inhibition of NO production is 57. 03 at a dose level of at least of 200 µg/ ml. 8. A pharmaceutical composition useful as an antioxidant with free radical scavenging activity, substantially as herein described with reference to the examples .

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1109-DEL-2001-Abstract.pdf

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1109-DEL-2001-Correspondence-Others.pdf

1109-DEL-2001-Correspondence-PO.pdf

1109-del-2001-description (complete).pdf

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1109-DEL-2001-Form-18.pdf

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