Title of Invention	"A PROCESS FOR ISOLATION OF NOVEL COMPOUND 2,6-DIHYDROXY-2-(P-HYDROXYBENZYL)-3(2H)-BENZOFURANONE-7-C-ß-D-GLUCOPYRANOSIDE PTEROCARPUS FROM MARSUPIUM"
Abstract	This invention relates to a process for the isolation of a novel compound 2,6-dihydroxy -{p-hydroxybenzyl)-3 (2H)- benzofuranone -7-C- ß -D-glucopyranoside useful as antidiabetic agent and having the formula 1 which comprises: (a) powdering of the heartwood of the plant Pterocarpus marsupium, (b) extracting the powdered plant material so prepared, with protic solvent such as herein described preferably water (c) concentrating the extract and partitioning the extract with different organic polar and non-polar solvents such as herein described followed by extracting the aqueous layer with polar solvent, such as ethanol, propanol, n-butanol, (d) subjecting the n-butanol extract to conventional chromatography to obtain desired 2,6-dihydroxybenzyl)-3(2H)-benzofuranone-7 C-ß-D- glucopyranoside,

Title of Invention

"A PROCESS FOR ISOLATION OF NOVEL COMPOUND 2,6-DIHYDROXY-2-(P-HYDROXYBENZYL)-3(2H)-BENZOFURANONE-7-C-ß-D-GLUCOPYRANOSIDE PTEROCARPUS FROM MARSUPIUM"

Abstract

This invention relates to a process for the isolation of a novel compound 2,6-dihydroxy -{p-hydroxybenzyl)-3 (2H)- benzofuranone -7-C- ß -D-glucopyranoside useful as antidiabetic agent and having the formula 1 which comprises: (a) powdering of the heartwood of the plant Pterocarpus marsupium, (b) extracting the powdered plant material so prepared, with protic solvent such as herein described preferably water (c) concentrating the extract and partitioning the extract with different organic polar and non-polar solvents such as herein described followed by extracting the aqueous layer with polar solvent, such as ethanol, propanol, n-butanol, (d) subjecting the n-butanol extract to conventional chromatography to obtain desired 2,6-dihydroxybenzyl)-3(2H)-benzofuranone-7 C-ß-D- glucopyranoside,

Full Text	This invention relates to a process for the isolation of novel compound 2,6-dihydroxy-2-(p-hydroxyben2yl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside from Pterocarpus marsupium. Particularly this invention relates to a process of isolation of 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside, designated by us as marsuposide from Pterocarpus marsupium. The compound 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside isolated by the process of the invention has the formula 1 shown in the drawing accompanying this specification. The 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside so isolated is found to possess poly oxygenated benzofuranone C -glucoside and has the formula 1 shown in the drawing accompanying the specification. Pterocarpus marsupium Roxb (Leguminosae) also known as Indian Kino tree or Bijasar, is common in the hilly regions of central and penisular India [Jain, S. K.., Medicinal Plants, National Book Trust, New Delhi, 1968, p. 116]. The extracts of leaves, flowers and gum of this tree have been used medicinally in the treatment of diarrhea, toothache, fever, urinary tract and skin infections [Chopra, R. N., Chopra, I. C., Handa, K. L. and Kapur, L. D., Indigenous Drugs of India, 2nd Ed., Dhar, U. N. and Sons Private Limited , Calcutta, 1958, p. 522.]. While extract of the bark has long been regarded as useful in the therapy of diabetes [Kirtikar, K. R. and Basu, B. D., Indian Medicinal Plants, 2nd Ed., edited by Blatter, E., Cailes, J. F. and Mhaskar, K. S., Singh and singh, Delhi, India, 1975, p. 2135], it has been reported by Chakravarthy et al [Chakravarthy, B. K., Gupta, S. and Gode, K. D., Lancet, 1982, 272 and reference cited therein] that the active hypoglycemic principle of the bark is (-)-epicatechin and that its effect is due to the regeneration of pancreatic beta cells. This claim has, however, been questioned by Kolb et al [Kolb, H., Kiesel, U., Grenlich, B. and Bosch, J. V. D., Lancet, 1982, 1303.] and Sheehan et al [Sheehan, E. W., Zemaitis, M. A., Slatkin, D. J. and Schiff, Jr., P. L., Journal of Natural Products, 1983, 46, 232] and it is now felt that further investigation are necessary before (-)-epicatechin can be considered a viable antidiabetic agent for use in human clinical studies. Medical practitioners of the Indian System of Medicine are, however, of the view that it is the heartwood rather than the bark of Pterocarpus marsupium which is useful for treatment of diabetic patients and that older the plant more efficacious is its heartwood. It is also claimed that only that heartwood which is distinctly red in colour and which imparts a red colouration with bluish green fluorescence to the water in which it is kept soaked is suitable for use as an antidiabetic drug. The hypoglycaemic effects of aqueous or alcoholic extract of the heartwood of Pterocarpus marsupium have been verified by several workers by experimental [Shah, D. S., Indian Journal of Medical Research, 1967, 55, 166 and references cited therein; Gupta, S. S., Indian Journal of Medical Research, 1963, 51, 716.] as well as by clinical studies [Sepha, G. C. and Bose, S. N., J. Ind. Med. Assoc., 1956, 27, 383; Kedar, P. and Chakrabarti, C. H., Maharastra Med. J., 1981,28,165.]. The heartwood of Pterocarpus marsupium is rich in phenolics. The chemical investigation on heartwood of P. marsupium dates back to 1946 but early works [Bhargava, P. N., Proc. Ind. Acad. Sci., 1946, 24A, 496] on this drug are rather fragmentary in nature. The previous studies on this plants have revealed the following chemical constituents. 1. The ether extract of the P. marsupium heartwood furnished isoflavonoid glycol, 4,4'-dihydroxy-a-methylhydrobenzoin designated as marsupol [Rao, A. V. S., Mathew, J., Phytochemistry, 1982, 21, 1837], a benzofurannone derivative, 2-benzyl-2,4',6-trihydroxy-4-methoxybenzo(b)furan-3(2H)-one named as carpusin [Mathew, J. and Rao, A. V. S., Phytochemistry, 1983, 22, 794], 1,3-bis (4-hydroxyphenyl)propan-2-ol , named as propterol [Rao, A. V. S., Mathew, J. and Shankaran, A. V. B., Phytochemistry, 1984, 23, 897] and l-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propan-2-ol, propterol B [Mathew, J. and Rao, A. V. S., [Phytochemistry, 1984, 23, 1814] and 6-hydroxy-7-O-methyl-3-(3-hydroxy-4-0-methyl benzyl) chroman-4-one [Jain, S. C., Sharma, S. K., Kumar, R., Rajwansh, V. K. and Babu, V. R., Phytochemistry, 1997, 44,765]. 2. Ethyl acetate soluble fraction of alcoholic extract of the heartwood furnished pterosupin ß, 2',4,4'-tetrahydroxy-3'(C-p-D-glucopyranoside) dihydrochalcone [Adinarayana, D., Syamasundar, K. V., Seligmann, 0. and Wagner, H., [Z Naturforsch. 1982,37C, 145], marsupinol [Trivedi, J. J., Indian J. Phys. Pharmacol, 1997,15, 51], 5, 4' -dimethoxy-8-methylisoflavone-7-O-a-L-rhamnopyranoside,retusin-0- ß-D- glucopyran-oside and irisolidine-7-O-a-L-rhamnopyranoside [Mitra, J. and Joshi, T., Phytochemistry, 1982, 21, 2429] and 5, 7'-dihydroxy-6-methoxyisoflavone-7-O-a-L- rhamnopyranoside [Mitra, J. and Joshi, T., Phytochemistry, 1983,22,2326]. 3. The ethyl acetate extract of the defatted heartwood furnished novel benzofuranone derivative, 2,6-dihydroxy-2-(p-hydroxybenzyl)-4-methoxy-3 (2H)- benzofuranone designated as marsupin [Maurya, R., Ray, A. B., Duah, F. K., Slatkin, D. J. and Schiff, P. L. Jr., Heterocycles, 1982, 19, 2103] along with pterostilbin, (2S)- hydroxyflavone, isoliquiritigenin, liquiritigenin, 7, 4'-dihydroxyflavone, 5- deoxykaempferol , 3, 7, 4'-trihydroxyflavone and (2R)-3-(p-hydroxyphenyl)-lactic acid [Maurya, R., Ray, A. B. Duah, F. K., Slatkin, D. J. and Schiff, P. L. Jr., J. Nat. Prod. 1984, 47, 179]. Two C-glycosides, 8-C-p-D-glucopyranosyl-3, 7, 4'-trihydroxy and 3, 7, 3', 4'-tetrahydroxy flavone and 3'-C-p-D-glucopyranosyl -a-hydrpxy dihydrochalcone [Bezuidenhoudt, B. C. B., Brandt, E. V., and Ferreira, E. V., Phytochemistry, 1987, 26, 531]. 4. The petrole extract of P. marsupium root wood afforded selin-4(15)-ene- 1ß, 11-diol, p-eudesmol, erythrodiol-3-monoacetate and pterostilbene [Adinarayana, D. and Syamasundar K. V., Phytochemistry, 1982, 22, 1083]. Ethanolic extract of P. marsupium flowers furnished 4, 6, 4'-trihydroxyaurone 6-O-rhamnopyranoside and 4, 6, 4'-trihydroxy-7-methylaurone 4-O-rhamnopyranoside [Mohan, P. and Joshi, T., Phytochemistry, 1989, 28, 1287] and ethanolic extract of P. marsupium bark furnished (-)-epicatechin [Charkravarthy, B. K. and Gode, K. D., Planta Medico, 1985, 56]. No details about their biological activities associated with them are available. Keeping in view the high reputation enjoyed by Pterocarpus marsupium in the traditional system of medicine and confirmed biological activity as antidiabetic agent, stimulated us to investigate the water extract of heartwood of Pterocarpus marsupium with special emphasis on the isolation of bioactive molecule from it. The main object of the present invention is to isolate a novel compound 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside from Pterocarpus marsupium. 2,6-Dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C-ß - D -glucopyranoside has been isolated from n-butanol soluble fraction of the water decoction of the heartwood of Pterocarpus marsupium which has shown antidiabetic activity both in animal and human. Other groups could not get 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside because they have worked on the ether extract, ethyl acetate extract and ethyl acetate soluble fraction of the alcoholic extract. The process of isolating the active principle from the Pterocarpus marsupium involves partition of aqueous extract of powdered heartwood with different organic solvents containing (1-6 carbon atoms in the molecule). 2,6-Dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C- ß - D - glucopyranoside, designated by us as marsuposide, is isolated from polar fraction by applying modern chromatographic techniques, such as, medium pressure liquid chromatography (MPLC) and flash chromatography, using silica gel (230 - 400 mesh) and gel chromatography . Accordingly, the present invention provides a process for the isolation of a novel compound 2,6 - dihydroxy -2-(p-hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside useful as antidiabetic agent and having the formula 1 as shown in the drawing accompanying this specification, which comprises: (a) powdering of the heartwood of the plant Pterocarpus marsupium, (b) extracting the powdered plant material so prepared, with protic solvent such as herein described preferably water (c) concentrating the extract and partitioning the extract with different organic polar and non-polar solvents such as herein described followed by extracting the aqueous layer with polar solvent, such as ethanol, propanol, n- butanol, (d) subjecting the n - butanol extract to conventional chromatography to obtain desired 2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D - glucopyranoside, In a preferred embodiment of the invention a process for the isolation of novel compound 2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D - glucopyranoside from Pterocarpus marsupium has been described in which the solvent used for preparing the extract may be water, methanol, ethanol, propanol and butanol and like or their mixtures. The organic solvent used in the step ( c ) to remove non polar component may be such as hexane, pet ether, chloroform. The polar solvent used to extract aqueous layer may be such as ethyl acetate, propanol, butanol. The chromatographic methods used for the isolation of 2,6- dihydroxybenzyl) - 3 (2H) - benzofuranone - 7 C -ß- D - glucopyranoside, may be Medium Pressure Liquid Chromatography (MPLC) technique, High performance Liquid Chromatography (HPLC), flash chromatography. In the MPLC method the solvent is pumped through the column and in the flash chromatography solvent is pushed with air pressure. The molecular formula of compound C21H22O10 was established on the basis of strong peak at m/z 435 [M+1]* in the FAB mass spectrum , together with the support of spectroscopic methods. The compound displayed diagnostic IR absorption atvmax 3300 , 1680 , 1510 , 1444cm-1 for hydroxyls , carbonyl group and aromatic ring. UV spectrum showed absorption maxima λ maxMeOH 211, 240, 282, 333 nm with a bathochromic shift on addition of NaOAc to 211,261,283,344 nm . The 1H NMR spectrum indicated the presence of one benzylic methylene group at δ 2.90 (1H, d, J = 13.7 Hz) and 3.11 (1H, d, J = 13.7 Hz), two ortho-coupled aromatic protons at 5 6.61 (1H, d, J = 8.5 Hz) and 7.03 (1H, d, J = 8.5 Hz) and one A2B2 aromatic system at δ 6.61 (2H, d, J = 8.5 Hz) and 7.13 (2H, d, J = 8.5 Hz). Further 'H and 13C NMR spectra showed signals attributed to one glucose moiety. The C-C-coupling was exemplified by 1H - 13C heteronuclear correlation of the anomeric proton at 8 4.68 (1H, d, J = 9.9 Hz) with a carbon doublet at 6 79.7 in the region characteristic of C,- substituted glucosides. The coupling constant (J = 9.9 Hz) of the signal resulting from the anomeric proton of the glucopyranoside indicated the glucosidic linkage to have p-configuration. A consideration of the spectral data suggested that marsuposide was benzofuranone derivative containing phenolic hydroxy group and glucose moiety in ring-A and one alcoholic hydroxy group in ring-B and one phenolic hydroxy group in ring-C. The FAB mass spectrum showed [M+l]+ at m/z 435 for C2IH22O10 with other significant fragment ion at m/z 457 [M+Na]+, 418, 327, 299, 107 93, further supported the hypothesis. On the basis of the above evidence the structure of compound was established as 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside, designated by us as marsuposide shown in the formula 1. 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 powdered heartwood of Pterocarpus marsupium (1kg) was percolated with 80% aqueous ethanol (3x3 lits.) for a period of 48 hours. The resultant concentrate was partitioned with hexane, chloroform, propanol, butanol in that order. The n-butanol polar extract was subjected to MPLC using silica gel (100 - 200 mesh) for gross fractions with hexane, chloroform, methanol, ethanol in that order. The compound was purified by repeated MPLC and flash chromatography over silca gel (230 - 400 mesh) using CHC13 -MeOH (9:1) as solvent, to furnish 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside of the formula 1, (yield 0.078.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225). Example - 2: The heartwood of Pterocarpus marsupium was extracted with hot water for a period of 4 hours. The resultant concentrate was partitioned between hexane, chloroform, propanol and butanol in that order. The n-butanol polar extract so obtained was subjected to flash chromatography employing silica gel (100 - 200 mesh) using hexane, chloroform, ethylacetate, methanol as solvent system to afford 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß- D - glucopyranoside rich fraction, which on repeated chromatography over silica gel (230 - 400 mesh) using EtOAc - MeOH (19:1) as solvent, furnished 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside of the formula 1, (yield 0.087.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225). Example-3: The heartwood of Pterocarpus marsupium was boiled with water (16 times) till 1/4 volume of water is left. Filtered, concentrated and partitioned between hexane, chloroform, ethyl acetate, propanol and n-butanol in that order. The n-butanol polar extract obtained was subjected to column chromatography employing silica gel (60-120 mesh) using hexane, chloroform, ethyl acetate and methanol as solvent system to afford 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside rich fraction. The 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside rich fraction on repeated column chromatography over silica gel (100-200 mesh) using mixture of ethyl acetate - acetone (7:3), furnished 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside of the formula 1, (yield 0.090.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225). Advantages: 1. This is the novel method by which we can isolate 2,6-dihydroxy-2-(p- hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside. 2. The method of isolation of 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)- benzofuranone -7 - C -ß - D - glucopyranoside is simple. 3. This novel method has been helpful in isolating a new compound namely 2,6- dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside. We ClSim: 1. A process for the isolation of a novel compound 2,6 - dihydroxy -2-(p-hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside useful as antidiabetic agent and having the formula 1 as shown in the drawing accompanying this specification, which comprises: (a) powdering of the heartwood of the plant Pterocarpus marsupium, (b) extracting the powdered plant material so prepared, with protic solvent such as herein described preferably water (c) concentrating the extract and partitioning the extract with different organic polar and non-polar solvents such as herein described followed by extracting the aqueous layer with polar solvent, such as ethanol, propanol, n- butanol, (d) subjecting the n - butanol extract to conventional chromatography to obtain desired 2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D - glucopyranoside, 2. A process as claimed in claim 1 wherein the protic solvent used for the extraction in step (b) is such as water, methanol, ethanol, propanol, n-butanol. 3. A process as claimed in claims 1 and 2 wherein the organic polar and non- polar solvent used in step ( c ) is selected from hexane, pet ether, chloroform, ethyl acetate, propanol and n-butanol. 4. A process as claimed in claims 1-3 wherein the isolation of 2,6-dihydroxy-2- (p-hydroxybenzyl)-3)2H)-benzofuranone-7-C-ß-D - glucopyranoside is effected by conventional chromatographic methods such as Medium Pressure Liquid Chromatography (MPLC) technique, High performance Liquid Chromatograpy (HPCL), flash chromatography. 5. A process as claimed in claim 4 wherein the solvent used for chromatography is such as chloroform, ethyl acetate, acetone, lower alcohal and mixture thereof. 6. A process for the isolation of a novel compound 2,6 - dihydroxy -2-(p- hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside having the formula 1 substantially as herein described with reference to the examples and drawing accompanying this specification.

Full Text

This invention relates to a process for the isolation of novel compound 2,6-dihydroxy-2-(p-hydroxyben2yl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside from Pterocarpus marsupium. Particularly this invention relates to a process of isolation of 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside, designated by us as marsuposide from Pterocarpus marsupium. The compound 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside isolated by the process of the invention has the formula 1 shown in the drawing accompanying this specification. The 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside so isolated is found to possess poly oxygenated benzofuranone C -glucoside and has the formula 1 shown in the drawing accompanying the specification.
Pterocarpus marsupium Roxb (Leguminosae) also known as Indian Kino tree or Bijasar, is common in the hilly regions of central and penisular India [Jain, S. K.., Medicinal Plants, National Book Trust, New Delhi, 1968, p. 116]. The extracts of leaves, flowers and gum of this tree have been used medicinally in the treatment of diarrhea, toothache, fever, urinary tract and skin infections [Chopra, R. N., Chopra, I. C., Handa, K. L. and Kapur, L. D., Indigenous Drugs of India, 2nd Ed., Dhar, U. N. and Sons Private Limited , Calcutta, 1958, p. 522.]. While extract of the bark has long been regarded as useful in the therapy of diabetes [Kirtikar, K. R. and Basu, B. D., Indian Medicinal Plants, 2nd Ed., edited by Blatter, E., Cailes, J. F. and Mhaskar, K. S., Singh and singh, Delhi, India, 1975, p. 2135], it has been reported by Chakravarthy et al [Chakravarthy, B. K., Gupta, S. and Gode, K. D., Lancet, 1982, 272 and reference cited therein] that the active hypoglycemic principle of the bark is (-)-epicatechin and that its effect is due to the regeneration of pancreatic beta cells. This claim has, however, been questioned by Kolb et al [Kolb, H., Kiesel, U., Grenlich, B. and Bosch, J. V. D., Lancet, 1982, 1303.] and Sheehan et al [Sheehan, E. W., Zemaitis, M. A., Slatkin, D. J. and Schiff, Jr., P. L., Journal of Natural Products, 1983, 46, 232] and it is now felt that further investigation

are necessary before (-)-epicatechin can be considered a viable antidiabetic agent for use in human clinical studies.
Medical practitioners of the Indian System of Medicine are, however, of the view that it is the heartwood rather than the bark of Pterocarpus marsupium which is useful for treatment of diabetic patients and that older the plant more efficacious is its heartwood. It is also claimed that only that heartwood which is distinctly red in colour and which imparts a red colouration with bluish green fluorescence to the water in which it is kept soaked is suitable for use as an antidiabetic drug.
The hypoglycaemic effects of aqueous or alcoholic extract of the heartwood of Pterocarpus marsupium have been verified by several workers by experimental [Shah, D. S., Indian Journal of Medical Research, 1967, 55, 166 and references cited therein; Gupta, S. S., Indian Journal of Medical Research, 1963, 51, 716.] as well as by clinical studies [Sepha, G. C. and Bose, S. N., J. Ind. Med. Assoc., 1956, 27, 383; Kedar, P. and Chakrabarti, C. H., Maharastra Med. J., 1981,28,165.].
The heartwood of Pterocarpus marsupium is rich in phenolics. The chemical investigation on heartwood of P. marsupium dates back to 1946 but early works [Bhargava, P. N., Proc. Ind. Acad. Sci., 1946, 24A, 496] on this drug are rather fragmentary in nature. The previous studies on this plants have revealed the following chemical constituents.
1. The ether extract of the P. marsupium heartwood furnished isoflavonoid glycol, 4,4'-dihydroxy-a-methylhydrobenzoin designated as marsupol [Rao, A. V. S., Mathew, J., Phytochemistry, 1982, 21, 1837], a benzofurannone derivative, 2-benzyl-2,4',6-trihydroxy-4-methoxybenzo(b)furan-3(2H)-one named as carpusin [Mathew, J. and Rao, A. V. S., Phytochemistry, 1983, 22, 794], 1,3-bis (4-hydroxyphenyl)propan-2-ol , named as propterol [Rao, A. V. S., Mathew, J. and Shankaran, A. V. B., Phytochemistry, 1984, 23, 897] and l-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propan-2-ol, propterol

B [Mathew, J. and Rao, A. V. S., [Phytochemistry, 1984, 23, 1814] and 6-hydroxy-7-O-methyl-3-(3-hydroxy-4-0-methyl benzyl) chroman-4-one [Jain, S. C., Sharma, S. K., Kumar, R., Rajwansh, V. K. and Babu, V. R., Phytochemistry, 1997, 44,765].
2. Ethyl acetate soluble fraction of alcoholic extract of the heartwood
furnished pterosupin ß, 2',4,4'-tetrahydroxy-3'(C-p-D-glucopyranoside) dihydrochalcone
[Adinarayana, D., Syamasundar, K. V., Seligmann, 0. and Wagner, H., [Z Naturforsch.
1982,37C, 145], marsupinol [Trivedi, J. J., Indian J. Phys. Pharmacol, 1997,15, 51], 5,
4' -dimethoxy-8-methylisoflavone-7-O-a-L-rhamnopyranoside,retusin-0- ß-D-
glucopyran-oside and irisolidine-7-O-a-L-rhamnopyranoside [Mitra, J. and Joshi, T.,
Phytochemistry, 1982, 21, 2429] and 5, 7'-dihydroxy-6-methoxyisoflavone-7-O-a-L-
rhamnopyranoside [Mitra, J. and Joshi, T., Phytochemistry, 1983,22,2326].
3. The ethyl acetate extract of the defatted heartwood furnished novel
benzofuranone derivative, 2,6-dihydroxy-2-(p-hydroxybenzyl)-4-methoxy-3 (2H)-
benzofuranone designated as marsupin [Maurya, R., Ray, A. B., Duah, F. K., Slatkin, D.
J. and Schiff, P. L. Jr., Heterocycles, 1982, 19, 2103] along with pterostilbin, (2S)-
hydroxyflavone, isoliquiritigenin, liquiritigenin, 7, 4'-dihydroxyflavone, 5-
deoxykaempferol , 3, 7, 4'-trihydroxyflavone and (2R)-3-(p-hydroxyphenyl)-lactic acid
[Maurya, R., Ray, A. B. Duah, F. K., Slatkin, D. J. and Schiff, P. L. Jr., J. Nat. Prod.
1984, 47, 179]. Two C-glycosides, 8-C-p-D-glucopyranosyl-3, 7, 4'-trihydroxy and 3, 7,
3', 4'-tetrahydroxy flavone and 3'-C-p-D-glucopyranosyl -a-hydrpxy dihydrochalcone
[Bezuidenhoudt, B. C. B., Brandt, E. V., and Ferreira, E. V., Phytochemistry, 1987, 26,
531].
4. The petrole extract of P. marsupium root wood afforded selin-4(15)-ene-
1ß, 11-diol, p-eudesmol, erythrodiol-3-monoacetate and pterostilbene [Adinarayana, D.
and Syamasundar K. V., Phytochemistry, 1982, 22, 1083]. Ethanolic extract of P.
marsupium flowers furnished 4, 6, 4'-trihydroxyaurone 6-O-rhamnopyranoside and 4, 6,
4'-trihydroxy-7-methylaurone 4-O-rhamnopyranoside [Mohan, P. and Joshi, T.,

Phytochemistry, 1989, 28, 1287] and ethanolic extract of P. marsupium bark furnished (-)-epicatechin [Charkravarthy, B. K. and Gode, K. D., Planta Medico, 1985, 56].
No details about their biological activities associated with them are available. Keeping in view the high reputation enjoyed by Pterocarpus marsupium in the traditional system of medicine and confirmed biological activity as antidiabetic agent, stimulated us to investigate the water extract of heartwood of Pterocarpus marsupium with special emphasis on the isolation of bioactive molecule from it.
The main object of the present invention is to isolate a novel compound 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside from Pterocarpus marsupium.
2,6-Dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C-ß - D -glucopyranoside has been isolated from n-butanol soluble fraction of the water decoction of the heartwood of Pterocarpus marsupium which has shown antidiabetic activity both in animal and human. Other groups could not get 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside because they have worked on the ether extract, ethyl acetate extract and ethyl acetate soluble fraction of the alcoholic extract.
The process of isolating the active principle from the Pterocarpus marsupium involves partition of aqueous extract of powdered heartwood with different organic solvents containing (1-6 carbon atoms in the molecule). 2,6-Dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C- ß - D - glucopyranoside, designated by us as marsuposide, is isolated from polar fraction by applying modern chromatographic techniques, such as, medium pressure liquid chromatography (MPLC) and flash chromatography, using silica gel (230 - 400 mesh) and gel chromatography .

Accordingly, the present invention provides a process for the isolation of a novel compound 2,6 - dihydroxy -2-(p-hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside useful as antidiabetic agent and having the formula 1 as shown in the drawing accompanying this specification, which comprises:
(a) powdering of the heartwood of the plant Pterocarpus marsupium,
(b) extracting the powdered plant material so prepared, with protic solvent such as
herein described preferably water
(c) concentrating the extract and partitioning the extract with different organic polar and
non-polar solvents such as herein described followed by extracting the aqueous layer
with polar solvent, such as ethanol, propanol, n- butanol,
(d) subjecting the n - butanol extract to conventional chromatography to obtain desired
2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D - glucopyranoside,
In a preferred embodiment of the invention a process for the isolation of novel compound 2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D - glucopyranoside from Pterocarpus marsupium has been described in which the solvent used for preparing the extract may be water, methanol, ethanol, propanol and butanol and like or their mixtures. The organic solvent used in the step ( c ) to remove non polar component may be such as hexane, pet ether, chloroform. The polar solvent used to extract aqueous layer may be such as ethyl acetate, propanol, butanol.
The chromatographic methods used for the isolation of 2,6- dihydroxybenzyl) - 3 (2H) - benzofuranone - 7 C -ß- D - glucopyranoside, may be Medium Pressure Liquid Chromatography (MPLC) technique, High performance Liquid Chromatography (HPLC), flash chromatography. In the MPLC method the solvent is pumped through the column and in the flash chromatography solvent is pushed with air pressure. The molecular formula of compound C21H22O10 was established on the basis of strong peak at m/z 435 [M+1]* in the FAB mass spectrum , together with the support of spectroscopic methods. The compound displayed diagnostic IR absorption atvmax 3300 , 1680 , 1510 , 1444cm-1 for hydroxyls , carbonyl group and aromatic ring. UV

spectrum showed absorption maxima λ maxMeOH 211, 240, 282, 333 nm with a bathochromic shift on addition of NaOAc to 211,261,283,344 nm .
The 1H NMR spectrum indicated the presence of one benzylic methylene group at δ 2.90 (1H, d, J = 13.7 Hz) and 3.11 (1H, d, J = 13.7 Hz), two ortho-coupled aromatic protons at 5 6.61 (1H, d, J = 8.5 Hz) and 7.03 (1H, d, J = 8.5 Hz) and one A2B2 aromatic system at δ 6.61 (2H, d, J = 8.5 Hz) and 7.13 (2H, d, J = 8.5 Hz). Further 'H and 13C NMR spectra showed signals attributed to one glucose moiety. The C-C-coupling was exemplified by 1H - 13C heteronuclear correlation of the anomeric proton at 8 4.68 (1H, d, J = 9.9 Hz) with a carbon doublet at 6 79.7 in the region characteristic of C,- substituted glucosides. The coupling constant (J = 9.9 Hz) of the signal resulting from the anomeric proton of the glucopyranoside indicated the glucosidic linkage to have p-configuration. A consideration of the spectral data suggested that marsuposide was benzofuranone derivative containing phenolic hydroxy group and glucose moiety in ring-A and one alcoholic hydroxy group in ring-B and one phenolic hydroxy group in ring-C. The FAB mass spectrum showed [M+l]+ at m/z 435 for C2IH22O10 with other significant fragment ion at m/z 457 [M+Na]+, 418, 327, 299, 107 93, further supported the hypothesis. On the basis of the above evidence the structure of compound was established as 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside, designated by us as marsuposide shown in the formula 1.
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 powdered heartwood of Pterocarpus marsupium (1kg) was percolated with 80% aqueous ethanol (3x3 lits.) for a period of 48 hours. The resultant concentrate was partitioned with hexane, chloroform, propanol, butanol in that order. The n-butanol polar extract was subjected to MPLC using silica gel (100 - 200 mesh) for gross fractions with hexane, chloroform, methanol, ethanol in that order. The compound was purified by

repeated MPLC and flash chromatography over silca gel (230 - 400 mesh) using CHC13 -MeOH (9:1) as solvent, to furnish 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside of the formula 1, (yield 0.078.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225).
Example - 2:
The heartwood of Pterocarpus marsupium was extracted with hot water for a period of 4 hours. The resultant concentrate was partitioned between hexane, chloroform, propanol and butanol in that order. The n-butanol polar extract so obtained was subjected to flash chromatography employing silica gel (100 - 200 mesh) using hexane, chloroform, ethylacetate, methanol as solvent system to afford 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß- D - glucopyranoside rich fraction, which on repeated chromatography over silica gel (230 - 400 mesh) using EtOAc - MeOH (19:1) as solvent, furnished 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -glucopyranoside of the formula 1, (yield 0.087.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225).
Example-3:
The heartwood of Pterocarpus marsupium was boiled with water (16 times) till 1/4 volume of water is left. Filtered, concentrated and partitioned between hexane, chloroform, ethyl acetate, propanol and n-butanol in that order. The n-butanol polar extract obtained was subjected to column chromatography employing silica gel (60-120 mesh) using hexane, chloroform, ethyl acetate and methanol as solvent system to afford 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside rich fraction. The 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside rich fraction on repeated column chromatography over silica gel (100-200 mesh) using mixture of ethyl acetate - acetone (7:3), furnished 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside of the formula 1, (yield 0.090.%), mp. 156 -158° C, [ α]D 26 + 8.44° (MeOH, c, 0.225).

Advantages:
1. This is the novel method by which we can isolate 2,6-dihydroxy-2-(p-
hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D - glucopyranoside.
2. The method of isolation of 2,6-dihydroxy-2-(p-hydroxybenzyl)-3(2H)-
benzofuranone -7 - C -ß - D - glucopyranoside is simple.
3. This novel method has been helpful in isolating a new compound namely 2,6-
dihydroxy-2-(p-hydroxybenzyl)-3(2H)-benzofuranone -7 - C -ß - D -
glucopyranoside.

We ClSim:
1. A process for the isolation of a novel compound 2,6 - dihydroxy -2-(p-hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside useful as antidiabetic agent and having the formula 1 as shown in the drawing accompanying this specification, which comprises:
(a) powdering of the heartwood of the plant Pterocarpus marsupium,
(b) extracting the powdered plant material so prepared, with protic solvent
such as herein described preferably water
(c) concentrating the extract and partitioning the extract with different organic
polar and non-polar solvents such as herein described followed by
extracting the aqueous layer with polar solvent, such as ethanol, propanol,
n- butanol,
(d) subjecting the n - butanol extract to conventional chromatography to
obtain desired 2,6-dihydroxybenzyl) - 3(2H)- benzofuranone - 7 C-ß- D -
glucopyranoside,

2. A process as claimed in claim 1 wherein the protic solvent used for the
extraction in step (b) is such as water, methanol, ethanol, propanol, n-butanol.
3. A process as claimed in claims 1 and 2 wherein the organic polar and non-
polar solvent used in step ( c ) is selected from hexane, pet ether, chloroform,
ethyl acetate, propanol and n-butanol.
4. A process as claimed in claims 1-3 wherein the isolation of 2,6-dihydroxy-2-
(p-hydroxybenzyl)-3)2H)-benzofuranone-7-C-ß-D - glucopyranoside is
effected by conventional chromatographic methods such as Medium
Pressure Liquid Chromatography (MPLC) technique, High performance Liquid
Chromatograpy (HPCL), flash chromatography.

5. A process as claimed in claim 4 wherein the solvent used for chromatography
is such as chloroform, ethyl acetate, acetone, lower alcohal and mixture
thereof.
6. A process for the isolation of a novel compound 2,6 - dihydroxy -2-(p-
hydroxybenzyl)-3(2H)- benzofuranone - 7 -C-ß-D-glucopyranoside having
the formula 1 substantially as herein described with reference to the
examples and drawing accompanying this specification.

Documents:

1402-del-1999-abstract.pdf

1402-del-1999-claims.pdf

1402-del-1999-correspondence-others.pdf

1402-del-1999-correspondence-po.pdf

1402-del-1999-description (complete).pdf

1402-del-1999-drawings.pdf

1402-del-1999-form-1.pdf

1402-del-1999-form-2.pdf

1402-del-1999-form-3.pdf

1402-del-1999-form-9.pdf

1402-del-1999-petition-138.pdf

« Previous Patent

Next Patent »

Patent Number

192163

Indian Patent Application Number

1402/DEL/1999

PG Journal Number

10/2004

Publication Date

06-Mar-2004

Grant Date

27-Jun-2005

Date of Filing

22-Oct-1999

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110067, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RAKESH MAURYA	SCIENTISTS OF REGIONAL RESEARCH LABORATORY, CANAL ROAD JAMMU, INDIA.
2	SUKHDEV SWAMI HANDA	SCIENTISTS OF REGIONAL RESEARCH LABORATORY, CANAL ROAD JAMMU, INDIA.
3	RAJINDER SINGH	SCIENTISTS, ICMR CENTRE FOR ADV ANCED RESEARCH AT REGIONAL RESEARCH LABORATORY, CANAL ROAD, JAMMU 180 001, JAMMU AND KASHMIR , INDIA.

PCT International Classification Number

A61K 31/70

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA