Title of Invention

"A PROCESS FOR THE ISOLATION OF NOVEL COMPOUND 8-(C-ß-D-GLUCOPYRANOSYL)-7,3',4'-TRIHYDROXYFLAVONE FROM PTEROCARPUS MARSUPIUM"

Abstract The invention relates to a process for the isolation of novel compound 8-(Cβ-D- glucopyranosyl )-7, 3",4"- trihydroxyflavone from Pterocarpus marsupium. The process steps are: 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, c) concentrating the extract to minimum volume and partitioning with different organic solvents such as herein described of increasing polarity to remove non polar components, extracting the aqueous layer with polar solvent, removing the solvent to get residue, d) isolating the 8-(C-β-D-glucopyranosyl)-7,3",4"-trihydroxyflavone from the residue by conventional chromatographic methods.
Full Text This invention relates to a process for the isolation of novel compound 8-(C-(3-D-glucopyranosyl)-7,3',4'-trihydroxyflavone from Pterocarpus marsupium. Particularly this invention relates to a process of isolation of 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone designated by us as vijayoside from Pterocarpus marsupium. The 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone isolated by the process of the invention has the formula 1 shown in the drawing accompanying this specification. The S-(C-P-D-glucopyranosyl)-7,3',4'-trihydroxyflavone so isolated is found to possess polyoxygenated flavone 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.,MaharastraMed. 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 plant 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-O-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 P, 2',4,4'-tetrahydroxy-3'(C-p-D-glucopyranoside) dihydrochalcone
[Adinarayana, D., Syamasundar, K. V., Seligmann, O. and Wagner, H., [Z. Naturforsch.

1982, 37C, 145], marsupinol [Trivedi, J. I, Indian J. Phys. Pharmacol, 1997, 15, 51], 5, 4'-dimethoxy-8-methylisoflavone-7-0-α-L-rhamnopyranoside,retusin-O-p-D-glucopyran-oside and irisolidine-7-O-α-L-rhamnopyranoside [Mitra, J. and Joshi, T., Phytochemistry, 1982, 21, 2429] and 5, 7'-dihydroxy-6-methoxyisoflavone-7-O-α-L-rhamnopyranoside [Mitra, J. and Joshi, T.; Phytochemistry, 1983, 22, 2326].
3. The ethyl acetate extract of the defatted heartwoood 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 -cc-hydroxy 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-
1β, 11-diol, β-eudesmol, erythrodiol-3-monoacetate and pterostilbene [Adinarayana, D.
and Syamasundar K. V., Phytochemistry, 1982, 22, 1083]. Ethanolic extract of P.
marsuphim flowers furnished 4, 6, 4'-trihydroxyaurone 6-O-rhamnopyranoside and 4, 6,
4'-trihydroxy-7-methylaurone 4-0-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., PlantaMedica, 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 S-(C-β-D-glucopyranosyl)-7,3',4'-trihydroxyflavone from Pterocarpus marsupium.
The compound 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone 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 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone 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). 8-(C-β-D- Glucopyranosyl)-7,3',4'-trihydroxyflavone, designated by us as vijayoside, 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 .
The compound has been evaluated for hypoglycaemic activity in 18 h. fasted Wistar rats. In the dose of 10 mg/ kg p. o., hypoglycaemic effect was recorded in all the treated rats. The mean fall recorded was 24 mg/dl blood serum, from initial mean of 92 to mean of 68 mg/dl blood serum. As compared to this, glipizide, the clinically used hypoglycaemic agent, employed as positive control in the study, showed mean fall of 23 mg/dl blood serum.
Accordingly, the present invention provides a process for the isolation of novel compound S-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone of the formula 1 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,

c) concentrating the extract to minimum volume and partitioning with different
organic solvents such as herein described of increasing polarity to remove non
polar components, extracting the aqueous layer with polar solvent, removing the
solvent to get residue,
d) isolating the 8-(C-β-D-glucopyranosyl)-7,3',4'-trihydroxyflavone from the
residue by conventional chromatographic methods.
In a preferred embodiment of the invention a process for the isolation of novel compound 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone from Pterocarpus marsupium has been described in which the solvent used fro 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 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone, may be, Medium Pressure Liquid Chromatography (MPLAC) technique, High Performance Liquid Chromatography (HPLC), flash chromatography. In the MPLC method the required solvent is pumped through the column and in the flash chromatography solvent is pushed with air pressure. The compound was assigned the molecular C21H2oO10 (FAB-MS, m/z 433[M+1]+). This conclusion was supported by the I3C NMR and DEPT spectra.
The compound was proved to be phenol from its positive phosphomolybedic acid test (blue), ferric chloride test (green). It was recognized to be flavone as it responded to Shinoda test. The presence of hydroxyl, carbonyl, and phenyl nucleus was indicated by

the IR absorption at 3228, 1615, 1554, 1448, 1422 cm"1. The UV spectrum of the compound showed absorption maxima atλmax MeOH 219, 238, 260, 320, 358 nm which underwent bathochromic shift 219, 238, 267, 320, 367 nm in presence of NaOAc. This observation suggested the presence of a free hydroxyl group located at C-7.
The !H NMR spectrum (200 MHz, in DMSO-d6) displayed broadening of signals in the aromatic regions presumably because of the steric crowding of the glucosyl and B-ring. The inspection of spectrum revealed singlet signal at 8 6.98 (IH) characteristic of proton at C-3 of flavone. A doublet proton signal at 8 8.28 (IH, d, J = 8.3 Hz) which is lowfield shifted due to the effect of neighbouring C=O, is ortho-coupled with the doublet signal atδ 6.95 (IH, d, J = 8.3 Hz), this ortho coupling system is assigned to the proton at C-5 and C-6 indicating that only these two protons belong to the A-ring and that C-8 is occupied by a glucosyl group. The proton signal at 8 7.84 (IH, br d, J = 2.1 Hz), 7.97 (IH, br dd, J = 2.1, 8.7 Hz) and 6.99 (IH, d, J = 8.7 Hz) are assigned to the protons at the B-ring. Further 1H 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 5.16 with a carbon doublet at 8 79.3 in the region characteristic of C1- substituted glucosides. Further the coupling constant (J = 9.5 Hz) of the signal resulting from the anomeric proton of the glucopyranoside indicated the glucosidic linkage to have β-configuration. Thus, the above analysis led to the structure 8-(C-β-D-glucopyranosyl)-7, 3', 4'-trihydroxyflavone, designated by us as vijayoside 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 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 CHCla - MeOH (19:1) as solvent, to furnish 8-(C-p-D- glucopyranosyl)-7,3',4'- trihydroxyflavone of the formula 1, (yield 0.046.%), mp. 202 -204° C, [ α]D 19 + 25.6° (MeOH, c, 0.5).
Example - 2:
The heartwood of Pterocarpus marsupium was extracted with hot water for a period of 4x4 hours. The resultant concentrate was partitioned between hexane, chloroform, propanol and butanol in that order.' The 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 8-(C-β-D-glucopyranosyl)-7,3',4'- trihydroxyflavone rich fraction, which on repeated chromatography over silica gel (230 - 400 mesh) using EtOAc - MeOH (19.5:0.5) as solvent, furnished 8-(C-β-D- glucopyranosyl)-7,3',4'- trihydroxyflavone of the formula 1, (yield 0.049.%), mp. 202 -204° C, [ α]D 19 + 25.6° (MeOH, c, 0.5).
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 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 8-(C-p-
D- glucopyranosyl)-7,3',4'- trihydroxyflavone rich fraction. The S-(C-P-D-
glucopyranosyi)-7,3',4'- trihydroxyflavone rich fraction on repeated column chromatography over silica gel (100-200 mesh) using mixture of ethyl acetate - acetone (8:2), furnished 8-(C-β-D-glucopyranosyl)-7,3',4'- trihydroxyflavone of the formula 1, (yield 0.051.%), mp. 202 -204° C, [ α]D 19 + 25.6° (MeOH, c, 0.5).

Advantages:
1. This is the novel method by which we can isolate 8-(C-β-D- glucopyranosyl)-
7,3 ',4'-trihydroxyflavone.
2. The method of isolation of 8-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone is
simple.
3. This novel method has been helpful in isolating a new compound namely 8-(C-
β-D- glucopyranosyl)-7,3 ',4'-trihydroxyflavone.







We Claim:
1. A process for the isolation of novel compound 8-(C-β-D- glucopyranosyl)-7, 3',4'- trihydroxyflavone from Pterocarpus marsupium of the formula 1 which comprises
(Formula Removed)
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,
c) concentrating the extract to minimum volume and partitioning with different
organic solvents such as herein described of increasing polarity to remove non
polar components, extracting the aqueous layer with polar solvent, removing the
solvent to get residue,
d) isolating the 8-(C-β-D-glucopyranosyl)-7,3',4'-trihydroxyflavone from the
residue by conventional chromatographic methods.

2. A process as claimed in claim 1 wherein the protic solvent used for the extraction
in step (b) is selected from water, methanol, ethanol, propanol, n-butanol or their
mixtures.
3. A process as claimed in claims 1 and 2 wherein the organic solvent used to
remove non polar component in step (c) is selected from hexane, petroleum ether,
chloroform and the polar solvent used for extraction of aqueous layer is selected
from ethyl acetate, propanol and n-butanol.
4. A process as claimed in the claims 1-3 wherein the isolation of S-(C-β-D-
glucopyranosyl)-7,3', 4 '-trihydroxyflavone is effected by conventional
chromatographic methods such as Medium Pressure Liquid Chromatography
(MPLC) technique, High Performance Liquid Chromatography (HPLC), flash
chromatography.
5. A process as claimed in claim 4 wherein the solvent used for chromatography is
selected from chloroform, ethyl acetate, acetone, lower alcohol and mixture
thereof.
6. A process for the isolation of S-(C-β-D- glucopyranosyl)-7,3',4'-trihydroxyflavone from Pterocarpus marsupium having formula 1 substantially as herein described with reference to the examples and drawing accompanying this specification.



Documents:

1405-del-1999-abstract.pdf

1405-del-1999-claims.pdf

1405-del-1999-correspondence-others.pdf

1405-del-1999-correspondence-po.pdf

1405-del-1999-description (complete).pdf

1405-del-1999-drawings.pdf

1405-del-1999-form-1.pdf

1405-del-1999-form-19.pdf

1405-del-1999-form-2.pdf

1405-del-1999-form-3.pdf


Patent Number 215658
Indian Patent Application Number 1405/DEL/1999
PG Journal Number 12/2008
Publication Date 21-Mar-2008
Grant Date 29-Feb-2008
Date of Filing 22-Oct-1999
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 RAKESH MAURYA REGIONAL RESEARCH LABORATORY, CANAL ROAD JAMMU.
2 SUKHDEV SWAMI HANDA REGIONAL RESEARCH LABORATORY, CANAL ROAD JAMMU.
3 RAJINDER SINGH ICMR CENTRE FOR ADVANCED RESEARCH AT REGIONAL RESEARCH LAVORATORY, CANAL ROAD, JAMMU 180 001, JAMMU AND KASHMIR.
PCT International Classification Number A61K 7/48
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA