Title of Invention	A PROCESS FOR THE PREPARATION OF (-)-EPICATECHIN FROM A NEW NATURAL SOURCE NAMELY DICHROSTACHYS CINEREA
Abstract	This invention relates to a process for preparation of (-)-epicatechin from Dichrostachys cinerea. Dichrostachys cinerea is a medicinal plant used in the traditional Indian system of medicine and is widely advocated in diuretic, lithotriptic, anodyne, digestive, constipating, and inflammatory conditions. Dichrostachys cinerea hence is a new plant source for (-)-epicatechin and its presence in this plant in good yields makes this invention more important. The process steps are: a) extracting the dried wood powder characterized in that of Dichrostachys cinerea with hexane; b) extracting the residue of step (a) with chloroform; c) extracting the residue of step (b) with methanol solution to obtain a methanolic extract; d)concentrating the methanolic extract of step (c) under vacuum; e) adsorbing the concentrated methanolic extract of step (d) on silica gel and loading on silica gel column ; f) eluting the column with chloroform methanol gradient, g) collecting the fraction eluted at 6 % methanol in chloroform, and concentrating the eluted fraction of step (g) to obtain pure (-)-epicatechin.

Title of Invention

A PROCESS FOR THE PREPARATION OF (-)-EPICATECHIN FROM A NEW NATURAL SOURCE NAMELY DICHROSTACHYS CINEREA

Abstract

This invention relates to a process for preparation of (-)-epicatechin from Dichrostachys cinerea. Dichrostachys cinerea is a medicinal plant used in the traditional Indian system of medicine and is widely advocated in diuretic, lithotriptic, anodyne, digestive, constipating, and inflammatory conditions. Dichrostachys cinerea hence is a new plant source for (-)-epicatechin and its presence in this plant in good yields makes this invention more important. The process steps are: a) extracting the dried wood powder characterized in that of Dichrostachys cinerea with hexane; b) extracting the residue of step (a) with chloroform; c) extracting the residue of step (b) with methanol solution to obtain a methanolic extract; d)concentrating the methanolic extract of step (c) under vacuum; e) adsorbing the concentrated methanolic extract of step (d) on silica gel and loading on silica gel column ; f) eluting the column with chloroform methanol gradient, g) collecting the fraction eluted at 6 % methanol in chloroform, and concentrating the eluted fraction of step (g) to obtain pure (-)-epicatechin.

Full Text	Technical Field This invention relates to the process of preparation of a (-)-epicatechin from Dichrostachys cinerea. The process provides good yield . Background Art Dichrostachys cinerea is a medicinal plant used in the traditional Indian system of medicine and is widely advocated in diuretic, lithotriptic, anodyne, digestive, constipating, and inflammatory conditions. Also it is useful in vitiated conditions of kapha and vata, arthralgia, elephantiasis, dyspepsia, diarrhea, vesicle calculi, strangury, nephropathy, vaginopathy and metropathy. (Indian Medicinal Plants, Vol.2 p.330).It is useful in opthalmia, rheumatism , urinary calculi and renal troubles. (Wealth of India Vol.3 p.56).It is further reported to possess protease inhibitor activity(CA, 90, 118086u), fungi toxic activity (Ind. J. plant. Physiol, 1986, 29(3), 278-80.), antibacterial (Fitoterapia, 1988, 59(1), 57-62.). Hence it becomes pertinent to look for the molecules possessing such important biological properties. In this connection, the phytochemical investigation of Dichrostachys cinerea has been taken up. The applicants made efforts for the isolation of a compound (-)-epicatechin in highly economical yield. Objects of the invention: The main object of the invention is to provide a process for preparation of (-)-epicatechin from Dichrostachys cinerea. Another object of invention is to provide a new source for obtaining (-)-epicatechin in good yield. (-)-Epicatechin known to posses several activities, which are shown in Table. 1 Table 1 (Table Removed) Summary of the invention: The present invention relates to a process for the isolation of (-)-epicatechin from D. cinerea. Detailed Description: Accordingly,present invention provides a process for preparation of (-)-epicatechin from Dichrostachys cinerea comprises of the following steps: (a) extracting dried wood powder characterized in that of Dichrostachys cinerea with hexane; (b) extracting the residue of step (a) with chloroform; (c) extracting the residue of step (b) with methanol; (d) concentrating the methanol solution from step (c) under vacuum; (e) adsorbing the methanol extract on silica gel (60-120 mesh) and loading on silica gel (60-120 mesh) column (5 cm diameter to height of 100 cm); (f) eluting the column with chloroform methanol gradient, (g) collecting the fraction eluted at 6 % methanol in chloroform, and (h) concentrating the eluted fraction to obtain pure (-)-epicatechin. In another embodiment of the present invention, the solvent used or selected from Hexane, Chloroform and Methanol. In another embodiment of the present invention, the yield of (-)-epicatechin obtained is about 0.45% of the dried material. The percentage recited herein is % by weight. The invention further provides a method for the isolation of (-)-epicatechin from a new plant source namely Dichrostachys cinerea. In accordance with this invention, it has been found that (-)- epicatechin is isolated from a new plant source, Dichrostachys cinerea in significant yield. In another invention the process of isolation of (-)-epicatechin is highly economical. Dichrostachys cinerea hence is a new plant source for (-)-epicatechin and its presence in this plant in good yields makes this invention more important. The different plant sources from where (-)-epicatechin is isolated are given in Table.2. Table.2 (Table Remove) Experimental protocol: A process for the isolation of compound (-)-epicatechin. The dried stem bark powder of Dichrostachys cinerea (2 Kg) was loaded on a soxhlet apparatus. The powder was first extracted with hexane. The residue from the extraction of hexane was further extracted with chloroform. After the chloroform extraction the residue was taken in a conical flask and soaked in methanol at room temperature. The methanol solution was filtered and concentrated under vacuum (50 g). The methanol extract (50 g) was adsorbed on silica gel (60-120 mesh) and loaded on silica gel (60-120 mesh) column. (5 cms diameter to a height of 100 cms). The column is subjected to elution with chloroform-methanol gradient. The chloroform-methanol gradient is so selected to obtain specific fraction and thereby the desired compound. In the present case, the fractions eluted at 6% methanol in chloroform are collected separately and concentrated. The above fractions are subjected to further purification using silica gel column (> 200 mesh, 3 cm. dia and 50 cm. length) using chloroform methanol gradient. The eluent at 6% methanol in chloroform gave pure (-)-epicatechin (9.0 g). The spectrochemical data of (-)-epicatechin are given below: (-)-Epicatechin 1. Molecular formula: C15H\|4O6 2. 1HNMR: [(CD3)z CO; 200 MHz]: 6 7.04 (IH, d, J2 Hz, H-21), 6.86 (IH, d, J 8 Hz, H-51), 6.78 (IH, dd, J2 and 8 Hz, H-61), 6.22 (IH, d, J2 Hz, H-8), 6.12 (IH, d, J 2 Hz, H-6), 4.88 (IH, s, H-2), 4.20 (IH, m, H-3), 2.80 (2H, m, H-4, H-4'); 3. 13C NMR [(CD3)2 CO]: 5 78.13 (C-2), 65.01 (C-3), 28.22 (C-4), 156.57 (C-5), 95.27 (C-6), 156.29 (C-7), 94.22 (C-8), 155.79 (C-9), 98.62 (C- 10), 130.69 (C-l1), 118.05 (C-21), 144.47 (C-31', 144.54 (C- 41), 114.97(0-5'), 114.90(0-6'). 4. EIMS: 290 (M*), 272, 152,139, and 124. 5. IRvmax(KBr)cm-':3306 6. [a]D25 -68°(c0.1,MeOH) Brief description of the accompanying drawings Fig-1 Represents the structure of (-)-epicatechin We Claim: 1. A process for preparation of (-)-epicatechin from Dichrostachys cinerea, said process comprising the steps of: (a) extracting the dried wood powder characterized in that of Dichrostachys cinerea with hexane; (b) extracting the residue of step (a) with chloroform; (c) extracting the residue of step (b) with methanol solution to obtain a methanolic extract; (d) concentrating the methanolic extract of step (c) under vacuum; (e) adsorbing the concentrated methanolic extract of step (d) on silica gel and loading on silica gel column ; (f) eluting the column with chloroform methanol gradient, (g) collecting the fraction eluted at 6 % methanol in chloroform, and (h) concentrating the eluted fraction of step (g) to obtain pure (-)-epicatechin. 2. A process as claimed in claim 1 wherein, the yield of (-)-epicatechin is about 0.45% of the dried material. 3. A process for preparation of (-)-epicatechin from Dichrostachys cinerea as herein described with reference to examples accompanying this specification.

Full Text

Technical Field
This invention relates to the process of preparation of a (-)-epicatechin from Dichrostachys cinerea. The process provides good yield . Background Art
Dichrostachys cinerea is a medicinal plant used in the traditional Indian system of medicine and is widely advocated in diuretic, lithotriptic, anodyne, digestive, constipating, and inflammatory conditions. Also it is useful in vitiated conditions of kapha and vata, arthralgia, elephantiasis, dyspepsia, diarrhea, vesicle calculi, strangury, nephropathy, vaginopathy and metropathy. (Indian Medicinal Plants, Vol.2 p.330).It is useful in opthalmia, rheumatism , urinary calculi and renal troubles. (Wealth of India Vol.3 p.56).It is further reported to possess protease inhibitor activity(CA, 90, 118086u), fungi toxic activity (Ind. J. plant. Physiol, 1986, 29(3), 278-80.), antibacterial (Fitoterapia, 1988, 59(1), 57-62.). Hence it becomes pertinent to look for the molecules possessing such important biological properties. In this connection, the phytochemical investigation of Dichrostachys cinerea has been taken up. The applicants made efforts for the isolation of a compound (-)-epicatechin in highly economical yield. Objects of the invention:
The main object of the invention is to provide a process for preparation of (-)-epicatechin
from Dichrostachys cinerea.
Another object of invention is to provide a new source for obtaining (-)-epicatechin in
good yield.
(-)-Epicatechin known to posses several activities, which are shown in Table. 1
Table 1

(Table Removed)

Summary of the invention:
The present invention relates to a process for the isolation of (-)-epicatechin from D. cinerea.
Detailed Description:
Accordingly,present invention provides a process for preparation of (-)-epicatechin from Dichrostachys cinerea comprises of the following steps:
(a) extracting dried wood powder characterized in that of Dichrostachys cinerea with hexane;
(b) extracting the residue of step (a) with chloroform;
(c) extracting the residue of step (b) with methanol;
(d) concentrating the methanol solution from step (c) under vacuum;
(e) adsorbing the methanol extract on silica gel (60-120 mesh) and loading on silica gel (60-120 mesh) column (5 cm diameter to height of 100 cm);
(f) eluting the column with chloroform methanol gradient,
(g) collecting the fraction eluted at 6 % methanol in chloroform, and
(h) concentrating the eluted fraction to obtain pure (-)-epicatechin.
In another embodiment of the present invention, the solvent used or selected from Hexane,
Chloroform and Methanol.
In another embodiment of the present invention, the yield of (-)-epicatechin obtained is
about 0.45% of the dried material. The percentage recited herein is % by weight.
The invention further provides a method for the isolation of (-)-epicatechin from a new
plant source namely Dichrostachys cinerea.
In accordance with this invention, it has been found that (-)- epicatechin is isolated from a
new plant source, Dichrostachys cinerea in significant yield.
In another invention the process of isolation of (-)-epicatechin is highly economical.
Dichrostachys cinerea hence is a new plant source for (-)-epicatechin and its presence in
this plant in good yields makes this invention more important. The different plant sources
from where (-)-epicatechin is isolated are given in Table.2.

Table.2
(Table Remove) Experimental protocol: A process for the isolation of compound (-)-epicatechin. The dried stem bark powder of Dichrostachys cinerea (2 Kg) was loaded on a soxhlet apparatus. The powder was first extracted with hexane. The residue from the extraction of hexane was further extracted with chloroform. After the chloroform extraction the residue was taken in a conical flask and soaked in methanol at room temperature. The methanol solution was filtered and concentrated under vacuum (50 g). The methanol extract (50 g) was adsorbed on silica gel (60-120 mesh) and loaded on silica gel (60-120 mesh) column. (5 cms diameter to a height of 100 cms). The column is subjected to elution with chloroform-methanol gradient. The chloroform-methanol gradient is so selected to obtain specific fraction and thereby the desired compound. In the present case, the fractions eluted at 6% methanol in chloroform are collected separately and concentrated.
The above fractions are subjected to further purification using silica gel column (> 200 mesh, 3 cm. dia and 50 cm. length) using chloroform methanol gradient. The eluent at 6% methanol in chloroform gave pure (-)-epicatechin (9.0 g). The spectrochemical data of (-)-epicatechin are given below:
(-)-Epicatechin
1. Molecular formula: C15H|4O6
2. 1HNMR: [(CD3)z CO; 200 MHz]: 6 7.04 (IH, d, J2 Hz, H-21), 6.86 (IH,
d, J 8 Hz, H-51), 6.78 (IH, dd, J2 and 8 Hz, H-61), 6.22 (IH, d, J2 Hz, H-8), 6.12 (IH, d, J 2 Hz, H-6), 4.88 (IH, s, H-2), 4.20 (IH, m, H-3), 2.80 (2H, m, H-4, H-4');
3. 13C NMR [(CD3)2 CO]: 5 78.13 (C-2), 65.01 (C-3), 28.22 (C-4), 156.57
(C-5), 95.27 (C-6), 156.29 (C-7), 94.22 (C-8), 155.79 (C-9), 98.62 (C-
10), 130.69 (C-l1), 118.05 (C-21), 144.47 (C-31', 144.54 (C- 41),
114.97(0-5'), 114.90(0-6').
4. EIMS: 290 (M*), 272, 152,139, and 124.
5. IRvmax(KBr)cm-':3306
6. [a]D25 -68°(c0.1,MeOH)
Brief description of the accompanying drawings Fig-1 Represents the structure of (-)-epicatechin

We Claim:
1. A process for preparation of (-)-epicatechin from Dichrostachys cinerea, said
process comprising the steps of:
(a) extracting the dried wood powder characterized in that of Dichrostachys cinerea with hexane;
(b) extracting the residue of step (a) with chloroform;
(c) extracting the residue of step (b) with methanol solution to obtain a methanolic extract;
(d) concentrating the methanolic extract of step (c) under vacuum;
(e) adsorbing the concentrated methanolic extract of step (d) on silica gel and loading on silica gel column ;
(f) eluting the column with chloroform methanol gradient,
(g) collecting the fraction eluted at 6 % methanol in chloroform, and
(h) concentrating the eluted fraction of step (g) to obtain pure (-)-epicatechin.
2. A process as claimed in claim 1 wherein, the yield of (-)-epicatechin is about 0.45% of the dried material.
3. A process for preparation of (-)-epicatechin from Dichrostachys cinerea as herein described with reference to examples accompanying this specification.

Documents:

462-DEL-2003-Abstract-(28-08-2008).pdf

462-del-2003-abstract.pdf

462-DEL-2003-Claims-(28-08-2008).pdf

462-del-2003-claims.pdf

462-DEL-2003-Correspondence-Others-(17-09-2008).pdf

462-DEL-2003-Correspondence-Others-(28-08-2008).pdf

462-del-2003-correspondence-others.pdf

462-del-2003-correspondence-po.pdf

462-del-2003-description (complete)-28-08-2008.pdf

462-del-2003-description (complete).pdf

462-del-2003-form-1.pdf

462-del-2003-form-18.pdf

462-DEL-2003-Form-2-(28-08-2008).pdf

462-del-2003-form-2.pdf

462-DEL-2003-Form-3-(28-08-2008).pdf

462-del-2003-form-3.pdf

462-DEL-2003-Petition-137-(28-08-2008).pdf

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Patent Number

225168

Indian Patent Application Number

462/DEL/2003

PG Journal Number

48/2008

Publication Date

28-Nov-2008

Grant Date

05-Nov-2008

Date of Filing

27-Mar-2003

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	JANASWAMY MADHUSUDANA RAO	CHEMICAL TECHNOLOGY (IICT), HYDRABAD 500 007,ANDHRA PRADESH INDIA
2	RAO JAGADLESHWAR RAO	CHEMICAL TECHNOLOGY (IICT), HYDRABAD 500 007,ANDHRA PRADESH INDIA
3	JILLU SINGH YADAV	CHEMICAL TECHNOLOGY (IICT), HYDRABAD 500 007,ANDHRA PRADESH INDIA
4	KONDAPURAM VIJAYA RAGHAVAN	CHEMICAL TECHNOLOGY (IICT), HYDRABAD 500 007,ANDHRA PRADESH INDIA

PCT International Classification Number

A61K 35/78

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA