Title of Invention	A PROCESS FOR THE PRODUCTION OF A HUMAN PLATELET AGGREGATION INHIBITOR FROM ASPERGILLUS NIGER.
Abstract	The present invention provides a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. In the process of present invention a simple liquid medium is employed for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property and employed a simple extraction and purification method to obtain compound of formula 1 .

Title of Invention

A PROCESS FOR THE PRODUCTION OF A HUMAN PLATELET AGGREGATION INHIBITOR FROM ASPERGILLUS NIGER.

Abstract

The present invention provides a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. In the process of present invention a simple liquid medium is employed for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property and employed a simple extraction and purification method to obtain compound of formula 1 .

Full Text	The present invention relates to a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. The invention particularly relates to a compound having formula 1 as shown below which is a inhibitor of human platelet aggregation having soybean lipoxygenase inhibiting property. (Formula Removed) Formula 1 Japan Patent No. 0625627.5 claimed a process for the production of a rabbit platelet aggregation inhibitor from streptomyces sps. However, a large number of medium constituents have been used for the production of the inhibitor. Japan Patent No. 04159274 claimed a benzoquinone derivative through fermentation as a rabbit platelet aggregation inhibitor. The activity claimed in the prior art is very low activity with an 1C 50 of 1 mm. United States Patent No. 5208364 claimed a 5-lipoxygenase inhibitor from Streptomyces sps. The prior art describes an unwieldy process of using HPLC for purifying the inhibitor from the broth. The preparation of lipoxygenase inhibitors is disclosed in Betina, V; Prog Ind Microbiol 30 (1994) 121-135. The prior art describes the production of these inhibitors, such as 3-methoxytropolone and kf 8940, from streptoverticillium sps and pseudomonas methanica, respectively. However, these metabolites have been reported to be produced from complex medium components. Studies on sulphasalazine metabolites, such as N-acetylaminosalicylic acid and and 5-amino salicylic acids showing effective soybean lipoxygenase inhibition is disclosed in Allgayer.H; Eisenburg.J ; Paumgartner.G. Eur J Clin Pharmacol. 26_(984) 449-451. However, this prior art has no disclosure or suggestion that soybean lipoxygenase inhibitors are inhibitors against platelet aggregation as well. In view of the above discussion describing the state of the art there were no known or apparently simple fermentation medium and conditions to obtain molecules with an activity against human platelet aggregation inhibitorsOr/spybean lipoxygenase inhibitors. Further, none of the above prior arts disclosed a preparation of Formula! as a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property. The objective of the present invention is to develop a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property of formula 1 as shown below from Aspergillus niger. (Formula Removed) Formula 1 In the process of present invention a simple liquid medium is employed for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property and employed a simple extraction and purification method to obtain compound of formula 1 . Further the method to produce the inhibitor of formula 1 employs a short fermentation time ( 5-8 days). The present invention is to develop a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property of formula 1 from Aspergillus niger Formula 1 Accordingly the present invention provides, a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. , which comprises developing an inoculum of Aspergillus sp. by conventional manner such as here in described , transferring 0.5 -2 litres of inoculum to a 500-2000 ml of medium followed by incubating at 28 - 37°C for 24-48 hours under agitation at 100-200 rpm thereby fermenting the medium , wherein the medium used for both inoculum development and fermentation is potato dextrose broth comprising of potatoes and glucose at a ratio of 10:1 for 100 ml of the medium , wherein pH of the medium ranges from 5.1 - 5.5 , transferring 1-5 litres of solvent preferably ethylacetate to the fermented medium as obtained above , incubating at room temperature for 1-3 hours , filtering the fermented broth by conventional method as described herein to separate the solvent , adding 10-50 gm of anhydrous sodium sulphate to remove any traces of residual water from the obtained broth, distilling the solvent under vacuum to get a crude extract , loading the obtained extract on 10-50 gm of silica gel (100-200 mesh) column in hexane having a bed volume of 20-60 ml , washing the column with 1-2 volumes of hexane followed by eluting using 1-2 volumes of hexane:chloroform (1:1) to get an orange coloured fraction, distilling the obtained fraction under vacuum to remove the solvent and to get semi purified inhibitor, adding 1-10 ml of hexane to the obtained inhibitor and stored at 1°-10°C for at least 2-3 days to obtain the purified inhibitor in the form of red crystals which has a final ic50 activity of 234 urn and 95.6 urn against human platelet aggregation and soybean lipoxygenase respectively. In an embodiment of the present invention the medium used for both inoculum and fermentation comprises boiled 15-20 gms of peeled and cut potatoes and 1-3 gms of glucose for 100 ml of the medium. In an another embodiment of the present invention the fungi used for the preparation of inhibitor is selected from Aspergillus and Penicillium species. In an another embodiment of the present invention the liquid medium used is potato dextrose broth consisting of potato and glucose in the ratio of 10:1 for 100 ml of the medium. In an another embodiment of the present invention the potato used is is preferably 20gm of per 100 ml of liquid medium. In an another embodiment of the present invention the glucose used is preferably 2gm of per 100 ml of liquid medium. In yet another embodiment of the present invention the fermented medium used is extracted with water immiscible solvent selected from chloroform and ethylacetate and the solvent extract is used as a source for purifying the inhibitor. The invention is illustrated but not restricted by the description in the following example Example I In a 15 litres capacity fermentor, 10 litres of fresh potato dextrose broth was was prepared by boiling 200 gms of peeled and sliced potatoes for 20 minutes and after cooling to room temperature, 200 gms of dextrose was added, the ph was adjusted to 5.1. the flask was then autoclaved at 121 °c for 15 minutes, to this 1 I of a 24 hour old fungal inoculum was added and then incubated for 7 days at 30 °c. at the end of the fermentation,4 litres of ethylacetate was added and incubated at room temperature for two hours, the solvent was filtered through whatman no 1 filter paper, subsequently, the solvent was separated from the broth using a separating funnel and 50 gms of anhydrous sodium sulphate was added to the solvent to remove any traces of residual water, the solvent was evaporated under vacuum to obtain 1.5 gms crude extract, with platelet aggregation inhibition activity of 22.5% was obtained, this was loaded on a 50 gms of silica gel (100-200 mesh) column in hexane to give a bed volume of 40 ml. after washing the column with 100 ml of hexane, the inhibitor was eluted using 600 ml of hexane:chloroform (1:1). the orange coloured fraction containing the semi purified said inhibitor was distilled under vacuum to remove the solvent, to 150 mgs of this semi purified inhibitor, 2 ml of hexane was added and stored in cold overnight to obtain 3.3 mgs of the purified inhibitor in the form of red crystals. 234 (am of the inhibitor gave 50 % inhibition against human platelet aggregation and 95.6 jam gave 50 % inhibition against soybean lipoxygenase Analytical data of formula I: Molecular Formula: C2o H22 O Molecular Weight: 278 Colour and physical appearance of inhibitor: red crystals Solubility: highly soluble in chloroform, dimethylsulfoxide, acetonitrile Absorption maxima (nm) at 30 µg/ml dmso: 255, 304 and 406 nm 1H NMR spectrum (cdcl3, 5tms=0 ppm): 7.4 2h d 9.03hz; 7.31 2h t 9.00hz; 7.25 3h m 9.03 hz; 6.92 1h dd 16.1hz; 6.67 2h dd 18.1hz; 6.96 1h dd 16.1hz; 6.39 2h dd 16.1hz; 6.21 1h d 18.1hz; 1.14 3h t 7.6hz; 1.97 3h s ; 2.6 2h q 7.6hz. Mass spectrum: m+ - ch3 = 263; m+ - ch3-ch2 = 249; m+ - ch3-ch2-co+ = 221;m+-91 =187; 187-26= 161;t91+ch= =105 Activity of purified inhibitor: IC50 of the inhibitor against platelet aggregation:234 \|am IC50 of the inhibitor against soybean lipoxygenase (ph 9) is :95.6 We Claim: 1. A process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp., which comprises developing an inoculum of Aspergillus sp. by conventional manner such as here in described , transferring 0.5 -2 litres of inoculum to a 500-2000 ml of medium followed by incubating at 28 - 37°C for 24-48 hours under agitation at 100-200 rpm thereby fermenting the medium , wherein the medium used for both inoculum development and fermentation is potato dextrose broth comprising of potatoes and glucose at a ratio of 10:1 for 100 ml of the medium and pH of the medium ranges from 5.1 - 5.5 , transferring 1-5 litres of solvent preferably ethylacetate, to ^ the fermented medium as obtained above , incubating at room temperature for 1-3 hours , filtering the fermented broth by conventional method as described herein to separate the solvent , adding 10-50 gm of anhydrous sodium sulphate to remove any traces of residual water from the obtained broth, distilling the solvent under vacuum to get a crude extract , loading the obtained extragt on 10-50 gm of silica gel (100-200 mesh) column in hexane having a bed volume of 20-60 ml , washing the column with 1-2 volumes of hexane followed by eluting using 1-2 volumes of hexane:chloroform (1:1) to get an orange coloured fraction, distilling the obtained fraction under vacuum to remove the solvent and to get semi purified inhibitor, adding 1-10 ml of hexane to the obtained inhibitor and stored at 1°-10°C for at least 2-3 days to obtain the purified inhibitor in the form of red crystals which has a final ic50 activity of 234 urn and 95.6 urn against human platelet aggregation and soybean lipoxygenase respectively. 2. A process as claimed in claims 1 wherein the Aspergillus sp. used for the preparation of inhibitor is selected from Aspergillus flavus, Aspergillus japonicus, Aspergillus niger, Aspergillus awamori. 3. A process as claimed in claims 1-2, wherein the potato used in potato-dextrose medium is preferably 20 gm of per 100 ml of liquid medium. 4. A process as claimed in claims 1-3, wherein solvent used is selected from water immiscible solvent selected from the group comprising chloroform and ethylacetate. 5. A process as claimed in claims 1-4, wherein solvent used for column chromatography is selected from hexane and chloroform. 6. A process for the production of a human platelet aggregation inhibitor substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. The invention particularly relates to a compound having formula 1 as shown below which is a inhibitor of human platelet aggregation having soybean lipoxygenase inhibiting property.
(Formula Removed)
Formula 1
Japan Patent No. 0625627.5 claimed a process for the production of a rabbit platelet aggregation inhibitor from streptomyces sps. However, a large number of medium constituents have been used for the production of the inhibitor.
Japan Patent No. 04159274 claimed a benzoquinone derivative through fermentation as a rabbit platelet aggregation inhibitor. The activity claimed in the prior art is very low activity with an 1C 50 of 1 mm.
United States Patent No. 5208364 claimed a 5-lipoxygenase inhibitor from Streptomyces sps. The prior art describes an unwieldy process of using HPLC for purifying the inhibitor from the broth.
The preparation of lipoxygenase inhibitors is disclosed in Betina, V; Prog Ind Microbiol 30 (1994) 121-135. The prior art describes the production of these inhibitors, such as 3-methoxytropolone and kf 8940, from streptoverticillium sps and pseudomonas methanica, respectively. However, these metabolites have been reported to be produced from complex medium components.
Studies on sulphasalazine metabolites, such as N-acetylaminosalicylic acid and and 5-amino salicylic acids showing effective soybean lipoxygenase inhibition is disclosed in Allgayer.H; Eisenburg.J ; Paumgartner.G. Eur J Clin Pharmacol. 26_(984) 449-451. However, this prior art has no disclosure or suggestion that soybean lipoxygenase inhibitors are inhibitors against platelet aggregation as well.
In view of the above discussion describing the state of the art there were no known or apparently simple fermentation medium and conditions to obtain molecules with an activity against human platelet aggregation inhibitorsOr/spybean lipoxygenase inhibitors. Further, none of the above prior arts disclosed a preparation of
Formula! as a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property.
The objective of the present invention is to develop a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property of formula 1 as shown below from Aspergillus niger.
(Formula Removed)
Formula 1
In the process of present invention a simple liquid medium is employed for the
production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property and employed a simple extraction and purification method to obtain compound of formula 1 . Further the method to produce the inhibitor of formula 1 employs a short fermentation time ( 5-8 days).
The present invention is to develop a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibitory property of formula 1 from Aspergillus niger
Formula 1
Accordingly the present invention provides, a process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp. , which comprises developing an inoculum of Aspergillus sp. by conventional manner such as here in described , transferring 0.5 -2 litres of inoculum to a 500-2000 ml of medium followed by incubating at 28 - 37°C for 24-48 hours under agitation at 100-200 rpm thereby fermenting the medium , wherein the medium used for both inoculum development and fermentation is potato dextrose broth comprising of potatoes and glucose at a ratio of 10:1 for 100 ml of the medium , wherein pH of the medium ranges from 5.1 - 5.5 , transferring 1-5 litres of solvent preferably ethylacetate to the fermented medium as obtained above , incubating at room temperature for 1-3 hours , filtering the fermented broth by conventional method as described herein to separate the solvent , adding 10-50 gm of anhydrous sodium sulphate to remove any traces of residual water from the obtained broth, distilling the solvent under vacuum to get a crude extract , loading the obtained extract on 10-50 gm of silica gel (100-200 mesh) column in hexane having a bed volume of 20-60 ml , washing the column with 1-2 volumes of hexane followed by eluting using 1-2 volumes of hexane:chloroform (1:1) to get an
orange coloured fraction, distilling the obtained fraction under vacuum to remove the solvent and to get semi purified inhibitor, adding 1-10 ml of hexane to the obtained inhibitor and stored at 1°-10°C for at least 2-3 days to obtain the purified inhibitor in the form of red crystals which has a final ic50 activity of 234 urn and 95.6 urn against human platelet aggregation and soybean lipoxygenase respectively.
In an embodiment of the present invention the medium used for both inoculum and fermentation comprises boiled 15-20 gms of peeled and cut potatoes and 1-3 gms of glucose for 100 ml of the medium.
In an another embodiment of the present invention the fungi used for the preparation of inhibitor is selected from Aspergillus and Penicillium species.
In an another embodiment of the present invention the liquid medium used is potato dextrose broth consisting of potato and glucose in the ratio of 10:1 for 100 ml of the medium.
In an another embodiment of the present invention the potato used is is preferably 20gm of per 100 ml of liquid medium.
In an another embodiment of the present invention the glucose used is preferably 2gm of per 100 ml of liquid medium.
In yet another embodiment of the present invention the fermented medium used is extracted with water immiscible solvent selected from chloroform and ethylacetate and the solvent extract is used as a source for purifying the inhibitor.
The invention is illustrated but not restricted by the description in the following example
Example I
In a 15 litres capacity fermentor, 10 litres of fresh potato dextrose broth was was prepared by boiling 200 gms of peeled and sliced potatoes for 20 minutes and after cooling to room temperature, 200 gms of dextrose was added, the ph was adjusted to 5.1. the flask was then autoclaved at 121 °c for 15 minutes, to this 1 I of a

24 hour old fungal inoculum was added and then incubated for 7 days at 30 °c. at the end of the fermentation,4 litres of ethylacetate was added and incubated at room temperature for two hours, the solvent was filtered through whatman no 1 filter paper, subsequently, the solvent was separated from the broth using a separating funnel and 50 gms of anhydrous sodium sulphate was added to the solvent to remove any traces of residual water, the solvent was evaporated under vacuum to obtain 1.5 gms crude extract, with platelet aggregation inhibition activity of 22.5% was obtained, this was loaded on a 50 gms of silica gel (100-200 mesh) column in hexane to give a bed volume of 40 ml. after washing the column with 100 ml of hexane, the inhibitor was eluted using 600 ml of hexane:chloroform (1:1). the orange coloured fraction containing the semi purified said inhibitor was distilled under vacuum to remove the solvent, to 150 mgs of this semi purified inhibitor, 2 ml of hexane was added and stored in cold overnight to obtain 3.3 mgs of the purified inhibitor in the form of red crystals. 234 (am of the inhibitor gave 50 % inhibition against human platelet aggregation and 95.6 jam gave 50 % inhibition against soybean lipoxygenase
Analytical data of formula I: Molecular Formula: C2o H22 O

Molecular Weight: 278
Colour and physical appearance of inhibitor: red crystals Solubility: highly soluble in chloroform, dimethylsulfoxide, acetonitrile
Absorption maxima (nm) at 30 µg/ml dmso: 255, 304 and 406 nm 1H NMR spectrum (cdcl3, 5tms=0 ppm): 7.4 2h d 9.03hz; 7.31 2h t 9.00hz; 7.25 3h m 9.03 hz; 6.92 1h dd 16.1hz; 6.67 2h dd 18.1hz; 6.96 1h dd 16.1hz; 6.39 2h dd 16.1hz; 6.21 1h d 18.1hz; 1.14 3h t 7.6hz; 1.97 3h s ; 2.6 2h q 7.6hz. Mass spectrum: m+ - ch3 = 263; m+ - ch3-ch2 = 249; m+ - ch3-ch2-co+ = 221;m+-91 =187; 187-26= 161;t91+ch= =105 Activity of purified inhibitor: IC50 of the inhibitor against platelet aggregation:234 |am
IC50 of the inhibitor against soybean lipoxygenase (ph 9) is :95.6

We Claim:
1. A process for the production of a human platelet aggregation inhibitor having soybean lipoxygenase inhibiting property using Aspergillus sp., which comprises developing an inoculum of Aspergillus sp. by conventional manner such as here in described , transferring 0.5 -2 litres of inoculum to a 500-2000 ml of medium followed by incubating at 28 - 37°C for 24-48 hours under agitation at 100-200 rpm thereby fermenting the medium , wherein the medium used for both inoculum development and fermentation is potato dextrose broth comprising of potatoes and glucose at a ratio of 10:1 for 100 ml of the medium and pH of the medium ranges from 5.1 - 5.5 , transferring 1-5 litres of solvent preferably ethylacetate, to
^
the fermented medium as obtained above , incubating at room temperature for 1-3 hours , filtering the fermented broth by conventional method as described herein to separate the solvent , adding 10-50 gm of anhydrous sodium sulphate to remove any traces of residual water from the obtained broth, distilling the solvent under vacuum to get a crude extract , loading the obtained extragt on 10-50 gm of silica gel (100-200
mesh) column in hexane having a bed volume of 20-60 ml , washing the column with 1-2 volumes of hexane followed by eluting using 1-2 volumes of hexane:chloroform (1:1) to get

an orange coloured fraction, distilling the obtained fraction under vacuum to remove the solvent and to get semi purified inhibitor, adding 1-10 ml of hexane to the obtained inhibitor and stored at 1°-10°C for at least 2-3 days to obtain the purified inhibitor in the form of red crystals which has a final ic50 activity of 234 urn and 95.6 urn against human platelet aggregation and soybean lipoxygenase respectively.
2. A process as claimed in claims 1 wherein the Aspergillus sp. used
for the preparation of inhibitor is selected from Aspergillus flavus,
Aspergillus japonicus, Aspergillus niger, Aspergillus awamori.
3. A process as claimed in claims 1-2, wherein the potato used in
potato-dextrose medium is preferably 20 gm of per 100 ml of liquid
medium.
4. A process as claimed in claims 1-3, wherein solvent used is
selected from water immiscible solvent selected from the group
comprising chloroform and ethylacetate.
5. A process as claimed in claims 1-4, wherein solvent used for
column chromatography is selected from hexane and chloroform.
6. A process for the production of a human platelet aggregation
inhibitor substantially as herein described with reference to the
examples.

Documents:

216-del-2001-abstract.pdf

216-del-2001-claims.pdf

216-del-2001-correspondence-others.pdf

216-del-2001-correspondence-po.pdf

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

216-del-2001-form-1.pdf

216-del-2001-form-19.pdf

216-del-2001-form-2.pdf

216-del-2001-form-3.pdf

216-del-2001-petition-137.pdf

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Patent Number

220105

Indian Patent Application Number

216/DEL/2001

PG Journal Number

28/2008

Publication Date

11-Jul-2008

Grant Date

15-May-2008

Date of Filing

28-Feb-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

Inventors:

#	Inventor's Name	Inventor's Address
1	AVINASH PRAHLAD SATTUR
2	THIRUMALAI PARTHASARATMY KRISHNAKANTH SURYANARAYANA VISHWANATHA
3	WESLEY JESSIESUNEETHA
4	NAIKANAKATTE GANESH KARANTH
5	SOUNDAR DIVAKAR
6	KADIYACA CHANDRA SEKHAR RAO

PCT International Classification Number

A61K31/522

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA