Title of Invention

"A PROCESS FOR THE PREPARATION OF A LIPOXYGENASE INHIBITOR USEFUL IN HUMAN BLOOD PLATELET AGGREGATION FROM PENICILLIUM FREQUENTANS"

Abstract The present invention relates to a process for the preparation of human platelet aggregation and lipoxygenase inhibitor. In particular human platelet aggregation inhibitor and a lipoxygenase inhibitor from a fungal source. Platelet aggregation and blood clotting results in the blockage of capillaries and arteries. This leads to heart attacks and paralytic attacks if the arteries in the brain are blocked. Inhibitors against platelet aggregation thus have a great potential application in the medical sector. Lipids are highly vulnerable to oxidation which can be initiated by enzymatic and non enzymatic processes. The enzymatic process is initiated by Lipoxygenases, which are responsible for the oxygenation of polyunsaturated fatty acids such as linoleic, linolenic and arachidonic acid. These enzymes are found to be responsible for just for the deterioration, rancidity and loss of flavour in food materials and also for various diseases in the human body, such as Parkinson's disease, cataractogenesis, endotoxin liver injury, and myocardial infarction. Inhibitors against these enzymes thus have a potential application in both the food and medical sector.
Full Text The present invention relates to a process for the preparation of human platelet aggregation and lipoxygenase inhibitor In particular human platelet aggregation inhibitor and a lipoxygenase inhibitor from a fungal source
The inhibitor is obtained by fermentation of Penicilhum frequentans CFTRI A-24 Platelet aggregation and blood clotting results in the blockage of capillaries and arteries This leads to heart attacks and paralytic attacks if the arteries in the brain are blocked Inhibitors against platelet aggregation thus have a great potential application in the medical sector
Lipids are highly vulnerable to oxidation which can be initiated by enzymatic and non enzymatic processes The enzymatic process is initiated by Lipoxygenases which are responsible for the oxygenation of polyunsaturated fatty acids such as linoleic, Imolenic and arachidonic acid These enzymes are found to be responsible for just for the deterioration, rancidity and loss of flavour in food materials and also for various diseases in the human body, such as Parkinson's disease, cataractogenesis, endotoxm liver injury, and myocardial infarction Inhibitors against these enzymes thus have a potential application in both the food and medical sector
Reference may be made to Madhavi, DL and Salunkhe, DK (1994) In Food Additive Toxicology, Maga, JA and TU, AT (eds) Marcel Dekker, NY 88 -177 wherein they have discussed the use of various antioxidants in food materials The drawback is that the antioxidants discussed such as Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and Tertiary butylhyroquinone (TBHQ) are chemically synthesized and are not reported from any fermentation method
Reference may be made to Betina, V, Prog Ind Microbiol 30 (1994) 121-135 wherein the author has reported lipoxygenase inhibitors, such as 3-methoxytropolone and KF 8940, from Streptoverticilhum sps and Pseudomonas methamca, respectively The drawback is that these metabolites have been reported to be produced from complex medium components
Reference may be made to Kamayama T, Sato T and Watanabe J (Patent JP 06256275 Sept 1994) wherein the authors have produced a rabbit platelet

aggregation inhibitor from Streptomyces sps. The drawback is that a large number of medium constituents have been used for the production of the inhibitor.
Reference may be made to Kawashima A, Kagamizono T, Kishimura Y and Hanada K (Patent JP 04159274 June 1992) wherein the authors have produced a benzoquinone derivative through fermentation as a rabbit platelet aggregation inhibitor. The drawback is the inhibitor has a very low activity with an IC50of 1 mM.
The main object of the present invention is to provide a process for the preparation of human platelet aggregation and lipoxygenase inhibitor which obviates the drawbacks as detailed above.
Accordingly the present invention provides a process for the preparation of human platelet aggregation and lipoxygenase inhibitor comprising:
a) developing inoculum for the fermentation by making a spore
suspension in 0.1-0.2% Tween 20 from a 3-5 day old potato dextrose
agar slant of Penicillium frequentans CFTRI A-24 ;
b) transferring the inoculum to a 500-2000 ml potato dextrose broth
solution;
c) incubating the inoculum obtained from the above step at a temperature
ranging 28 -37°C for 24-48 hours under agitation at 100-200 rpm;
d) fermenting the above inoculum in a fermentor of 10-15 L capacity,
wherein the medium used for both inoculum development and main
fermentation is potato dextrose broth comprising boiled 15-20 gm of
peeled and cut potatoes and 1-3 gm of glucose for 100 ml of the
medium., with the final pH being 5.1-5.5 and the medium being
sterilized at 121°C for 15-20 minutes and allowed to cool to 30°C before
inoculation;
e) transferring 0.5-2 L of inoculum to the liquid medium and incubating at a
temperature ranging 28-30 °C for a period ranging 5-8 days at a100-200
rpm;

f) adding 1 -8 L of solvent such as ethyl acetate at the end of fermentation
to the fermented medium and incubated at room temperature for a
period of 1-3 hours at 100-200 rpm,
g) filtering the fermented broth by known methods and separating the
using a separating funnel, adding 10-50 gm of anhydrous sodium
sulphate to remove any traces of residual water,
h) distilling the solvent under vacuum to give a crude extract of 1 -5 gm which is loaded on 10-50 gm of silica gel (60-120 mesh) column in hexane, wherein the column has a bed volume of 20-60 ml,
i) washing the column with 1-2 volumes of hexane ,
j) eluting the inhibitor using 1-2 volumes chloroform ethyl acetate (9 1) in the form of an yellowish orange colored fraction, which is distilled under vacuum to remove the solvent and obtain 0 4 -2 gm of this semi purified inhibitor,
k) adding 10-50 ml of warm methanol and storing at a temperature ranging 1-10°C for a period of 2-3 days to obtain 50-100 mg of purified inhibitor in the form of yellowish orange crystals having final IC50 activity of 614 |xM against human platelet aggregation,
In an embodiment of the process the fungi such as Pemcillium species is used for the preparation of the inhibitor and the Pemalium species selected is from a group of P notatum, P sclerotiorum, P multicolor, P hiryamae, P cyclosponum P fumiculosum particularly Pemalium frequentans CFTRI A-24
In an another embodiment of the process the liquid medium used is potato dextrose broth consisting of 20 gm of potato and 2 gm of glucose for 100 ml of the medium
In an another embodiment of the process the cultivation of the fungi is for 3-5 days and the age of the inoculum is 24-48 hours grown under agitation at 100-200 rpm

In yet an another embodiment of the process the fermented medium is extracted with water immiscible solvents like chloroform, ethyl acetate and the solvent extract is used for purifying the inhibitor.
In yet an another embodiment of the process the purification of a human platelet aggregation inhibitor comprises recovering the said inhibitor by conventional column chromatography using combinations of polar and non polar solvents.
A general process for the production of the said inhibitor is given in the flow sheet:


The structure of the isolated inhibitor was determined by UV 1H NMR and mass spectrometry
Pemcillium frequentans CFTRI A-24, ascomycetes belong to the order eurotiales poduces conidial fructification in the form of branches or penicills The filtrate was assayed for human platelet aggregation inhibitor using the standard method (Kitagawa S, Endo J and Kametani F, Biochimica Biophysica Acta 818 (1985) 391-397) and is given as follows
The inhibition studies was carried out by incubating human platelets (platelet rich plasma (PRP) obtained from 10 ml human blood mixed with 2 ml of 3 8% tnsodium citrate and centnfuging at 1100 rpm for 20 minutes) with the microbial inhibitor (2-5 nl) at 30 °C for 1 minute, followed by the addition of 8-10 nl of inducer, ADP (Sigma chemicals, USA) in a total volume of 0 45 ml PRP The aggregation was recorded for at least 3 minutes Inhibition was calculated relative to rate of platelet aggregation induced by ADP as a control in the absence of the inhibitor Platelet aggregation in followed in PRP with PPP (Platelet poor plasma) in the reference cell
The inhibitor was assayed for lipoxygenase inhibition using the standard method (Methods in Enzymology 71 (1981) 441- 451) and is given as follows The inhibition studies was earned out by incubating soybean lipoxygenase enzyme (crude preparation of lipoxygenase enzyme was obtained from defatted powdered soybean in 0 1 M phosphate buffer, pH 6 8, and centnfuging at 1000 x g) with the microbial extract filtrate (2-10 \i\) at 30°C for 15 minutes, followed by the addition of 30 \i\ of substrate, linoleic acid (Sigma chemicals, USA) and in a total volume of 3 0 ml of 0 1M borate buffer pH 9 0 Absorbance change at 234 nm was recorded every 30 seconds for at least 3 minutes Inhibition was calculated relative to solvent control

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the invention
EXAMPLE -1
In a 15 L fermentation capacity container, 10 L of fresh potato dextrose broth was prepared by boiling 2 Kg of peeled and sliced potatoes for 20 minutes and after cooling to room temperature, 200 gm 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 L of a 24 hour old fungal inoculum was added and then incubated for 7 days at 30°C At the end of the fermentation, 5 L of ethyl acetate 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 gm of anhydrous sodium sulphate was added to the solvent to remove any traces of residual water The solvent was evaporated under vacuum to obtain 6 0 g of the concentrated extract This was used as a source of the enzyme inhibitor
EXAMPLE - 2
From 8 L of fermentation medium extracted with 5 L of ethyl acetate as given accordingly in Example 1, 1 5 gm crude extract With platelet aggregation inhibition activity of 50% was obtained This was loaded on a 50 gm of silica gel (60-120 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 200 ml of chloroform ethylacetate (9 1) The orange coloured fraction containing the semi purified said inhibitor was distilled under vacuum to remove the solvent To 550 mg of this semi purified inhibitor, 20 ml of hexane was added and stored in cold overnight to obtain 150 mg of the purified inhibitor in the form of yellowish orange crystals 614 ^iM of the inhibitor gave 50 % inhibition against human platelet aggregation and 39 |xM gave 50% inhibition against soybean lipoxygenase

Thus activity of purified inhibitor IC50 of the inhibitor against platelet aggregation 614|xm IC50 of the inhibitor against soybean lipoxygenase (pH 9) is 39 urn
Physico chemical property Molecular formula C21H23O5CI, Molecular weight 390
Colour and physical appearance of inhibitor yellowish orange red crystals Solubility highly soluble in chloroform, ethyl acetate, dimethylsulfoxide, acetonitnle Absorption maxima (nm) at 30 ^g/ml DMSO 284,361 nm
MS 390 [M+], 348 [M+ - COCHJ, 306[M+ - 2 X COCH2], 291 [306 - CH3],
1H NMR and 13CNMR spectral assignments of the formula 1 are presented in the
following table
Table 1H and 13C NMR chemical shifts of compound

H* Chemical shifts MULTIPLICI TY J(Hz) c**
1 7 94 s 153 3
2
3 158 8
4 6 60 s 107 0
4a 139 3
5 1152
6 192 4
7 85 2
8 186 6
8a 111 4
9 6 08 d 15 66 1163
10 7 06 Ld 15 66 143 5
11 132 6
12 5 71 d 9 74 149 5
13 2 49 m 35 8
14 1 34, 1 44 m,m 30 7
15 0 87 t 7 30 126
16 1 01 d 6 60 20 8
17 1 85 s 130
18 1 57 s 23 2
COCH3 170 7
COCH3 2 17 s 20 7
* 500 MHz
125 MHz


The novelty of the present invention is that an effective human platelet aggregation inhibitor may be obtained using a fungal culture grown on simple liquid medium
The main advantages of the present invention are
1 It employs a simple liquid medium for the production of a human platelet
aggregation inhibitor with lipoxygenase inhibition
2 It employs a short fermentation time (5-8 days)
3 It provides a novel human platelet aggregation inhibitor and a lipoxygenase
inhibitor from a fungal source that is not known to produce such inhibitors
4 It employs a simple extraction and purification method of a novel human platelet
aggregation inhibitor, produced by fungal fermentation











We Claim:
1. A process for the preparation of human platelet aggregation and lipoxygenase
inhibitor from Penicillium frequentans, wherein the steps comprising:
a) culturing Penicillium frequentans CFTRI A-24 in potato dextrose broth solution having a pH between 5.1 - 5.5 at a temperature ranging between 28 -37°C for 3-8 days under agitation at 100-200 rpm,
b) adding 1-8 L of a water immiscible solvent to the fermented medium and incubating at 28 -37°C for a period of 1-3 hours at 100-200 rpm followed by filtration;
c) distilling the filtrate obtained from step [b] under vacuum to obtain a crude extract;
d) washing the crude extract obtained from step [c] with 1-2 volumes of hexane;
e) eluting the crude extract obtained in step [d] using 1-2 volumes of chloroform: ethyl acetate (9:1) to obtain a yellowish orange colored fraction, which is distilled under vacuum to remove the solvent and obtain the semi purified inhibitor;
f) adding 10-50 ml of methanol to the semi purified inhibitor as obtained in step [e] and storing at a temperature ranging between 1-10°C for a period of 2-3 days to obtain the purified inhibitor in the form of yellowish orange crystals having final IC50 activity of 614 µM against human platelet aggregation.
2. A process as claimed in claim 1, wherein the water immiscible solvent is selected
from the group consisting of chloroform and ethyl acetate.
3. A process for the preparation of human platelet aggregation and lipoxygenase inhibitor substantially as herein described with reference to the foregoing examples.

Documents:

551-DEL-2004-Abstract-(08-10-2010).pdf

551-DEL-2004-Abstract-(19-11-2010).pdf

551-del-2004-abstract.pdf

551-DEL-2004-Claims-(08-10-2010).pdf

551-DEL-2004-Claims-(19-11-2010).pdf

551-del-2004-claims.pdf

551-DEL-2004-Correspondence-Others-(08-10-2010).pdf

551-DEL-2004-Correspondence-Others-(19-11-2010).pdf

551-del-2004-correspondence.pdf

551-DEL-2004-Description (Complete)-(19-11-2010).pdf

551-del-2004-description.pdf

551-DEL-2004-Form-1-(08-10-2010).pdf

551-DEL-2004-Form-1-(19-11-2010).pdf

551-DEL-2004-Form-2-(19-11-2010).pdf

551-DEL-2004-Form-3-(08-10-2010).pdf

551-del-2004-form1.pdf

551-del-2004-form2.pdf

551-del-2004-form3.pdf

551-del-2004-form5.pdf


Patent Number 244123
Indian Patent Application Number 551/DEL/2004
PG Journal Number 47/2010
Publication Date 19-Nov-2010
Grant Date 18-Nov-2010
Date of Filing 22-Mar-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address Rafi Marg, New Delhi
Inventors:
# Inventor's Name Inventor's Address
1 AVINASH PRAHLAD SATTUR Central Food Technological Research Institute, Mysore
2 LINGAMALLU JAGAN MOHAN RAO Central Food Technological Research Institute, Mysore
3 NAIKANAKATTE GANESH KARANTH Central Food Technological Research Institute, Mysore
4 TUMKUR RAMACHANDRAIAH SHAMALA Central Food Technological Research Institute, Myosre
5 APPURAO GOPALRAO APPU RAO Central Food Technological Research Institute, Mysore
6 THIRUMALAI PARTHASARATHY KRISHNAKANTHA Central Food Technological Research Institute, Mysore
7 WESLEY JESSIE SUNEETHA Central Food Technological Research Institute, Mysore
PCT International Classification Number A 61 K 35/66
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA