Title of Invention	A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2, 12-dihydroxy-dodeca-4-one inhibitor
Abstract	A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor by extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent; filtering the extract through a cloth or Whatman filter paper to obtain a clear solution; evaporating the solution under reduced pressure to obtain a crude extract; purifying the crude extract by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity; pooling active eluted fractions and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent; repooling the active eluted fractions; evaporating the pooled fractions to get a residue and dissolving the residue in ethyl acetate to yield 12-(2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one.

Title of Invention

A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2, 12-dihydroxy-dodeca-4-one inhibitor

Abstract

A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor by extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent; filtering the extract through a cloth or Whatman filter paper to obtain a clear solution; evaporating the solution under reduced pressure to obtain a crude extract; purifying the crude extract by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity; pooling active eluted fractions and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent; repooling the active eluted fractions; evaporating the pooled fractions to get a residue and dissolving the residue in ethyl acetate to yield 12-(2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one.

Full Text	The present invention relates to a process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor. The invention in particular describes the compound 12- (2'-CARBOXY-5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone", main- ly as acetylcholinesterase (AchE) inhibitor. The present invention also relates to a process for the isolation of said compound from fungus Sporotrichum species. Enzyme inhibitors are important class of molecules that are used as drugs and pesticides. The enzyme acetylcholinesterase (AchE) is involved in the synaptic transmission of the nerve impulse and its inhibition leads to accumulation of the neurotransmitter, acetylcholine leading to overexcitation of the postsynaptic neuron. This property of the inhibitor has been exploited to develop newer insecticides against a wide range of insect pests as well as drugs effective against worms, and, recently a new class of neuroactive drugs against dementia (Alzheimer's). Although earlier authors have isolated metabolites such asasteric acis, questin and questinol from Sorortricum sp., no AchE inhibitor activity has been reported (Slater, GP, Haskins, RH and Hogge, L.R. Can J Mirobiol 17 (1971), 1576-79). The fungi, Aspergillus terreus (Ling, KH, Liou.HH, Yang, CM and Yang CK, Appl. Env. Microbiol, 37 (1979) 355-57) and Penicillium sp. (Omura, Skuno,F, Otoguro.K, Shiomi.K. Mauma.R and Iwai.Y. J. Antibiot. 48(1995) 745-46 ) have been reported to produce an AchE inhibitor named Arusigacin. However, the AchE inhibitor of the present invention is isolated from Sporotrichum having distinct chemical structure and properties and therefore a novel inhibitor molecule. After screening of various microorganisms, a fungal culture is selected which shows inhibition against a serine esterase/ protease/cholinesterase enzyme. This imperfect deuteromycetes, Sporotrichum species and was first isolated in 1966. The taxonomic features of Sporotrichum species (deuteromyces) are broad hyphae and septate in nature; has hyalline conidiophores with little differentiation from vegetatative hyphae and solitary conidia with broad attachment to the hyphae. This culture has previously been a subject of research investigation at the Central Food Technological Research Institute (CFTRI) India, for its ability to grow on lignocellulosic wastes for the production of enzymes and organic acids (Sreekantaiah, KR, PhD thesis (1976) University of Mysore; Manonmani, HK, PhD thesis (1986) University of Mysore) This culture has now been used in the present invention to produce a fermented extract containing a serine esterase / protease/ cholinesterase inhibitor. The conditions of fermentation have been described earlier in Indian Patent Application No. 303/DEL/2000 Sattur, AP, Shivanandappa, T, Divakar, S and Karanth, NG. The main object of the present invention is to provide a process for the preparation and isolation of cholinesterase inhibitor which obviates the drawbacks as detailed above. An object of the invention is to have a bioactive compound 12- (2'-CARBOXY-5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone" having inhibitory activity against acetylcholinesterase (AchE). Another object of the present invention is to provide a compound having inhibitory activity against serine esterase and protease. Yet another object of the present invention is to provide a compound having insecticidal properties. Still another object of the present invention is to provide a compound having enhanced cholinergic activity. Yet another object of the present invention is to provide a process for the isolation of Sporotricolone. Accordingly, the present invention provides a process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor of formula I as given below: Formula (1) (Formula Removed) wherein the method for the isolation of the compound comprising the steps of: (a) extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent; (b) filtering the extract of step (a) through a cloth or Whatman filter paper to obtain a clear solution; (c) evaporating the solution of step (b) under reduced pressure to obtain a crude extract; (d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity such as herein described; (e) pooling active eluted fractions of step (d) and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent such as herein described; (f) repooling the active eluted fractions of step (e); (g) evaporating the pooled fractions of step (f) to get a residue and (h) dissolving the residue in step (g) in ethyl acetate to yield 12- (2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one. In an embodiment of the present invention wherein the compound 12- (2'- CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone" of formula (I) having the following characteristic properties: Solubility: Highly soluble in ethyl acetate, methanol and acetone. UV (ethyl acetate) X max: 265 nm, 312 nm. 1HNMR spectrum (DMSO, 6TMS = 0.00 ppm); δ 1.03(3H, d, J=6.3Hz, -CH-CH3) δ 1.2-2.7 (14H,m,7x-CH2) δ 3.6-3.8 (m, Ar-0-CH3 and Ar-CH-OH) δ 7.20(1H, d, J=2.5Hz, C6-H) δ 7.30 (2H, d, J = 7.1 Hz, C3—H and C4-H) Mass spectrum (El, 70 eV, 25° C, 200-ul amp): m/e: 336 (M+), 279(366-87), 167(274-112), 57 (CH2COCH3), 43, 29. In another embodiment of the present invention, the purity of the compound may be established by TLC and RP HPLC. Yet in another embodiment of the present invention, the said compound may be named as 12- (2'-CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone". Still in another embodiment of the present invention, the said compound may be an inhibitor of the enzyme acetylcholinesterase from the rat brain as well as erythrocytes with a IC5o value of 20 x 10"6 M. Yet in another embodiment of the present invention, the said compound may act as an inhibitor of serine esterase of the rat liver serum. Still in another embodiment of the present invention, wherein said compound having insecticidal properties. Yet in another embodiment of the present invention, the said compound may be effective against mosquito larvae at an optimum concentration of 70 µg/ml water (70 ppm) when exposed for 24 hrs. Still in another embodiment of the present invention, the insecticidal activity of the compound against mosquito larvae may be selected from culex quinquifasciatus. Yet in another embodiment of the present invention, the said compound as acetylcholineesterase inhibitor may have potential application as a drug for Alzheimer's disease or dermentia. The present invention also provides a process for the isolation of 12- (2'- CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE Sporotricolone from the fungus Sporotrichum species, said process comprising the steps of: (a) extractingtfielermented soljd)with an organic solvent; (b) filtering the extract of step (a) through a cloth or Whatman filter paper to 4__ obtain a clear solution; %r. (c) evaporating the solution of step (b) under reduced pressure to obtain a } crude extract; (d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity; (e) pooling active eluted fraction of step (d) and further subjected to column chromatography over silica gel by eluting with mixture of organic solvents with increasing polarity; (f) repooling the active eluted fractions of step (e); (g) evaporating the pooled fractions of step (f) to get a residue; and (h) dissolving the residue in step (g) in ethyl acetate to yield the pure compound "Sporotricolone". Yet in another embodiment of the present invention, a process wherein in step (a) the organic solvent may be selected from a group consisting of ethyl acetate, acetone or methanol and preferably ethyl acetate. Still in another embodiment of the present invention, a process wherein in step (d) the mixture of organic solvents may be selected from the combination of hexane: diethyl ether and chloroform: methanol mixtures. In another embodiment of the present invention, a process wherein in step (e) the mixture of organic solvent used may be chloroform: ethyl acetate mixture. Yet in another embodiment of the present invention, the said compound may be separated and purified by column chromatography on silica gel and RP HPLC. Still in another embodiment of the present invention, the said compound may have UV absorption at 265 and 312 nm. In the present invention a process for the isolation of an acetylcholinesterase inhibitor, which comprises the extraction of the fermented broth culture with solvents such as ethyl acetate. The crude extract is further extracted with 10-20 ml of methanol and subjected to column chromatography using silica gel and eluted with various combinations of solvents such as hexane: diethyl ether (85:15, 50:50) followed by chloroform: methanol (95:5, 50:50, 10:90). Fractions are evaporated under nitrogen, dissolved in ethyl acetate and assayed for acetylcholinesterase (AchE) inhibition. The active fractions are pooled and further subjected to purification on silica gel column chromatography and eluted with chloroform: ethyl acetate (90:10, 50:50, 0:100). The active fractions pooled and the solvent evaporated and dissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed a single spot and HPLC on reverse phase column (C18) with chloroform and methanol as mobile phase. The yield is about 10 mg. The purified inhibitor showed inhibitor potency against rat brain AchE with an IC50 of 15-20 x10"6M. A general process for the production of the novel Acetylcholinesterase inhibitor is given in the flow sheet: (Structure Removed)12- (2'-CARB0XY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-D0DECA-4-0NE "Sporotricolone" The structure of the isolated inhibitor is determined by UV, 1H NMR and mass spectrometry. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the invention. EXAMPLE 1 The fermentation culture is extracted with 100 ml of ethyl acetate, filtered through a cotton filter and concentrated in vacuo to obtain 1 ml of the crude extract. This is used as a source of the enzyme inhibitor and 20 \i\ of the extract gave 60-90 % inhibition of rat brain acetylcholinesterase enzyme. EXAMPLE 2 The crude extract is further extracted with 10-20 ml of methanol and subjected to column chromatography using silica gel and eluted with various combinations of solvents such as hexane: diethyl ether (85:15, 50:50) followed by chloroform: methanol (95:5, 50:50, 10:90). Fractions are evaporated under nitrogen, dissolved in ethyl acetate and assayed for acetyl cholinesterase (AchE) inhibitor. The active fractions (# 11-19) are pooled and further subjected to purification on silica gel column chromatography and eluted with chloroform: ethyl acetate (90:10, 50:50, 0:100). The active fractions pooled and the solvent evaporated and dissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed a single spot. RP HPLC also ascertained the purity on a C18 column with chloroform and methanol as the mobile phase wherein it is a single peak. The yield is about 10 mg. EXAMPLE 3 The purified inhibitor showed inhibitor potency against rat brain AchE with an IC50 of 15-20 x 10"6 M. as assayed according to Ellman et al., (Biochem. Pharmacol. 7(1961), 88-95) and is given as follows: The enzyme inhibition is carried out by pre- incubating the enzyme (rat brain acetylcholinesterase) with 2-20ul of the culture extract or the column fraction at room temperature (25 ° C) for 15 minutes followed by the addition of the substrate, acetyl thiocholine iodide (0.5mM), in 3ml phosphate buffer (0.1.M, pH.7.4) containing 0.25mM dithiobisnitrobenzoic acid. Absorbance change at 412 nm is monitored every 30 seconds for 2 min in an UV-VIS Spectrophotometer. Inhibition is calculated relative to the solvent control. IC50 is determined by regression analysis. The main advantages of the present invention are: 1. The present invention provides an AchE /serine esterase/protease inhibitor from a microbial source. 2. The present invention provides a simple extraction and chromatographic procedure to purify the AchE inhibitor from the crude mixture. 3. In the present invention the isolated inhibitor is a novel bioactive molecule We claim : 1. A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor of formula I as given below: Formula (1) (Formula Removed) wherein the method for the isolation of the compound comprising the steps of: (a) extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent; (b) filtering the extract of step (a) through a cloth or Whatman filter paper to obtain a clear solution; (c) evaporating the solution of step (b) under reduced pressure to obtain a crude extract; (d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity such as herein described; (e) pooling active eluted fractions of step (d) and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent such as herein described; (f) repooling the active eluted fractions of step (e); (g) evaporating the pooled fractions of step (f) to get a residue and (h) dissolving the residue in step (g) in ethyl acetate to yield 12- (2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one. 2. A process as claimed in claim 1 wherein in step (a) the organic solvent is selected from the group consisting of ethyl acetate, acetone or methanol and preferably ethyl acetate. 3. A process as claimed in claims 1-2 wherein in step (d) the mixture of organic solvents is selected from the combination of hexane: diethyl ether and chloroform: methanol mixtures. 4. A process as claimed in claims 1-3 wherein in step (e) the mixture of organic solvent used is chloroform: ethyl acetate mixture. 5. A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor.
The invention in particular describes the compound 12- (2'-CARBOXY-5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone", main- ly as acetylcholinesterase (AchE) inhibitor. The present invention also relates to a process for the isolation of said compound from fungus Sporotrichum species. Enzyme inhibitors are important class of molecules that are used as drugs and pesticides. The enzyme acetylcholinesterase (AchE) is involved in the synaptic transmission of the nerve impulse and its inhibition leads to accumulation of the neurotransmitter, acetylcholine leading to overexcitation of the postsynaptic neuron. This property of the inhibitor has been exploited to develop newer insecticides against a wide range of insect pests as well as drugs effective against worms, and, recently a new class of neuroactive drugs against dementia (Alzheimer's).
Although earlier authors have isolated metabolites such asasteric acis, questin and questinol from Sorortricum sp., no AchE inhibitor activity has been reported (Slater, GP, Haskins, RH and Hogge, L.R. Can J Mirobiol 17 (1971), 1576-79). The fungi, Aspergillus terreus (Ling, KH, Liou.HH, Yang, CM and Yang CK, Appl. Env. Microbiol, 37 (1979) 355-57) and Penicillium sp. (Omura, Skuno,F, Otoguro.K, Shiomi.K. Mauma.R and Iwai.Y. J. Antibiot. 48(1995) 745-46 ) have been reported to produce an AchE inhibitor named Arusigacin. However, the AchE inhibitor of the present invention is isolated from Sporotrichum having distinct chemical structure and properties and therefore a novel inhibitor molecule.
After screening of various microorganisms, a fungal culture is selected which shows inhibition against a serine esterase/ protease/cholinesterase enzyme. This imperfect deuteromycetes, Sporotrichum species and was first isolated in 1966. The taxonomic features of Sporotrichum species (deuteromyces) are broad hyphae and septate in nature; has hyalline conidiophores with little differentiation from vegetatative hyphae and solitary conidia with broad attachment to the hyphae.
This culture has previously been a subject of research investigation at the Central Food Technological Research Institute (CFTRI) India, for its ability to grow on lignocellulosic wastes for the production of enzymes and organic acids (Sreekantaiah, KR, PhD thesis (1976) University of Mysore; Manonmani, HK, PhD thesis (1986) University of Mysore)
This culture has now been used in the present invention to produce a fermented extract containing a serine esterase / protease/ cholinesterase inhibitor.
The conditions of fermentation have been described earlier in Indian Patent Application No. 303/DEL/2000 Sattur, AP, Shivanandappa, T, Divakar, S and Karanth, NG.
The main object of the present invention is to provide a process for the preparation and isolation of cholinesterase inhibitor which obviates the drawbacks as detailed above.
An object of the invention is to have a bioactive compound 12- (2'-CARBOXY-5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone" having inhibitory activity against acetylcholinesterase (AchE).
Another object of the present invention is to provide a compound having inhibitory activity against serine esterase and protease.
Yet another object of the present invention is to provide a compound having insecticidal properties.
Still another object of the present invention is to provide a compound having enhanced cholinergic activity.
Yet another object of the present invention is to provide a process for the isolation of Sporotricolone.
Accordingly, the present invention provides a process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor of formula I as given below:
Formula (1) (Formula Removed)
wherein the method for the isolation of the compound comprising the steps of:
(a) extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent;
(b) filtering the extract of step (a) through a cloth or Whatman filter paper to obtain a clear solution;
(c) evaporating the solution of step (b) under reduced pressure to obtain a crude extract;
(d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity such as herein described;
(e) pooling active eluted fractions of step (d) and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent such as herein described;
(f) repooling the active eluted fractions of step (e);
(g) evaporating the pooled fractions of step (f) to get a residue and
(h) dissolving the residue in step (g) in ethyl acetate to yield 12- (2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one.
In an embodiment of the present invention wherein the compound 12- (2'-
CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE
"Sporotricolone" of formula (I) having the following characteristic properties:
Solubility: Highly soluble in ethyl acetate, methanol and acetone. UV (ethyl acetate) X max: 265 nm, 312 nm. 1HNMR spectrum (DMSO, 6TMS = 0.00 ppm);
δ 1.03(3H, d, J=6.3Hz, -CH-CH3)
δ 1.2-2.7 (14H,m,7x-CH2)
δ 3.6-3.8 (m, Ar-0-CH3 and Ar-CH-OH)
δ 7.20(1H, d, J=2.5Hz, C6-H)
δ 7.30 (2H, d, J = 7.1 Hz, C3—H and C4-H)
Mass spectrum (El, 70 eV, 25° C, 200-ul amp):
m/e: 336 (M+), 279(366-87), 167(274-112), 57 (CH2COCH3), 43, 29.
In another embodiment of the present invention, the purity of the compound may be established by TLC and RP HPLC.
Yet in another embodiment of the present invention, the said compound may be named as 12- (2'-CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE "Sporotricolone".
Still in another embodiment of the present invention, the said compound may be an inhibitor of the enzyme acetylcholinesterase from the rat brain as well as erythrocytes with a IC5o value of 20 x 10"6 M.
Yet in another embodiment of the present invention, the said compound may act as an inhibitor of serine esterase of the rat liver serum.
Still in another embodiment of the present invention, wherein said compound having insecticidal properties.
Yet in another embodiment of the present invention, the said compound may be effective against mosquito larvae at an optimum concentration of 70 µg/ml water (70 ppm) when exposed for 24 hrs.
Still in another embodiment of the present invention, the insecticidal activity of the compound against mosquito larvae may be selected from culex quinquifasciatus.
Yet in another embodiment of the present invention, the said compound as acetylcholineesterase inhibitor may have potential application as a drug for Alzheimer's disease or dermentia.
The present invention also provides a process for the isolation of 12- (2'-
CARBOXY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE
Sporotricolone from the fungus Sporotrichum species, said process comprising the steps of:
(a) extractingtfielermented soljd)with an organic solvent;
(b) filtering the extract of step (a) through a cloth or Whatman filter paper to 4__ obtain a clear solution;
%r. (c) evaporating the solution of step (b) under reduced pressure to obtain a } crude extract;
(d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity;
(e) pooling active eluted fraction of step (d) and further subjected to column chromatography over silica gel by eluting with mixture of organic solvents with increasing polarity;
(f) repooling the active eluted fractions of step (e);
(g) evaporating the pooled fractions of step (f) to get a residue; and
(h) dissolving the residue in step (g) in ethyl acetate to yield the pure
compound "Sporotricolone".
Yet in another embodiment of the present invention, a process wherein in step (a) the organic solvent may be selected from a group consisting of ethyl acetate, acetone or methanol and preferably ethyl acetate.
Still in another embodiment of the present invention, a process wherein in step (d) the mixture of organic solvents may be selected from the combination of hexane: diethyl ether and chloroform: methanol mixtures.
In another embodiment of the present invention, a process wherein in step (e) the mixture of organic solvent used may be chloroform: ethyl acetate mixture.
Yet in another embodiment of the present invention, the said compound may be separated and purified by column chromatography on silica gel and RP HPLC.
Still in another embodiment of the present invention, the said compound may have UV absorption at 265 and 312 nm.
In the present invention a process for the isolation of an acetylcholinesterase inhibitor, which comprises the extraction of the fermented broth culture with solvents such as ethyl acetate. The crude extract is further extracted with 10-20 ml of methanol and subjected to column chromatography using silica gel and eluted with various combinations of solvents such as hexane: diethyl ether (85:15, 50:50) followed by chloroform: methanol (95:5, 50:50, 10:90).
Fractions are evaporated under nitrogen, dissolved in ethyl acetate and assayed for acetylcholinesterase (AchE) inhibition. The active fractions are pooled and further subjected to purification on silica gel column chromatography and eluted with chloroform: ethyl acetate (90:10, 50:50, 0:100). The active fractions pooled and the solvent evaporated and dissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed a single spot and HPLC on reverse phase column (C18) with chloroform and methanol as mobile phase. The yield is about 10 mg. The purified inhibitor showed inhibitor potency against rat brain AchE with an IC50 of 15-20 x10"6M.
A general process for the production of the novel Acetylcholinesterase inhibitor is given in the flow sheet:
(Structure Removed)12- (2'-CARB0XY- 5'-METHOXYPHENYL)-2,12-DIHYDROXY-D0DECA-4-0NE "Sporotricolone" The structure of the isolated inhibitor is determined by UV, 1H NMR and mass spectrometry.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the invention.
EXAMPLE 1
The fermentation culture is extracted with 100 ml of ethyl acetate, filtered through a cotton filter and concentrated in vacuo to obtain 1 ml of the crude extract. This is used as a source of the enzyme inhibitor and 20 \i\ of the extract gave 60-90 % inhibition of rat brain acetylcholinesterase enzyme.
EXAMPLE 2
The crude extract is further extracted with 10-20 ml of methanol and subjected to column chromatography using silica gel and eluted with various combinations of solvents such as hexane: diethyl ether (85:15, 50:50) followed by chloroform: methanol (95:5, 50:50, 10:90). Fractions are evaporated under nitrogen, dissolved in ethyl acetate and assayed for acetyl cholinesterase (AchE) inhibitor. The active fractions (# 11-19) are pooled and further subjected to purification on silica gel column chromatography and eluted with chloroform: ethyl acetate (90:10, 50:50, 0:100). The active fractions pooled and the solvent evaporated and dissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed a single spot. RP HPLC also ascertained the purity on a C18 column with chloroform and methanol as the mobile phase wherein it is a single peak. The yield is about 10 mg.
EXAMPLE 3
The purified inhibitor showed inhibitor potency against rat brain AchE with an IC50 of 15-20 x 10"6 M. as assayed according to Ellman et al., (Biochem. Pharmacol. 7(1961), 88-95) and is given as follows: The enzyme inhibition is carried out by pre- incubating the enzyme (rat brain acetylcholinesterase) with 2-20ul of the culture extract or the column fraction at room temperature (25 ° C) for 15 minutes followed by the addition of the substrate, acetyl thiocholine iodide (0.5mM), in 3ml phosphate buffer (0.1.M, pH.7.4) containing 0.25mM dithiobisnitrobenzoic acid. Absorbance change at 412 nm is monitored every 30 seconds for 2 min in an UV-VIS Spectrophotometer. Inhibition is calculated relative to the solvent control. IC50 is determined by regression analysis.
The main advantages of the present invention are:
1. The present invention provides an AchE /serine esterase/protease inhibitor from a microbial source.
2. The present invention provides a simple extraction and chromatographic procedure to purify the AchE inhibitor from the crude mixture.
3. In the present invention the isolated inhibitor is a novel bioactive molecule

We claim :
1. A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor of formula I as given below:
Formula (1) (Formula Removed)
wherein the method for the isolation of the compound comprising the steps of:
(a) extracting fermented solid containing fungus sporotricolone deutromyces with an organic solvent;
(b) filtering the extract of step (a) through a cloth or Whatman filter paper to obtain a clear solution;
(c) evaporating the solution of step (b) under reduced pressure to obtain a crude extract;
(d) purifying the crude extract of step (c) by column chromatography over silica gel and eluting with mixture of organic solvents of increasing polarity such as herein described;
(e) pooling active eluted fractions of step (d) and subjected to column chromatography over silica gel by eluting with a mixture of organic solvent such as herein described;
(f) repooling the active eluted fractions of step (e);
(g) evaporating the pooled fractions of step (f) to get a residue and (h) dissolving the residue in step (g) in ethyl acetate to yield 12- (2'-carboxy-5'-methoxyphenyl)-2,12-dihydroxy-dodeca-4-one.
2. A process as claimed in claim 1 wherein in step (a) the organic solvent is selected from the group consisting of ethyl acetate, acetone or methanol and preferably ethyl acetate.
3. A process as claimed in claims 1-2 wherein in step (d) the mixture of organic solvents is selected from the combination of hexane: diethyl ether and chloroform: methanol mixtures.
4. A process as claimed in claims 1-3 wherein in step (e) the mixture of organic solvent used is chloroform: ethyl acetate mixture.
5. A process for the isolation of 12-(2'-carboxy-5'-methoxyphenyl) 2,12-dihydroxy-dodeca-4-one inhibitor substantially as herein described with reference to the examples.

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391-del-2002-form-2.pdf

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Patent Number

228258

Indian Patent Application Number

391/DEL/2002

PG Journal Number

36/2008

Publication Date

05-Sep-2008

Grant Date

24-Nov-2006

Date of Filing

28-Mar-2002

Name of Patentee

Council of Scientific and Industrial Research

Applicant Address

Rafi Marg

Inventors:

#	Inventor's Name	Inventor's Address
1	N.g. Karanth	Central Food Technological Research Institute Mysore
2	T. Shivanandappa	Central Food Technological Research Institute Mysore
3	A.P.Sattur	Central Food Technological Research Institute Mysore
4	Shereen	Central Food Technological Research Institute Mysore
5	S.Divakar	Central Food Technological Research Institute Mysore

PCT International Classification Number

c07c 39/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA