Title of Invention

A PROCESS FOR THE PREPARATION OF AN ACETYLCHOLINESTERASE INHIBITOR

Abstract

A process for the preparation of an acetylcholinesterase inhibitor by developing Inoculum of sporotrichem species for fermentation by scraping the mycelia from a 3-7 day s old potato dextrose agar slant, transferring it to a 50-100 ml commercially available potato dextrose broth solution and incubating at a temperature ranging between 28-37 °C for 24-48 hours under agitation between 150-200 rpm, wherein carrying out fermentation in a medium comprising 20-50 gms of commercial wheat bran and 20-60 ml of salt solution containing 0.001-0.010 gms of copper sulphate, ferrous sulphate and zinc sulphate in 100 ml of 0.2 N HCI and is sterilized at 121 °C for 40-60 minutes and allowing it to cool before inoculation of 5-10 ml of inoculum, incubating at a temperature ranging between 28-37 °C for 4-8 days, adding an organic solvent to the fermented bran at the end of fermentation and incubating 20-35°C for 1-3 hours followed by filtration through a cloth filter, Whatman No 1 filter, finally filtering the filtrate containing the solvent through a cotton filter followed by distillation under vacuum to obtain a crude extract of 1-5 gms, re-dissolving the above abstract in 20-100 ml of ethylacetate and sequentially washing it in 5-50 ml of 5%-10% sodium bicarbonate and 5%-10% sodium carbonate, pooling these base washings and adjusting the pH to 2.0-4.0 and again extracting it in 20-100 ml ethylacetate, purifying this active fraction containing the inhibitor by conventional column chromatography, using chloroform and methanol (9:2-1:1) as the solvent system to obtain about 500 mgs of semi purified inhibitor, dissolving the semi purifies inhibitor in 2-50 ml benzene to obtain about 200 mgs of methanol soluble active residue, adding 20- 50 ml activated charcoal to the above said active residue and heating for 2-10 minutes followed by filtration to obtain about 50 mgs of purified inhibitor having a final IC50 of 21.3 X 10-5 M against crude rat brain acetylcholinesterase enzyme.

Full Text

The present invention relates to A process for the preparation of an acetylcholinesterase inhibitor from Sporotrichum species. The process particularly relates to the production of an acetylcholinesterase inhibitor produced by solid state fermentation of Sporotrichum sps. One of the many systems of classification of enzymes is on the main amino acid present on the binding site, such as serine, histidine, aspartic acid. Inhibitors of enzymes are also classified based on their action on the amino acid present in the active site. One such serine enzyme acetylcholinestrase which is responsible for the inactivation of the neurotransmitter, acetylcholine. Inhibitors against this enzyme have potential in being used as leads for development of insecticides against a wide range of household pests and for development of drugs against dementia and as beta lactamase antibacterials. Reference may be made to Slater, GP, Haskins, RH and Hogge.LR, Can J Microbiol 17 (1971)1576-1579) wherein the authors have isolated some metabolites such as asterric acid, questin and questinol from Sporotrichum sps. The drawback is that these metabolites have not been reported to possess any acetylcholinesterase inhibitory activity. Reference may be made to Ling.KH, Liou.HH, YangCM and Yang CK Appl Env Microbiol 37 (1979) 355-357 wherein they have reported the production of Arusigacin from Aspergillus terreus. The drawbacks are that the time taken to produce the inhibitor from Aspergillus terreus is very long, about 21 days. Reference may be made to Omura.S, Kuno.F, Otoguro.K, Sunazaka.T, Shiomi.K, Masuma.R and Iwai.Y J.Antibiot. 48 (1995) 745-746 wherein they have reported the production of an acetylcholinesterase inhibitor, Arusigacin, from Penicillium sp FO 4259 from a medium that contains saccharose, glucose, corn steep liquor, meat extract, agar, potassium phosphate and calcium carbonate. The drawbacks are that the medium is extremely complex with a large number of medium constituents. Reference may be made to (Slater.GP, Haskins.RH and Hogge.LR, Can J Microbiol 17 (1971) 1576-1579 wherein the authors have isolated some metabolites such as asterric acid, questin and questinol from Sporotrichum sps. The drawback is that these metabolites have not been reported to possess any acetylcholinesterase inhibitor activity or serine esterase/ protease inhibitor activity. The main object of the present invention is to provide a process for the production of an acetylcholinesterase inhibitor which obviates the drawbacks as detailed above. After screening of various microrganisms, a fungal culture was selected which has inhibition against a serine esterase/ protease enzyme. This imperfect deutromycetes, Sporotrichum sps., numbered as 1106 and deposited at the Central Food Technological Research Institute Culture Collection Center, Mysore, India, was first isolated in 1966. This culture has previously been a subject of research investigation at CFTRI 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). Using this culture, we now have been able to produce a fermented extract containing a serine esterase/ protease inhibitor under suitable fermentation conditions. The taxonomic features of the deuteromycete, Sporotrichum sps, are summarized as follows: The hyphae are broad and septate in nature. The deutromycete has hyaline conidiophores with little differentiation from vegetative hyphae and solitary conidia with a broad attachment to the hyphae. Accordingly the present invention provides a process for the preparation of an acetylcholinesterase inhibitor from Spirotrichum species which comprises developing Inoculum of Sporotrichum species for fermentation by scraping the mycelia from a 3-7 day old potato dextrose agar slant, transferring it to a 50-100 ml commercially available potato dextrose broth solution and incubating at a temperature ranging between 28-37 °C for 24-48 hours under agitation between 150-200 rpm, wherein carrying out fermentation in a medium comprising 20-50 gms of commercial wheat bran and 20-60 ml of salt solution containing 0.001-0.010 gms of copper sulphate, ferrous sulphate and zinc sulphate in 100 ml of 0.2 N HCI and is sterilized at 121 °C for 40-60 minutes and allowing it to cool before temperature ranging between 28-37 °C for 4-8 days, adding an organic solvent to the fermented bran at the end of fermentation and incubating 20-35°C for 1-3 hours followed by filtration through a cloth filter, Whatman No 1 filter, finally filtering the filtrate containing the solvent through a cotton filter followed by distillation under vacuum to obtain a crude extract of 1-5 gms, re-dissolving the above abstract in 20-100 ml of ethylacetate and sequentially washing it in 5-50 ml of 5%-10% sodium bicarbonate and 5%-10% sodium carbonate, pooling these base washings and adjusting the pH to 2.0-4.0 and again extracting it in 20-100 ml ethylacetate, purifying this active fraction containing the inhibitor by conventional column chromatography, using chloroform and methanol (9:2-1:1) as the solvent system to obtain about 500 mgs of semi purified inhibitor, dissolving the semi purifies inhibitor in 2-50 ml benzene to obtain about 200 mgs of methanol soluble active residue, adding 20- 50 ml activated charcoal to the above said active residue and heating for 2-10 minutes followed by filtration to obtain about 50 mgs of purified inhibitor having a final IC50 of 21.3 X 10-5 M against crude rat brain acetylcholinesterase enzyme In an embodiment of the present invention the age of the slant used is between 3-7 days and the age of the inoculum is preferably between 12-48 hours grown under agitation at preferably 50-200 rpm. In an another embodiment of the present invention the fermentation time used for the production of the said inhibitor is preferably between 3 to 8 days. In yet another embodiment of the present invention the fermented solid mass used is extracted with organic solvents selected from ethylacetate and acetone the solvent extract is used as a source of the inhibitor. In yet another embodiment of the present invention the said inhibitor is separated from the crude extract by weak bases and purified by column chromatography In yet another embodiment of the present invention the said inhibitor is soluble in ethylacetate, and methanol. A general process for the production of the novel lipoxygenase inhibitor is given in the flow sheet: (Flow Sheet Removed) The structure of the isolated inhibitor was 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 To 25 gms of wheat bran, 25 ml of an acidic salt solution comprising 0.007 gm of zinc sulphate, ferrous sulphate and copper sulphate in 100 ml of 0.2 N hydrochloric acid was added in a 500 ml conical flask.. The flask was then autoclaved at 121 °C for 60 minutes. To this flask, 10 mL of a 72 hour old inoculum was added and then incubated for 7 days at 30 °C. At the end of the fermentation, 100 mL of ethylacetate was added to each flask containing the fermented bran. The bran was macerated and incubated at room temperature for two hours. The solvent was filtered through a cheese cloth followed by Whatman no 1 filter paper. To the filtrate anhydrous sodium sulphate was added and left overnight at room temperature. Subsequently, the solvent was filtered through a cotton filter and concentrated under vacuum to obtain 1 mL of the extract. This was used as a source of the enzyme inhibitor and 20 µl of the extract gave 65-80 % inhibition of rat brain acetylcholinesterase enzyme. EXAMPLE -2 3 gms of the crude extract, as prepared according to Example 1, was redissolved with 25 ml of ethylacetate and sequentially washed in 10 ml of 5% sodium bicarbonate(Loba chemicals, India) followed by 10 ml of 5% sodium carbonate (Loba chemicals, India). The base washings were pooled and the pH adjusted to 2.0 and 1 gm of the crude extract containing the inhibitor was extracted in 25 ml ethylacetate. This active fraction containing the inhibitor was further purified by conventional column chromatography, using chloroform and methanol (8.5-1.5) as the solvent system to obtain about 100 mgs of the purified inhibitor. This was dissolved in 10 ml benzene to obtain about 60 mgs of residue which was highly active. To this residue which was soluble in methanol, 30 mgs of activated charcoal was added and heated for 5 minutes and filtered to obtain 51.4 mgs of purified inhibitor. The purified inhibitor shows an IC50 of 21.3 X 10-5 M against crude rat brain acetylcholinesterase enzyme as assayed according to Ellman GL et al (Biochem Pharmacol 7 (1961) 88-95) and is given as follows: The enzyme inhibition study was carried out by incubating electric eel acetylcholinesterase (Sigma,USA) or rat brain acetylcholinesterase enzyme (crude preparation of acetylcholinesterase enzyme was obtained by homogenizing rat brain in 0,1M phosphate buffer, pH 7.4, and centrifuging at 1000 x g ) with the microbial extract or the active fraction (2-20 µl) at 37 °C in water bath for 15 minutes, followed by the addition of 500 µM of the substrate, acetylcholine iodide (Sigma chemicals, USA) and 250 µM of dithiobisnitrobenzoic acid (Sigma chemicals, USA) in a total volume of 3.0 ml of 0.1 M phosphate buffer, pH 7.4. Absorbance change at 412 nm was recorded every 30 seconds for at least 5 minutes. Inhibition was calculated relative to the solvent control. The characteristic spectroscopic data obtained: UV Spectrum (methanol, 0.5 mg/ml) Max : 228,257, 300 nm Solubility: highly soluble in ethylacetate and methanol 1H NMR spectrum (DMSO, δTMS= 0.00 ppm, J/Hz): 1.01 3H,d, 6.2 Hz CH3 1.23 2H,m 10 CH2 (Formula Removed) 1.34 2H CH2 2.33 2H -CH2-Ar 3.52 1H -CH-O 5.99 2H -Ar-H Mass Spectrum (El, 70eV,25 C, 200ul amp) Characteristic ions: 336 (M+ 338-2H), 137 (Ar-CH2-2H), 124 (Ar unit 30H ), 45 (-CH3-CHOH) The novelty of the present invention is to purify an enzyme inhibitor which has been previously grown on simple solid state medium. The main advantages of the present invention are: 1. It employs a simple and inexpensive medium for the production of a serine esterase/protease inhibitor 2. It provides a AchE /serine esterase/protease inhibitor from a microbial source 1. It employs a simple combination of base washings and chromatographic procedure to purify the AchE inhibitor from the crude mixture WE CLAIM: 1. A process for the preparation of an acetylcholinesterase inhibitor from Sporotrichum species which comprises developing Inoculum of Sporotrichum species for fermentation by scraping the mycelia from a 3-7 day s old potato dextrose agar slant, transferring it to a 50-100 ml commercially available potato dextrose broth solution and incubating at a temperature ranging between 28-37 °C for 24-48 hours under agitation between 150-200 rpm, wherein carrying out fermentation in a medium comprising 20-50 gms of commercial wheat bran and 20-60 ml of salt solution containing 0.001-0.010 gms of copper sulphate, ferrous sulphate and zinc sulphate in 100 ml of 0.2 N HCI and is sterilized at 121 °C for 40-60 minutes and allowing it to cool before inoculation of 5-10 ml of inoculum, incubating at a temperature ranging between 28-37 °C for 4-8 days, adding an organic solvent to the fermented bran at the end of fermentation and incubating 20-35°C for 1-3 hours followed by filtration through a cloth filter, Whatman No 1 filter, finally filtering the filtrate containing the solvent through a cotton filter followed by distillation under vacuum to obtain a crude extract of 1-5 gms, re-dissolving the above abstract in 20-100 ml of ethylacetate and sequentially washing it in 5-50 ml of 5%-10% sodium bicarbonate and 5%-10% sodium carbonate, pooling these base washings and adjusting the pH to 2.0-4.0 and again extracting it in 20-100 ml ethylacetate, purifying this active fraction containing the inhibitor by conventional column chromatography, using chloroform and methanol (9:2- 1:1) as the solvent system to obtain about 500 mgs of semi purified inhibitor, dissolving the semi purifies inhibitor in 2-50 ml benzene to obtain about 200 mgs of methanol soluble active residue, adding 20- 50 ml activated charcoal to the above said active residue and heating for 2-10 minutes followed by filtration to obtain about 50 mgs of purified inhibitor having a final IC50 of 21.3 X 10-5 M against crude rat brain acetylcholinesterase enzyme 2. A process, as claimed in claim 1, wherein the age of the slant used is between 3-7 days and the age of the inoculum is preferably between 12-48 hours grown under agitation at preferably 50-200 rpm. 3. A process, as claimed in claims 1&2, wherein the fermentation time used for the production of the said inhibitor is preferably between 3 to 8 days. 4. A process, as claimed in claims 1-3, wherein the fermented solid mass used is extracted with organic solvents selected from ethylacetate and acetone the solvent extract is used as a source of the inhibitor. 5. A process, as claimed in claims 1-4, wherein the said inhibitor is separated from the crude extract by weak bases and purified by column chromatography 6. A process, as claimed in claims 1-5, wherein the said inhibitor is soluble in ethylacetate, and methanol. A process for the preparation of an acetylcholinesterase inhibitor from Spirotrichum species substantially as herein described with reference to the examples accompanying this specification.

Documents:

219-del-2001-abstract.pdf

219-del-2001-claims.pdf

219-del-2001-correspondence-others.pdf

219-del-2001-correspondence-po.pdf

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

219-del-2001-form-1.pdf

219-del-2001-form-19.pdf

219-del-2001-form-2.pdf