Title of Invention	A PROCESS FOR THE PREPARATION OF A BIOACTIVE COMPOUND FROM THE CULTURE OF ASPERGILLUS NIGER
Abstract	The present invention relates to a novel compound having a molecular formula C13H15NO5 and a process for the isolation of said compound. The present invention also relates to a method of inhibiting 13-lipoxygenase and having free radical scavenging activity. To meet the above objectives, the present invention provides a novel compound having molecular formula C13H-15NO5. The present invention also provides a process for the isolation of said compound from AspergiHus niger and a method treatment in inhibiting 13-Lipoxygenase inhibitor, in scavenging of free radicals, for the treatment of asthma, hypersensitivity, pson'asis, inflammatory conditions and complications arising out of diabetes.

Title of Invention

A PROCESS FOR THE PREPARATION OF A BIOACTIVE COMPOUND FROM THE CULTURE OF ASPERGILLUS NIGER

Abstract

The present invention relates to a novel compound having a molecular formula C13H15NO5 and a process for the isolation of said compound. The present invention also relates to a method of inhibiting 13-lipoxygenase and having free radical scavenging activity. To meet the above objectives, the present invention provides a novel compound having molecular formula C13H-15NO5. The present invention also provides a process for the isolation of said compound from AspergiHus niger and a method treatment in inhibiting 13-Lipoxygenase inhibitor, in scavenging of free radicals, for the treatment of asthma, hypersensitivity, pson'asis, inflammatory conditions and complications arising out of diabetes.

Full Text	The present invention relates to a process for the preparation of a bioactive compound from the culture of Aspergillus niger with particular reference to the isolation and method of treatment for lipoxygenase inhibition and as free radical scavenging agent. The present invention relates to a novel compound having a molecular formula C13H15NO5 and a process for the isolation of said compound. The present invention also relates to a method of inhibiting 13-lipoxygenase and having free radical scavenging activity. 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 arachadonic acid. These enzymes are found to be responsible for the deterioration, rancidity and loss of flavor in food materials and also for various diseases in the human body, such as Parkinson's disease, cataractogenesis, endotoxin liver injury, and myocardial infarction. (Began G, Sudharshan E and Appu Rao AG Lipids 33 (1998) 1223-1228; Sics H (cd.) Oxidative State, Oxidants and Antioxidants, pp-8, Academic Press, London (1991)). Inhibitors against these enzymes thus have a potential application in both the food and medical sector. The use and manufacture of various antioxidants have been reviewed by Madhavi, DL and Salunkhe, DK (1994) In Food Additive Toxicology, Maga, JA and Tu, AT (eds) Marcel Dekker, NY 88-177. Allgayer et al (1984) have reported that therapeutically active compound such as sulphasalazine metabolites are soybean lipoxygenase inhibitors (Allgayer H, Eisenburg J and Paumgartner G Eur J Clin Pharmacol 26 (1984) 449-451) The main object of the present invention is to provide a process for the preparation of a bioactive compound from the culture of Aspergillus niger which obviates the drawbacks detailed above. An object of the present invention is to isolate a bioactive compound having a molecular formula C13H15NO5. Another object of the present invention is to provide a process for the isolation of said compound. Yet another object of the present invention is to provide a method of treatment to inhibit lipoxygenase enzyme and for the treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes and also as free radical scavenging activity. Accordingly, the present invention provides a process for the preparation of a bioactive compound from the culture of Aspergillus niger, the said compound having a molecular formula C13H15NO5. An embodiment of the present invention, wherein said compound having a basic skeleton of benzene ring having substituents hydroxyl, methyl, carboxyl, carboxamide, methoxyl and propenyl groups. Yet another embodiment of the present invention, wherein said compound is soluble in an organic solvent selected from the group consisting of ethanol, methanol, ethyl acetate, and dimethyl sulphoxide. Still another embodiment of the present invention, wherein said compound is sparingly soluble in chloroform and hexane, but insoluble in water. Yet another embodiment of the present invention, wherein said compound is soluble in aqueous alkaline solution selected from sodium bicarbonate, sodium carbonate and potassium bicarbonate and potassium carbonate and sodium hydroxide, lithium hydroxide and potassium hydroxide. Further embodiment of the present invention, wherein said compound having the physical characteristics as given below: Nature: yellow amorphous powder. Melting Point: 253°C  max nm)(6) in methanol: 235(20,700), 292(11,600), 358 (4,400) IR: 3499, 1657,2994cm'1. Molecular formula: C13H15NO5 EI-MS m/z: 265 (M+) 263[M+-2H, 60%] 235 [M+-(CH3-CH-), 45%] 207 [235-(CH3-CAr, 30%] 163(207-C02,,49%) 161 [100%] 99 [45%] 81 [37%] 1H NMR spectra (δ. ppm): 2.04 (3H, d, J= 6.6 Hz, CH3-CH=CH-) 6.61 (1H, dq, J= 16.4 Hz, 6.9 Hz, CH3-CH=C) 6.69 (1H, d, J= 16.4 Hz, HC=CH-Ar) 2.02 (S) (3H, s, Ar-CH3) 3.43 (S) (3H, s, Ar-OCH3) 10.3 (Ar-OH) 11.5 (Ar-COOH) 13C NMR snectra (δ. poml: CH3 15.0 =C-CAr 167 =CH 122 -COOH 161 =CH 134 CAT-O-CH3 149.5 -CH3 15.0 CArOH 148 CArCH3 117 CONH2 168 The present invention also provides a pharmaceutical composition for Lipoxygenase inhibition and free radical scavenging activity, in subjects, said composition comprising an effective amount of the said bioactive compound having a molecular formula C13H15NO5 along with pharmaceutically accepted excipients. An embodiment of the present invention wherein said composition is used to treat asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes. An embodiment of the present invention, the pharmaceutical composition wherein the excipients are selected from the group consists of carriers, colorants, flow modifiers and stabilizers. Yet another embodiment of the present invention, the pharmaceutical composition wherein the excipients used are in the suitable amounts ranging between 0.001 -0.99wt%. Still another embodiment of the present invention, wherein said composition is used in the form of oral, parental, nasal, topical, buccal and ocular. Yet another embodiment of the present invention, wherein the subject is selected from mammals." Accordingly the present invention provides a process for preparation of a bioactive compound from the culture of Aspergillus niger, useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions, having a molecular formula C13H15NO5 the said process comprising the steps of: a) isolating the strain of Aspergillus niger Teigh , from honey bee wax; b) growing the strain obtained from step(a) on a Potato Dextrose Agar medium and incubating for 4 days at 30°C; c) transferring the culture of step (b) in liquid medium and incubating at 30°C on a rotary shaker at 250 rpm, d) transferring the culture of step (c) into a medium containing wheat bran, mineral acid, sulfates and incubated for 5 days at 30°C to obtain fermented wheat bran; e) treating the fermented wheat bran of step (d) with an organic solvent, for two hours to obtain an organic solvent extract; f) separating the organic solvent extract of step (e) from the wheat bran by cheese cloth filtration, followed by drying over anhydrous sodium sulfate to obtain a solid, g) suspending the solid of step (f) in an organic solvent, and centrifuging to obtain a residue; h) drying the residue of step (g) to obtain an orange solid; i) dissolving the solid of step (h) in an alcoholic solvent, treating the solution with active charcoal, filtering; and concentrating to obtain the compound having a molecular formula C13H15NO5 as yellow powder. compound of molecular formula C13H15NO5 as yellow amorphous powder. An embodiment of the present invention, the process wherein the seed liquid medium is selected from Czapex solution agar for Carbon source and Czapex solution agar replacing sodium nitrate for nitrate source. Another embodiment of the present invention, the process wherein the mineral acid that is used for flask fermentation in step (e) is hydrochloric acid. Yet another embodiment of the present invention, the process wherein the organic solvent used in step (f) is selected from the group consisting of dichloromethane, chloroform, ethylacetate, methylisobutyl ketone and preferably ethylacetate. Still another embodiment of the present invention, the process wherein the organic solvent used for suspending the residue in step (i) is chloroform. The present invention further provides a method of treating subjects with pharmaceutical composition comprising a bioactive compound of molecular formula C13H15NO5 along with pharmaceutically accepted excipients used for treatment of 13- Lipoxygenase inhibition and having free radical scavenging activity. An embodiment of the present invention the method wherein said composition is used to treat asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes. Yet another embodiment of the present invention the method wherein said compound having 13-lipoxygenase and crude rat lens aldose reductase inhibitory activity. Still another embodiment of the present invention the method wherein the subject is selected from mammals. Yet another embodiment of the present invention the method wherein the IC50 value of the compound against purified soybean lipoxygenase and crude rat lens aldose reductase inhibitory activity is 79µ moles and 69 µ moles respectively. Further embodiment of the present invention the method wherein ED50 value of the composition for free radical scavenging activity is 66 µM. The invention is further explained in the form of following embodiments Applicants have discovered a antibiotic 13-LOX inhibitory compound which is derived via the fermentation of certain microorganisms. Aspergillus niger CFR-W-105 isolated from a wax sample is found to produce new biologically active substance. This substance is extracted from the fermented wheat bran using ethyl acetate followed by crystallization. The purified compound is identified as compound having a molecular formula C13H- 15NO5 Thus the fermentation of Aspergillus niger CFR-W-105 or a mutant thereof and suitable isolation techniques may be used to produce the compound of the investigation. A biologically pure culture of Aspergillus niger CFR-W-105, from which the compound of investigation is derived, has been deposited with the American Type Culture Collection (ATCC) in Rockville, MD, and has been added to its permanent collection under its accession code ATCC. To meet the above objectives, the present invention provides a novel compound having molecular formula C13H15NO5 The present invention also provides a process for the isolation of said compound from Aspergillus niger and a method treatment in inhibiting 13-Lipoxygenase inhibitor, in scavenging of free radicals, for the treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes. TAXONOMY Morphology The fungal mycelium on czapek's solution agar had abundant erect and crowded conidial structures, which is brownish black or dark brown covering the entire colony except for the narrow peripheral growing area. Conidial heads are globose initially, which slowly split into radiating columns at the end of 10 days. Sclerotia are not observed. Cultural and physiological characteristics The growth characteristics of CFR-W-105 on malt-extract-agar are similar to that of Czapek's solution agar. But the colonies are deeper brownish black, reverse of the plate is uncolored and unwrinkled and conidiophores are less dense. Growing margin is less prominent. The culture grew well at ambient temperature of 26°-30°C. conidia is born by biseriate sterigmata (25-30A) and are 2.5-4.5n in size and globose at maturity. The walls are smooth arid thick. The length of conidiophores is 0.7-1.0 mm. Table 1 shows the growth of CRF- W-105 in the presence of various carbon and nitrogen sources. The basal medium of the above experiments contained Czapex solution agar for Carbon source and Czapex solution agar replacing sodium nitrate for nitrate source. Table 1: Growth of Aspergillus niger CFR-W-105 in the presence of various carbon and nitrogen sources. (Table Removed) Taxonomic position The strain CFR-W-105 is isolated from honeybee wax from local region. The characteristics indicated that the strain belongs to Aspergillus Niger group. According to the descriptions of Raper and Fennell ( Raper KB and Fennell DT (1965) The Genus Aspergillus; The Williams and Wilkins Co, Baltimore, pp 293-310), the strain CFR-W-105 is related to Aspergillus niger V. Tiegh. The taxonomic relationships between the strains and four other cultures that did not give the bioactive molecules during screening are compared and given in Table 2. Table 2. Comparison between the strains CFR-W-105 and three other related species which did not give the compound I (as on Czapex solution agar, 26- 28°C 10 days) (Table Removed) FERMENTATION Seed Culture Strain CFR W-105 is propagated on Potato Dextrose Agar (Hi Media Mumbai, India) slant composed of soluble starch 0.4 % and glucose 2 %. After incubation for 4 days at 30°C, a portion of the mature agar slant is inoculated into 100 ml of a seed liquid medium of the same medium composition in a 500-ml Erlenmeyer flask and incubated at 30°C on a rotary shaker at 250 rpm. Flask Fermentation A 5-ml of the seed culture is transferred into 250-ml Erlenmeyer flasks each containing 10 gm of wheat bran, 10 ml of 0.2 N HC1 comprising 2.1 mg each of ferrous sulfate, zinc sulfate and copper sulfate and 5 ml distilled water. The inoculated flasks are incubated for 5 days at 30°C. Isolation and Purification. The fermented wheat bran (450 g) is treated with ethyl acetate (1 liter) for two hours. The organic extract is separated from the wheat bran by cheesecloth filtration. This extract is dried over anhydrous sodium sulfate and concentrated in vacuo to afford a crude solid (1.36 gm) and the residue is resuspended in chloroform (25 ml) and gently centrifuged (2000 rpm, 20 minutes, 27°C). The residue is dried to afford an orange colored active fraction (230-mg). This is dissolved in ethyl alcohol (50 ml) by gentle warming and treated with activated charcoal (200 mg) filtered (Whatman No.l) while warm. The filtrate is concentrated in vacuo to obtain 30 mg of yellow amorphous powder. Physico-chemical properties The compound is amorphous yellow powder. It is soluble in ethanol, methanol, ethyl acetate, dimethyl sulphoxide, sodium bicarbonate solution, sodium carbonate solution and sodium hydroxide solution, slightly soluble in chloroform and hexane, but insoluble in water. The EI-MS spectra of the compound showed the molecular ions at m/z 265. Compositions and Methods The novel compounds of the invention can be used in a variety of pharmaceutical dosage forms. Thus, oral, parental, nasal, topical, buccal, ocular and other forms can be used. When such forms are formulated they will include pharmaceutically acceptable excipients such as colorants, carriers, perfumes, stabilizers, flow modifiers and the like in suitable amounts (i.e., from 0.001 to 0.99wt%). The compound of the invention is useful in methods of inhibiting the effects of 13-LOX. The compound is also used to treat a host, preferable a mammal, which is suffering from a disorder associated with a metabolism of 13-LOX, such as asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising from diabetes. The following example illustrates the 13-Lipoxygenase inhibitor effects of the novel compound, however this shall not be considered as limiting the scope of the present invention. EXAMPLE Soybean lipoxygenase (LOX-1) is purified according to the method of Axelrod et al., lipoxygenase from soybeans. Methods in Enzymology 1981, 71, 441-451, using the Hardee variety of soybean, after dehulling and defatting by hexane. The substrate, sodium linoleate (10 mM) is prepared as follows: To 70 mg of linoleic acid an equal weight of Tween-20 and 4 ml of oxygen free water is added and homogenized by drawing back and forth in a pasture pipette, avoiding air bubbles. Sufficient quantity of 0.5 N NaOH is added to this to yield a clear solution. The resulting sodium linoleate is divided into 1-2 ml portions in small screw cap vials, flushed with N2 before closing, and keep frozen until needed. Enzyme reaction is carried out at 25°C in a quartz cuvette with a 1 cm light path. The assay mixture contained (2.975 - x) ml of borate buffer (0.2 M, pH 9.0), 0.025 ml of sodium linoleate substrate, and x mL of enzyme. After each addition the mixture is stirred with a few strokes of a plastic paddler. The reference cuvette contained no enzyme. Absorption at 234 nm is recorded, and the reaction rate is determined from the slope of the straight-line portion of the curve. Lens are collected from rat eyes and homogenised with sodium, potassium phosphate buffer (0.135 M, pH 7.0) containing 0.5 mM of phenyl methyl sulphonyl fluoride and 10 mM of p-mercaptoethanol. The homogenate is centrifuged at 10,000 r.p.m for 30 minutes at 4°C. The supernatant is taken as enzyme source. Enzyme reaction is carried out at 25°C in a quartz cuvette with a 1-cm light path. The assay mixture contained 100 µL of nicotinamide adenine dinucleotide phosphate tetra sodium salt (from a stock solution of 9.6 mM), 100 µL of DL-glyceraldehyde (from a stock solution of 250 mM), 10 µL of inhibitor dissolved in dimethyl sulphoxide, sodium-potassium-phosphate buffer (0.135 M, pH 7.0) to make up the total volume to 3 µL and the reaction is initiated by the addition of 200 µL of enzyme solution. The enzyme reaction is monitored by the decrease in absorbency at 340 nm and compared with enzyme reaction without inhibitor. Results The bioactive compound having molecular formula C13H15NO5 is discovered in the fermented bran of a species of Aspergillus Niger CFR W-105 (= ATCC...). The compound is successfully purified to homogeneity. The IC50 value of the compound against purified soybean lipoxygenase and crude rat lens aldose reductase inhibitory activity is determined to be 79n moles and 69n moles respectively. We Claim: 1. A process for preparation of a bioactive compound from the culture of Aspergillus niger useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions, having a molecular formula Ci3Hi5NO5, the said process comprising the steps of: (a) isolating the strain of Aspergillus niger Teigh , from honey bee wax; (b) growing the strain obtained from step(a) on a Potato Dextrose Agar medium and incubating for 4 days at 30°C; (c) transferring the culture of step (b) in liquid medium and incubating at 30°C on a rotary shaker at 250 rpm, (d) transferring the culture of step (c) into a medium containing wheat bran, mineral acid, sulfates and incubated for 5 days at 30°C to obtain fermented wheat bran; (e) treating the fermented wheat bran of step (d) with an organic solvent, for two hours to obtain an organic solvent extract; (f) separating the organic solvent extract of step (e) from the wheat bran by cheese cloth filtration, followed by drying over anhydrous sodium sulfate to obtain a solid, (g) suspending the solid of step (f) in an organic solvent , and centrifuging to obtain a residue; (h) drying the residue of step (g) to obtain an orange solid; (i) dissolving the solid of step (h) in an alcoholic solvent, treating the solution with active charcoal, filtering; and concentrating to obtain the compound having molecular formula C13H15NO5 as yellow powder. 2. A process as claimed in claim 1, wherein the seed liquid medium used is selected from ezapek's solution agar as Carbon source and ezapek's solution agar replacing sodium nitrate for nitrate source. 3. A process as claimed in claims 1, wherein the mineral acid used in fermentation of step (d) is hydrochloric acid. 4. A process as claimed in claims 1, wherein the organic solvent used in step (e) is selected from the group consisting of dichloromethane, chloroform, ethylacetate, methylisobutyl ketone and preferably ethylacetate. 5. A process as claimed in claims 1, wherein the organic solvent used for suspending the residue in step (g) is chloroform. 6. A process for the preparation of a bioactive compound from culture of Aspergillus niger useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions from the culture of Aspergillus niger substantially as herein described with reference to the examples,

Full Text

The present invention relates to a process for the preparation of a bioactive compound from the culture of Aspergillus niger with particular reference to the isolation and method of treatment for lipoxygenase inhibition and as free radical scavenging agent. The present invention relates to a novel compound having a molecular formula C13H15NO5 and a process for the isolation of said compound. The present invention also relates to a method of inhibiting 13-lipoxygenase and having free radical scavenging activity.
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 arachadonic acid.
These enzymes are found to be responsible for the deterioration, rancidity and loss of flavor in food materials and also for various diseases in the human body, such as Parkinson's disease, cataractogenesis, endotoxin liver injury, and myocardial infarction. (Began G, Sudharshan E and Appu Rao AG Lipids 33 (1998) 1223-1228; Sics H (cd.) Oxidative State, Oxidants and Antioxidants, pp-8, Academic Press, London (1991)). Inhibitors against these enzymes thus have a potential application in both the food and medical sector.
The use and manufacture of various antioxidants have been reviewed by Madhavi, DL and Salunkhe, DK (1994) In Food Additive Toxicology, Maga, JA and Tu, AT (eds) Marcel Dekker, NY 88-177.
Allgayer et al (1984) have reported that therapeutically active compound such as sulphasalazine metabolites are soybean lipoxygenase inhibitors (Allgayer H, Eisenburg J and Paumgartner G Eur J Clin Pharmacol 26 (1984) 449-451)
The main object of the present invention is to provide a process for the preparation of a bioactive compound from the culture of Aspergillus niger which obviates the drawbacks detailed above.
An object of the present invention is to isolate a bioactive compound having a molecular formula C13H15NO5.
Another object of the present invention is to provide a process for the isolation of said compound.
Yet another object of the present invention is to provide a method of treatment to inhibit lipoxygenase enzyme and for the treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes and also as free radical scavenging activity.
Accordingly, the present invention provides a process for the preparation of a bioactive compound from the culture of Aspergillus niger, the said compound having a molecular formula C13H15NO5.
An embodiment of the present invention, wherein said compound having a basic skeleton of benzene ring having substituents hydroxyl, methyl, carboxyl, carboxamide, methoxyl and propenyl groups.
Yet another embodiment of the present invention, wherein said compound is soluble in an organic solvent selected from the group consisting of ethanol, methanol, ethyl acetate, and dimethyl sulphoxide.
Still another embodiment of the present invention, wherein said compound is sparingly soluble in chloroform and hexane, but insoluble in water.
Yet another embodiment of the present invention, wherein said compound is soluble in aqueous alkaline solution selected from sodium bicarbonate, sodium carbonate and potassium bicarbonate and potassium
carbonate and sodium hydroxide, lithium hydroxide and potassium hydroxide.
Further embodiment of the present invention, wherein said compound having the physical characteristics as given below: Nature: yellow amorphous powder. Melting Point: 253°C  max nm)(6) in methanol: 235(20,700),
292(11,600), 358 (4,400)
IR: 3499, 1657,2994cm'1. Molecular formula: C13H15NO5 EI-MS m/z: 265 (M+)
263[M+-2H, 60%]
235 [M+-(CH3-CH-), 45%]
207 [235-(CH3-CAr, 30%]
163(207-C02,,49%)
161 [100%]
99 [45%]
81 [37%] 1H NMR spectra (δ. ppm):
2.04 (3H, d, J= 6.6 Hz, CH3-CH=CH-)
6.61 (1H, dq, J= 16.4 Hz, 6.9 Hz, CH3-CH=C)
6.69 (1H, d, J= 16.4 Hz, HC=CH-Ar)
2.02 (S) (3H, s, Ar-CH3) 3.43 (S) (3H, s, Ar-OCH3) 10.3 (Ar-OH) 11.5 (Ar-COOH)
13C NMR snectra (δ. poml:
CH3 15.0 =C-CAr 167
=CH 122 -COOH 161
=CH 134 CAT-O-CH3 149.5
-CH3 15.0 CArOH 148
CArCH3 117 CONH2 168
The present invention also provides a pharmaceutical composition for Lipoxygenase inhibition and free radical scavenging activity, in subjects, said composition comprising an effective amount of the said bioactive compound having a molecular formula C13H15NO5 along with pharmaceutically accepted excipients.
An embodiment of the present invention wherein said composition is used to treat asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes.
An embodiment of the present invention, the pharmaceutical composition wherein the excipients are selected from the group consists of carriers, colorants, flow modifiers and stabilizers.
Yet another embodiment of the present invention, the pharmaceutical composition wherein the excipients used are in the suitable amounts ranging between 0.001 -0.99wt%.
Still another embodiment of the present invention, wherein said composition is used in the form of oral, parental, nasal, topical, buccal and ocular.
Yet another embodiment of the present invention, wherein the subject is selected from mammals."
Accordingly the present invention provides a process for preparation of a bioactive compound from the culture of Aspergillus niger, useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions, having a molecular formula C13H15NO5 the said process comprising the steps of:
a) isolating the strain of Aspergillus niger Teigh , from honey bee wax;
b) growing the strain obtained from step(a) on a Potato Dextrose Agar medium
and incubating for 4 days at 30°C;
c) transferring the culture of step (b) in liquid medium and incubating at 30°C on a
rotary shaker at 250 rpm,
d) transferring the culture of step (c) into a medium containing wheat bran,
mineral acid, sulfates and incubated for 5 days at 30°C to obtain fermented
wheat bran;
e) treating the fermented wheat bran of step (d) with an organic solvent, for two
hours to obtain an organic solvent extract;
f) separating the organic solvent extract of step (e) from the wheat bran by
cheese cloth filtration, followed by drying over anhydrous sodium sulfate to
obtain a solid,
g) suspending the solid of step (f) in an organic solvent, and centrifuging to obtain
a residue;
h) drying the residue of step (g) to obtain an orange solid;
i) dissolving the solid of step (h) in an alcoholic solvent, treating the solution with
active charcoal, filtering; and concentrating to obtain the compound having a
molecular formula C13H15NO5 as yellow powder.
compound of molecular formula C13H15NO5 as yellow amorphous
powder.
An embodiment of the present invention, the process wherein the seed liquid medium is selected from Czapex solution agar for Carbon source and Czapex solution agar replacing sodium nitrate for nitrate source.
Another embodiment of the present invention, the process wherein the mineral acid that is used for flask fermentation in step (e) is hydrochloric acid.
Yet another embodiment of the present invention, the process wherein the organic solvent used in step (f) is selected from the group consisting of dichloromethane, chloroform, ethylacetate, methylisobutyl ketone and preferably ethylacetate.
Still another embodiment of the present invention, the process wherein the organic solvent used for suspending the residue in step (i) is chloroform.
The present invention further provides a method of treating subjects with pharmaceutical composition comprising a bioactive compound of molecular formula C13H15NO5 along with pharmaceutically accepted excipients used for treatment of 13- Lipoxygenase inhibition and having free radical scavenging activity.
An embodiment of the present invention the method wherein said composition is used to treat asthma, hypersensitivity, psoriasis, inflammatory conditions and complications arising out of diabetes.
Yet another embodiment of the present invention the method wherein said compound having 13-lipoxygenase and crude rat lens aldose reductase inhibitory activity.
Still another embodiment of the present invention the method wherein
the subject is selected from mammals.
Yet another embodiment of the present invention the method wherein the IC50 value of the compound against purified soybean lipoxygenase and crude rat lens aldose reductase inhibitory activity is 79µ moles and 69 µ moles respectively.
Further embodiment of the present invention the method wherein ED50 value of the composition for free radical scavenging activity is 66 µM.
The invention is further explained in the form of following embodiments
Applicants have discovered a antibiotic 13-LOX inhibitory compound which is derived via the fermentation of certain microorganisms.
Aspergillus niger CFR-W-105 isolated from a wax sample is found to produce new biologically active substance. This substance is extracted from the fermented wheat bran using ethyl acetate followed by crystallization. The purified compound is identified as compound having a molecular formula C13H-
15NO5
Thus the fermentation of Aspergillus niger CFR-W-105 or a mutant thereof and suitable isolation techniques may be used to produce the compound of the investigation.
A biologically pure culture of Aspergillus niger CFR-W-105, from which the compound of investigation is derived, has been deposited with the American Type Culture Collection (ATCC) in Rockville, MD, and has been added to its permanent collection under its accession code ATCC.
To meet the above objectives, the present invention provides a novel compound having molecular formula C13H15NO5 The present invention also provides a process for the isolation of said compound from Aspergillus niger
and a method treatment in inhibiting 13-Lipoxygenase inhibitor, in scavenging
of free radicals, for the treatment of asthma, hypersensitivity, psoriasis,
inflammatory conditions and complications arising out of diabetes.
TAXONOMY
Morphology
The fungal mycelium on czapek's solution agar had abundant erect and
crowded conidial structures, which is brownish black or dark brown covering
the entire colony except for the narrow peripheral growing area. Conidial heads
are globose initially, which slowly split into radiating columns at the end of 10
days. Sclerotia are not observed.
Cultural and physiological characteristics
The growth characteristics of CFR-W-105 on malt-extract-agar are similar to
that of Czapek's solution agar. But the colonies are deeper brownish black,
reverse of the plate is uncolored and unwrinkled and conidiophores are less
dense. Growing margin is less prominent. The culture grew well at ambient
temperature of 26°-30°C. conidia is born by biseriate sterigmata (25-30A) and
are 2.5-4.5n in size and globose at maturity. The walls are smooth arid thick.
The length of conidiophores is 0.7-1.0 mm. Table 1 shows the growth of CRF-
W-105 in the presence of various carbon and nitrogen sources. The basal
medium of the above experiments contained Czapex solution agar for Carbon
source and Czapex solution agar replacing sodium nitrate for nitrate source.
Table 1: Growth of Aspergillus niger CFR-W-105 in the presence of various
carbon and nitrogen sources.
(Table Removed)
Taxonomic position
The strain CFR-W-105 is isolated from honeybee wax from local region. The characteristics indicated that the strain belongs to Aspergillus Niger group. According to the descriptions of Raper and Fennell ( Raper KB and Fennell DT (1965) The Genus Aspergillus; The Williams and Wilkins Co, Baltimore, pp 293-310), the strain CFR-W-105 is related to Aspergillus niger V. Tiegh. The taxonomic relationships between the strains and four other cultures that did not give the bioactive molecules during screening are compared and given in Table 2.
Table 2. Comparison between the strains CFR-W-105 and three other related species which did not give the compound I (as on Czapex solution agar, 26- 28°C 10 days)
(Table Removed)
FERMENTATION Seed Culture
Strain CFR W-105 is propagated on Potato Dextrose Agar (Hi Media Mumbai, India) slant composed of soluble starch 0.4 % and glucose 2 %. After incubation for 4 days at 30°C, a portion of the mature agar slant is inoculated into 100 ml of a seed liquid medium of the same medium composition in a 500-ml Erlenmeyer flask and incubated at 30°C on a rotary shaker at 250 rpm. Flask Fermentation
A 5-ml of the seed culture is transferred into 250-ml Erlenmeyer flasks each containing 10 gm of wheat bran, 10 ml of 0.2 N HC1 comprising 2.1 mg each of ferrous sulfate, zinc sulfate and copper sulfate and 5 ml distilled water. The inoculated flasks are incubated for 5 days at 30°C. Isolation and Purification. The fermented wheat bran (450 g) is treated with ethyl acetate (1 liter) for two hours. The organic extract is separated from the wheat bran by cheesecloth filtration. This extract is dried over anhydrous sodium sulfate and concentrated
in vacuo to afford a crude solid (1.36 gm) and the residue is resuspended in
chloroform (25 ml) and gently centrifuged (2000 rpm, 20 minutes, 27°C). The
residue is dried to afford an orange colored active fraction (230-mg). This is
dissolved in ethyl alcohol (50 ml) by gentle warming and treated with activated
charcoal (200 mg) filtered (Whatman No.l) while warm. The filtrate is
concentrated in vacuo to obtain 30 mg of yellow amorphous powder.
Physico-chemical properties
The compound is amorphous yellow powder. It is soluble in ethanol, methanol,
ethyl acetate, dimethyl sulphoxide, sodium bicarbonate solution, sodium
carbonate solution and sodium hydroxide solution, slightly soluble in chloroform
and hexane, but insoluble in water. The EI-MS spectra of the compound
showed the molecular ions at m/z 265.
Compositions and Methods
The novel compounds of the invention can be used in a variety of
pharmaceutical dosage forms. Thus, oral, parental, nasal, topical, buccal,
ocular and other forms can be used. When such forms are formulated they will
include pharmaceutically acceptable excipients such as colorants, carriers,
perfumes, stabilizers, flow modifiers and the like in suitable amounts (i.e., from
0.001 to 0.99wt%).
The compound of the invention is useful in methods of inhibiting the effects of
13-LOX.
The compound is also used to treat a host, preferable a mammal, which is
suffering from a disorder associated with a metabolism of 13-LOX, such as
asthma, hypersensitivity, psoriasis, inflammatory conditions and complications
arising from diabetes.
The following example illustrates the 13-Lipoxygenase inhibitor effects of the novel compound, however this shall not be considered as limiting the scope of the present invention.
EXAMPLE
Soybean lipoxygenase (LOX-1) is purified according to the method of Axelrod et al., lipoxygenase from soybeans. Methods in Enzymology 1981, 71, 441-451, using the Hardee variety of soybean, after dehulling and defatting by hexane. The substrate, sodium linoleate (10 mM) is prepared as follows: To 70 mg of linoleic acid an equal weight of Tween-20 and 4 ml of oxygen free water is added and homogenized by drawing back and forth in a pasture pipette, avoiding air bubbles. Sufficient quantity of 0.5 N NaOH is added to this to yield a clear solution. The resulting sodium linoleate is divided into 1-2 ml portions in small screw cap vials, flushed with N2 before closing, and keep frozen until needed. Enzyme reaction is carried out at 25°C in a quartz cuvette with a 1 cm light path. The assay mixture contained (2.975 - x) ml of borate buffer (0.2 M, pH 9.0), 0.025 ml of sodium linoleate substrate, and x mL of enzyme. After each addition the mixture is stirred with a few strokes of a plastic paddler. The reference cuvette contained no enzyme. Absorption at 234 nm is recorded, and the reaction rate is determined from the slope of the straight-line portion of the curve.
Lens are collected from rat eyes and homogenised with sodium, potassium phosphate buffer (0.135 M, pH 7.0) containing 0.5 mM of phenyl methyl sulphonyl fluoride and 10 mM of p-mercaptoethanol. The homogenate is centrifuged at 10,000 r.p.m for 30 minutes at 4°C. The supernatant is taken as enzyme source. Enzyme reaction is carried out at 25°C in a quartz cuvette with
a 1-cm light path. The assay mixture contained 100 µL of nicotinamide adenine dinucleotide phosphate tetra sodium salt (from a stock solution of 9.6 mM), 100 µL of DL-glyceraldehyde (from a stock solution of 250 mM), 10 µL of inhibitor dissolved in dimethyl sulphoxide, sodium-potassium-phosphate buffer (0.135 M, pH 7.0) to make up the total volume to 3 µL and the reaction is initiated by the addition of 200 µL of enzyme solution. The enzyme reaction is monitored by the decrease in absorbency at 340 nm and compared with enzyme reaction without inhibitor. Results
The bioactive compound having molecular formula C13H15NO5 is discovered in the fermented bran of a species of Aspergillus Niger CFR W-105 (= ATCC...). The compound is successfully purified to homogeneity. The IC50 value of the compound against purified soybean lipoxygenase and crude rat lens aldose reductase inhibitory activity is determined to be 79n moles and 69n moles respectively.

We Claim:
1. A process for preparation of a bioactive compound from the culture of Aspergillus niger useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions, having a molecular formula Ci3Hi5NO5, the said process comprising the steps of:
(a) isolating the strain of Aspergillus niger Teigh , from honey bee
wax;
(b) growing the strain obtained from step(a) on a Potato Dextrose
Agar medium and incubating for 4 days at 30°C;
(c) transferring the culture of step (b) in liquid medium and
incubating at 30°C on a rotary shaker at 250 rpm,
(d) transferring the culture of step (c) into a medium containing
wheat bran, mineral acid, sulfates and incubated for 5 days at
30°C to obtain fermented wheat bran;
(e) treating the fermented wheat bran of step (d) with an organic
solvent, for two hours to obtain an organic solvent extract;
(f) separating the organic solvent extract of step (e) from the wheat
bran by cheese cloth filtration, followed by drying over
anhydrous sodium sulfate to obtain a solid,
(g) suspending the solid of step (f) in an organic solvent , and
centrifuging to obtain a residue;
(h) drying the residue of step (g) to obtain an orange solid; (i) dissolving the solid of step (h) in an alcoholic solvent, treating
the solution with active charcoal, filtering; and concentrating to
obtain the compound having molecular formula C13H15NO5 as
yellow powder.
2. A process as claimed in claim 1, wherein the seed liquid medium
used is selected from ezapek's solution agar as Carbon source
and ezapek's solution agar replacing sodium nitrate for nitrate
source.
3. A process as claimed in claims 1, wherein the mineral acid used
in fermentation of step (d) is hydrochloric acid.
4. A process as claimed in claims 1, wherein the organic solvent
used in step (e) is selected from the group consisting of
dichloromethane, chloroform, ethylacetate, methylisobutyl ketone
and preferably ethylacetate.
5. A process as claimed in claims 1, wherein the organic solvent
used for suspending the residue in step (g) is chloroform.
6. A process for the preparation of a bioactive compound from
culture of Aspergillus niger useful for treatment of asthma, hypersensitivity, psoriasis, inflammatory conditions from the culture of Aspergillus niger substantially as herein described with reference to the examples,

Documents:

325-del-2002-abstract.pdf

325-del-2002-claims.pdf

325-del-2002-correspondence-others.pdf

325-del-2002-correspondence-po.pdf

325-del-2002-description (complete).pdf

325-del-2002-form-1.pdf

325-del-2002-form-2.pdf

325-del-2002-form-3.pdf

325-del-2002-form-4.pdf

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Patent Number

217483

Indian Patent Application Number

325/DEL/2002

PG Journal Number

15/2008

Publication Date

11-Apr-2008

Grant Date

26-Mar-2008

Date of Filing

27-Mar-2002

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	AVINASH PRAHLAD SATTUR	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
2	KADIYALA CHANDRA SEKHAR RAO	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
3	SOUNDAR DIVAKAR	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
4	NAIKANAKATTE GANESH KARANTH	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
5	TUMUKUR RAMACHANDRAIAH SHAMALA	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
6	APPURAO GOPAL RAO	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.

PCT International Classification Number

C09K 15/22

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA