JPH11140091A - Physiologically active substance na32176a, its production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new substance having the ability to differentiate neurocytes, and useful as a neurocyte differentiation promoter, antidemential agent, therapeutic agent for peripheral nerve disorders, by culturing microorganisms belonging to the genus Streptomyces and having the ability to produce the aimed physiologically active substance NA32176A. SOLUTION: This new physiologically active substance NA32176A has the following characteristics: having the structural formula shown by the formula, being white powder in appearance, having a molecular weight of 418, soluble to water and dimethyl sulfoxide, insoluble to hexane and petroleum ether, Rf- value determined by silica gel thin-layer chromatography being 0.7 with n- butanol-acetic acid-water (4:1:2) as developing solvent, and showing positive color reaction to phosphomolybdic acid and palladium chloride. This new substance is useful as a neurocyte differentiation promoter, antidemential agent, therapeutic agent for peripheral nerve disorders or neurocyte protective agent, and is obtained by culturing Streptomyces sp. NA32176 (FERM BP-6411) in a medium mixed with pantetheine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a novel physiologically active substance NA32176A and its use. The compound of the present invention has a neurite outgrowth effect on PC12 cells, and is expected as a physiologically active substance to be used as, for example, an anti-dementia agent, a therapeutic agent for peripheral neuropathy, or a nerve cell protective agent.

[0002]

2. Description of the Related Art Nerve growth factor (hereinafter referred to as NGF) has an effect of regulating neurite outgrowth and neurotransmitter production, and has a regenerative effect on nerve cells of aged animals in vitro. (Age, Volume 8,
19 (1985)). However, NGF does not have a cerebrovascular barrier (hereinafter, referred to as BBB) permeability, and therefore cannot be transferred into the brain by peripheral administration. Therefore, a complex of NGF and an anti-transferrin receptor antibody was prepared,
When the permeability of GF to BBB was increased, peripheral NGF administration was found to regenerate cholinergic and non-cholinergic neurons in the central nervous system of rats (Science, 259, 373-3).
77 (1993)). However, NGF itself has B
It has no BB penetration effect and cannot be an anti-dementia agent. Therefore,
Unlike NGF, compounds having a BBB penetration effect are expected as anti-dementia agents.

[0003] On the other hand, by adding NGF to PC12 cells, a cell line cloned from rat adrenal medulla pheochromocytoma, the PC12 cells stop growing and differentiate into sympathetic cells having neurites. It is known. Since PC12 cells respond to NGF and extend neurites, they are now widely used in many laboratories as model cells for neural differentiation.

[0004] Using these PC12 cells, it has been clarified that, in addition to NGF, fibroblast growth factor and interleukin 6 also promote neurite outgrowth. Also recently,
Staurosporine, a low molecular weight compound derived from microorganisms, has also been shown to cause neurite outgrowth to PC12 cells (Neurochemistry, 26, 200-220 (1987)).

[0005]

The above-mentioned staurosporine, unlike NGF, is a very low molecular weight compound and is extremely valuable in medical treatment, but has high toxicity and therefore needs to be studied in actual use. it is conceivable that. Under such circumstances, providing a low molecular weight compound having lower toxicity and a neurite outgrowth effect at a low concentration is considered to be extremely important in medical treatment.

[0006]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of various searches for metabolites of microorganisms, it was found that one strain belonging to the genus Streptomyces produces a novel bioactive substance NA32176A having a neuronal differentiation promoting action. Furthermore, it was found that the productivity of the physiologically active substance NA32176A was improved by adding pantethein to the culture solution. In addition, the physiologically active substance NA3217
Although sarcomycin, which is considered to be a precursor of 6A, is an unstable substance that is instantaneously reduced to dihydrosarcomycin due to its high reactivity when isolated, the present inventors have found that sarcomycin has a high level of reactivity. It was found to be very stable in a 0.1 to 10 normal saline solution.
Therefore, they have found that NA32176A can be obtained in high yield by producing sarcomycin using a sarcomycin-producing bacterium and adding pantethein to the crudely extracted sarcomycin. That is, the present invention relates to the following (1) to (11).

(1) Equation (1)

Embedded image Or NA32176A having the formula: or a pharmaceutically acceptable salt thereof. (2) A physiologically active substance NA32 having the following physicochemical properties:
176A or a pharmaceutically acceptable salt thereof. 1) Appearance; white powder 2) Molecular weight; 418 3) Molecular formula; C ₁₈ H ₃₀ N ₂ O ₇ S (measured by high-resolution mass spectrum) 4) Solubility: Soluble in lower alcohol, water, and dimethyl sulfoxide. Insoluble in hexane and petroleum ether. 5) Rf value by silica gel thin layer chromatography;
The developing solvent of n-butanol-acetic acid-water (4: 1: 2) shows 0.7. 6) Ultraviolet absorption spectrum; shows terminal absorption in water. 7) Infrared absorption spectrum; FIG. 1 shows a spectrum measured with a potassium bromide tablet. 8) Hydrogen nuclear magnetic resonance spectrum; FIG. 2 shows a spectrum measured in heavy water. 9) Carbon nuclear magnetic resonance spectrum; FIG. 3 shows a spectrum measured in heavy water. The chemical shift values are shown below. δ (ppm) 223.5 (s), 180.1 (s), 1
76.4 (s), 175.1 (s), 77.0
(D), 69.6 (t), 53.4 (d), 47.
7 (d), 39.8 (s), 39.7 (t), 3
8.9 (t), 36.7 (t), 36.5 (t),
32.5 (t), 31.0 (t), 25.7 (t),
21.8 (q), 20.3 (q) 10) Color reaction; positive for phosphomolybdic acid and palladium chloride. (3) A medicament comprising a physiologically active substance NA32176A or a pharmaceutically acceptable salt thereof as an active ingredient. (4) The medicament according to the above (3), which has a neurite outgrowth effect on PC12 cells. (5) The medicament according to the above (3), which is a nerve cell differentiation promoting agent. (6) The medicament according to the above (3), which is any one of an anti-dementia agent, a therapeutic agent for peripheral neuropathy, or a nerve cell protective agent.

(7) Streptomyces
myces) belonging to the genus and a bioactive substance NA32176A
A microorganism having the ability to produce. (8) Streptomyces sp. NA 32176
(Streptomyces sp. NA 3217
6) Strain (Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, Accession Number: FERM BP-6411) and its mutants. (9) Streptomyces
A microorganism that belongs to the genus and has the ability to produce the physiologically active substance NA32176A is cultured in a medium, the physiologically active substance NA32176A is produced and accumulated in the culture, and the physiologically active substance NA32176A or a pharmaceutical thereof is collected. For the production of chemically acceptable salts. (10) Streptomyces
s) A microorganism belonging to the genus belonging to the genus and capable of producing the physiologically active substance NA32176A is cultured in a medium, and pantethein (p
antetheine) is added to the culture solution to produce and accumulate the physiologically active substance NA32176A in the culture, which is collected.
A method for producing 6A or a pharmaceutically acceptable salt thereof. (11) when culturing sarcomycin-producing bacteria, or
A sarcomycin-producing bacterium is cultured to produce sarcomycin, and if necessary, pantethein is added to the crudely extracted sarcomycin, thereby obtaining a physiologically active substance NA32176.
A, a biologically active substance NA32176
A. A process for producing A or a pharmaceutically acceptable salt thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

The physiologically active substance NA32176A is obtained by culturing NA32176 strain belonging to the genus Streptomyces,
Alternatively, it is obtained by adding and culturing pantetheine in a medium to produce and accumulate the physiologically active substance NA32176A, and collecting the physiologically active substance NA32176A from the culture. Bioactive substance NA3217
One representative example of a 6A producing bacterium has the following mycological and physiological properties.

[0010] 1. Morphological properties As a result of observation after culturing at 27 ° C. for 2 weeks, the aerial hyphae are simply branched, and the tip is spiral or hook-shaped. No formation of sporangia or torus is observed. There are no zoospores. The spore surface is smooth or rough, and the spores are cylinder-shaped and have a size of 0.7-0.9 × 0.9-1.
3 μm. In addition, spores are formed in a chain of 20 or more.

2. Growth in various media Table 1 below shows the growth state of each medium at 27 ° C for 2 weeks.

[0012]

[Table 1]

3. Physiological properties 1 Moderate growth range: 24 to 37 ° C 2 Nitrate reduction: Negative 3 Liquefaction of gelatin (on glucose, peptone, gelatin medium, 20 ° C): False positive 4 Starch hydrolysis (Start Inorganic salt agar medium): Positive 5 Coagulation of skim milk: Negative 6 Peptoneization of skim milk: Positive 7 Production of melanin-like pigment: Negative 4. Utilization of carbon source (on Pridham-Godrib agar medium) L-arabinose + D-xylose + D-glucose + D-fructoose + sucrose + inositol-L-rhamno -Surfinose + D-mannitol +

5. Diaminopimelic acid in cell wall LL-diaminopimelic acid. To summarize the above,
In this strain, the cell wall is LL-diaminopimelic acid, and according to the method of the International Streptomyces Project (abbreviated as ISP), the spore-forming hypha belongs to the section Spirales, and the spore surface is Smooth or rough, mature mycelium is gray color-series and wettable (hygroscopic). It does not produce melanin-like pigments. The color of the underlying hyphae is pale yellow or pale brown. Examples of carbon sources include L-arabinose, D-glucose, D-fructose, sucrose, raffinose, and D-mannite.
And D-xylose.

Based on the above properties, the version of Bergey's Manual of Varieties Manual of Deterministic Bacteriology, edited by Al-Buffanand and N. Gibbons. Determinative Bacteriolog
y) A search was carried out in accordance with the eighth edition, 1974, and it was found that the above NA 32176 strain belongs to the genus Streptomyces.
(Streptomyces sp.) NA 3217
No. 6. The strain was deposited on August 8, 1997, with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry,
Thereafter, it was transferred to an international deposit based on the Budapest Treaty on July 7, 1998, and the accession number was FERM BP-641.
1 is given.

The physiologically active substance NA32176A producing bacterium having a neuronal differentiation promoting action according to the present invention belongs to the genus Streptomyces, and for example, Streptomyces sp. NA isolated by the present inventors.
32176 (Streptomyces sp. NA
32176) strain (Accession number: FERM BP-6411, National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology) is an example of a strain most effectively used in the present invention.

The strain belonging to the genus Streptomyces used in the present invention, like other strains of the genus Streptomyces, is liable to change its properties, and is easily mutated by, for example, an artificial mutating means using ultraviolet rays, X-rays, drugs and the like. Any mutant strain having the ability to produce the physiologically active substance NA32176A of the present invention can be used in the present invention.

In one method for producing NA32176A according to the present invention, first, the above strain is aerobically cultured in a medium containing nutrients that can be used by the bacteria. As the nutrient source, known ones conventionally used for culturing bacteria can be used. For example, glucose, fructose,
Glycerin, sucrose, dextrin, galactose, organic acids and the like can be used alone or in combination.

The inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate,
Sodium nitrate, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soy flour, cottonseed oil residue,
Casamino acid, bactosoytone, oatmeal and the like can be used alone or in combination.

In addition, if necessary, inorganic salts such as salt, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, phosphate and the like can be added, and organic substances such as amino acids, vitamins, and nucleic acids can be added. And inorganic substances can be appropriately added. The most suitable culture method is a liquid culture method, particularly a submerged stirring culture method.
It is desirable that the culturing is performed at a culturing temperature of 20 ° C. to 45 ° C. and a pH of slightly acidic to slightly alkaline.

In liquid culture, NA32176A substance is usually produced and accumulated in the culture solution after culturing for 3 to 5 days. Further, on the second and / or third day of the culture, pantethein is added to the culture solution to thereby prepare the physiologically active substance NA32176.
The productivity of A is improved. The amount of pantethein to add
It is not particularly limited as long as it does not adversely affect the growth of the bacterium.
ml is preferable, and 10 mg / ml to 300 mg / ml is more preferable. When the amount of NA32176A produced in the culture reaches the maximum, the culture is stopped, the cells are separated from the culture, and the target substance is purified and isolated from the filtrate. The purification and isolation of the substance from the filtrate generally employs the separation and purification method used to isolate the microbial metabolite from the culture filtrate.

That is, the culture solution is separated into the filtrate and the cells by a usual filtration method. The obtained filtrate is passed through a Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei) column under alkaline conditions to adsorb the target substance, washed with water, and then eluted with a linear gradient from water to 80% methanol water. The eluted active fraction is concentrated, methanol is distilled off, and the concentrated solution is extracted with n-butanol under acidic conditions of hydrochloric acid. About the vacuum concentrate of the n-butanol layer, Sephadex LH-20 (trade name;
The active fraction was concentrated and then dissolved in a mixture of ethyl acetate-water (1: 1), and the lower layer of the mixture was used as a stationary phase in advance. Centrifugal liquid partition chromatography (MPC Engineering, CPC-LLB
-M), and after washing with the upper layer solution of the mixture, the active fraction is reversed and eluted with the lower layer solution. Further, Sephadex LH-20 column chromatography (mobile layer: methanol) is performed to obtain NA32176A.

Alternatively, the culture solution is separated into the filtrate and the cells by a usual filtration method. The obtained filtrate is passed through a Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei) column under acidic conditions to adsorb the target substance, washed with water, and eluted with 20% acetone water. The acetone of the eluted active fraction is distilled off. The concentrated solution was subjected to QAE Sephadex A25 (trade name; manufactured by Pharmacia Biotech, Cl.
(Type) Perform column chromatography (mobile layer: saline), concentrate the active fraction, pass through a Diaion HP-20 column to adsorb the target substance, wash with water, and elute with 20% acetone water. After the acetone of the eluted active fraction was distilled off, NA32176A was obtained by freeze-drying.
Get.

As another method for producing NA32176A, a method using sarcomycin-producing bacteria was described in the literature (Sumiki Yusuke, Antibiotic, Vol. 908-921 (196)).
In accordance with the method described in 1)), culturing is performed by liquid culture to produce and accumulate sarcomycin in the culture solution. After the culture is stopped, the bacterial cells and the culture solution are filtered off, and the method described in the literature is applied from the filtrate. There is a method in which sarcomycin is extracted and purified by applying the same, and pantethein is added to the roughly-purified sarcomycin saline solution to obtain a physiologically active substance NA32176A in high yield. At this time, pantetheine is used in an amount of usually 1 to 3 equivalents, preferably 1.0 to 1.5 equivalents to the produced sarcomycin. The reaction temperature is usually 0 ° C to 5
It is carried out at 0 ° C, preferably at 10 ° C to 30 ° C. The reaction time varies depending on the reaction temperature, but is usually about 5 minutes to 2 hours, preferably about 10 minutes to 1 hour. The sarcomycin saline solution is stable under the above reaction conditions, and no decomposition products are generated. NA32176A obtained by the reaction can be purified by applying a method of isolating it from the culture filtrate. Incidentally, as described above, pantethein may be added to the crudely purified sarcomycin, but may be added instead, when culturing a sarcomycin-producing bacterium, or after culturing, but before crudely purifying sarcomycin, these methods But the target NA3
2176A can be obtained.

The physiologically active substance N obtained as described above
The structural formula and physicochemical properties of A32176A are shown below. 1) Structural formula (1)

Embedded image 2) Appearance; white powder 3) Molecular weight; 418 4) Molecular formula; C ₁₈ H ₃₀ N ₂ O ₇ S (measured by high-resolution mass spectrum) 5) Solubility: Soluble in lower alcohol, water, and dimethyl sulfoxide. Insoluble in hexane and petroleum ether. 6) Rf value by silica gel thin layer chromatography;
The developing solvent of n-butanol-acetic acid-water (4: 1: 2) shows 0.7. 7) Ultraviolet absorption spectrum; shows terminal absorption in water. 8) Infrared absorption spectrum; spectrum measured with potassium bromide tablet is shown in FIG. 9) Hydrogen nuclear magnetic resonance spectrum; FIG. 2 shows a spectrum measured in heavy water. 10) Carbon nuclear magnetic resonance spectrum; FIG. 3 shows a spectrum measured in heavy water. The chemical shift values are shown below. δ (ppm) 223.5 (s), 180.1 (s), 1
76.4 (s), 175.1 (s), 77.0
(D), 69.6 (t), 53.4 (d), 47.
7 (d), 39.8 (s), 39.7 (t), 3
8.9 (t), 36.7 (t), 36.5 (t),
32.5 (t), 31.0 (t), 25.7 (t),
21.8 (q), 20.3 (q) 10) Color reaction; positive for phosphomolybdic acid and palladium chloride.

NA32176A or a pharmaceutically acceptable salt thereof can be produced by a generally known method. For example, NA32176A or a pharmaceutically acceptable salt thereof is prepared in a solution containing sodium hydroxide, potassium hydroxide and hydrochloric acid, sulfuric acid and the like. By treating an acceptable salt.

Various methods known in the art can be applied to the preparation and administration of the composition when used as a pharmaceutical. That is, injection, oral, rectal administration and the like can be used as an administration method. The preparation may take the form of injections, powders, granules, tablets, suppositories and the like. NA32 at the time of formulation
As long as it does not adversely affect 176A, various auxiliaries used for medicine, that is, carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, and emulsifiers, can be used as necessary. In the preparation, the content of NA32176A can be varied widely depending on the form of the preparation and the like. In general, NA32176A contains 0.01 to 100% (by weight), preferably 0.1 to 70% (by weight), and Consists of carriers and other auxiliaries commonly used for pharmaceuticals.

The dose of NA32176A varies depending on symptoms and the like, but is about 0.01 to 800 mg per adult per day. When continuous throwing is required, it is preferable to reduce the daily consumption.

[0029]

The effect of NA32176A on promoting neuronal differentiation is described below with reference to experimental examples. Experimental example

PC12 of the physiologically active substance NA32176A
The neurite outgrowth effect on cells is green (Green).
en) et al. (Ann. Rev. Neurosc)
i. , Vol. 3, p. 353 (1980)) was partially modified and judged by morphological change.

Specifically, PC12 cells were inoculated at a density of 2 × 10 ⁴ / ml in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10% horse serum, and cultured on a collagen-coated 96-well multiplate overnight at 37 ° C. The cells were cultured at 5 ° C. under 5% CO ₂ . Here, a sample is added and the morphological change is observed.

As a result, the physiologically active substance NA32176A
The effective concentration that causes neurite outgrowth on PC12 cells ranged from 25 μg / ml to 1.56 μg / ml. As a control, the activity of staurosporine having the same effect was measured by the above method.
Extension of neurites was observed in the range of μg / ml to 0.08 μg / ml.

The concentration at which NA32176A kills PC12 cells by cytotoxicity by the above method was 50 μg / ml or more, while that of staurosporine was 1.25 μg / ml or more. Therefore, NA
32176A was found to be less toxic than staurosporine.

[0034]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below, but these are merely examples and do not limit the present invention at all, and various modifications are possible.

Production method 1 2.0% glycerin, 1.0% glucose, 0.5% soybean powder in a 500 ml Erlenmeyer flask for rotary shaker.
%, Peptone 0.3%, yeast extract 0.5%, calcium carbonate 0.2%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05% medium (pH 7.0) 100m
1 was dispensed and autoclaved at 120 ° C. for 20 minutes. One platinum loop of NA32176 strain (FERM BP-6411, National Institute of Bioscience and Biotechnology) was inoculated into this, and shaking culture was performed at 27 ° C. and 220 rpm for 2 days to obtain a seed culture. Subsequently, glucose 1.0%, starch syrup 4.0%, corn steep liquor 1.0%, yeast extract 0.2%, gluten meal 1.0%, iron sulfate-7 water were placed in a 500 ml Erlenmeyer flask for rotary shaker. 0.00011% of sum, 0.00064 of copper sulfate pentahydrate
%, Zinc sulfate heptahydrate 0.00015%, manganese chloride tetrahydrate 0.00079%, cobalt chloride 0.00
01%, 0.2% calcium carbonate medium (pH 7.0)
After dispensing 100 ml and autoclaving at 120 ° C. for 20 minutes, 1 ml of the seed culture was transplanted and
Shaking culture was performed for 4 days at 220 ° C. and 220 ° C.
The obtained culture solution (10 liters) was separated into a filtrate and cells by a usual filtration method. The obtained filtrate was adjusted to pH 8 with 4N caustic soda, absorbed on a Diaion HP-20 column (1 liter), washed with water, and eluted with a linear gradient from water (2 liter) to 80% methanol (2 liter). . After distilling off the eluted fraction with methanol, p with 1N hydrochloric acid
The mixture was combined with H2 and extracted with n-butanol. The vacuum concentrate (491 mg) of the n-butanol layer was subjected to Sephadex LH-20 column chromatography (φ3.5).
× 90 cm, mobile phase: methanol) to obtain an active fraction (267 mg). After dissolving this active fraction in a mixed solution of ethyl acetate-water (1: 1), centrifugal liquid-liquid chromatography (250 ml capacity, 1500 rotations / minute, flow rate 3 ml) using the lower layer of the mixed solution as a stationary phase in advance.
/ Min), and the mixture was washed with the upper layer solution, and then the active fraction was reversed and eluted with the lower layer solution to obtain a crude active substance (16 mg). Further, NA32176 was obtained by purification by Sephadex LH-20 column chromatography (φ1.8 × 85 cm, mobile layer: methanol).
A (13 mg) was obtained. Using purified NA32176A, appearance, molecular weight, solubility, Rf value by silica gel thin layer chromatography, ultraviolet absorption spectrum, infrared absorption spectrum, ¹ H-NMR spectrum, ¹³ C-N
The MR spectrum was measured. The physicochemical properties of NA32176A were as described above.

Production method 2 In a 500 ml Erlenmeyer flask for rotary shaker, glycerin 2.0%, glucose 1.0%, soybean powder 0.5
%, Peptone 0.3%, yeast extract 0.5%, calcium carbonate 0.2%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05% medium (pH 7.0) 100m
1 was dispensed and autoclaved at 120 ° C. for 20 minutes. One platinum loop of NA 32176 strain (FERM BP-6411, National Institute of Bioscience and Biotechnology) was inoculated into this, and shake-cultured at 27 ° C. and 220 rpm for 2 days to obtain a seed culture. Subsequently, glucose 1.0%, starch syrup 4.0%, corn steep liquor 1.0%, yeast extract 0.2%, gluten meal 1.0%, iron sulfate-7 water were placed in a 500 ml Erlenmeyer flask for rotary shaker. 0.00011% of sum, 0.00064 of copper sulfate pentahydrate
%, Zinc sulfate heptahydrate 0.00015%, manganese chloride tetrahydrate 0.00079%, cobalt chloride 0.00
01%, 0.2% calcium carbonate medium (pH 7.0)
After dispensing 100 ml and autoclaving at 120 ° C. for 20 minutes, 1 ml of the seed culture was transplanted and
Shaking culture was performed at 220 ° C. and 220 rpm for 4 days. Pantethein (50 mg / ml) was added on the second and third days (two times in total) after the start of the culture.

The obtained culture (200 ml) was separated into a supernatant and cells by centrifugation. The obtained supernatant is 6N
After adjusting the pH to 3 with hydrochloric acid, adsorb it onto a Diaion HP-20 column (Mitsubishi Kasei; φ1.2 × 20 cm), wash with water,
Elution was performed with 20% acetone water. After distilling off acetone from the eluate, the residue was adjusted to pH 6 and QAE-Sephadex (Pharmacia Biotech, Cl type, φ1.8 × 100 cm)
To water, wash with water, and add 0.5 ml of water (600 ml).
Elution was carried out with a linear gradient to an aqueous M sodium chloride solution (600 ml). The eluted fraction was adsorbed on a Diaion HP-20 column (manufactured by Mitsubishi Kasei; φ1.2 × 20 cm), washed with water, and eluted with 20% acetone water. The acetone in the eluted active fraction was distilled off, followed by freeze-drying to obtain NA32176A (20 mg). Purified NA3
Using 2176A, appearance, molecular weight, solubility, Rf value by silica gel thin layer chromatography, ultraviolet absorption spectrum, infrared absorption spectrum, ¹ H-NMR spectrum, and ¹³ C-NMR spectrum were measured. NA32
The structural formula and physicochemical properties of 176A were as described above.

Production method 3 2.0% glycerin, 1.0% glucose, 0.5% soy flour in a 500 ml Erlenmeyer flask for rotary shaker.
%, Peptone 0.3%, yeast extract 0.5%, calcium carbonate 0.2%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05% medium (pH 7.0) 100m
1 was dispensed and autoclaved at 120 ° C. for 20 minutes. One platinum loop of a sarcomycin-producing strain (Fermentation Research Institute IFO3304) was inoculated into this, and the temperature was 27 ° C and 220 ° C.
Shaking culture was performed for 2 days under the condition of rotation / min to obtain seed culture. Subsequently, glucose 1.0%, starch syrup 4.0%, corn steep liquor 1.0%, yeast extract 0.2%, gluten meal 1.0%, iron sulfate-7 water were placed in a 500 ml Erlenmeyer flask for rotary shaker. 0.00011% of Japanese hydrate, 0.00064% of copper sulfate pentahydrate, 0% of zinc sulfate heptahydrate.
00015%, manganese chloride tetrahydrate 0.00079
%, Cobalt chloride 0.0001%, calcium carbonate 0.
100 ml of 2% medium (pH 7.0) was dispensed, and
1 ml of the above seed culture was transplanted into a flask autoclaved at 20 ° C. for 20 minutes, and cultured with shaking at 27 ° C. and 220 rpm for 4 days.

The obtained culture solution (5 liters) was separated into a supernatant and cells by centrifugation. The obtained supernatant is 6N
The pH was adjusted to 4.5 with hydrochloric acid, and activated carbon column (500 m
1), washed with water, and eluted with 20% acetone water. After acetone was distilled off from the eluate, freeze-drying was performed to obtain crude sarcomycin (22.2 g). 5.0 g thereof was adsorbed on QAE-Sephadex (Pharmacia Biotech, Cl type, φ1.8 × 100 cm),
The extract was washed with water and eluted with a linear gradient from water (600 ml) to a 0.5 M aqueous sodium chloride solution (600 ml) to obtain a sarcomycin-containing fraction (80 ml). 4 of them
Pantetein (220 mg / 2.2) with stirring for 0 ml
ml) was added and stirred for 20 minutes. The reaction solution was adjusted to pH 6 using hydrochloric acid, adsorbed on QAE-Sephadex (manufactured by Pharmacia Biotech, Cl type, φ1.8 × 100 cm), washed with water, and then 0.5M sodium chloride from water (600 ml). Elution was performed with a linear gradient to an aqueous solution (600 ml). The eluted fraction of NA32176A was applied to a Diaion HP-20 column (Mitsubishi Kasei; φ1.2 × 20 cm).
After washing with water, the mixture was eluted with 20% acetone water.
After evaporating acetone from the eluted active fraction, lyophilization was performed to obtain NA32176A (60 mg).

[0040]

As is clear from the above, the physiologically active substance NA32176A of the present invention or a pharmaceutically acceptable salt thereof has a neurite outgrowth-promoting action.
It can be expected as an active ingredient such as a therapeutic agent for peripheral neuropathy or a nerve cell protective agent.

[Brief description of the drawings]

FIG. 1 Infrared absorption spectrum measured with potassium bromide tablet of NA32176A

FIG. 2. Hydrogen nuclear magnetic resonance spectrum of NA32176A measured in heavy water

FIG. 3. Carbon nuclear magnetic resonance spectrum of NA32176A measured in heavy water

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12P 13/00 C12R 1: 465)

Claims (11)

[Claims] (1) Formula (1) Or NA32176A having the formula: or a pharmaceutically acceptable salt thereof. 2. A physiologically active substance N having the following physicochemical properties:
A32176A or a pharmaceutically acceptable salt thereof. 1) Appearance; white powder 2) Molecular weight; 418 3) Molecular formula; C ₁₈ H ₃₀ N ₂ O ₇ S (measured by high-resolution mass spectrum) 4) Solubility: Soluble in lower alcohol, water, and dimethyl sulfoxide. Insoluble in hexane and petroleum ether. 5) Rf value by silica gel thin layer chromatography;
The developing solvent of n-butanol-acetic acid-water (4: 1: 2) shows 0.7. 6) Ultraviolet absorption spectrum; shows terminal absorption in water. 7) Infrared absorption spectrum; FIG. 1 shows a spectrum measured with a potassium bromide tablet. 8) Hydrogen nuclear magnetic resonance spectrum; FIG. 2 shows a spectrum measured in heavy water. 9) Carbon nuclear magnetic resonance spectrum; FIG. 3 shows a spectrum measured in heavy water. The chemical shift values are shown below. δ (ppm) 223.5 (s), 180.1 (s), 1
76.4 (s), 175.1 (s), 77.0
(D), 69.6 (t), 53.4 (d), 47.
7 (d), 39.8 (s), 39.7 (t), 3
8.9 (t), 36.7 (t), 36.5 (t),
32.5 (t), 31.0 (t), 25.7 (t),
21.8 (q), 20.3 (q) 10) Color reaction; positive for phosphomolybdic acid and palladium chloride. 3. A medicament comprising a physiologically active substance NA32176A or a pharmaceutically acceptable salt thereof as an active ingredient. 4. The medicament according to claim 3, which has a neurite outgrowth effect on PC12 cells. 5. The medicament according to claim 3, which is a nerve cell differentiation promoting agent. 6. The medicament according to claim 3, which is any one of an anti-dementia agent, a therapeutic agent for peripheral neuropathy, and a nerve cell protective agent. 7. Streptomyces (Streptomyc)
es) A microorganism belonging to the genus and capable of producing the physiologically active substance NA32176A. 8. Streptomyces sp. NA 32
176 (Streptomyces sp. NA 3
2176) strain (Accession No .: FERM BP-6411, National Institute of Bioscience and Biotechnology). 9. Streptomyces (Streptomyc)
es) A biologically active substance NA32176A characterized by culturing a microorganism belonging to the genus and having the ability to produce the physiologically active substance NA32176A in a medium, producing and accumulating the physiologically active substance NA32176A in the culture, and collecting the same.
Or a method for producing a pharmaceutically acceptable salt thereof. 10. Streptomyces (Streptomyces)
ces) A microorganism belonging to the genus belonging to the genus and having the ability to produce the bioactive substance NA32176A is cultured in a medium, and pantetheine is added to the culture solution to produce and accumulate the bioactive substance NA32176A in the culture. Physiologically active substance NA3
A method for producing 2176A or a pharmaceutically acceptable salt thereof. 11. A method for culturing a sarcomycin-producing bacterium, the method comprising:
Alternatively, sarcomycin-producing bacteria are cultured to produce sarcomycin, and if necessary, pantethein is added to the crudely extracted sarcomycin, whereby the physiologically active substance NA321 is added.
Bioactive substance NA321 characterized by obtaining 76A
A method for producing 76A or a pharmaceutically acceptable salt thereof.