JP2993767B2 - FO-1289A substance and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an acyl coenzyme A
Novel substance F having cholesterol acyltransferase inhibition
The present invention relates to an O-1289A substance and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, several hyperlipidemic drugs have been known, but no effective substance has been obtained yet.

[0003]

In recent years, symptoms caused by cholesterol accumulation such as hyperlipidemia and arteriosclerosis in adults have been regarded as a modern disease with the improvement of dietary habits. Cholesterol is converted into a cholesterol ester by acyl group transfer from acyl coenzyme A, and is accumulated in lipoproteins in cells and blood. The enzyme that catalyzes the acyl transfer reaction is acyl coenzyme A cholesterol acyl transferase, which is closely involved in the absorption of cholesterol from the intestinal tract and the formation of foam cells in coronary arteries. Therefore, it is presumed that a substance that inhibits acyl coenzyme A cholesterol acyltransferase is effective for such diseases.

Under such circumstances, it is useful to provide a substance having acylcoenzyme A cholesterol acyltransferase inhibitory activity in the treatment of adult diseases such as hyperlipidemia and arteriosclerosis.

[0005]

The present inventors have continued their research on metabolites produced by microorganisms and found that acyl coenzyme A cholesterol acyl was contained in a culture of FO-1289 strain isolated from fresh soil. It has been found that a substance having a translocation enzyme inhibitory activity is produced. Next, the substance having acylcoenzyme A cholesterol acyltransferase inhibitory activity was separated and purified from the culture, and as a result, no substance having the physicochemical properties described below has been known at all. Therefore, this substance is referred to as FO-1289A substance. It was to be. The present invention has been completed on the basis of the above findings, and is a FO-1289A represented by a molecular formula of C ₃₁ H ₃₇ NO _10.
To provide the substance.

Further, the present invention relates to a method of culturing a microorganism belonging to the genus Aspergillus and having the ability to produce a FO-1289A substance in a culture medium to accumulate the FO-1289A substance in a culture, and then producing the FO-1289A substance from the culture. And a method for producing a FO-1289A substance.

[0007] Microorganisms capable of producing the FO-1289A substance (hereinafter referred to as FO-1289A substance-producing bacteria) belong to the genus Aspergillus, and for example, FO-1289 belonging to the genus Aspergillus isolated by the present inventors.
The strain is an example of the most effectively used strain of the present invention, and the microbiological properties of the strain are as follows.

The strain used for producing the FO-1289A substance of the present invention includes, for example, Aspergillus sp. Isolated from the soil by the present inventors.
( Aspergillus sp.) FO-1289 strain.

I. This strain is a malt extract agar medium, Tupetsk yeast extract agar medium,
It grows well on a 20% sucrose-tupetsk-yeast extract agar medium and has good conidium formation. When the colonies grown on the Tupetsk yeast extract agar medium are observed with a microscope, the hyphae are transparent and have partition walls, the conidiophores mainly grow straight from the basal hyphae, and the surface is smooth.

The tips of the conidiophores bulge, forming a flask-shaped apex (10-25 μm in diameter). No metres are formed, and the fluoride grows directly on the top. Conidia are 2-3 μm in diameter and are spherical and subspherical.

II. Characteristics of culture (1) Table 1 shows the culture findings of this strain. This finding is the result of macroscopic observation when cultured on various media at 25 ° C. for 7 days.

[0012]

[Table 1]

(2) The growth state when cultured at 37 ° C. for 7 days on a Tupetsk yeast extract agar medium is very vigorous (80 mm or more). 5
When the cells were cultured at 7 ° C. for 7 days, they did not grow. No formation of secretions and sclerotium was observed in all of the above-mentioned media as the bacteria grew.

III. Physiological properties (1) Growth temperature range: 15 ° C to 47 ° C, optimal growth temperature range 27 ° C to 40 ° C (2) Growth pH range: 3 to 11, optimal growth pH range 5 to 5
8 (3) Aerobic and anaerobic distinction: aerobic

[0015] Based on the morphological characteristics, culture characteristics, and physiological characteristics of the FO-1289 strain, an attempt was made to compare it with known bacterial strains. As a result, this strain was identified as Aspergillus ( Asperg).
illus ), identified as one strain belonging to the genus Aspergillus sp. FO-1289. This strain is
Aspergillus sp. FO-1289 ( Aspe
rgillus sp. FO-1289). (FER
MP-12194).

The FO-1289A producing bacteria have been described above. However, as a general property of the bacteria, the mycological properties are extremely liable to vary, are not constant, and are naturally or normally performed by ultraviolet irradiation or mutated derivatives. , For example N
-Methyl-N-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc., it is a well-known fact that mutation occurs by artificial mutation means. Any strain belonging to the genus and capable of producing the FO-1289A substance can be used in the present invention. In addition, strains mutated by cell engineering such as cell fusion and genetic manipulation are also included as substance FO-1289A substance-producing bacteria.

In the present invention, a FO-1289A substance-producing bacterium belonging to the genus Aspergillus is first cultured in a medium. In culturing the fungus, a fungal culture method is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be assimilated, and, if necessary, an inorganic acid salt or the like is used. As the assimilable carbon source, glucose, sucrose, molasses, dextrin, cellulose and the like are used alone or in combination.

Examples of nitrogen sources that can be assimilated include organic nitrogen such as peptone, meat extract, yeast extract, dried yeast, soy flour, corn steep liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, and urea. Sources, inorganic nitrogen compounds such as nitrates and ammonium salts are used alone or in combination. In addition, if necessary, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate, and heavy metal salts are added. Furthermore, if necessary, micronutrients, growth promoting substances, precursors, and the like that promote the growth of the present bacterium and the production of the FO-1289A substance may be appropriately added to the medium.

The culture is usually carried out under aerobic conditions such as shaking or aeration and stirring culture. Industrially, deep aeration stirring culture is preferred. The culture is preferably performed at a pH around neutrality. The cultivation temperature can be 20-37 ° C,
Usually, it is good to keep at 24 to 30 ° C. In the case of liquid, the FO-128 is usually cultured for 2 to 3 days.
Since the substance 9A is accumulated, the culture may be terminated when the accumulated amount during the culture reaches the maximum.

The culture conditions such as the medium composition, the liquid properties of the medium, the culture temperature, the culture rate and the aeration rate are appropriately adjusted and selected so as to obtain preferable results according to the kind of the strain used and the external conditions. Needless to say. If there is foaming in the liquid culture, an antifoaming agent such as silicone oil, vegetable oil, or a surfactant can be used as appropriate.

Since the FO-1289A substance accumulated in the culture thus obtained is contained in the cells and in the culture filtrate, the culture is centrifuged to separate the culture filtrate and the cells. , It is advantageous to collect the present FO-1289A material from each.

To collect the FO-1289A substance from the culture filtrate, the culture filtrate is first extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, benzene, etc., and the extract is concentrated under reduced pressure to obtain crude FO-1289A. The substance is obtained. The crude substance is further purified by a known method usually used for purification of a fat-soluble substance, for example, FO-128 by column chromatography using a carrier such as silica gel or alumina.
9A substance can be separated and purified.

To collect the FO-1289A substance from the cells, the cells are extracted with a water-containing hydrophilic organic solvent such as water-containing acetone and water-containing methanol, and the obtained extract is concentrated under reduced pressure. The extract is extracted with a non-hydrophilic organic solvent such as ethyl, butyl acetate, benzene, etc., and the obtained extract is separated and purified in combination with the extract obtained from the culture filtrate, or the FO-1289A substance is obtained by the same method as described above. Can be separated and purified.

Next, the physicochemical properties of the substance F-1289A of the present invention will be described. (1) molecular formula; C ₃₁ H ₃₇ NO ₁₀ (m / z 583 was observed in the high-resolution spectrum) (2) molecular weight; 583 (m / z 58 from the mass spectrum)
((3) (M ⁺ ) was observed) (3) Specific rotation; [α] ²² _D = +73.2 (C = 1, chloroform) (4) Ultraviolet absorption spectrum (in methanol); ) Infrared absorption spectrum (in carbon tetrachloride);

(6) Solubility in a solvent: methanol,
Soluble in ethanol, acetonitrile, ethyl acetate, benzene, insoluble in water (7) Distinguishing between basic, acidic and neutral; neutral (8) Color and shape of substance; yellow powder (9) Proton nuclear magnetic resonance spectrum ( In deuterated chloroform); as in FIG.

Next, the biological properties of the FO-1289A substance of the present invention will be described. (1) rats from acyl-coenzyme A influence on the inhibitory action acyl-coenzyme A cholesterol acyltransferase activity against cholesterol acyltransferase is used crude enzyme prepared from rat liver microsome fraction 300μM ^[1-14 C] Oleoyl-
CoA; 20 μl each of 3 mg / ml cholesterol (0.
02 μCi); 6.67 μl was added thereto, and reacted at 37 ° C. for 30 minutes.
1) After extraction with the mixture, TLC (Kieselgel GF ₂₅₄ ,
After separating each lipid with petroleum ether: diethyl ether: acetic acid (90: 10: 1) as a developing solvent, the cholesterol ester fraction was scraped off, and acyl coenzyme A cholesterol acyltransferase activity was measured with a liquid scintillation counter. The result of calculating the concentration that inhibits the enzyme activity by 50% is 0.03 μm for the FO-1289A substance.
g / ml.

[0027]

As described above, the FO-1289 of the present invention has been described.
Substance A exhibits a remarkable inhibitory activity on acyl coenzyme A cholesterol acyltransferase and is therefore useful for prevention and treatment of diseases caused by cholesterol accumulation in humans.

Next, the present invention will be described specifically with reference to examples.

[Example] 0.1 g of glucose was placed in a 500 ml Erlenmeyer flask.
100%, starch 2.4%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4% (adjusted to pH 7.0) 100
ml of Aspergillus sp., which had been cotton-plugged, steam-sterilized, and grown on an agar medium. FO-1289 (FE
RM P-12194) aseptically with a platinum loop.
A seed culture was obtained by shaking culture at 27 ° C. for 48 hours.

On the other hand, one 50 l jar armamenter contains 1.0% glucose, 0.5% tryptone, 0.3% yeast extract, 0.3% malt extract, 0.1% agar (pH
6.0), steam-sterilized and cooled, and then aseptically transplanted with 200 ml of a seed culture solution subjected to seed culture, and stirred at 250 rpm.
The mixture was stirred under aeration at 27 ° C. for 72 hours under culture conditions of 10 pm / min.

After the cultivation, 30 l of the culture was added to 18 l of ethyl acetate.
And the extract was concentrated under reduced pressure to obtain a crude product. This crude product was silica gel (250 g, Merck, Art. 9).
385), and column chromatography was performed, eluting with chloroform-methanol (99: 1). Each fraction was fractionated in 100 ml portions, and the fractions containing the active ingredient were collected and dried under reduced pressure to obtain 1.5 g of a crude active substance.

This was separated and purified by high-performance liquid chromatography in five times. The device used was Trirotor V (manufactured by JASCO Corporation), and the column was YMC-Pack A-3.
43 (ODS resin, manufactured by Yamamura Chemical Laboratory), the solvent system was 55% acetonitrile water, and the detection was UV2.
At 80 nm, the flow rate was 8 ml / min. As a result, FO-
50 mg of 1289A substance was isolated.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of a FO-1289A substance. (Measured as methanol solution)

FIG. 2 is an infrared absorption spectrum of a FO-1289A substance. (Measured as carbon tetrachloride solution)

FIG. 3 is a proton nuclear magnetic resonance spectrum of the FO-1289A substance. (Measured as deuterated chloroform solution)

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ identification code FI C12R 1:66) (58) Investigated field (Int.Cl. ⁶ , DB name) C07G 17/00 C12N 1/14 C12P 1 / 02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. FO-128 having the following physicochemical properties:
9A substance. (1) molecular formula; C ₃₁ H ₃₇ NO ₁₀ (m / z 583 was observed in the high-resolution spectrum) (2) molecular weight; 583 (m / z 58 from the mass spectrum)
((3) (M ⁺ ) was observed) (3) Specific rotation; [α] ²² _D = +73.2 (C = 1, chloroform) (4) Ultraviolet absorption spectrum (in methanol); ) Infrared absorption spectrum (in carbon tetrachloride); as shown in FIG. 2 (6) Solubility in solvent; methanol, ethanol,
Soluble in acetonitrile, ethyl acetate, benzene, insoluble in water (7) Basic, acidic, neutral distinction; neutral (8) Color and shape of substance; yellow powder 2. FO-128 belonging to the genus Aspergillus
A method for producing a FO-1289A substance, comprising culturing a microorganism capable of producing a 9A substance in a medium, accumulating the FO-1289A substance in the culture, and collecting the FO-1289A substance from the culture. 3. FO-128 belonging to the genus Aspergillus
A microorganism capable of producing the 9A substance is Aspergillus sp. FO-1289 ( Aspergillu)
s sp. FO-1289 FERM P-1219
3. The method according to claim 2, wherein the method is 4). 4. FO-128 belonging to the genus Aspergillus
A microorganism capable of producing 9A substances. 5. The method according to claim 5, wherein the microorganism is Aspergillus sp. F
The microorganism according to claim 4, which is O-1289.