JPH04360895A - Fo-1289a substance and production thereof - Google Patents

Abstract

PURPOSE:To provide the subject new substance showing a remarkable inhibitory effect for acylcoenzyme A cholesterol acyltransferase and useful for prevention and theraphy of diseases caused by accumulation of cholesterol, e.g. hyperlipidemia therefor. CONSTITUTION:FO-1289A substance having following properties. Molecular formula: C31H37NO10; Molecular weight: 583; Solubility: soluble in methanol, benzene, etc., and insoluble in water; Classification of basic, acid or neutral: neutral; Color and appearance: yellow powder. The above-mentioned substance can be obtained by culturing a microorganism belonging to Aspergillus genus and capable of producing FO-1289 substance and subsequently collecting it from the cultured material. As the above-mentioned micro-organism, e.g. a new microorganism, Aspergillus sp. FO-1289 (FERM P-12194) is used. In addition, culture is recommendably carried out in an aerobic condition at pH around neutrality and 24-30 deg.C for 2-3 days.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides acyl coenzyme A
Novel substance F that inhibits cholesterol acyltransferase
This invention relates to O-1289A substance and its manufacturing method.

0002

BACKGROUND OF THE INVENTION Several hyperlipidemia drugs have been known, but no effective substance has yet been obtained.

0003

[Problems to be Solved by the Invention] In recent years, as dietary habits have improved, symptoms caused by cholesterol accumulation, such as hyperlipidemia and arteriosclerosis in adults, have become problematic as modern diseases. Cholesterol becomes cholesterol ester by acyl group rearrangement from acyl coenzyme A, and is accumulated in cells and in blood lipoproteins. The enzyme that catalyzes this acyl group transfer reaction is acylcoenzyme A cholesterol acyltransferase, which is deeply involved in the absorption of cholesterol from the intestinal tract and the formation of foam cells in coronary arteries. Therefore, it is presumed that substances that inhibit acyl-coenzyme A cholesterol acyltransferase are effective against such diseases.

[0004] Under these circumstances, it would be useful to provide a substance having acyl-coenzyme A cholesterol acyltransferase inhibitory activity in the treatment of adult diseases such as hyperlipidemia and arteriosclerosis based thereon.

[0005]

[Means for Solving the Problems] As a result of continuing research on metabolites produced by microorganisms, the present inventors discovered that acyl-coenzyme A cholesterol acyl was present in a culture of the FO-1289 strain isolated from fresh soil. It was discovered that a substance with transposase inhibitory activity was produced. Next, an acyl-coenzyme A cholesterol acyltransferase inhibitory active substance was separated and purified from the culture, and as a result, this substance was designated as FO-1289A substance, since no substance having the physical and chemical properties described below was previously known. It was to be. The present invention was completed based on such knowledge, and is based on FO-12 represented by the molecular formula C31H37NO10.
89A substance.

Furthermore, the present invention involves culturing a microorganism belonging to the genus Aspergillus and having the ability to produce the FO-1289A substance in a medium, accumulating the FO-1289A substance in the culture, and producing the FO-1289A substance from the culture. The present invention provides a method for producing a substance FO-1289A, which is characterized by collecting FO-1289A substance.

Microorganisms having the ability to produce the FO-1289A substance (hereinafter referred to as FO-1289A substance-producing bacteria) belong to the genus Aspergillus, and for example, FO-1289, which belongs to the genus Aspergillus and was isolated by the present inventors,
This strain is an example of the strain most effectively used in the present invention, and the mycological properties of this strain are as follows.

The bacterial strain used to produce the FO-1289A substance of the present invention is, for example, Aspergillus sp., which was isolated from soil by the present inventors.
(Aspergillus sp.) FO-1289
Stocks are one example.

I. This strain can be used on malt extract agar, Czapetsk yeast extract agar,
It grows well on a 20% sucrose-Zapetsk yeast extract agar medium, and conidia are well attached to it. When a colony grown on a Tuapetsk yeast extract agar medium is observed under a microscope, the hyphae are transparent and have septated walls, the conidiophores mainly grow straight from the basal hyphae, and their surfaces are smooth.

The tip of the conidiophore swells to form a flask-shaped apical capsule (10-25 μm in diameter). Metoles are not formed, and phialides grow directly on the apical sac. Conidia have a diameter of 2 to 3 μm and are spherical or subspherical in shape.

II. Various properties on 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) When cultured on a Czapek yeast extract agar medium at 37°C for 7 days, the growth state is very vigorous (more than 80 mm). On the other hand, 5
When cultured at ℃ for 7 days, no growth occurred. In all of the above-mentioned media, secretion and sclerotia formation accompanying bacterial growth were not observed.

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

Based on the morphological characteristics, various culture properties, and physiological properties of the above-mentioned FO-1289 strain, we attempted to compare it with known bacterial species.
It was identified as a strain belonging to the genus Aspergillus sp. It was named FO-1289. This strain is Aspergillus sp. FO-1289(As
pergillus sp. FO-1289) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. (
FERM P-12194).

[0016] The FO-1289A producing bacteria have been explained above, but as a general property of the bacteria, the mycological properties are extremely variable and are not constant, and may be affected by natural or normal ultraviolet irradiation or mutated derivatives. , for example N
It is a well-known fact that mutations occur through artificial mutation means using -methyl-N-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc., and not only such artificial mutant strains but also natural mutant strains, Aspergillus All strains belonging to the genus and having the ability to produce the FO-1289A substance can be used in the present invention. In addition, strains mutated by cell engineering such as cell fusion or genetic manipulation are also included as FO-1289A substance producing bacteria.

In the present invention, first, a FO-1289A substance-producing bacterium belonging to the genus Aspergillus is cultured in a medium. In culturing this bacterium, a normal 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 by microorganisms, and, if necessary, an inorganic acid salt is used. Assimilable carbon sources include glucose, sucrose, molasses, dextrin,
Cellulose and the like can be used alone or in combination.

Assimilable nitrogen sources include organic nitrogen sources such as peptone, meat extract, yeast extract, dried yeast, soybean flour, corn staple liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, and urea. Inorganic nitrogen compounds such as salts, nitrates, and ammonium salts are used alone or in combination. In addition, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, and heavy metal salts are added as necessary. Furthermore, micronutrients, growth-promoting substances, precursor substances, etc. that promote the growth of the present fungus and the production of the FO-1289A substance may be appropriately added to the medium, if necessary.

[0019] Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. It is preferable to culture at a pH around neutrality. The culture temperature can be 20 to 37°C,
It is usually best to maintain the temperature at 24 to 30°C. In the case of liquid, the culture time is usually 2 to 3 days, and this FO-128
Since the 9A substance is accumulated, the culture may be terminated when the amount accumulated during culture reaches the maximum.

[0020] These culture conditions, such as medium composition, medium liquid properties, culture temperature, culture speed, and aeration volume, should be adjusted and selected as appropriate to obtain preferable results depending on the type of bacterial strain used and external conditions. Needless to say, it will be done. When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants can be used as appropriate.

[0021] Since the FO-1289A substance accumulated in the culture thus obtained is contained in the bacterial cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the bacterial cells. , it is advantageous to collect the present FO-1289A material from each of them.

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, or benzene, and the extract is concentrated under reduced pressure to obtain crude FO-1289A. Substances are obtained. This crude material was further purified by a known method commonly used for the purification of fat-soluble substances, such as column chromatography using a carrier such as silica gel or alumina to obtain FO-128.
9A substance can be separated and purified.

To collect the FO-1289A substance from bacterial cells, the bacterial cells are extracted with a hydrophilic organic solvent containing water such as aqueous acetone or methanol, the resulting extract is concentrated under reduced pressure, and the concentrate is diluted with acetic acid. Extraction is performed with a non-hydrophilic organic solvent such as ethyl, butyl acetate, or benzene, and the resulting extract is separated and purified by combining it with the extract obtained from the culture filtrate, or
Alternatively, the FO-1289A substance can be separated and purified by the same method described above.

Next, the physical and chemical properties of the F-1289A substance of the present invention will be described. (1) Molecular formula; C31H37NO10 (m/z 583 observed in high-resolution spectrum) (2) Molecular weight; 583 (m/z 58 from mass spectrum)
3 (M+) was observed) (3) Specific optical rotation; [α]22D = +73.2 (C = 1
, chloroform) (4) Ultraviolet absorption spectrum (in methanol); as shown in Figure 1 (5) Infrared absorption spectrum (in carbon tetrachloride); as in Figure 2

(6) Solubility in solvent; methanol,
Soluble in ethanol, acetonitrile, ethyl acetate, benzene, insoluble in water (7) Basic, acidic, neutral; neutral (8) Color and shape of substances; yellow powder (9) Proton nuclear magnetic resonance spectrum ( in deuterated chloroform); as shown in Figure 3.

Next, the biological properties of the FO-1289A substance of the present invention will be described. (1) Inhibitory effect on rat-derived acyl-coenzyme A cholesterol acyltransferase The effect on acyl-coenzyme A cholesterol acyltransferase activity was determined using 300 μM [1-14C] Oleoyl using crude enzyme prepared from rat liver microsomal fraction.
-CoA; 20 μl each of 3 mg/ml cholesterol
(0.02 μCi); 6.67 μl added, 37°C
The total lipids were extracted with a chloroform:methanol (2:1) mixture, followed by TLC (Kieselgel G
After separating each lipid using F254 (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 using a liquid scintillation counter. Reduces enzyme activity by 50%
The inhibitory concentration was calculated to be 0.03 μg/ml for the FO-1289A substance.

[0027]

Effects of the Invention As described above, FO-1289 of the present invention
Since Substance A exhibits significant inhibitory activity against acyl-coenzyme A cholesterol acyltransferase, it is useful for the prevention and treatment of diseases caused by cholesterol accumulation in humans.

Next, the present invention will be specifically explained with reference to Examples.

[Example] 0.0 g of glucose in a 500 ml Erlenmeyer flask.
1%, 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 was plugged with cotton, steam sterilized, and grown on an agar medium. FO-1289(
FERM P-12194) was aseptically inoculated using a platinum loop, and cultured with shaking at 27°C for 48 hours to obtain a seed culture.

On the other hand, glucose 1.0%, tryptone 0.5%, yeast extract 0.3%, malt extract 0.3%, agar 0.1% (p
After steam sterilization and cooling, 200 ml of the seed culture solution was aseptically transplanted and stirred at a stirring speed of 25.
The mixture was aerated and stirred at 27° C. for 72 hours under culture conditions of 0 rpm and an aeration rate of 10 l/min.

After culturing, 30 liters of the culture solution was diluted with 18 liters of ethyl acetate.
The extract was concentrated under reduced pressure to obtain a crude product. This crude product was mixed with silica gel (250 g, manufactured by Merck & Co., Ltd., Art
．． 9385) column, and column chromatography was performed by eluting with chloroform-methanol (99:1). Each fraction was divided into 100 ml fractions, and the fractions containing the active ingredient were collected and dried under reduced pressure to obtain 1.5 g of the crude active substance.

[0031] This was separated and purified by high performance liquid chromatography in 5 parts. The device used was Trirotor V (manufactured by JASCO Corporation), and the column was YMC-Pack A-.
343 (ODS resin, manufactured by Yamamura Chemical Research Institute), the solvent system was 55% acetonitrile water, and the detection was by UV.
The wavelength was 280 nm and the flow rate was 8 ml/min. As a result, F
50 mg of O-1289A material was isolated.

[Brief explanation of the drawing]

Claims (5)

[Claims] Claim 1: FO-12 having the following physical and chemical properties
89A substance. (1) Molecular formula; C31H37NO10 (m/z 583 observed in high-resolution spectrum) (2) Molecular weight; 583 (m/z 58 from mass spectrum)
3 (M+) was observed) (3) Specific optical rotation; [α]22D = +73.2 (C = 1
, chloroform) (4) Ultraviolet absorption spectrum (in methanol); as shown in Figure 1 (5) Infrared absorption spectrum (in carbon tetrachloride); as shown in Figure 2 (6) Solubility in solvents; methanol, ethanol,
Soluble in acetonitrile, ethyl acetate, benzene, insoluble in water (7) Basic, acidic, neutral; neutral (8) Color and shape of substance; yellow powder Claim 2: Belongs to the genus Aspergillus and FO-12
A method for producing an FO-1289A substance, which comprises culturing a microorganism capable of producing an 89A substance in a medium, accumulating the FO-1289A substance during the culture, and collecting the FO-1289A substance from the culture. Claim 3: Belongs to the genus Aspergillus and FO-12
A microorganism that has the ability to produce 89A substance is Aspergillus sp. FO-1289 (Aspergil
lus sp. FO-1289 FERM P-
12194).The manufacturing method according to claim 2. Claim 4: Belongs to the genus Aspergillus and FO-12
Microorganisms that have the ability to produce 89A substances. [Claim 5] The microorganism is Aspergillus sp. The microorganism according to claim 4, which is FO-1289.