JPH04360894A - Fo-1513a substance and production thereof - Google Patents

Abstract

PURPOSE:To obtain the title new substance useful for preventing and treating diseases such as hyperlipemia or arteriosclerosis caused by accumulation of cholesterol because of possession of extremely inhibitory activity against acylcoenzyme A cholesterol acyltransferase. CONSTITUTION:FO-1,513 substance having the following properties. Molecular formula: C45H82O5. Molecular weight: 702. Solubility: soluble in methanol, benzene, etc.: insoluble in water. Distinction of basicity, acidity and neutrality: neutrality. Color and property: colorless oily state. The substance is prepared by culturing a fungus [e.g. Gliocladium sp. FO-1,513 (FERM P-12,220), new fungus] belonging to the genus Gliocladium, capable of producing FO-1,513A substance and collecting the substance from the culture mixture. The culture is preferably carried out under an aerobic condition at pH near neutrality at 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-1513A 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 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 found that acyl-coenzyme A cholesterol acyltransferase was present in a culture of the FO-1513 strain isolated from fresh soil. It was found that a substance with 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-1513A substance, since no substance having the physicochemical properties described below was previously known. It was to be. The present invention was completed based on this knowledge, and is based on FO-15 represented by the molecular formula C45H82O5.
13A substance.

Furthermore, the present invention involves culturing a microorganism belonging to the genus Gliocladium and having the ability to produce the FO-1513A substance in a medium, accumulating the FO-1513A substance in the culture, and extracting FO-1513A from the culture. The present invention provides a method for producing FO-1513A substance, which comprises collecting FO-1513A substance.

Microorganisms that have the ability to produce the FO-1513A substance (hereinafter referred to as FO-1513A substance-producing bacteria) belong to the genus Gliocladium. -15
Strain No. 13 is an example of the strain most effectively used in the present invention, and the mycological properties of this strain are as follows.

[0008] Examples of the bacterial strain used to produce the FO-1513A substance of the present invention include Gliocladium sp., which was isolated from soil by the present inventors. (Gliocladium sp.) FO-15
There are 13 stocks listed. This strain shows the culture findings shown in Table 1. This finding is the result of culturing on various media at 25°C for 14 days and observing.

[0009]

[Table 1]

[0010] The growth temperature range of strain FO-1513 is 10°C.
~32°C, with an optimal temperature range of 16.5°C to 27°C. In addition, the growth pH range is pH 3 to 9, and the optimal pH range is p
It is H5-7. No sclerotia formation or chlamydospores accompanying bacterial growth were observed in any of the above-mentioned media.

[0011] The conidiophore is almost smooth and has a size of 80 to 200 x 4 to 6 microns, and forms a penicillium at its tip. Penicilli exhibit a symmetrical or asymmetrical body, and there are usually 2 to 4 penicilli, and the size is 15 to 25 ×
It is 2 to 3 microns. The shape of Fireride is an elongated pen tip. The shape of the conidia is oval, and the size is 3 to 7 x 3 microns.The conidia stick together and form a sticky ball with a diameter of 20 to 40 microns, forming a mass at the tip of the metre. do.

Based on the culture findings, morphological characteristics, etc. of the above-mentioned strain FO-1513, we attempted to compare it with known bacterial species, and as a result, we identified this strain as a strain belonging to the genus Gliocladium. Radium Sp. It was named FO-1513. This bacterial strain is Gliocladium sp. It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FO-1513. (FERM P
-12220).

[0013] The bacteria producing the FO-1513A substance 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 caused by natural or normal ultraviolet irradiation or mutation. derivatives such as N-methyl-N-nitro-N-nitrosoguanidine,
It is a well-known fact that mutations occur through artificial mutation methods using ethyl methanesulfonate, etc., and such artificial mutant strains as well as natural mutant strains belong to the genus Gliocladium and produce the FO-1513A substance. Any strain that has the ability to do so 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-1513 substance-producing bacteria.

In the present invention, first, FO-1513A substance-producing bacteria belonging to Gliocladium are 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 are 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 this fungus and the production of the FO-1513A substance may be appropriately added to the medium, if necessary.

[0016] Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. The pH of the culture is preferably near neutral. The culture temperature may be 20 to 37°C, but it is usually best to maintain the culture at 24 to 30°C. In the case of liquid, the culture time is usually 2 to 3 days, and this FO-1513
Since Substance A is accumulated, the culture may be terminated when the amount accumulated during culture reaches the maximum.

[0017] Culture conditions such as medium composition, medium liquid properties, culture temperature, 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. When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants can be used as appropriate.

Since the FO-1513A 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-1513A material from each of them.

To collect the FO-1513A 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-1513A. 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 FO1513, respectively.
Substance A can be separated and purified.

To collect the FO-1513A 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-1513A substance can be separated and purified by the same method as described above.

Next, the physical and chemical properties of the F-1513A substance of the present invention will be described. (1) Molecular formula; C45H82O5 (m/z 630 (M-4H2 O)+ was observed in the high-resolution spectrum) (2) Molecular weight; 702 (m/z from the FAB mass spectrum)
/z703(M+1)+ was observed) (3) Specific optical rotation; [α]22D = +0.4 (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 solvent; methanol,
Soluble in ethanol, acetonitrile, ethyl acetate, benzene, insoluble in water (7) Basic, acidic, neutral; neutral (8) Color and shape of substances; colorless oil (9) Proton nuclear magnetic resonance spectrum ( in deuterated chloroform); as shown in Figure 3.

Next, the biological properties of the FO-1513A substance of the present invention will be described. 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 mixture was allowed to react for 30 minutes, and the total lipids were extracted with a chloroform:methanol (2:1) mixture.

After extraction, TLC (Kieselgel GF254
, petroleum ether as developing solvent: diethyl ether:
After separating each lipid with acetic acid (90:10:1), the cholesterol ester fraction was scraped off, and acyl coenzyme A cholesterol acyltransferase activity was measured using a liquid scintillation counter. As a result of calculating the concentration that inhibits this enzyme activity by 50%, the FO-1513A substance was 25 μg.
/ml.

[0025]

Effects of the Invention As described above, FO-1513 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 Gliogradium sp., which was grown on an agar medium after being steam-sterilized after being plugged with cotton. FO-1513
(FERM P-12220) 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, 1.0% glucose, 3.0% soluble starch, 2.0% soybean flour, 0.3% dry yeast, 0 potassium chloride in one 50l jar fermenter.
．． 3%, calcium carbonate 0.2%, magnesium sulfate 0
．． 05%, potassium phosphate 0.5%, 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 (9:1). Each fraction was divided into 100 ml,
Fractions containing the active ingredient were collected and dried under reduced pressure to obtain 1.5 g of crude active substance.

[0029] This was divided into 5 times and separated and purified by high performance liquid chromatography. 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 85% 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
200 mg of O-1513A material was isolated.

[Brief explanation of the drawing]

Claims (5)

[Claims] Claim 1: FO-15 having the following physical and chemical properties
13A substance. (1) Molecular formula; C45H82O5 (m/z 630 (M-4H2 O)+ was observed in the high-resolution spectrum) (2) Molecular weight; 702 (m/z from the FAB mass spectrum)
/z703(M+1)+ was observed) (3) Specific optical rotation; [α]22D = +0.4 (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; colorless oil Claim 2: Belongs to the genus Gliocladium, and FO-1
A method for producing a FO-1513A substance, which comprises culturing a microorganism capable of producing a 513A substance in a medium, accumulating the FO-1513A substance during the culture, and collecting the FO-1513A substance from the culture. Claim 3: Belongs to the genus Gliocladium, FO-1
A microorganism that has the ability to produce 513A substance is Gliocladium sp. FO-1513 (Gliocl
adium sp. FO-1513 FERM
P-12220).The manufacturing method according to claim 3. Claim 4: Belongs to the genus Gliocladium, and FO-1
Microorganisms capable of producing 513A substances. [Claim 5] The microorganism is Gliocladium sp. The microorganism according to claim 5, which is FO-1513.