JPH0352884A - Fo-608a substance and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula having the following physical and chemical properties. Molecular formula: C23H26O7 (observed by high- resolution spectrum at 414m/z). Molecular weight: 414 (mass spectrum). Specific rotatory power: [alpha] =-57.6 (c=1, chloroform). Solubility: soluble in methanol, ethanol and benzene and insoluble in water. Slightly acidic. Color and shape: white powder, etc. USE:Useful for preventing and treating diseases caused by human cholesterol accumulation. Having inhibitory action on acylcoenzyme A cholesterol transferase. PREPARATION:A fungus such as Penicillium sp. FO606 (FERM P-10776) is cultured preferably in neutrality at 24-30 deg.C for 3-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention pertains to the FO-608A substance and its manufacturing method. More specifically, FO-, a new substance with acyl coenzyme A cholesterol acyl transferase inhibitory
608A substance and its manufacturing method.

[Conventional technology]

Although several antihyperlipidemic drugs have been known in the past, no effective substance has yet been obtained.

[Problem R that the invention attempts to solve]

In recent years, as dietary habits have improved, symptoms caused by cholesterol accumulation, such as hyperlipidemia and arteriosclerosis, have become a problem as modern diseases. Cholesterol becomes cholesterol ester through acyl group rearrangement of acyl coenzyme A, and is accumulated in cells and in blood riboproteins. The enzyme that catalyzes this acyl group transfer reaction is acylco-A cholesterol acyltransferase, which is deeply involved in the absorption of cholesterol from the intestinal tract and the formation of foam terminal cells in coronary arteries. Therefore, it is presumed that substances that inhibit acyl-coenzyme A cholesterol acyltransferase are effective against such diseases. Under these circumstances, providing a substance having acyl-coenzyme A cholesterol acyltransferase inhibitory activity is useful in the treatment of hyperlipidemia and Kaito's diseases such as arteriosclerosis based thereon.

[Means to solve the problem]

As a result of continuing research on metabolites produced by microorganisms, the present inventors discovered that a substance with acyl-coenzyme A cholesterol acyltransferase inhibitory activity was produced during cultivation of the FO-608 strain newly isolated from soil. I found out. Then, the azircoenzyme A is extracted from the culture.
As a result of separating and purifying the cholesteryl acyltransferase inhibitory activity, we decided to name this substance FO-608A because no substance with such a chemical structure was previously known. The present invention was completed based on this knowledge, and relates to a new substance, FO-608A substance, represented by the formula. Furthermore, the present invention belongs to the genus Penicillium, F.
A novel product 1iFo-608A characterized in that a microorganism capable of producing an O-608A substance is cultured in a medium, FO-608A is accumulated in the culture, and the FO-608A substance is collected from the culture. It provides methods for producing substances or their salts. Microorganisms capable of producing FO-608A substances (hereinafter referred to as FO-60
8A substance producing bacteria> belongs to the genus Penicillium, and for example, the FO-608 strain belonging to the genus Benicillium isolated by the present inventors is an example of a strain most effectively used in the present invention, The mycological properties of this strain are as follows. The bacterial strain used to produce the FO-608A9J material of the present invention includes, for example, Benicillium sp., which was newly isolated from soil by the present inventors.
Penicillium sp. ) FO-608 strain is mentioned. The mycological properties of this strain are as follows.

■． Morphological Properties This strain grows relatively well on wort agar, potato-dextrose agar, Zurbaek agar, oat ξ-le agar, YpSs agar, etc., and has good conidial settlement. ．． When a colony grown on a Zuavec agar medium is observed under a microscope, the hyphae are transparent and have septa, the conidiophores grow straight from the basal hyphae, and their surfaces are smooth. Penicillas is a two-wheeled, symmetrical creature composed of Metre and Phialide. The size of the metre is 9~
11 x 2-2.5μ and 3-6 pieces grow. Phialides are pen-shaped and grow in clusters of 3 to 6 pieces, and the size is 10 to 14X0.
．． It is 9 to 1.7μ. At first, a single phialoid conidium attaches to the apex of the phialide, and as the culture time progresses, it forms a chain, and eventually this chain reaches around 150 μm. When observed with an electron microscope, the conidia are oval in shape and have a size of 2.5 to 3X.
It has a diameter of 1.8 to 2.2μ, and its surface is smooth.

■． Various culture properties <11 Table 1 shows the results of macroscopic observation when cultured on various media at 25° C. for 12 days.

(2) When cultured in the above medium at 37°C for 12 days, the growth state was suppressive (colony diameter, 20-30 mm).
), the hyphae did not spread and were velvety. Also, 5℃
The growth condition when cultured for 12 days was extremely suppressive (less than 10 mm), and no conidia were formed. In all of the above-mentioned media, no secretions or sclerotia accompanying growth of the fungus were observed. ■． Physiological and ecological properties (1) Optimal growth conditions The optimal growth conditions for this strain are wort agar medium with pH
4-7, temperature 18-33°C.

(2) Growth range The growth range of this strain is pH 2 to 9 on wort agar medium.
, the temperature is 15-39°C. (3) Distinction between aerobic and anaerobic Among the various properties beyond aerobic, morphological observation revealed that this strain belongs to the genus Penicillium. This strain is Penicillium sp. FO-608 (Penici
11 1um sp. It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FO-608). (FERM P-10776). That’s all, FO-60
Although we have explained the 8A substance-producing bacteria, the general properties of bacteria are that their mycological properties are extremely variable and not constant, and that they are susceptible to natural or commonly used ultraviolet irradiation or mutated derivatives, such as N-methyl-N. -Nitoro N-
It is a well-known fact that mutations occur through artificial mutation methods using nitrosoguanidine, ethyl methanesulfonate, etc., and not only such artificial mutant strains but also natural mutant strains belong to the genus Penicillium and the FO-608A substance Any strain that has the ability to produce 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-608A substance-producing bacteria. In the present invention, first, a substance belonging to Penicillium FO
-608A producing bacteria are cultured in a medium. In culturing Honkan, 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 oxidized, and, if necessary, an inorganic salt is used. Assimilable carbon sources include glucose, shi! I-sugar, molasses, dextrin, cellulose, etc. may be used alone or in combination. Digestible nitrogen sources include Bebutone, meat extract, yeast extract, dried yeast, soy flour, corn stave liquor, cottonseed meal, casein, soy protein hydrolyzate, amino acids, organic nitrogen sources such as urea, and nitrates. , inorganic nitrogen compounds such as ammonium salts are used alone or in combination. Other inorganic salts and heavy metal salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and phosphates may be added as necessary. Furthermore, micronutrients, growth-promoting substances, precursor substances, etc. that promote the growth of this bacterium and the production of the FO-608A substance may be added to the medium as necessary. Cultivation is usually carried out under aerobic conditions such as shaking or aerated culture. Industrially, deep aeration agitation culture is preferred. It is preferable to culture at a pH around neutrality. The culture temperature can be 20-37°C, but usually 24-37°C.
It is best to keep it at 30°C. Regarding the culture time, in the case of a liquid, the FO-608A substance is usually produced or accumulated after 3 to 6 days of culture, so the culture should be terminated when the amount accumulated during culture reaches the maximum. ．． These culture conditions such as culture medium composition, medium liquid properties, culture temperature, culture speed, aeration amount, etc. are adjusted and selected as appropriate to obtain favorable 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.

FO-6 accumulated in the cultures thus obtained
Since the 08A substance is contained in the bacterial cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the bacterial cells,
Advantageously, the present material FO-6 0 8A material is taken from each.

To collect the F○-608A substance from the culture filtrate, first extract the culture filtrate with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, or benzene, and concentrate the extract under reduced pressure to obtain the crude substance, FO-608A. Substances are obtained. The crude substance can be further purified to separate and purify the FO-608A substance by a known method commonly used for purifying fat-soluble substances, such as column chromatography using a carrier such as silica gel or alumina. To collect the FO-608A substance from bacterial cells, the bacterial cells are extracted with a hydrophilic organic solvent containing water such as aqueous acetone or methanol, and the resulting extract is concentrated under reduced pressure. Extraction is performed with a non-hydrophilic organic solvent such as butyl or benzene, and the resulting extract is separated and purified by combining it with the extract obtained from the culture filtrate, or by the same method as above.
Substances can be separated and purified. Next, the FO-
6 Describe the physical and chemical properties of the 8A substance. [l) FO-608A substance (1) Molecular formula: Cz+HgiOr (m/z 414 was observed in the high-resolution spectrum) (2) Molecular weight: 414 (m/z 4 from the mass spectrum
(3) Specific optical rotation: (α)D-57.6 (C=1,
(4) Ultraviolet absorption spectrum (in ethanol): As shown in Figure 1 (5) Infrared absorption spectrum (in carbon tetrachloride): As shown in Figure 2 (6) Solubility in solvents: methanol, ethanol, acetonitrile , ethyl acetate, soluble in benzene, insoluble in water (7) Basic, acidic, neutral: acidic (8) Color and shape of substance: white powder (9) Proton nuclear magnetic resonance spectrum (in deuterochloroform) ): aφ chemical structure as shown in Figure 3: Next, the biological properties and toxicity of the F○-608A substance of the present invention will be described. ? The effect on silcoenzyme A cholesterol acyltransferase activity was determined using crude enzyme prepared from rat liver microsomal fraction at 300 μM (1-”C)0 1 e.
o y 1-COA: 3 mg/mj! 20μl each (0.02μCl) of cholesterol i6.67μ
After extracting the cholesterol fraction with chloroform, TLC (Kieselgel GF 4, petroleum ether: diethyl ether: acetic acid as a developing solvent, 90%
The cholesterol fraction was separated using 10:1) and the activity of acyl coenzyme A cholesterol acyltransferase was measured using a liquid scintillation counter. The concentration that inhibits this enzyme by 50% was calculated and the result was 50μg/
It was ml. (2) 100 mg/kg of toxic FO-608A substance
was administered intraperitoneally to mice, but no toxic changes were observed. As described above, the FO-608A substance of the present invention has low toxicity and exhibits significant inhibitory activity against acyl-coenzyme A cholesterol acyltransferase, so it is useful for the prevention and treatment of diseases caused by cholesterol accumulation in humans. It is considered useful.

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

〔Example〕

0.1 glucose in a 500 ml Erlenmeyer flask
%, starch 2.4%, pebtone 0.3%, meat extract 0
．． 100 m of medium containing 3% yeast extract, 0.5% yeast extract, and 0.4% calcium carbonate (adjusted to pH 7.0)
Benicillium S.B. was prepared with It, plugged with cotton, steam sterilized, and grown on an agar medium. FO-608 (F
ERM P-10776) 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 glucose per 30 liter jar fermentor
．． 0%, glycerol 3%, pebtone 0.5%, sodium chloride 0.2%, agar 0.1% (adjusted to p87.0), steam sterilized, cooled, and seed cultured Seed culture solution 2
Transfer 0 0 ml aseptically and stir at 250 rpm.
The cells were cultured with aeration and agitation for 160 hours at 27°C under culture conditions with an aeration rate of 10 l/min. After culturing, the culture solution is centrifuged to separate supernatant 201 and bacterial cells, and the bacterial cells are mixed with 80% acetone water 1.5
1, the extract was concentrated under reduced pressure to about 1 liter, and the concentrated solution was added to the supernatant. This was extracted with 18 liters of ethyl acetate, and the extract was condensed under reduced pressure to obtain 10.3 g of a crude product. This crude product was suspended in 30 mj+ of ethyl acetate, and silica gel (
250 g1 Merck & Co., Art. Column chromatography was performed by charging a column of 9385) and eluting with chloroform. Each fraction was divided into 50 mJ fractions, and fractions containing active fractions were collected and dried under reduced pressure to obtain 1.5 g of crude active substance. 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 YM.
C-Pack A-343 (ODS resin, manufactured by Yamamura Chemical Research Institute) was used, the solvent system was 65% acetonitrile water, detection was at UV 280 nm, and the flow rate was 8 ml.
I went there in minutes. As a result, 60 mg of FO-608A substance was isolated.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the FO-608A substance, Figure 2 shows the infrared absorption spectrum of the substance, and Figure 3 shows the proton nuclear magnetic resonance spectrum of the substance.

Claims (3)

[Claims] (1), Eq. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ FO-608A substance represented by or a salt thereof. (2) A microorganism belonging to the genus Penicillium and having the ability to produce FO-608A substance is cultured in a medium, FO-608A substance is accumulated during the culture, and FO-608A substance is accumulated from the culture.
FO-608 characterized by collecting 608A substance
A method for producing substances or their salts. (3) A microorganism that belongs to the genus Penicillium and has the ability to produce the FO-608A substance is Penicillium sp.
FO-608 (Penicililum sp. FO-6
08FERMP-10776
Manufacturing method described in section.