JPH03197477A - Substances fo-608b and c and its production - Google Patents

Abstract

NEW MATERIAL:Substances FO-608B expressed by formula I and FO-608C expressed by formula II or salts thereof. USE:A treating agent, having inhibitory activity against acylcoenzyme A cholesterol acyltransferase and useful for hyperlipemia and adult diseases such as arteriosclerosis caused thereby. PREPARATION:A mirroorganism, belonging to the genus Penicillium and having the ability to produce the substance FO-608B or FO-608C or both thereof is cultured in a culture medium to accumulate the substance FO-608B or FO-608C or both thereof in the resultant culture. The above-mentioned substances are then collected from the culture. The culture is normally carried out in a nutrient culture medium containing carbon sources, nitrogen sources, etc., capable of being anabolized or assimilated by the microorganism according to a method for culturing fungi.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to novel FO-608 substances that inhibit acyl-coenzyme A cholesterol acyltransferase and a method for producing the same.

[Conventional technology]

Although several hyperlipidemia drugs have been known, no effective substance has been obtained yet.

[Problem 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 riboproteins. The enzyme that catalyzes this acyl group transfer reaction is acylco-A enzyme A.
It is a cholesterol acyltransferase and 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.

Under these circumstances, providing a substance having acyl-coenzyme A cholesterol acyltransferase inhibitory activity is useful in the treatment of adult diseases such as hyperlipidemia and arteriosclerosis that are exposed thereto.

[Failure 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 in a culture of strain F0608, which was newly isolated from soil. I discovered that. Next, an acyl-coenzyme A cholesterol acyltransferase inhibitory substance was isolated and purified from the culture, and as a result, this substance was classified as FO160B substance B, since no substance with such a chemical structure was previously known.
and F0608C substance.

The present invention was completed based on such knowledge, and
The present invention provides FO-608 substances selected from the group consisting of the FO-608B substance represented by the formula and the FO-608C substance represented by the formula or a salt thereof.

Furthermore, the present invention belongs to the genus Penicillium, and FO-608B
FO-608C substance and/or microorganisms capable of producing the FO-608C substance are cultured in a culture medium to form a culture with FO-608C substance.
FO-608B substance and/or FO-608C substance are accumulated from the culture.
-FO-60 characterized by collecting 608C substance
The present invention provides a method for producing 8B substance and /FO608C substance or salts thereof.

Microorganisms having the ability to produce the FO-608B substance and/or the FO-608C substance (hereinafter referred to as FO608 substance-producing bacteria) belong to the genus Penicillium.
The 608 strain is an example of the strain most effectively used in the present invention, and the mycological properties of this strain are as follows.

FO"-6'08B substance and/or FO6 of the present invention
Examples of bacterial strains used to produce 08C substances (hereinafter collectively referred to as FO-608 substances) include Penicillium sp., which was isolated from soil by the present inventors.
p, ) FO-608 strain.

■ Morphological properties This strain grows relatively well on wort agar, potato-dextrose agar, Czapek agar, oatmeal agar, Y p S, S agar, etc., and does not exhibit conidial settlement. is also good. When a colony grown on a Zapek agar medium is observed under a microscope, the hyphae are transparent and have septa, the conidiophores arise directly from the basal hyphae, and their surfaces are smooth. Benicillus is a double-wheeled, monosymmetric type consisting of metho and phialide. The size of the methos is 9-11 x 2-2.5μ and 3-6 pieces are attached. Phialides are pen-shaped and grow in clusters of 3 to 6 pieces, and the size is 10 to 10.
1'4×0.9 to 1°7 μm.

At first, one phialoid conidia settles on 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 under an electron microscope, the conidia are oval in shape, measuring 2.5-3 x 1.8-2.2 μm, and have a smooth surface.

(2) Various properties on culture (],) 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 were not diffused and velvety.

Furthermore, when cultured at 5° C. for 12 days, the growth state was extremely suppressed (10 mm or less), and no conidia were formed.

In all of the above-mentioned media, secretion and sclerotia formation accompanying bacterial growth were not 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 from pH 2 to 2 on wort agar medium.
9. 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., FO608 (
Penicillium sp, FO-608
) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. (FERM l” No. 10776).

The bacteria that produce the FO-608B substance and FO-608C substance have been explained above, but as a general property of bacteria, the properties according to Dutch science are extremely variable and are not constant.
It is a well-known fact that mutations occur naturally or by artificial mutation means using conventional ultraviolet irradiation or mutation derivatives such as N-methyl-Nnitro-N-nitrosoguanidine, ethyl methanesulfonate, etc. Not only artificial mutant strains but also natural mutant strains belong to the genus Penicillium 1, and the FO-608B substance and FO-6
Any strain capable of producing 08C substances can be used in the present invention. In addition, strains mutated through cell engineering such as cell fusion and genetic manipulation can also be used as a substance.
Included as substance-producing bacteria.

In the present invention, first, FO-6 belonging to Penicillium
08B substance and FO-608C substance producing bacteria 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. As assimilable carbon sources, glucose, sucrose, molasses 1, dextrin, cellulose, etc. are used alone or in combination. Nitrogen sources that can be assimilated include peptone, meat extract, yeast extract, dried yeast, soy flour, corn staple,
Organic nitrogen sources such as liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, urea, and inorganic nitrogen compounds such as nitrates and ammonium salts are used alone or in combination. In addition, inorganic salts and heavy metal salts such as thorium salts, potassium salts, calcium salts, magnesium salts, and phosphates are added as necessary.

Furthermore, the culture medium may contain FO-
Micronutrients, growth promoters, precursors, etc. that promote the production of 608B and FO-608C substances may be added as appropriate.

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 20-37°C, but usually 24-37°C.
It is best to keep it at 30°C. Incubation time is usually 2 if using liquid.
When cultured for ~3 days, trees FO-608B and FO-6
Since the 08C substance accumulates, the culture may be terminated when the amount accumulated during culture reaches the maximum. These culture conditions such as medium composition, medium liquid properties, culture temperature, culture speed, and aeration amount 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. Since the PCI-608B substance and FO608C substance accumulated in the culture thus obtained are contained in the bacterial cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the bacterial cells, Zero FO from each
-608B material and FO) -6080 material is advantageously taken.

F, C)-608 substances and FO-608 from culture filtrate
To collect substance C, first, the culture filtrate is 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-608B substance and FO-608C. Substances are obtained. The crude substance can be further separated and purified into the FO-608B substance and the FO-608C 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 PCI-608B and FO-608C substances from bacterial cells, the bacterial cells are extracted with a hydrophilic organic solvent containing water such as aqueous acetone and methanol, and the resulting extract is concentrated under reduced pressure. ethyl acetate, butyl acetate,
Extraction is performed with a non-hydrophilic organic solvent such as benzene, and the resulting extract is combined with the extract obtained from the culture filtrate and separated and purified, or the same method as described above is used to separate and purify each FO)-
608B substance and FO-608C proboscis can be separated and purified.

Next, the FO-608B substance and FO608C of the present invention
Describe the physical and chemical properties of substances.

(1) FO-608B substance (1) Molecular formula: CzsHzbC)r (m/z 414 was observed in the high-resolution spectrum) (2) Molecular weight: 4'14 (m/z 4 from the mass spectrum)
14 (M”) was observed) (3) Specific optical rotation: [α)20-+355.6 (C=
(1° chloroform) (4) Ultraviolet absorption spectrum (in ethanol): As shown in Figure 1 (5) Infrared absorption spectrum (in carbon tetrachloride): As shown in Figure 3 (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 (9) Proton nuclear magnetic resonance spectrum (deuterochloroform (middle): 001 chemical structure as shown in Figure 5: (2) FO-608C substance 111 molecular formula: C2:1H2407 (m/2412 was observed in the high-resolution spectrum) (2) Molecular weight: 412 (m/2412 from the mass spectrum) z4
12 (M”) was observed) (3) Specific optical rotation: [α] Min-1476.2 (C=1
(4) Ultraviolet absorption spectrum (in ethanol): As shown in Figure 2 (5) Infrared absorption spectrum (in carbon tetrachloride): As shown in Figure 4 (6) Solubility in solvents: methanol, ethanol, acetonitrile , ethyl acetate, benzene? Is it bad for water? (7) Distinction between basic, acidic, and neutral: Neutral (8) Color and shape of substance: Yellow powder (9) Proton nuclear magnetic resonance spectrum (in deuterium chloroform): Q19 chemical structure as shown in Figure 6: 5 6 Next, the FO-608B substance and FO608C of the present invention
Describe the biological properties and toxicity of the substance.

The effect on acyl-coenzyme A cholesterol acyltransferase activity was determined using crude enzyme prepared from rat liver microsomal fraction at 300 μM (1-14C)01eoy.
l-CoA; 3mg/m7! 20 each of cholesterol
μJ (0,02μ42ci); 6.67μ! Added 3
React at 7°C for 30 minutes, and convert total lipids to chlorophor J1:
After extraction with a methanol (2:1) mixture, each lipid was separated using TLC (Kieselgel GF2S4, petroleum ether:diethyl ether:acetic acid, 90:10:1 as a developing solvent), and the cholesterol ester fraction was scraped off and subjected to liquid scintillation. Azylcoenzyme A cholesterol acyltransferase activity was measured using a counter. The results of calculating the concentration that inhibits this enzyme by 50% are FO-608
B substance is 50μg/mp, FO-608C substance is 52
μg/m7! Met.

(2) Toxic FO-608B substance and FO-608C substance 10 each
Although 0 mg/kg was administered intraperitoneally to mice, no toxic changes were observed.

As described above, the FO-608B substance of the present invention and FO
-608C substance has low toxicity, acylcoenzyme 1, A
Since it shows remarkable inhibitory activity against cholesterol acyltransferase, it is considered to be 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〕

In a 500mβ Erlenmeyer flask, glucose 0.1%, starch 2.4%, peptone 0.3%, meat extract 0.3%,
Yeast extract 0.5%, calcium carbonate 0.
Penicillium sp., FO608 (FERM P-
10776> was aseptically inoculated using a platinum loop, and incubated at 27℃ for 4 hours.
A seed culture solution was obtained by culturing for 8 hours.

On the other hand, Gluco-7 in one 50r jar fermenter!
, 1.0%, Chrycerol 3%, peptone 0.5%, sodium chloride 0.2%, agar 0.1% (adjusted to pH 7.0), steam sterilized and cooled, and then seed cultured.
00ml was transplanted aseptically, stirring speed 250 rpm,
The mixture was aerated and stirred at 27° C. for 44 hours under culture conditions with an aeration rate of 10 β/min.

After culturing, the culture solution 30β was extracted with 18% ethyl acetate, and 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.

Column chromatography was performed by charging a column of 9385) and eluting with n-hexane ethyl acetate (4:1 to 2:1). Each fraction was divided into 100 mβ 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. This was separated and purified by high performance liquid chromatography in 5 parts. The device used is Trirotor V (manufactured by JASCO Corporation), and the column was YMC-P.
Ack A-343 (ODS resin, Yamamura Kagaku Institute type) was used, the solvent system was 65% acetonitrile water, detection was performed at UV 280 nm, and the flow rate was 8 ml 7 minutes. As a result, 40 mg of FO-608B substance, FO
550 mg of -608C material was isolated.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectra of FO-608'B material.
Figure 2 is the ultraviolet absorption spectrum of the FO-608C material, Figure 3 is the infrared absorption spectrum of the FO-608B material, Figure 4 is the infrared absorption spectrum of the FO-608C material, and Figure 5 is the infrared absorption spectrum of the FO-608B material. Proton Nuclear Magnetic Resonance Spectrum Figure 6 shows the proton nuclear magnetic resonance spectrum of the FO-608C material. 9 2 〇- Unexamined Japanese Patent Publication No. 3 197477 (8n C ward 6ri

Claims (1)

[Claims] (1) Consists of the FO-608B substance represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ and the FO-608C substance represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ FO-608 substance or a salt thereof selected from the group. (2) FO-608B substance and/or FO-60 belonging to the genus Penicillium;
FO-608B substance and/or FO-6 during culture by culturing a microorganism capable of producing 8C substance in a medium.
08C material was allowed to accumulate and FO-608B was removed from the culture.
FO-608B substance and/or FO-608B substance and/or FO-608C substance characterized by collecting the substance and/or FO-608C substance
Method for producing 608C substances or their salts. (3) Microorganisms belonging to the genus Penicillium and having the ability to produce the FO-608B substance and/or the FO-608C substance are Penicillium sp. FO-608 (Pen
icilium sp. FO-608FERMP-10
776. The manufacturing method according to claim 2. (4) A microorganism belonging to the genus Penicillium and having the ability to produce a substance FO-608B and/or a substance FO-608C. (5) The microorganism is Penicillium sp. FO-608
The microorganism according to claim 4.