JP2872311B2 - FO-608B, C substance and method for producing the same - Google Patents

Description

The present invention relates to a novel substance FO-608 having acylcoenzyme A cholesterol acyltransferase inhibition and a method for producing the same.

[Conventional technology]

Heretofore, several hyperlipidemic drugs have been known, but no effective substance has been obtained yet.

[Problems to be solved by the invention]

In recent years, symptoms resulting from cholesterol accumulation such as hyperlipidemia and arteriosclerosis in adults have been regarded as a modern illness 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 this acyl transfer reaction is acylcoenzyme A enzyme 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, substances that inhibit acyl coenzyme A cholesterol acyltransferase
It is presumed to be effective for such diseases.

Under such circumstances, providing a substance having acylcoenzyme A cholesterol acyltransferase inhibitory activity is useful for treating adult diseases such as hyperlipidemia and arteriosclerosis based on it.

[Means for solving the problem]

The present inventors have continued their research on metabolites produced by microorganisms. As a result, a substance having acylcoenzyme A cholesterol acyltransferase inhibitory activity was produced in a culture of FO-608 strain newly isolated from soil. Found to be. Next, an acyl coenzyme A cholesterol acyltransferase inhibitory active substance was separated and purified from the culture, and as a result, no substance having such a chemical structure has been known at all. FO-608C material.

The present invention has been completed based on this finding, FO-608B substance represented by FO-60 selected from the group consisting of FO-608C substances represented by
8 substances or salts thereof are provided.

Further, the present invention further comprises culturing a microorganism belonging to the genus Penicillium and having the ability to produce FO-608B and FO-608C substances in a medium to accumulate FO-608B and FO-608C substances in the culture, FO-608B substance and FO-60
FO-608B substance characterized by collecting 8C substance and
The present invention provides a method for producing a FO-608C substance or a salt thereof.

Microorganisms capable of producing FO-608B and FO-608C substances (hereinafter referred to as FO-608 substance producing bacteria)
The FO-608 gold strain belonging to the genus Penicillium but belonging to the genus Penicillium isolated by the present inventors is an example of the most effectively used gold strain of the present invention. Is as follows.

Examples of the strain used for producing the FO-608B substance and the FO-608C substance (hereinafter, collectively referred to as FO-608 substance) of the present invention include, for example, those isolated from soil by the present inventors. Penicillium sp. (Penicill
ium sp.) FO-608 strain.

I. Morphological properties This strain is composed of wort agar medium, potato / glucose agar medium, Tzapek agar medium, oatmeal agar medium, Yp
It grows relatively well on Ss agar medium, etc., and has good conidiation. When observing the colony grown on Fapec agar medium with a microscope, the hyphae are transparent and have septum,
The conidiophores grow straight from the basal hyphae, and the surface is smooth. Penicillus is a bicyclic-symmetric type composed of metre and phialide. The size of metre is 9-11 × 2
3-6 plants grow at ~ 2.5μ. Fialide is a nib type and grows in groups of 3 to 6, and the size is 10 to 14 × 0.9 to 1.7 μm.

Initially, a phiaroid conidium is placed at the top of the phialide.
Individually, they grow into a chain as the culture time elapses, and finally this chain reaches around 150 μm. Observation with an electron microscope shows that the conidium is elliptical and has a size of 2.5 to 3 × 1.8.
2.22.2 μm, the surface of which is smooth.

II. Properties of Culture (1) Table 1 shows the results of macroscopic observation when cultured at 25 ° C for 12 days on various media.

(2) When cultured in the above medium at 37 ° C. for 12 days, the growth state was repressive (colony diameter 20 to 30 mm), the hypha did not spread, and was velvety. The growth state when cultured at 5 ° C for 12 days is extremely suppressed (less than 10mm)
And conidium did not form.

No secretion fluid or sclerotium formation was observed with the growth of the bacteria in all of the above media.

III. Physiological and ecological properties (1) Optimum growth conditions The optimum growth conditions for this strain are determined in wort agar medium at pH
4-7, temperature 18-33 ° C.

(2) Range of growth The growth range of the strain is from pH 2 to wort agar medium.
9. The temperature is 15-39 ° C.

(3) Distinguishing between aerobic and anaerobic aerobic Among the above properties, morphological observation revealed that this strain belongs to the genus Penicillium.

The strain was Penicillium sp. FO-608 (Pe
nicillium sp. FO-608). (FERM P-10776
issue).

Above, the FO-608B substance and FO-608C substance producing bacteria have been described, but as a general property of the bacteria, the mycological properties are extremely liable to vary, are not constant, and are naturally or normally irradiated with ultraviolet light. Or a mutated derivative such as N-methyl-N-nitro-N-nitrosoguanidine,
It is a well-known fact that mutations are made by artificial mutation using ethyl methanesulfonate or the like, and such artificial mutants belong to the genus Penicillium, including naturally occurring mutants, as well as FO-608B substances and FO-608C. Any strain capable of producing the 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-608 substance-producing bacteria.

In the present invention, first, FO-60 belonging to penicillium
The 8B substance and the FO-608C substance producing bacterium are cultured in the 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 or the like is used alone or in combination. Nitrogen sources that can be assimilated include peptone, meat extract, yeast extract, dried yeast, soy flour, corn stap liquor,
Organic nitrogen sources such as cottonseed meal, casein, soybean protein hydrolysate, amino acids and urea, and inorganic nitrogen compounds such as nitrates and ammonium salts are used alone or in combination. In addition, sodium salts, potassium salts, calcium salts, magnesium salts, inorganic salts such as phosphates, and heavy metal salts are added as needed. A micronutrient that promotes the production of -608B substance and FO-608C substance, a growth promoting substance, a precursor, and the like may be appropriately added.

Culture is preferably performed 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, but usually 24-30 ° C
Good to keep. When the culture time is a liquid, the FO-608B and FO-608C substances are accumulated when culturing is usually performed for 2 to 3 days. When the accumulated amount during the culturing reaches the maximum, the culturing may be terminated. . The culture conditions such as the culture medium composition, the liquid properties of the culture medium, the culture temperature, the culture rate, and the aeration rate are appropriately adjusted and selected so as to obtain preferable results depending on the type of the strain used, external conditions, and the like. 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-608B and FO-608C substances accumulated in the culture thus obtained are contained in the cells and in the culture filtrate, the culture is separated by centrifugation into the culture filtrate and the cells. It is advantageous to collect the FO-608B and FO-608C materials from each.

To collect FO-608 and FO-608C substances from the culture filtrate, first extract the culture filtrate with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, and benzene, and concentrate the extract under reduced pressure to obtain crude FO- -608B material and FO-608C material are obtained. The crude substance is further purified by a known method generally used for purification of a fat-soluble substance, for example, column chromatography using a carrier such as silica gel or alumina.
608B substance and FO-608C substance can be separated and purified.

To collect the FO-608B and FO-608C substances 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. Is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, benzene, etc., and the obtained extract is separated and purified together with the extract obtained from the above-mentioned culture filtrate, or FO is prepared by the same method as described above. -608B substance and FO-608C substance can be separated and purified.

Next, the physicochemical properties of the FO-608B substance and the FO-608C substance of the present invention will be described.

[1] FO-608B substance (1) Molecular formula: C ₂₃ H ₂₆ O ₇ (with a high resolution spectrum m / z 414
(2) Molecular weight: 414 (m / z 414 (M ⁺ ) was observed from the mass spectrum) (3) Specific rotation: ▲ [α] ²⁰ _D ▼ = +355.6 (C = 1, (Chloroform) (4) Ultraviolet absorption spectrum (in ethanol): as in FIG. 1 (5) Ultraviolet absorption spectrum (in carbon tetrachloride): as in FIG. 3 (6) Solubility in solvent: methanol, ethanol, acetonitrile, Soluble in ethyl acetate and benzene, insoluble in water (7) Basic, acidic, and neutral: neutral (8) Color and shape of substance: yellow powder (9) Proton nuclear magnetic resonance spectrum (in deuterated chloroform) : As shown in Fig. 5 (10) Chemical structure: [2] FO-608C substance (1) Molecular formula: C ₂₃ H ₂₆ O ₇ (m / z 412 in high resolution spectrum
(2) Molecular weight: 412 (m / z 412 (M ⁺ ) was observed from the mass spectrum) (3) Specific rotation: ▲ [α] ²⁰ _D ▼ = +476.2 (C = 1, (Chloroform) (4) Ultraviolet absorption spectrum (in ethanol): as in FIG. 2 (5) Infrared absorption spectrum (in carbon tetrachloride): as in FIG. 4 (6) Solubility in solvent: methanol, ethanol, acetonitrile, Soluble in ethyl acetate and benzene, insoluble in water (7) Basic, acidic, and neutral: neutral (8) Color and shape of substance: yellow powder (9) Proton nuclear magnetic resonance spectrum (in deuterated chloroform) : As shown in Fig. 6 (10) Chemical structure: Next, the biological properties and toxicity of the FO-608B and FO-608C substances of the present invention will be described.

(1) rats from acyl-coenzyme A cholesterol acyl rearrangement effect on the inhibitory activity acyl-coenzyme A cholesterol acyltransferase activity against the enzyme using a crude enzyme prepared from rat liver microsome fraction 300μM ^[1-14 C] oleoyl-CoA; 3 mg /
ml cholesterol 20μ (0.02μCi) each; 6.67μ
After adding and reacting at 37 ° C. for 30 minutes, the total lipid is extracted with a mixed solution of chloroform: methanol (2: 1), and then TLC (Kieselgel GF ₂₅₄ , petroleum ether: diethyl ether: acetic acid as a developing solvent, 90: 10: 1) After separating each lipid in the above, 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 by 50% is 50 μg / ml for the FO-608B substance, and
The 608C material was at 52 μg / ml.

(2) Toxicity 100 mg / kg of each of the FO-608B substance and the FO-608C substance was intraperitoneally administered to the mice, but no change in toxicity was observed.

As described above, the FO-608B substance and FO-608C of the present invention
Since the substance has low toxicity and shows remarkable inhibitory activity against acyl coenzyme A cholesterol acyltransferase, it is considered to be useful for prevention and treatment of diseases caused by cholesterol accumulation in humans.

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

〔Example〕

0.1% glucose, starch 2.
Medium containing 4%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4% (pH 7.
100 ml), and after sterilization with a cotton plug, steam-sterilized and grown on an agar medium Penicillium sp. FO-608 (FE
RM P-10776) is aseptically inoculated with a platinum loop, and
A seed culture was obtained by shaking culture for 8 hours.

On the other hand, glucose is added to one 50 jar fermenter.
0%, glycerol 3%, peptone 0.5%, sodium chloride 0.2%, agar 0.1% (adjusted to pH 7.0), steam sterilized and cooled, then aseptically transplanted with 200 ml of seed culture, and stirred Under culture conditions of 250 rpm and aeration rate of 10 / min, 27
The mixture was stirred under aeration at 44 ° C. for 44 hours.

After the culture, the culture 30 was extracted with ethyl acetate 18, and the extract was concentrated under reduced pressure to obtain a crude product. This crude product was charged into a column of silica gel (250 g, Merck, Art.9385) and subjected to column chromatography eluting with n-hexane-ethyl acetate (4: 1 to 2: 1). Each fraction is
Fractionation was performed in 100 ml portions, the fraction containing the active ingredient was thickened, and dried under reduced pressure to obtain 1.5 g of a crude active substance. This was separated into 5 times and separated and purified by high performance liquid chromatography. The device used was Trirotor V (manufactured by JASCO Corporation), the column used was YMC-Pack A-343 (ODS resin, manufactured by Yamamura Chemical Laboratory), the solvent system was 65% acetonitrile water, and the detection was UV280 nm. The flow rate was 8 ml / min. As a result F
40 mg of O-608B substance and 550 mg of FO-608C substance were isolated.

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of the FO-608B substance, FIG. 2 shows an ultraviolet absorption spectrum of the FO-608C substance, and FIG.
FIG. 4 shows the infrared absorption spectrum of the FO-608C substance, FIG. 5 shows the proton nuclear magnetic resonance spectrum of the FO-608B substance, and FIG. 6 shows the proton nuclear magnetic resonance spectrum of the FO-608C substance. Show.

Continuation of the front page (51) Int.Cl. ⁶ identification code FI C12R 1:80) (C12P 17/04 C12R 1:80) (58) Investigated field (Int.Cl. ⁶ , DB name) C07D 307/94 C12P 17/00-17/18 C12N 1/14 REGISTRY (STN) CA (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] (1) Expression FO-608B substance and formula represented by FO-60 selected from the group consisting of FO-608C substances represented by
8 substances or their salts. 2. A microorganism belonging to the genus Penicillium described in claim 1 and having an ability to produce FO-608B and FO-608C substances is cultured in a medium, and FO-60 is cultured during the culture.
8B substance and FO-608C substance were accumulated and
A method for producing a FO-608B substance and a FO-608C substance or a salt thereof, comprising collecting a FO-608B substance and a FO-608C substance. 3. A microorganism belonging to the genus Penicillium described in claim 1 and having an ability to produce a FO-608B substance and a FO-608C substance, is Penicillium sp. FO-.
608 (Penicillium sp. FO-608 FERM P-10776). 4. A microorganism belonging to the genus Penicillium and capable of producing FO-608B and FO-608C substances is Penicillium sp. FO-608 (Penicillium sp. FO-6).
08 FERM P-10776).