JPH0815435B2 - OM-4842A, a novel platelet aggregation inhibitor, its production method and its use - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a novel platelet aggregation inhibitor OM-4842A, a blood pressure lowering agent or a thrombosis therapeutic agent, and use as a carcinostatic agent.

[Conventional technology]

Conventionally, antiplatelet active substances are mostly limited to chemically synthesized substances, and it is extremely rare to report that a microorganism of the genus Streptomyces produces a platelet aggregation inhibitor.

[Problems to be solved by the invention]

In recent years, active substances have been actively screened from microbial metabolites. The present inventors isolated strains from various soils for the purpose of searching for novel platelet aggregation inhibitors, and as a result of continuing research on metabolites produced by the strains, platelets were found in the culture of Actinomyces OM-4842 strain. It was found that a substance having an aggregation suppressing activity was produced. Therefore,
The active substance was separated and purified from the culture, and its physicochemical properties were examined.
The substance was designated as platelet aggregation inhibitor OM-4842A. The present invention has been completed based on such findings.

[Means for solving problems]

The present invention belongs to the platelet aggregation inhibitor OM-4842A and Streptomyces having the physicochemical properties described below,
Characterized in that the platelet aggregation inhibitor OM-4842A producing bacterium is cultured in a medium to produce and accumulate the platelet aggregation inhibitor OM-4842A in culture, and the platelet aggregation inhibitor OM-4842A is collected from the culture. New Platelet Aggregation Inhibitor, OM-
A method for producing 4842A and a platelet aggregation inhibitor comprising the platelet aggregation inhibitor OM-4842A as an active ingredient.

Microorganisms having the ability to produce the platelet aggregation inhibitor OM-4842A of the present invention (hereinafter sometimes simply referred to as the substance OM-4842A) belong to the genus Streptomyces, for example, the present inventors have found that Tateyama City, Chiba Prefecture The OM-4842 strain belonging to the genus Streptomyces newly isolated from the soil is an example of the strain most effectively used in the present invention, and shows the mycological properties of this strain as follows. .

(A). Morphological properties Vegetative hyphae develop well on various agar media and no fragmentation is observed. The aerial mycelium abundantly grows on yeast extract / malt extract agar or starch inorganic salt agar, and exhibits a gray or pink system. When observed under a microscope, the aerial mycelium has a spiral shape and 20 or more spore chains are observed. The size of spores is 1.1 × 0.7μ, and it is cylindrical. The surface of spores is smooth. No sclerotia, sporangia and zoospores are found.

(B). Properties on various media EB Shirling and D
The method of D. Gottlieb (Internals of Sistimatic Bacteriology, 16
Vol. 1, p. 313, 1966) shows the culture properties of the presently produced strain. The color tone was determined using the Color Harmony Manual Fourth Edition (Container Corporation of America Chicago, 1958) as the standard color, and the code is also shown in parentheses along with the name of the color chart. The following are the results of observation in each medium at 27 ° C. for 2 weeks unless otherwise specified.

(C) Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar; positive (b) Peptone-yeast iron agar; positive (c) Glucose-peptone-gelatin medium (21-23)
℃); Positive (d) Tryptone-Yeast solution; Negative (2) Tyrosinase reaction; Positive (3) Hydrogen sulfide production; Negative (4) Nitrate reduction; Positive (5) Gelatin liquefaction (21-23 ° C) ( (Glucose / peptone / gelatin medium); Negative (6) Hydrolysis of starch; Positive (7) Coagulation of skim milk (37 ℃); Negative (8) Peptonization of skim milk (37 ℃); Positive (9) Growth temperature Range 15-41 ° C (d) Utilization of carbon source (Pleedam Gottlieb agar medium) Utilized; glucose, raffinose, melibiose,
D-mannitol, D-fructose, L-rhamnose, i-inositol, sucrose Some use; D-xylose not used; L-arabinose cellulose degradation; negative (e) Cell wall composition The cell wall diaminopimelic acid is LL type .

The following is a summary of the mycological properties of this bacterium. Diaminopimelic acid in the cell wall is LL type. The aerial hyphal morphology is helical and forms long spore chains. The surface of spores is smooth. As the properties of the culture, the vegetative mycelium has a brown or wine color tone, and the aerial mycelium has a gray or pink color tone. A brown dye is produced as a soluble dye.

From these results, this strain belongs to the genus Streptomyces and is classified into Pridham and Tresner (Virgin's Manual of Determinant Bacteriology, 8th edition, 748-829, 1974). It is considered to be a bacterial species belonging to the Gray or Red series according to.

This strain is Streptomyces sp. OM-
4842 (Streptmyces sp. OM-4842) has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology
No. 9427).

The OM-4842A-producing bacterium of this substance has been described above.
As a general property of actinomycetes, the bacteriological properties are extremely variable and are not constant, and UV irradiation, X-ray irradiation, or a mutagenesis agent such as N, which is carried out naturally or normally, is used.
It is a well-known fact that mutation is carried out by an artificial mutagenesis method using -methyl-N-nitro-N-nitrosoguanidine, ethyl methanesulfonate and the like. Such strains that belong to the genus Streptomyces and have the ability to produce the substance OM-4842A can be used in the present invention, including naturally occurring mutants, as well as such artificial mutation means.

In the present invention, first, the OM-4842A-producing bacterium belonging to the genus Streptomyces is cultured in an appropriate medium. In culturing the bacterium, actinomycete culture 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 digested, and if necessary, an inorganic salt is cultured. As the assimilable carbon source, glucose, molasses, starch, dextrin, cellulose, corn steep liquor, glycerin, organic acid or the like is used alone or in combination. As the digestible nitrogen source, peptone, meat extract, yeast extract, dry yeast, soybean powder, corn steep liquor, cottonseed meal, casein, soybean protein hydrolyzate, amino acids, organic nitrogen sources such as urea, nitrates, Inorganic nitrogen compounds such as ammonium salts are used alone or in combination. In addition, if necessary, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate are added. Further, if necessary, a micronutrient, a growth promoting substance, or a precursor substance that promotes the growth of the bacterium and the production of the substance OM-4842A may be appropriately added to the medium.

The medium is usually preferably aerated under aerobic conditions such as shaking or aeration-agitation culture. Industrially, deep aeration stirring culture is preferred. The pH of the medium is preferably slightly acidic or around neutral. The culturing temperature is in the range of 20 to 40 ° C., but it is usually maintained at 24 to 30 ° C., preferably around 27 ° C.

In the case of liquid culture, the culture time may be usually 1 to 8 days, but preferably the culture is terminated when the accumulated amount of the platelet aggregation inhibitor during culture reaches the maximum. It goes without saying that culture conditions such as medium composition, medium liquidity, medium temperature, stirring speed, and aeration amount are appropriately adjusted and selected so as to obtain preferable results depending on the type of strain to be used and external conditions. Nor. When the liquid is foamed, a defoaming agent such as silicone oil, vegetable oil, or a surfactant is used appropriately. Platelet aggregation inhibitor OM-4842A accumulated in the culture thus obtained is contained in the microbial cells and the culture filtrate, and thus centrifuged to separate the culture filtrate and the microbial cells from each. It is advantageous to collect the present platelet aggregation inhibitor OM-4842A.

To collect OM-4842A from the culture filtrate, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate or benzene. The obtained extract or eluate is concentrated under reduced pressure, and the crude substance obtained is further subjected to a known method usually used for purification of a fat-soluble substance, for example, column chromatography using a carrier such as silica gel or alumina to obtain the substance OM- 4842A
Can be separated and purified.

To collect this substance OM-4842A from the bacterial cells, the bacterial cells are extracted with a hydrous hydrophilic organic solvent such as hydrous acetone or hydrous methanol, and the obtained extract is concentrated under reduced pressure, and the concentrate is extracted with ethyl acetate. This ethyl acetate extract can be separated and purified together with the ethyl acetate extract obtained from the culture filtrate of the previous stage, or can be separated and purified by the same method as described above.

Next, the physicochemical properties of the platelet aggregation inhibitor OM-4842A will be described.

Molecular formula: C ₃₇ H ₄₆ O ₁₄ (by ¹ H and ¹³ C-NMR spectrum and elemental analysis), molecular weight: 714 (by ¹³ C-NMR spectrum and elemental analysis), specific optical rotation; [α] + 60 ° (C = 0.2, methanol) UV absorption spectrum (in methanol); λ max n
m (E) 220 (416.75), 321.5 (63.25), 427.5 (83.2)
5), infrared absorption spectrum (in potassium bromide); as shown in Fig. 2 Solubility in solvent; soluble in acetone, methanol, ethanol, ethyl acetate, chloroform, benzene, dimethyl sulfoxide, insoluble in water, n-hexane , Basic, acidic, neutral; weakly acidic lipophilic quinone substance color of substance; orange red proton nuclear magnetic resonance spectrum (in deuterated chloroform); as shown in Fig. 3, carbon nuclear magnetic resonance spectrum (in deuterated chloroform) As shown in FIG. 4, [Effect of the invention] Next, the biological properties of the platelet aggregation inhibitor OM-4842A of the present invention will be described.

(1) Platelet aggregation inhibitory effect Platelet-rich plasma (400μ) prepared from rabbit blood was placed in a cuvette, and OM-4842A (50μg / ml ~ 5
μg / ml) and incubate at 37 ° C for 10 minutes. Next, each concentration of ADP, arachidonic acid, PAF, and collagen was added as a platelet aggregating agent, the reaction solution was incubated for 5 minutes, and the degree of platelet aggregation inhibition was examined in a 24-well. The minimum inhibitory concentration of this substance OM-4842A for various aggregating agents was as follows.

ADP (5 μM): 12.5 μg / ml Arachidonic acid (100 μM): 5.0 μg / ml PAF (5 × 10 ^-8 M): 25.0 μg / ml Collagen (100 μg / ml):> 25.0 μg / ml (2) Toxicity book The anti-platelet aggregation inhibitor OM-4842A was intraperitoneally administered to 200 mg / kg mice, but no toxicity was observed.

As described above, the anti-platelet aggregation inhibitor OM-4842A,
Since it has low toxicity and exhibits a remarkable aggregation-suppressing effect on platelet aggregation agents such as ADP, arachidonic acid, and PAF, it is useful as a blood pressure-lowering effect in humans and a preventive agent for thrombosis.

〔Example〕

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Glucose 0.1%, starch 2.4%, in a 500 ml Sakaguchi flask,
Dispense 100 ml of liquid medium (pH 7.0) containing 0.3% peptone, 0.3% meat extract, 0.5% yeast extract, 0.4 calcium carbonate, steam sterilize at 121 ° C for 15 minutes, and add glycerol to them.
1.0%, calcium malate 1.0, ammonium chloride 0.05%,
Dipotassium phosphate 0.05%, yeast extract 0.1%, agar 1.5%
1 platinum loop each was inoculated from the slant culture of Streptomyces OM-4842 strain cultured at 27 ° C on an agar slant culture medium containing, and shake-cultured at 27 ° C for 2 days using a rotary shaker. Got a mother Oatmeal on a 50-volume jarfamentor 2.
Charge 30% liquid medium (pH 7.0) at 15 ℃ at 121 ℃.
Steam sterilized for minutes. The above 9 seeds were transplanted into this,
The cells were agitated and cultured at 27 ° C. for 96 hours under the conditions of agitation speed of 250 rpm and aeration rate of 15 / min. The culture broth was centrifuged (10000 rpm) with a Sharpless centrifuge to separate into cells and culture supernatant.

Ethyl acetate 30 was added to the obtained supernatant, and the mixture was stirred and extracted. The ethyl acetate layer was separated and concentrated under reduced pressure to obtain 28 g of a dark red substance.

28 g of the extracted substance was subjected to column chromatography using a column of Kiselgel 60 silica gel (manufactured by Merck) (50 × 7 cm).

The oil was first eluted with the developing solvent chloroform-methanol (100: 1) and then with chloroform-methanol (50: 1-20: 1) to collect the fractions containing the substance OM-4842A and reduce the pressure. After concentration to dryness, 3 g of a reddish brown substance was obtained. Soluble this in as little chloroform as possible and use Kiselg
El60F254 was charged on a silica gel thin layer plate (manufactured by Merck), chloroform-methanol (developed with 10: 1), and a portion having an Rf value of 0.32 (yellow-orange band) was scraped off.
Pack the column with silica gel powder containing the substance OM-4842A,
Using the elution solvent chloroform-methanol (3: 1),
The active substance was eluted and concentrated under reduced pressure to give substance OM-4842A 13
4.6 mg was obtained as an orange-red powder.

Sephadex LH for further purification and separation of this powder
Column chromatography using -20 (developing solvent: methanol) was performed to obtain this substance OM-4842A as orange-red powder.
83.6 mg was obtained.

[Brief description of drawings]

Fig. 1 shows the ultraviolet absorption spectrum of the platelet aggregation inhibitor OM-4842A (in methanol); Fig. 2 shows the infrared absorption spectrum of the platelet aggregation inhibitor OM-4842A (in potassium bromide); Fig. 3 shows the platelet aggregation inhibitor. OM-4842A proton nuclear magnetic resonance spectrum (in deuterated chloroform); FIG. 4 is a carbon magnetic resonance spectrum (platelet in deuterated chloroform) of OM-4842A, a platelet aggregation inhibitor.

Claims (4)

[Claims] 1. A platelet aggregation inhibitor OM-4842A having the following physicochemical properties. Molecular formula: C ₃₇ H ₄₆ O ₁₄ (by ¹ H and ¹³ C-NMR spectrum and elemental analysis), molecular weight: 714 (by ¹³ C-NMR spectrum and elemental analysis), specific optical rotation; [α] + 60 ° (C = 0.2, methanol) UV absorption spectrum (in methanol); λ max n
m (E) 220 (416.75), 321.5 (63.25), 427.5 (83.2)
5), infrared absorption spectrum (in potassium bromide); as shown in Fig. 2 Solubility in solvent; soluble in acetone, methanol, ethanol, ethyl acetate, chloroform, benzene, dimethyl sulfoxide, insoluble in water, n-hexane , Basic, acidic, neutral; weakly acidic lipophilic quinone substance color of substance; orange red proton nuclear magnetic resonance spectrum (in deuterated chloroform); as shown in Fig. 3, carbon nuclear magnetic resonance spectrum (in deuterated chloroform) As shown in FIG. 2. A platelet aggregation inhibitor OM-4842A-producing bacterium belonging to the genus Streptomyces is cultured in a medium to produce and accumulate the platelet aggregation inhibitor OM-4842A in the culture, and the platelet aggregation is obtained from the culture. A method for producing a novel platelet aggregation inhibitor OM-4842A, which comprises collecting the inhibitor OM-4842A. 3. A platelet-aggregation-inhibiting substance OM-4842A-producing bacterium is Streptomyces species OM-4842 (FERM-P).
No.9427) and the production method according to claim 2. 4. A platelet aggregation inhibitor comprising a platelet aggregation inhibitor OM-4842A as an active ingredient.