JPH0359911B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel physiologically active substance SS48727E and a method for producing the same. [Prior Art] It has been known that microorganisms produce various physiologically active substances. For example, Streptomyces pactum 0-1-10 (Streptomyces pactum0-1
-10) is known to produce compounds of the piericidins group [Nobutaka Takahashi et al.
Agricultural and Biological Chemistry
Chemistry), Vol. 41, pp. 849-853 and pp. 855-862 (1977); Nobutaka Takahashi et al., JP-A No. 132183/1983]. [Problems to be Solved by the Invention] An object of the present invention is to provide a new substance useful as a medicine, an animal drug, an agricultural chemical, a reagent, or a material for conversion thereto. [Means for solving the problem] The present inventors isolated a large number of microorganisms from natural soil and conducted various studies on their products. As a result, a strain isolated from soil in Namie Town, Futaba District, Fukushima Prefecture was , a novel physiologically active substance of the piericidin group
It was discovered that SS48727E could be produced and the present invention was completed. That is, the present invention relates to a novel physiologically active substance SS48727E.
and a manufacturing method thereof. The strain S48727 that produces the physiologically active substance SS48727E of the present invention has the following mycological properties. (1) Morphology Cultured on various agar media at 28℃ for 10 to 14 days,
The morphology of the S48727 strain was observed using an optical microscope or an electron microscope, and the results showed the following characteristics. The spore-forming hyphae are simply branched from the aerial hyphae, and their tips are spiral-shaped. Ring-grown silk is not permitted. Mature conidia are a chain of 10 or more,
The shape of the spore is short cylindrical to oval, and its size is 0.5 to 0.8 x 0.7 to 1.4 μm. The spore surface structure is hair-like. No sporangia, flagellated spores, or sclerotia are observed. In addition, no division of basal hyphae was observed. (2) Growth status on various agar media The growth status of strain S48727 on various media is shown in the table below. Observations were made after culturing at 28°C for 14 days. The colors are described using the systematic color names from the "Small Dictionary of Color Names" published by Nippon Shikiken Business Co., Ltd. (1981). In addition, the number in parentheses in the table indicates the color standard number.

[Table] (3) Physiologically active substances Growth temperature range (yeast/malt agar medium,
(Culture for 14 days) Optimum temperature for growth: 26-34°C Growth temperature: 18-37°C Liquefaction of gelatin Positive Hydrolysis of starch Positive Coagulation of skim milk Positive Peptonization of skim milk Positive Production of melanin-like pigment Negative Nitrate reduction Negative Cellulose decomposition Negative (4) Carbon source assimilation (Pridham-Gottlieb agar medium, 28℃, 14 days culture) D-glucose, raffinose, galactose are used, and salicin is also used to a small extent. L-
Arabinose, D-xylose, D-fructose, sucrose, inositol, L-rhamnose, D-mannite, cellulose, lactose, D-sorbitol, D-mannose, and inulin are not used. (5) Diaminopimelic acid in all bacterial cells As a result of analyzing diaminopimelic acid in all bacterial cells, LL-diaminopimelic acid was detected. Based on the above morphological characteristics and containing LL-diaminopimelic acid, strain S48727 is judged to be a strain belonging to the genus Streptomyces. The mycological properties of strain S48727 can be summarized as follows. The tips of the aerial hyphae are spiral-shaped, and the conidia consist of 10 or more spores linked together, and the surface of each spore is hair-like. The color tone of the aerial mycelium is gray color series, and the color of the underside is colorless to yellow or brown, and on oatmeal agar medium, yellow with green is observed. No soluble pigments are observed and no melanin-like pigments are produced. Based on the above properties, Schearling and Gottlieb's ISP report “International Journal of Systematic Bacteriology”
Bacteriology) Volume 18, pp. 69, 279, (1968
), Vol. 19, p. 391 (1969), Vol. 22, p. 265,
(1972) and Bergey’s Manual of Determinative Bacteriology.
As a result of searching from the 8th edition of "Bacteriology", Streptomyces karnatakensis and Streptomyces pactum are
It is listed as a close relative of strain S48727. These 2
The results of comparison with bacterial strains are shown in the table below.

【table】

[Table] When using carbon sources, + means use, ± means weak use, - means not use.
As shown in the table above, the S48727 strain and Streptomyces carnatakensis IFO 13051 strain are
They differ in the color tone of aerial mycelia, the ability to utilize raffinose, D-fructose, and salicin, and the ability to utilize malic acid and lime. On the other hand, it differs from Streptomyces pactum IFO 13433 strain in its ability to utilize raffinose, D-fructose, and salicin, but has almost the same other mycological properties. Therefore, S48727
The strain was identified as a strain belonging to Streptomyces pactum. In addition, the S48727 strain has the above-mentioned mycological properties and Piericidin A ₁ (Piericidin A 1 ).
_A1 ), Streptomyces
Streptomyces pactum0-1-10 (Streptomyces pactum0-
1-10) [Nobutaka Takahashi et al., Agricultural and Biological Chemistry, Vol. 41, 849-
pp. 853 and 855-862, (1977)], [Nobutaka Takahashi et al.
A close relationship with JP-A-50-132183] is suggested. However, since strain 0-1-10 does not produce the compound of interest, strain S48727 is considered to be a new strain different from this strain. Therefore, in order to distinguish strain S48727 from known strains, the present inventor named it Streptomyces pactum S48727 and assigned it to the National Institute of Microbiology, Agency of Industrial Science and Technology with accession number. Deposited as No. 8117 (FERM P-8117). The physiologically active substance SS48727E of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the physiologically active substance SS48727E-producing strain, not only the above-mentioned S48727 strain, but also any artificial or natural mutant strain thereof that has the ability to produce the physiologically active substance SS48727E can be used in the present invention. . the above
The artificial mutant strain S48727 can be easily obtained by, for example, ultraviolet irradiation, cobalt-60 irradiation, chemical mutagen treatment, etc., as in the case of other actinomycetes. Next, the cultivation of the bacterial strain in the production of the physiologically active substance SS48727E will be explained. That is, a normal actinobacterial culture method is used to culture the physiologically active substance SS48727E-producing strain belonging to the genus Streptomyces. As the nutrient medium, any synthetic medium, semi-synthetic medium, or natural medium can be used as long as it contains appropriate amounts of assimilable carbon sources, nitrogen sources, inorganic substances, and the like. As the carbon source, for example, glycose, raffinose, starch, etc. may be used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, and organic acids may also be used. Nitrogen sources include inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, monosodium glutamate, etc., or natural products, such as soybean flour, yeast extract, peptone, meat extract, dried yeast, cottonseed meal. , proteose peptone, casamino acid, corn steep liquor, etc. may be used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, copper sulfate, manganese sulfate, zinc sulfate, iron sulfate, etc. are used alone or in combination. In addition, it helps the growth of S48727 strain.
Substances that promote the production of SS48727E or common antifoaming agents such as silicone oil or Adekanol (trade name) can also be added to the medium as appropriate. As the culture method, methods commonly used for the production of physiologically active substances are employed, but liquid culture methods, particularly deep agitation culture methods, are most suitable. Cultivation is performed under aerobic conditions, and the appropriate temperature for cultivation is 26 to 34°C, but it is generally preferable to culture at around 27°C. The production amount of the physiologically active substance SS48727E reaches its maximum after ~5 days of culture in both shaking culture and deep stirring culture methods. Physiologically active substances in culture medium
When the accumulated amount of SS48727E reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution. SS48727E is isolated from the culture solution by using various methods alone or in appropriate combinations, taking into consideration the physicochemical properties of the physiologically active substance, as shown in the Examples below. That is, since the physiologically active substance SS48727E normally exists in the culture filtrate and bacterial cells, the bacterial cells are separated from the culture solution by centrifugation or filtration, and the bacterial cells and culture filtrate are separated by conventional separation means such as solvent oil. Physiologically active substances can be purified using methods such as extraction method, ion exchange resin method, gel filtration method, adsorption or distribution column chromatography method, high performance liquid chromatography method, dialysis method, precipitation method, etc. alone or in appropriate combinations.
Separate and purify SS48727E. Examples of preferable separation and purification include the following methods. After fermentation, the culture solution is centrifuged,
Separate into culture filtrate and bacterial cells. The bacterial cells are extracted with a suitable solvent such as acetone or methanol, and this is concentrated under reduced pressure to remove the solvent and form an aqueous solution. Together with the filtrate obtained by treating the above-mentioned bacterial cells, the mixture is extracted with a suitable solvent such as ethyl acetate at a pH of around 7. After distilling off the solvent of the extract and washing with n-hexane or petroleum ether, the residue is subjected to silica gel column chromatography. Next, elution with an appropriate eluent, such as a chloroform/methanol mixture, will result in
SS48727E fraction is obtained. Furthermore, after distilling off the solvent of the SS48727E fraction, the residue was
Submit to LH20 (manufactured by Pharmacia) column chromatography. Further elution and purification with a suitable eluent, such as methanol or a chloroform/methanol mixture, yields slightly yellow amorphous crystals of the physiologically active substance SS48727E. The physiologically active substance SS48727E obtained as described above has the following physicochemical properties. <Physical and chemical properties> Elemental analysis C H N Experimental value (%) 64.21 8.34 2.51 Theoretical value (%) 64.45 8.20 2.42 Molecular formula C ₃₁ H ₄₇ O ₉ N Mass spectrum (FAB-MS) m/z 578 (M+H) ⁺ ratio Optical rotation [α] ²⁰ _D = -10.0° (C = 0.9, methanol) Ultraviolet absorption spectrum Fig. 1 233 (40050) λ ^HeOH _Max (ε) nm, 237 (40200) 275 (8300) Infrared absorption spectrum (KBr method) ) Figure 2 ¹ H-NMR spectrum (90MHz) Figure 3 Measurements were made using TMS in deuterated chloroform solution as a reference substance. ¹³ C-NMR spectrum (22.5MHz) Measured using TMS in deuterated chloroform solution as a reference substance. δ (ppm): 155.3 (s) 154.8 (s), 151.2 (s), 135.8 (d), 135.8 (2s), 135.0 (s), 133.2 (d)
, 133.2(s), 126.5(d), 123.2(d), 122.0(d)
, 118.6(s), 104.1(d), 82.8(d), 76.5(d), 75.9(d), 74.3(d), 70.0(d), 61.9(t), 60
.9 (q), 53.2 (q), 43.1 (t), 36.9 (d), 34.5
(t), 17.4 (q), 16.6 (q), 13.1 (q), 13.0 (q), 11
.6 (q), 10.7 (q) Solubility Soluble in chloroform, dimethyl sulfoxide, ethyl acetate, methanol, pyridine, ether, and acetone. n-hexane, insoluble in water. Distinction between basic, acidic and neutral Color and properties of the substance Slightly yellow amorphous crystals Color reaction Positive with vanillin sulfuric acid test solution (heated). Negative in ferric chloride test solution and Dragendorff test solution. Thin layer chromatography Support: Silica gel plate F254 (manufactured by Merck)

[Table] Structural formula [Effect] The physiologically active substance SS48727E of the present invention has the following pharmacological activities. Antibacterial activity Table 1 shows the minimum inhibitory concentration (MIC) of the physiologically active substance SS48727E against various microorganisms.

【table】

[Table] Hypotensive effect The hypotensive effect of the present compound SS48727E was investigated as follows. i.e. Wistar
After a male rat was anesthetized with urethane (1 g/Kgi.p.), it was fixed in a dorsal position, and a tracheal cannula was inserted to secure the airway. The compound of the present invention SS48727E was dissolved in a small amount of dimethyl sulfoxide, diluted appropriately with physiological saline, and administered into the femoral vein of the rat as a test drug. The blood pressure was measured from the femoral artery of the test rat via a pressure transducer, the electrocardiogram was measured using the first lead method, and the heart rate was measured using an instantaneous heart rate monitor using the R-R interval of the electrocardiogram as a trigger. rate) and recorded on a polygraph. The results are shown in Table 2.

〔Effect of the invention〕

When compared structurally with the piericidins discovered so far,
Since SS48727E has D-glucose in the hydroxyl group at the _C3 ' position, it is expected to have reduced toxicity. On the other hand, SS48727E has an antibacterial effect against Gram-positive bacteria, Trichophyton, and Grocerycus, and is therefore useful as an antibiotic or agricultural chemical. It is also useful as a medicine because it exhibits a sustained hypotensive effect. [Example] Next, an example will be given and explained. Example 1% glucose, 1% soluble starch, polypeptone
0.5%, meat extract 0.5%, yeast extract 0.3%, sodium chloride 0.3%, magnesium sulfate 0.1%, calcium carbonate 0.3%, copper sulfate 0.0007%, ferrous sulfate
0.0001%, manganese sulfate 0.0008%, zinc sulfate
A liquid medium with a composition of 0.0002% has a pH of 7.0,
Dispense 100ml into a 500ml volume Sakaguchi Lalasco and sterilize.
This is inoculated with Streptomyces pactum S48727 strain (Feikoken Bacterium No. 8117) and cultured with shaking at 27°C for 2 days to prepare a seed culture. Next, glucose 2%, proteose peptone 1%, copper sulfate
0.0007%, ferrous sulfate 0.0001%, manganese sulfate
A liquid medium with a composition of 0.0008% and 0.0002% zinc sulfate was adjusted to pH 6.5 and placed in a 30 volume jar fermenter.
18Prepare and sterilize. Add 180% of the above seed culture solution to this.
ml was inoculated and cultured for 90 hours at a culture temperature of 27°C, stirring rate of 350 rpm, and air flow rate of 18/min. In addition,
In order to prevent the pH from increasing during culture, the pH was adjusted to 7.5 using 5N acetic acid. In addition, ADEKA NOL (trade name) was appropriately added to prevent foaming. The culture solution obtained in this way is separated into bacterial cells and culture filtrate by centrifugation, and the bacterial cells are mixed with 5 methanol and filtered after stirring.
The mixture was concentrated under reduced pressure to volume. Next, an equal amount of water was added to this concentrated solution, and the mixture was extracted twice with 2 portions of ethyl acetate. On the other hand, the culture filtrate was also extracted twice with twice the amount of ethyl acetate, combined with the previous bacterial cell extract, and the solvent was distilled off under reduced pressure at 40°C to obtain an oily crude extract. This crude extract was washed with n-hexane, and 15.6 g of the oily crude extract obtained was subjected to silica gel (Merck Kiesel Gel 60, 230-400 mesh) column chromatography 4.0 (φ) x 50 cm, and chloroform/ Piericidin A ₁ can be quickly eluted with methanol (98:2) mixture 2.
(Piericidin A ₁ ) is eluted. Furthermore, if you elute with the same (96:4) mixture 4, SS48727B will appear first.
The fraction is eluted and then well separated from
45 mg of SS48727E fraction was eluted. Further SS48727E
Sephadex LH20 (manufactured by Pharmacia)
Attach to column chromatography 2.0 (φ) x 55cm,
Elution with methanol yields slightly yellow amorphous crystals.
20 mg of pure SS48727E was obtained. It was confirmed that this substance has the above-mentioned physical and chemical properties.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum, FIG. 2 shows the infrared absorption spectrum, and FIG. 3 shows the ¹ H-NMR spectrum of the physiologically active substance SS48727E of the present invention.

Claims (1)

[Claims] 1. SS48727E is a novel physiologically active substance expressed by SS48727E. 2. A method for producing a novel physiologically active substance SS48727E, which comprises culturing a novel physiologically active substance SS48727E-producing bacterium belonging to the genus Streptomyces and collecting the physiologically active substance SS48727E from the culture solution. 3. The production method according to claim 2, wherein the SS48727E producing bacterium is Streptomyces pactum S48727.