JPH0361675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic SS9816C. 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 the soil of Shikanoyama, Chiba Prefecture, has excellent antibacterial activity against Gram-positive bacteria. The present invention was completed by discovering the production of a new antibiotic SS9816C that also has antitumor effects. That is, the present invention provides a novel antibiotic SS9816C. The S9816 strain that produces the antibiotic SS9816C of the present invention has the following mycological properties. (1) Morphology The branches of aerial hyphae are simple branches, and no axle branches are observed. The tip of the aerial hyphae is spiral-shaped. Basal hyphae usually do not divide. Sporangia and flagellated spores are not observed. According to electron microscopy, the spores have a smooth surface and a cylindrical shape. The size of the spores is 0.7-0.9 x 0.8-1.5 μm, and 10 or more spores are usually linked together. (2) Growth status on various media The growth status of strain S9816 on various media is shown in the following table. Observation was performed after culturing at 27°C for 14 days. Colors were described using the ``Color Name Dictionary'' published by Nippon Shikiken Business Co., Ltd., and indicated by their systematic color names.

[Table] (3) Physiological substance growth temperature range (yeast starch agar medium,
14-day culture) Growth temperature: 8-33℃ Optimum growth temperature: 24-30℃ Liquefaction of gelatin Positive Hydrolysis of starch Positive Coagulation of skim milk Positive Peptonization of skim milk Positive Production of melanin-like pigments Negative Reduction of nitrate Negative Cellulose Degradation Negative Salt tolerance: Grows in medium supplemented with 7% NaCl, but 10
% will not grow. (4) Availability of carbon source (Pridham-Godelive agar medium, 27°C, 14 days culture) L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol - L-rhamnose + raffinose + D- Mannite - Galactose + Salicin + Lactose + D-mannose + (Note) + means to use, - means not to use. (5) Cell wall composition Analysis of diaminopimelic acid in the cell wall composition revealed that it was LL type. Based on the above mycological properties, this strain S9816 is judged to belong to the genus Streptomyces. Furthermore, the tip of the aerial hyphae of this strain is spiral-shaped and has 10 or more spores chained together, and the spore surface is smooth; the color of the aerial hyphae is gray color series or green color series, and the color of the underside is colorless to pale yellow. Yellowish brown~
It is a grayish olive; the soluble pigment is pale yellow ~
Brownish olive ~ Asai and reddish braum ~
It is dull red; the color and soluble pigment on the back side is not a PH indicator color, and it does not produce melanin-like pigments. The present inventors have the above mycological properties.
S9816 strain Streptomyces sp.
(Streptomyces sp.S9816) and was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM P-6886. The antibiotic SS9816C of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS9816C-producing strain, not only the above-mentioned S9816 strain, but also any artificial or natural mutant strain thereof can be used as long as it has the ability to produce the antibiotic SS9816C. The artificial mutant strain of the S9816 strain can be easily obtained using, for example, ultraviolet rays, cobalt-60 irradiation, chemical mutagenic agents, etc., as in the case of other actinomycetes. Next, culturing of bacterial strains in the production of antibiotic SS9816C will be explained. That is, the usual method for culturing actinomycetes is used to culture the strain producing the antibiotic SS9816C 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, glucose, fructose, sucrose, arabinose, starch, glycerin, dextrin, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. As nitrogen sources, inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulphate, ammonium nitrate, urea, sodium nitrate, sodium glutamate, etc., or natural products, such as soybean extract,
Soybean flour, yeast extract, dried yeast, cottonseed meal, vegetable protein, soytone, etc. may be used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, copper sulfate, manganese chloride, zinc sulfate, etc. are used alone or in combination. Other substances that help the growth of the S9816 strain and promote the production of SS9816C or general 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 antibiotics are employed, but liquid culture methods, especially shaking culture methods, are most suitable. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 24 to 30°C.
Generally, it is preferable to culture at around 27°C. The production of antibiotic SS9816C reaches its maximum after 5 to 8 days of culture in both shaking culture and deep stirring culture. When the amount of antibiotic SS9816C accumulated in the culture reaches its maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution. Antibiotics from culture medium
SS9816C is isolated by using various methods alone or in appropriate combinations, taking into account the physicochemical properties of this antibiotic, as shown in the Examples below. In other words, since the antibiotic SS9816C is normally present in the bacterial cells and the culture solution, the bacterial cells are separated from the culture by centrifugation or filtration, and then the bacterial cells and the culture solution are separated by conventional separation means such as precipitation. Antibiotic SS9816C is separated and purified using methods such as method, solvent extraction method, ion exchange resin method, gel filtration method, adsorption or distribution column chromatography method, dialysis method, etc. alone or in an appropriate combination. Examples of preferred separation and purification methods include the following methods. Dilute hydrochloric acid is added to the fermented culture to adjust the pH to 2.0, and a precipitate containing bacterial cells is obtained by centrifugation. A suitable solvent such as acetone is added to this precipitate, mixed well and extracted. Next, extract the excess residue with chloroform, combine the liquids,
Add an appropriate amount of water to this extract, divide it, and remove the lower layer. This lower layer was distilled off under reduced pressure, and the residue was washed with n-hexane and then subjected to silica gel column chromatography. After collecting the active fractions and concentrating under reduced pressure, the residue is recrystallized from a suitable solvent such as ethanol.
Dark purple fine crystals of SS9816C are obtained. SS9816C obtained as described above has the following physicochemical and biological properties. (1) Physical and chemical properties Elemental analysis C H N Experimental value (%) 67.29 4.13 2.38 Theoretical value (%) 67.27 4.19 2.53 Molecular weight 553.52 Melting point 270℃ (decomposed) Ultraviolet absorption spectrum Figure 1 λ ^EtOH _nax (E1% 1cm) 257nm (265), 280nm (285), 503nm (94), 539nm (178), 568nm
(180), 580nm (209), Infrared absorption spectrum (KBr method) Figure 2 ¹ H-NMR spectrum (90MHz) Deuterochloroform/deuterium trifluoroacetic acid (10:1)
^Figure 3 Mass spectrum measured using TMS as a reference substance in a mixed solution Melt. n-hexane, poorly soluble in water. Distinction between basic, acidic, and neutral Acid Color and properties of substance Dark purple microcrystals Blue in alkaline water Red in acidic water Thin layer chromatography Support: Silica gel plate F ₂₅₄ (manufactured by Merck & Co.)

[Table] Molecular formula C ₃₁ H ₂₃ NO ₉ (2) Biological properties Antibacterial action Table 1 shows the minimum inhibitory concentration (MIC) of antibiotic SS9816C against various microorganisms.

【table】

[Table] Test bacteria culture conditions: Inoculum size
For 10 ⁶ cells/ml bacteria, culture in Mueller-Hinton Agar (Difco) at 37°C for 18-20 hours. For yeast and mold, culture in glucose/peptone medium at 28°C for 120 hours. Antitumor Effect The therapeutic effect of antibiotic SS9816C on murine leukemia P-388, Sarcoma 180 (S-180), and Ehrlich carcinoma (Ehrlich) was tested by the following method. The results are shown in Table 2. The survival effects in the table are expressed as a percentage of the ratio of survival days (T) in the treated group to survival days (C) in the untreated group. Experimental method P-388: 10 ⁶ P-388 cells were transplanted into the peritoneal cavity of CDF (Charles River, Japan).
After 24 hours, administer antibiotic SS9816C once a day.
It was administered intraperitoneally 10 times. S-180 and Ehrlich: 5 x ¹⁰⁶ tumor cells were transplanted intraperitoneally into ICR mice (♀, CLEA Japan), and 24 hours later, antibiotic SS9816C was intraperitoneally administered once a day for a total of 10 times. .

[Table] The properties described above were compared with those of known antibiotics similar to the compound of the present invention, but no corresponding substances were found, and SS9816C was judged to be a new antibiotic. As is clear from these results, antibiotics
SS9816C has strong antibacterial activity against Gram-positive bacteria. Also murine leukemia P-388, Sarcoma 180
It has also shown remarkable therapeutic effects on Ehrlichi carcinoma and ascites cancer. Based on these antibacterial and antitumor activities, antibiotics
SS9816C is a substance useful as a medical antibiotic. Next, the present invention will be explained with reference to Examples. Example SS9816C producing bacterium Streptomyces sp.
4.0% soluble starch,
Glucose 4.0%, Soyton 1.0%, Yeast Extract 1.0
%, sodium chloride 0.25%, calcium carbonate 0.32
%, copper sulfate 0.0005%, manganese chloride 0.0005%, zinc sulfate 0.005% (PH 7.0) in a liquid medium, 27
A seed culture solution is prepared by culturing with shaking at ℃ for 2 days. Pour 120 ml of a liquid medium with the same medium composition into a 500 ml sloping flask, inoculate it with 0.6 ml of the above seed culture solution, and shake in a reciprocating shaking culture machine with a shaking width of 9 cm and a rotation speed.
Cultivate for 7 days at 110 rpm and a culture temperature of 27°C. After culturing, add diluted hydrochloric acid to culture solution 10,
The pH was adjusted to 2.0, and a precipitate containing bacterial cells was obtained by centrifugation. Acetone 1 was added to this precipitate, and the mixture was thoroughly stirred, extracted and filtered, and separated into a liquid and a residue. Add 1 part of chloroform to the residue, stir well for extraction, and filter to obtain a liquid. This operation was repeated several times and the liquids were combined. Add 2 parts of water to this extract.
was added and distributed, and the lower layer was separated. This lower layer was concentrated to dryness under reduced pressure, washed with a small amount of n-hexane, and dried to obtain 800 mg of red powder. The above red powder was dissolved in chloroform containing 1% acetic acid, passed through a 100 g column of Kieselgel 60 (manufactured by Merck & Co., Ltd.) filled in advance with chloroform containing 1% acetic acid, eluted with the same solvent, and the eluted fraction was removed. After that, acetic acid-chloroform-methanol (1:
100:2) Elute with a mixture. Among the eluted fractions, the fractions containing antibiotic SS9816C were collected and concentrated under reduced pressure. The concentrate was left in a cool place, and the precipitated crystals were collected (35 mg). This was recrystallized with ethanol,
20 mg of dark purple fine crystals of SS9816C were obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic SS9816C (in ethanol solution). Figure 2 shows the infrared absorption spectrum of antibiotic SS9816C (KBr tablet). Figure 3 shows ¹ H− of the antibiotic SS9816C.
The NMR spectrum [in a mixed solution of deuterochloroform/deuterotrifluoroacetic acid (10:1)] is shown.

Claims (1)

[Claims] 1. A novel antibiotic having the following physicochemical properties:
SS9816C. Elemental analysis C H N Experimental value (%) 67.29 4.13 2.38 Theoretical value (%) 67.27 4.19 2.53 Molecular weight 553.52 Melting point 270℃ (decomposition) Ultraviolet absorption spectrum Figure 1 λ ^EtoH _nax (E1% 1cm) 257nm (265), 280nm ( 285), 503nm (94), 539nm (178), 568nm (180), 580nm (209) Infrared absorption spectrum (KBr method) Figure 2 ¹ H-NMR spectrum (90MHz) Deuterochloroform/deuteroacetic acid (10:1 ) TMS in the mixed solution was measured as a reference substance. Figure 3 Mass spectrum (EI70eV) M ⁺ m/z553 Solubility in solvents Soluble in chloroform, acetone, ethanol, methanol, tetrahydrofuran, dioxane, pyridine, dimethyl sulfoxide, and tetrahydrofuran-water mixture. n-hexane, poorly soluble in water. Distinction between basic, acidic, and neutral Acid Color and properties of substance Dark purple microcrystals Blue in alkaline water Red in acidic water Thin layer chromatography Support: Silica gel plate F ₂₅₄ (manufactured by Merck & Co., Ltd.) [Table] Molecular formula C ₃₁ H ₂₃ NO ₉