JPH0361677B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic SS9816B. 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 and fungi. has
SS9816B, a novel antibiotic with antitumor activity
The present invention was completed by discovering that it can be produced. That is, the present invention provides a novel antibiotic SS9816B. The S9816 strain that produces the antibiotic SS9816B 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. Observations were made 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 properties Growth temperature range (yeast starch agar medium, 14-day culture) Growth temperature 8-33°C Optimal growth temperature 24-30°C Liquefaction of gelatin Positive Hydrolysis of starch Positive Skim milk Coagulation Positive Peptonization of skim milk Positive Production of melanin-like pigments Negative Reduction of nitrate Negative Decomposition of cellulose Negative Salt tolerance: Grows in medium supplemented with 7% NaCl, but 10
% will not grow. (4) Availability of carbon source (Bridham-Godelive agar medium, cultured at 27°C for 14 days) 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 more than 10 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 reddish brown ~
It is dull red: the color and soluble pigment on the back side is PH
It has characteristics such as not having an indicator color and not producing melanin-like pigment. 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 SS9816B of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. As the antibiotic SS9816B-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 SS9816B. The artificial mutant strain of the S9816 strain can be easily obtained, for example, by ultraviolet cobalt-60 irradiation, chemical mutagenic agents, etc., as in the case of other actinomycetes. Next, the culture of bacterial strains in the production of antibiotic SS9816B will be explained. That is, a normal method for culturing actinomycetes is used to culture the strain producing the antibiotic SS9816B 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 a nitrogen source, inorganic or organic nitrogen compounds,
For example, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, monosodium glutamate, etc., or natural products such as soybean extract, soybean flour, yeast extract, dried yeast, cottonseed meal, vegetable protein, soytone, etc. alone or in combination. used. As the inorganic substance, for example, calcium carbonate, sodium chloride, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, etc. are used alone or in combination. Helps the growth of other S9816 strains
A substance that promotes the production of SS9816B or a general antifoaming agent 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 SS9816B reaches its maximum after 5 to 8 days of culture in both shaking culture and deep stirring culture. When the accumulation of antibiotic SS9816B 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
SS9816B 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. That is, since the antibiotic SS9816B normally exists in bacterial cells and culture solution, the bacterial cells are separated from the culture by centrifugation or filtration, and then the bacterial cells and culture solution are separated by conventional separation means such as precipitation. Antibiotic SS9816B 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. The active fractions are collected and concentrated under reduced pressure, and the residue is recrystallized from a suitable solvent such as a chloroform-acetic acid mixture to obtain purple needle crystals of SS9816B. SS9816B obtained as described above has the following physicochemical and biological properties. (1) Physical and chemical properties Elemental analysis C H N Experimental value (%) 67.17 4.18 2.54 Theoretical value (%) 67.27 4.19 2.53 Molecular weight Actual value measured by mass spectrum (EI 70eV) 553 Calculated value as molecular formula C ₃₁ H ₂₃ NO ₉ 553.52 Melting point 300℃ or higher Ultraviolet absorption spectrum Figure 1 λtetrahydrofuran max (E1% 1cm) 259nm (492) 330nm (sh) 349nm (232) 373nm (sh) 390nm (312) 410nm (sh) 514nm (sh) 556nm (324) 596nm (350) Infrared absorption spectrum (KBr method) Figure 2 ¹ H-NMR spectrum (90MHz) Deuterochloroform/deuterium trifluoroacetic acid (1:
1) Measured using TMS in a mixed solution as a reference substance Figure 3 Soluble in chloroform, tetrahydrofuran, dioxane, pyridine, dimethyl sulfoxide, and chloroform-acetic acid mixture. n-hexane, poorly soluble in water. Distinction between basic, acidic and neutral Acid Color and properties of substance Purple needle crystals (recrystallized from a chloroform-acetic acid mixture) 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 SS9816B 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. (2) Antitumor effect The therapeutic effect of antibiotic SS9816B on murine leukemia P-388 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 ^Six P-388 cells were intraperitoneally transplanted into CDF (Charles River, Japan), and 24 hours later, antibiotic SS9816B was intraperitoneally administered once a day for a total of 10 times. Ehrlich: 5×10 ⁶ tumor cells were intraperitoneally transplanted into ICR mice (♀, CLEA Japan), and 24 hours later, antibiotic SS9816B 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 SS9816B was judged to be a new antibiotic. As is clear from these results, antibiotics
SS9816B has strong antibacterial activity against crumb-positive bacteria and fungi. It has also shown remarkable therapeutic effects on murine leukemia p-388 and Ehrlichi carcinoma ascites cancer. In view of these antibacterial and antitumor activities, antibiotic SS9816B is a useful substance as a medically effective substance. Next, the present invention will be explained with reference to Examples. Example 1 The SS9816B producing bacterium Streptomyces sp.
%, glucose 4.0%, soytone 1.0%, yeast extract 1.0%, sodium chloride 0.25%, calcium carbonate 0.32%, copper sulfate 0.0005%, manganese chloride 0.0005
%, lead sulfate 0.005% (PH7.0) liquid medium, and cultured with shaking at 27°C for 2 days to prepare a seed culture. 500ml of 120ml liquid medium with the same medium composition
Pour into a Sakaguchi flask and add the above seed culture solution to it.
Inoculate 0.6ml and use a reciprocating shaking culture machine to a width of 9cm.
Cultivated under the conditions of rotation speed 110r.pm and culture temperature 27℃,
On the fourth day, 1.2 ml of 2% cobalt chloride aqueous solution was added to each Sakaguchi flask and cultured for an additional 3 days.
After culturing, add dilute hydrochloric acid to culture solution 10 to adjust the pH to 2.0.
Then, a precipitate containing bacterial cells was obtained by centrifugation. Acetone 1 was added to this precipitate, and the mixture was thoroughly stirred for extraction and separated into a liquid and a liquid residue by filtration. Add 1 chloroform to the liquid residue, stir well for extraction, and filter to obtain a liquid. This operation was repeated several times and the liquids were combined. Water 2 was added to this extract for partition, 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 600 mg of reddish-purple powder. The reddish-purple powder was dissolved in chloroform containing 1% acetic acid, passed through a 100 g column of Kieselgel 60 (manufactured by Merck & Co., Ltd.) previously filled with chloroform containing 1% acetic acid, and eluted with the same solvent. Antibiotic SS9816B
Fractions containing 1 were collected and concentrated under reduced pressure. The concentrate was left in a cool place and the precipitated crystals were collected. (120mg)
This was recrystallized from a chloroform-acetic acid mixture,
90 mg of purple needle crystals of SS9816B were obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of antibiotic SS9816B (in tetrahydrofuran solution). Second
The figure shows the infrared absorption spectrum of antibiotic SS9816B (KBr tablet). Figure 3 shows antibiotic SS9816B
The ¹ H-NMR spectrum of [in a mixed solution of deuterochloroform/deuterotrifluoroacetic acid (1:1)] is shown.

Claims (1)

[Claims] 1. A novel antibiotic having the following physicochemical properties:
SS9816B Elemental analysis C H N Experimental value (%) 67.17 4.18 2.54 Theoretical value (%) 67.27 4.19 2.53 Molecular weight Actual value by mass spectrum (EI70eV) 553 Calculated value as molecular formula C ₃₁ H ₂₃ NO ₉ 553.52 Melting point 300℃ or more Ultraviolet absorption Spectrum Figure 1 λ ^THF _nax (E1% 1cm) 259nm (492) 330nm (sh) 349nm (232) 373nm (sh) 390nm (312) 410nm (sh) 514nm (sh) 556nm (324) 596nm (350) Infrared absorption Spectrum (KBr method) Figure 2 ¹ H-NMR spectrum (90MHz) Deuterochloroform/deuterium trifluoroacetic acid (1:
1) TMS in the mixed solution was measured as a reference substance. Figure 3 Solubility in solvents Soluble in chloroform, tetrahydrofuran, dioxane, pyridine, dimethyl sulfoxide, and chloroform-acetic acid mixture. Intolerant to n-hexane and water. Distinction between basic, acidic, and neutral Acid Color and properties of substance Purple needle crystals (recrystallized from a chloroform-acetic acid mixture) Thin layer chromatography Support: Silica gel plate F ₂₅₄ (Merck & Co., Ltd.) [Table] Molecular formula C ₃₁ H ₂₃ NO ₉