JPH01289491A - S-632-b1 and s-632-b2 substance - Google Patents

Abstract

NEW MATERIAL:S-632-B1 material. outlook: light-yellow, oily material; molecular weight (electron ionization mass spectrum method): 323; element analysis value (%): C 62.76, H 7.71, N 4.12; solubility: readily soluble in methanol and acetone, etc., hardly soluble in hexane and ether, insoluble in water; color reaction: positive to iodine gas and Ehrlich's reaction, negative to Ninhydrin reaction the Tollen's reaction; distinction of basic, acidic or neutral: weak acidic. USE:Antibiotic agent. PREPARATION:Production fungus of S-632 material [e.g., streptomyces species S-632 (FERM-P 1849), etc.] is cultured and the aimed material is collected from said cultured solution.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel substance useful as an antibiotic or the like.

Prior Art and its Problems The 8 632 B+ and S-632-B2 substances of the present invention are novel compounds that have not been described in any literature.

An object of the present invention is to provide a novel substance useful as an antibiotic or the like.

Means for Solving the Problems The above objects are achieved by S-632-B and S-632-B2 substances having the following properties.

(S-632B+substance) (1) Properties: Pale yellow oily substance.

(2) Molecular weight: 323 (according to electron impact (EI) mass spectroscopy).

(3) Elemental analysis value (%): C62,76, H7,71,
N4.12° (4) Specific rotation: [α] Suga = +68° (C = 1.0
) in chloroform).

(5) Ultraviolet absorption spectrum: in ethanol, λma
x (stop) (ε): 206 (14500), 282
(450). The chart is shown in Figure 1.

(6) Infrared absorption spectrum: liquid film method (in chloroform)
, νmax (cm1): 3490.2915.1
710.1697.1370.1245° chart is shown in Figure 3.

(7) Nuclear magnetic resonance spectrum in double chloroform, 10
A chart measured at 0M) (z is shown in FIG. 5).

(8) Solubility: Well soluble in methanol, ethanol, chloroform, acetone, ethyl acetate, and dimethyl sulfoxide, slightly soluble in hexane and ether, and insoluble in water.

(9) Color reaction: iodine vapor, sulfuric acid, Ehrlich,
Positive for phosphomolybdic acid reaction. Negative for ninhydrin and Tollens reaction.

<II Distinction between basic, acidic, and neutral Weakly acidic.

(S-632-82 substance) (1) Properties: Pale yellow oily substance.

(2) Molecular weight: 323 (according to electron impact (EI) mass spectroscopy).

(3) Elemental analysis value (%): C62,59, H7,91,
N4.23° (4) Specific rotation: [α] = +73° (C = 1.0
) in chloroform).

(5) Ultraviolet absorption spectrum: in ethanol, λmax
(nm) (ε): 207 (14800), 280
(610). The chart is shown in Figure 2.

(6) Infrared absorption spectrum: liquid film method (in chloroform), νwax (cm”): 3350) 2920.
The 1725.1705.1250° chart is shown in Figure 4.

(7) Nuclear magnetic resonance spectrum: in deuterated chloroform, 10
A chart measured at 0 MHz is shown in FIG.

(8) Solubility: Well soluble in methanol, ethanol, chloroform, acetone, ethyl acetate, and dimethyl sulfoxide, slightly soluble in hexane and ether, and insoluble in water.

(9) Color reaction: Positive for iodine vapor, sulfuric acid, Ehrlich, and phosphomolybdic acid reactions. Negative for ninhydrin and Tollens reaction.

(101 Distinction between basic, acidic, and neutral: weak acidity.

The S-632B+ and S-632-82 substances of the present invention are derived from Saccharomyces.
It has antibacterial activity against fungi such as yeasts of the genus.

In addition, S-632B+ and S-632-B2 substances are
It also has a cytocidal effect on cultured cell lines (KB cells) derived from human nasopharyngeal carcinoma.

The S-632B+ and S-632-82 substances of the present invention can be obtained by culturing microorganisms. That is, this substance can be obtained by culturing S-632-B and a strain capable of producing S-632-B2 substance (hereinafter referred to as S-632 substance-producing bacteria) under appropriate conditions, and then collecting the culture fluid. can do.

S-632 substance-producing bacteria that can be used to produce the present substance include strains belonging to the genus Streptomyces. As an example, Streptomyces sp. S-632 (S trcptom
yccs 5pecies S-632) strain. This strain belongs to the genus Streptomyces, which the present inventors newly isolated from soil in Shandong Province, People's Republic of China. ,
Accession number [FERM BP No. 1849 (FERM BP)
-1849) has been deposited as J.

Its mycological properties are as follows.

(a) Form Branching method of spore-forming hyphae: simple branching.

Morphology of sporulation: spirals (spore shape is cylindrical).

Number of spores: 10-50 spores or more.

Spore surface structure: markedly wrinkled, warty, partially smooth
).

Spore size: 0.8~1.0X1.1~1.2μm (
However, in many cases the individual spore shapes are unclear).

Presence or absence of flagellated spores: None.

Presence or absence of sporangia: None.

Spore stalk epiphyte position: aerial hyphae.

Presence or absence of sclerotia formation: None.

(b) Growth status in various media Growth conditions in various media are shown in Table 1.

The observation method was in accordance with the ISP method manual, and the classification color names were shown as "Color Standards" (Japan Color Research Institute). For detailed colors, please refer to the color code of "Color Harmony Manual" ()
I added it inside.

(c) Physiological properties Growth temperature range: Grows well in a temperature range of 27 to 30°C.

Liquefaction of gelatin (glucose/peptone φ gelatin medium, 27°C): Positive (weak).

Same (simple gelatin medium, 20°C): Negative.

Milk coagulation (37°C): Positive.

Peptonization of milk (37°C): Positive.

Production of melanin-like pigment: on tyrosine agar (ISP-7) medium, on peptone/yeast extract/iron agar (ISP-6) medium, and on tryptone/yeast extract/iron agar (ISP-6) medium.
Negative in yeast extract (lSP-1) liquid medium.

Production of hydrogen sulfide (peptone, yeast extract, iron agar (IS)
P-6) with 0.5% yeast extract added): Positive.

Hydrolysis of starch (starch/inorganic salt agar, l5P-
4 medium): Positive.

Nitrate reduction (bouillon containing 1% potassium nitrate, l5P
-8 medium): Positive.

Cellulose degradation: negative.

(d) Availability of carbon sources (Breedham-Gotlieb agar, 15P-9 medium) L-arabinose, D-xylose, D-7 lactose, sucrose, L-rhamnose, raffinose, inositol, D-mannitol, D- It thrives on galactose, soluble starch, dextrin, glycerol and maltose, and utilizes D-glucose and salicin. Slight growth is observed even on a medium without carbon source addition.

(e) Bacterial cell composition The method of Becker et al. [Appl, Microbiol, Inc.]
12.421-423 (1964)), LL type diaminopimelic acid was detected.

The above mycological properties, especially the characteristics that this strain forms aerial hyphae with more chains of spores than basal hyphae, that the amino acid in the cell wall composition is LL-diaminopimelic acid, and that it does not form flagellar spores or sporangia. It is clear that the strain belongs to the genus Streptomyces.

Therefore, this strain can be classified as Streptomyces sp.
-632 (Streptomyces 5pecies
S-632).

S-632-B+ and S-632-B2 of the present invention
The substance can be produced by culturing various S-632 substance-producing bacteria belonging to the genus Streptomyces, such as the above-mentioned S-632 strain or its mutant strains, in an appropriate medium.

The cultivation of the above-mentioned microorganisms is in principle similar to the cultivation of general microorganisms, and is preferably carried out under aerobic conditions, such as a shaking culture method using liquid culture or an aerated agitation culture method.

The medium used for culture may be any medium containing a nutrient source that can be used by the S-632 substance-producing bacteria, and any of various synthetic media, semi-synthetic media, natural media, etc. can be used. As for the medium composition, carbon sources such as glucose, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, and organic acids can be used alone or in combination. Nitrogen sources include organic nitrogen sources such as Pharmamedia, peptone, meat extract, yeast extract, soybean flour, casein, amino acids, and urea.
Inorganic nitrogen sources such as sodium nitrate, ammonium sulfate, etc. may be used alone or in combination. In addition, sodium salts, potassium salts, magnesium salts, phosphates, other heavy metal salts, and the like may be added to the medium as appropriate.

If there is significant foaming during culture, add antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol, and heptadecanol, and various silicon compounds to the medium as appropriate. It can also be added.

The pH of the medium is preferably slightly acidic to near neutral. The culture temperature is a temperature at which S-632 substance-producing bacteria grow well, usually 20 to 37°C, particularly preferably about 27°C.
It is best to keep the temperature around ~30°C. The culture time is generally about 3 to 8 days in both cases of liquid shaking culture and aerated agitation culture. The target S-63 obtained by the above culture
2-B and S-632B2 substances are produced and accumulated.

Of course, the various culture conditions described above can be changed as appropriate depending on the type and characteristics of the microorganism used, external conditions, etc., and optimal conditions can be selected and adjusted from the above ranges according to each.

S- produced by this culture is useful as an antibiotic, etc.
The 632B+ and S-632-82 substances can be isolated according to a general method for collecting fermentation products. Various means such as solvent extraction, liquid exchange, or crystallization can be used alone or in combination in any order.

More specifically, S-632- produced by the above culture
B, and S-632-82 substances are mainly cultured liquid (
Since S-632-81 and S-632-B2 are present in the culture furnace liquid), first perform p-filtration, centrifugation, etc. according to the usual method to separate the culture furnace liquid from the bacterial solids. For fermentation fluids containing substances, ethyl acetate, immiscible with water,
S-632 using a solvent such as chloroform or butanol
-B and S-632-82 substances are transferred to an organic solvent layer, Glauber's salt is added to the obtained solvent layer, and after dehydration, the solvent is distilled off under reduced pressure to produce S-632B+ and S-632-82.
A crude extract containing two substances can be obtained. If necessary, methods such as adjusting the pH with hydrochloric acid or sulfuric acid, increasing the extraction efficiency by adding industrial salt, etc., and preventing emulsion can be taken.

For further purification, conventional means for purifying fat-soluble low molecular weight substances can be applied. That is, various types of adsorption chromatography using adsorbents such as silica gel, alumina, and macroporous nonionic adsorption resins, and reverse phase chromatography using ODS-bonded silica gel and the like can be used.

Of these, silica gel chromatography uses a mixed solvent system such as chloroform/acetone or ethyl acetate/benzene as an elution solvent, and reversed phase chromatography uses a mixed solvent system such as acetonitrile/water or methanol/water for elution. Available.

If further purification is required, the above chromatography may be repeated or Sephadex LH-20 (manufactured by Pharmacia) using methanol as the elution solvent.
Highly pure S-632B+ and S-632-82 substances can be isolated and purified by performing gel filtration chromatography in appropriate combinations.

Example Next, a more detailed explanation will be given with reference to examples.

In addition, confirmation of active substances during the purification process is performed using S-632B+.
and microorganisms whose growth is inhibited by the S-63282 substance, such as Satucharomyces cerevisiae IF.
o 0304 (Saccharomyces e
erevisiae I F O0304) and a method in which the test bacteria are developed using thin layer chromatography or paper chromatography and then detected by bioautography using the above-mentioned test bacteria or a method in which color development is performed using iodine vapor is used in combination. It's good.

Furthermore, the effective fraction can be confirmed by examining the cell killing effect on cultured human nasopharyngeal cancer cell lines (KB cells).

Example 1 Production of s-632B+ and S-632-B2 substances Glucose 0.1%, glycerol 4.0%, potato starch 0.2%, soy flour 2.0%, peptone 0.5%,
Medium (pH 7.0) consisting of 0.5% dry yeast, 0.5% sodium chloride, and 0.2% calcium carbonate 100mQ
was dispensed into a 500 mG Erlenmeyer flask, and after sterilization, a platinum loopful of Streptomyces sp. strain S-632 (Feikoken Jokyo No. 1849) was inoculated and cultured with rotational shaking at 27°C for 72 hours. (220 revolutions per minute, amplitude 7 cm).

Next, glycerol 3.0%, glucose 0.2%, potato starch 0.2%, peptone 0.3%, dry fermentation 13
A medium (p
H6,4) in a 500 mQ Erlenmeyer flask at 100 mQ.
After sterilization, add the above inoculum at a rate of 5%,
Rotary shaking culture was carried out at 27°C for 144 hours. After culturing, collect the culture solution (12.6Ω, pH 8.2-8.4), centrifuge, filter, adjust the pH to 7.6 with dilute sodium hydroxide solution, and stir 3 times with ethyl acetate (2ρ). Extracted. This ethyl acetate extracted fraction was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellowish brown oil (6 g). This oily substance was mixed with chloroform/acetone (approximately 20 mQ
), adsorbed on silica gel column chromatography (Merck Kieselgel, 6, OX 45 cm), and eluted with chloroform/acetone (4:1).

Elution fraction of Satucharomyces cerevisiae I
By bioassay using Fo 0304, S-
The active fractions containing both 632-B and S-632-82 substances were collected, and the solvent was distilled off and dried to give a pale yellow oil (429).

The resulting oil was dissolved in acetonitrile/water (20:80).
After removing insoluble matter by centrifugation, 1/3 of the volume was adsorbed on an ODS-bonded silica gel column (Yamazen Co., Ltd. rMM1515J, 1.5 x 50 cm), and acetonitrile/water (20:80 ) was eluted.

By bioassay of elution fractions, S-632
The active fractions containing B+ and S-632B2 substances were collected, the organic solvent was distilled off under reduced pressure, and the aqueous solution was freeze-dried to obtain a pale yellow oily substance, 19 mg (S-632-B+
) and 6 mg (8632B2) were obtained, respectively.

The obtained 3 632 B+ and S-632-B2
Substance series Sabo by agar plate dilution method
The minimum inhibitory concentration (MIC) against fungi on agar medium (uraud) is shown in Table 2.

Substances S-632B+ and S-63282 showed activity against yeasts of the genus Saceharomyccs.

In addition, the S-632B+ and S-632-B2 substances have a
The 0% growth inhibitory concentration (ICso) was 0.03 and 3 μg/mQ, respectively.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of S-632B+ substance.
FIG. 2 shows the ultraviolet absorption spectra of the S-632-82 substance. FIG. 3 shows the infrared absorption spectrum of the S-632-B material, and FIG. 4 shows the infrared absorption spectrum of the S-63282 material. FIG. 5 shows the nuclear magnetic resonance spectrum of the S-632B+ material, and FIG. 6 shows the nuclear magnetic resonance spectrum of the S-632-82 material. (that's all)

Claims (1)

[Claims] [1] S-632-B_1 substance having the following properties. (1) Properties: Pale yellow oily substance. (2) Molecular weight: 323 (according to electron impact (EI) mass spectroscopy). (3) Elemental analysis values (%): C62.76, H7.71, N4.12. (4) Specific optical rotation: [α]^2^5_D=+68°(C=
1.0) in chloroform). (5) Ultraviolet absorption spectrum: in ethanol, λmax(
nm) (ε): 206 (14500), 282 (450
). The chart is shown in Figure 1. (6) Infrared absorption spectrum: liquid film method (in chloroform)
, νmax (cm^-^1): 3490, 2915, 1
710, 1697, 1370, 1245. The chart is shown in Figure 3. (7) Nuclear magnetic resonance spectrum: in deuterated chloroform, 10
A chart measured at 0 MHz is shown in FIG. (8) Solubility: Well soluble in methanol, ethanol, chloroform, acetone, ethyl acetate, and dimethyl sulfoxide, slightly soluble in hexane and ether, and insoluble in water. (9) Color reaction: Positive for iodine vapor, sulfuric acid, Ehrlich, and phosphomolybdic acid reactions. Negative for ninhydrin and Tollens reaction. (10) Distinction between basic, acidic, and neutral: Weakly acidic. [2] S-632-B_2 substance having the following properties. (1) Properties: Pale yellow oily substance. (2) Molecular weight: 323 (according to electron impact (EI) mass spectroscopy). (3) Elemental analysis values (%): C62.59, H7.91, N4.23. (4) Specific rotation: [α]^2^5_D=+73°(C=
1.0 in chloroform). (5) Ultraviolet absorption spectrum: in ethanol, λmax(
nm) (ε): 207 (14800), 280 (610
). The chart is shown in Figure 2. (6) Infrared absorption spectrum: liquid film method (in chloroform)
, νmax (cm^-^1): 3350, 2920, 1
725, 1705, 1250. The chart is shown in Figure 4. (7) Nuclear magnetic resonance spectrum: in deuterated chloroform, 10
A chart measured at 0 MHz is shown in FIG. (8) Solubility: Well soluble in methanol, ethanol, chloroform, acetone, ethyl acetate, and dimethyl sulfoxide, slightly soluble in hexane and ether, and insoluble in water. (9) Color reaction: Positive for iodine vapor, sulfuric acid, Ehrlich, and phosphomolybdic acid reactions. Negative for ninhydrin and Tollens reaction. (10) Distinction between basic, acidic, and neutral: Weakly acidic.