JPS6135788A - Physiologically active substance ss20846a and its preparation - Google Patents

Abstract

PURPOSE:To prepare a physiologically active substance SS20846A effective to suppress the peristaltic motion, by culturing a microbial strain belonging to Streptomyces genus and capable of producing the physiologically active subtance SS20846A. CONSTITUTION:A microbial strain belonging to Streptomyces genus and capable of producing physiologically active substance SS20846A [e.g. Streptomyces Sp S20846 (FERM-P No.7729)] is separated from the soil collected in the Narita City, Chiba Prefecture, Japan, inoculated in a medium containing a nutrient source, and cultured aerobically at 25-30 deg.C for 3-5 days to obtain the physiologically active substance SS20846A having the following physical and chemical properties. EI-mass spectrum (70eV), M<+>m/z 167; specific rotation [alpha]D<21> =-16.5 deg. (C=0.85, chloroform); ultraviolet absorption spectrum, shown in Fig.1; infrared absorption spectrum (liquid film), shown in Fig.2; <1>H-NMR spectrum (90MHz) measured in [<2>H]chloroform using TMS as the standard substance, shown in Fig.3, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel physiologically active substance SS20846A and a method for producing the same.

[Conventional technology]

Conventionally, various physiologically active substances produced by microorganisms have been known.

[Problem that the invention seeks to solve]

The purpose of the present invention is to obtain a novel physiologically active substance useful for medical use.

[Means to solve the problem]

The present inventors isolated numerous microorganisms from natural soil,
As a result of conducting various studies on the product, it was discovered that a bacterial strain isolated from the soil of Narita City, Chiba Prefecture, produces SS20846A, a new physiologically active substance that has the effect of suppressing gastrointestinal motility, and the present invention was completed.

In other words, the present invention relates to a novel physiologically active substance SS20846.
A and a method for producing the same.

8 producing physiologically active substance SS20846A of the present invention
Strain 20846 has the following mycological properties.

(1) Form Cultured on various agar media at 28℃ for 10 to 14 days, 8208
The morphology of the 46 strains was observed using an optical microscope or a scanning microscope, and the results showed the following characteristics.

Spore-forming hyphae are simply branched from aerial hyphae, and their tips are spiral-shaped. This can be observed well on yeast/malt agar, oatmeal agar, starch agar, etc. No axle branching is observed. When mature conidia are observed on oatmeal agar medium, chains of 10 or more spores are observed, and the shape of each spore is oval to cylindrical, and the size is 0.5 to 0.7 x 0.8 to 1.2 pm. The spore surface structure is smooth. We observed this strain grown on various agar media, but no special structures such as spore paste, electrospores, or sclerotia were observed. Also,
No division of basal hyphae was observed.

(2) Growth status on various media The growth status of strain 820846 on various media is shown in the following table. Observations were made after culturing at 28°C for 14 days. In addition, the color description is published by Nippon Shikiken Business Co., Ltd. (1981)
I used the systematic color names from the ``Small Dictionary of Color Names''.

(3) Physiological properties ■ Growth temperature range (yeast/malt agar medium, 14-day culture) Optimum growth temperature 27-30°C Growth temperature 13-39°C ■ Liquefaction of gelatin Positive ■ Hydrolysis of starch Positive ■ Skimmed milk Coagulation of positive Peptonization of skimmed milk [iE ■ Production of melanin-like pigments Positive ■ Reduction of nitrates Negative ■ Degradation of cellulose Negative (bleeding) Assimilation of carbon sources (Pritno-Mudegodrieb agar medium, 28°C, 14 days culture) L- Arabinose, D-xylose, D-glucose,
D-7 Lactose, Sucrose, Inositol, L-
Rhamnose, raffinose, and D-mannite are all commonly 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 820846 is a strain belonging to the genus Streptomyces. Therefore, we have determined that 320
846 strains include Streptomyces sp. 820846 (
Streptomyces sp, 820846).

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-7729.

The physiologically active substance SS20846A of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. The physiologically active substance SS20846A producing strain is not only the above-mentioned 820846 strain, but also its artificial mutants or natural mutants.
Any material capable of producing A can be used in the present invention. The above-mentioned artificial mutant strain 820846 can be easily obtained using, for example, ultraviolet rays, cobalt-60 irradiation, or chemical mutagenic agents, as in the case of other actinomycetes.

Next, the cultivation of the bacterial strain in the production of the physiologically active substance SS20846A will be explained. That is, a normal actinomycete culture method is used to culture the physiologically active substance S S 20846A-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, glucose, galactose, glycerin, dextrin, starch, 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. Nitrogen sources include inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, raw wood, sodium nitrate, monosodium glutamate, etc., and also natural products, such as soybean extract, soybean flour, defatted soybean, cornstarch liquor, peptone, nitrogen. , yeast extract, cottonseed meal, etc. may be used alone or in combination.

As the inorganic substance, for example, lesium carbonate, sodium chloride, steel sulfate, manganese chloride, zinc sulfate, and cobalt chloride "I" are used alone or in combination.

Other substances that help the growth of the 820846 strain and promote the production of SS20846A or general antifoaming agents such as silicone oil or Azonol (trade name) can also be added to the medium as appropriate.

As the culture method, methods generally used for the production of physiologically active substances are employed, but liquid culture methods, particularly deep agitation culture methods, are most suitable. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 25 to 30°C, but generally 27°C.
It is preferable to culture nearby. Physiologically active substance SS208
In both cases of shaking culture and submerged agitation culture, the production of 46A reaches its maximum after 3 to 5 days of culture. When the accumulated amount of physiologically active substance SS20846A in the culture solution reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution. Physiologically active substance SS20846A from culture solution
Isolation is carried out 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 SS20846A normally exists in the culture solution, the bacterial cells are separated from the culture by centrifugation or filtration, and the cultured solution is subjected to conventional separation methods such as solvent extraction, ion exchange resin method, etc. Physiologically active substances SS20846 can be prepared by gel filtration method, adsorption or distribution column chromatography method, dialysis method, precipitation method, etc. alone or in appropriate combinations.
Separate and purify A.

Examples of preferred separation and purification methods include the following methods. After fermentation, the culture is centrifuged to obtain lachrymal fluid.

Next, the pH of the tear fluid is adjusted to an alkaline range, and a suitable solvent,
For example, extract with ethyl acetate.

The resulting extract is then concentrated and further extracted with dilute hydrochloric acid solution. The pH of this extract was adjusted to the alkaline range again.
After extraction with a suitable solvent such as ethyl acetate, the extraction solvent is distilled off and the residue is subjected to silica gel column chromatography. The active fractions are collected and concentrated under reduced pressure, and the residue is subjected to column chromatography using Sephadex LH20 (manufactured by Pharmacia Fine Chemicals). The active fractions are collected and concentrated under reduced pressure to yield SS20846A, a colorless oil.

8820846A obtained as described above has the following physical and chemical properties.

<Physical and chemical properties> ■EI--e soot vector (70ev) M+ fovea 167 ■Specific optical rotation [α] 21 = -16.5° (C = 0.85.Chloroform) ■Ultraviolet absorption spectrum Figure 1 λEjOH ( E 1%): 228.5 (3, lX10
") max ltM ■ Infrared absorption spectrum Figure 2 ■ "H-NMR spectrum (90 MHz) Measured using TMS in a deuterated chloroform solution as a reference material.

Figure 3 δ (ppm): 1.63 (m, 7H), 2.50
(s, 2H), 2.88 (m.

2H), 3.55 (m, IH), 4.08 (m, I
H).

5.30-6.40 (m, 4H)■13C-NM
R spectrum (22.5 MHz) was measured using TMS in deuterium chloroform as a standard substance.

δ (ppm): 133.3 (dL131.0 (d), 1
29.9 (d) + 128.6 (d), 63.8 (d),
52.4(d), 40.3(t), 39.5(t), 3
2.9 (t), 17.7 (q) ③ Color reaction A purple color appears in the vanillin-sulfuric acid test solution (heated).

Also, after spraying with dilute sulfuric acid, it turns brown when heated.

In addition, the Dragendorff test solution was negative.

■Distinction between basic, acidic, and neutral Basic ■Color and properties of the substance Colorless oily substance ■Thin layer chromatography carrier Nijiri gel spray) F254 (manufactured by Merck & Co.)
■Molecular formula % Formula % ■Structural formula H [Operation] The physiologically active substance SS20846A of the present invention has the following biological activities.

■Gastrointestinal motility inhibitory effect Experimental method: ddY male mice (5 weeks old) were set at 6 mice per group, and the physiologically active substance SS20846A of the present invention was intraperitoneally administered.
Minute powder emulsion (5-carbon powder, 10% gum arabic)
0.2 mt was administered orally. A separate control group was established, and 30
After 1 minute, the intestine was removed and the distance to the advanced part of the carbon terminal was measured.
The small intestine transit rate was determined relative to the control group. The results are shown in Table 1.

Table 1 [Effects of the Invention] As described above, the physiologically active substance of the present invention S S 20846A
has the effect of suppressing transport capacity based on gastrointestinal motility, and is useful as an antispasmodic system.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 Liquid medium having a composition of 1.0% glucose, 2.0% soluble starch, cottonseed meal i, os, 0.5% cornstarch liquor, 0.2% ammonium sulfate, and 0.3% calcium carbonate. Adjust the pH to 7°O, dispense 100ml into a 500ml Yosaka flask, and sterilize. This is inoculated with Streptomyces sp. strain 820846 (Feikoken Bacterium No. 7729) and cultured with shaking at 27°C for 2 days to prepare a seed culture. 301 liters of liquid medium with the same medium composition
320 ml of the above-mentioned seed culture solution was inoculated into a jar fermentor, and the culture temperature was 27°C and the number of stirring was 45 O.
Culture is carried out for 96 hours under conditions of r, p, m, and aeration rate of 16 t/min.

After culturing, the culture solution is centrifuged, and the resulting ν solution is adjusted to pH
10, added 9 t of ethyl acetate, and extracted twice. Then, the obtained ethyl acetate extract was concentrated under reduced pressure (approximately 2.
OL-! t) and further diluted with 300 m of 2N-hydrochloric acid.
I! Extracted twice. Thereafter, the pH of this hydrochloric acid layer was adjusted to 10 with a 5N sodium hydroxide bath solution under ice cooling, and this was extracted three times with 2 t of ethyl acetate.The solvent was concentrated under reduced pressure, and the basicity of the dark brown oil was Fraction 2.36 f was obtained.

This dark brown oil was subjected to silica gel (Wako Pure Chemical Industries, Ltd. Wako Gel C-200) column chromatography (φ3 cm).
x 45 cm), eluted with a mixture of chloroform/methanol/water (7:7:2), removed the eluted fraction, and then eluted with a mixture of methanol/triethylamine (4:1) to remove the active fraction. collected. The oily substance obtained by concentrating these two fractions under reduced pressure was dissolved in a small amount of chloroform/methanol (1:1) mixture and subjected to Sephadex LH-20 (manufactured by Pharmacia Fine Chemicals) column chromatography (φ
3α
42 hits were obtained.

[Brief explanation of drawings]

Fig. 1 shows the ultraviolet absorption spectrum of the physiologically active substance SS20846A of the present invention, Fig. 2 shows its infrared absorption spectrum, and Fig. 31 shows its 'H-NMR spectrum. that's all

Claims (1)

[Claims] 1. Physiologically active substance SS20 having the following physicochemical properties
846A. [1] EI-mass spectrum (70eV) M^+m/z167 [2] Specific rotation [α]^2^1_D=-16.5° (C=0.85, chloroform) [3] Ultraviolet absorption spectrum No. Figure 1 λ^E^t^O^H_m_a_x(E^1^%_1_c
_m): 228.5 (3.1 x 10^3) [4] Infrared absorption spectrum (liquid film method) Figure 2 [5]^1H-NMR spectrum (90MHz) Measured in deuterium chloroform solution using TMS as a reference substance did. Figure 3 δ (ppm): 1.63 (m, 7H), 2.50 (s,
2H), 2.88 (m, 2H), 3.55 (m, 1H)
, 4.08 (m, 1H), 5.30-6.40 (m, 4
H) [6]^1^3C-NMR spectrum (22.5MHz
) TMS in deuterated chloroform solution was used as a reference substance. δ (ppm): 133.3 (d), 131.0 (d),
129.9(d), 128.6(d), 63.8(d)
, 52.4(d), 40.3(t), 39.5(t),
32.9 (t), 17.7 (q) [7] Color reaction Vanillin-sulfuric acid test solution (heated) shows purple color. Also, after spraying with dilute sulfuric acid, it turns brown when heated. still,
Dragendorff test solution was negative. [8] Distinction between basic, acidic and neutral Basic [9] Color and properties of substance Colorless oily substance [10] Thin layer chromatography carrier: Silica gel plate F_2_5_4 (manufactured by Merck & Co.) [11] Molecular formula C_1_0H_1_7NO 2, structure The novel physiologically active substance SS20846A according to claim 1 is represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼. 3. Physiologically active substance SS20 belonging to the genus Streptomyces
1. A method for producing a novel physiologically active substance SS20846A, which comprises culturing 846A-producing bacteria and collecting the physiologically active substance SS20846A from the culture. 4. The SS20846A producing bacterium is Streptomyces sp. S2084
6 (KAIKOKEN BIYORI NO. 7729).