JPS61189280A - Novel physiologically active substance ss43405e and production thereof - Google Patents

Abstract

NEW MATERIAL:The physiologically active substance SS43405E of formula having the following physical and chemical properties. Molecular formula, C22H18O5; mass spectrum, (EI 70eV)m/z 362(M<+>); melting point, 210-212 deg.C; specific rotation, [alpha]<25>D=-14 deg.C (c=0.50 methanol); solubility, soluble in chloroform, dimethyl sulfoxide, ethyl acetate and methanol, hardly soluble in n-hexane and water; pH, neutral; color and nature of the substance, yellow crystal; color reaction, positive to ferric chloride reaction; etc. USE:Pharmaceuticals promoting the function to exclude the foreign materials in reticuloendothelial system. PREPARATION:Streptomyces sp. S43405 (FERM-P 7956) which is a bacterial strain belonging to Streptomyces genus and capable of producing the physiologically active substance SS43405E is cultured by conventional liquid cultivation process for actinomycetes by submerged technique under agitation and the objective compound of formula I is separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel physiologically active substance 8843405E 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]

An object of the present invention is to provide a novel physiologically active substance useful as a pharmaceutical.

[Means for solving problems]

The present inventors isolated numerous microorganisms from natural soil,
As a result of conducting various studies on the product, it was discovered that a strain isolated from the soil of Hara Village, Suwa District, Nagano Prefecture produces a novel physiologically active substance 5S43405E, and the present invention was completed.

That is, the present invention provides novel physiologically active substance 5S43405
E and its manufacturing method.

84 producing the physiologically active substance 5S43405F of the present invention
The 3405 strain has the following mycological properties.

α) Cultured on various agar media at 28°C for 10 to 14 days.
As a result of observing the morphology of strain 05 using an optical microscope or an electron microscope, it showed the following characteristics.

The spore-forming hyphae are simply branched from the aerial hyphae, and their tips are planar or wavy. This can be observed well on yeast/malt agar, O'meal agar, starch agar, etc. No axle branching is observed. When mature conidia are observed on an oatmeal agar medium, a chain of 10 or more spores is observed, and the shape of each spore is oval to cylindrical, and the size is 0.5 to 0.7 × O,8 to 1.3μ
It is m. The surface structure of the spore is smooth. We observed this strain grown on various agar media, but found no special structures such as sporangia, flagellated spores, or sclerotia. Furthermore, no division of basal hyphae was observed.

(2) Growth status on various media The growth status of strain 843405 on various media is as shown in the following table. 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 Shokuken Business ■ (1981). In addition, the number in parentheses in the table indicates the color standard number.

Blank space below (3) Physiological properties■ Growth temperature range (yeast/malt agar medium, 14-day culture) Optimum growth temperature 25-35℃ Possible growth temperature 12-35℃ ■ Liquefaction of gelatin Negative■ Hydrolysis of starch Positive■ Coagulation of skimmed milk
Negative ■ Pezotonization of skim milk Positive Production of melanin-like pigment Negative ■ Reduction of nitrate Negative ■ Decomposition of cellulose Negative (4) Assimilation of carbon sources (Siritono 1/Godlieb agar medium, cultured at 28℃, 14 days) L- Arabinose, D-xylose, D-glucose,
D-7 lactose, inosyl), L-rhamnose, raffinose, and D-mannitol are used, and sucrose is not used.

(5) Cyamopimelic acid in all bacterial cells As a result of analyzing cyamino pimelic acid in all bacterial cells, LL-
Cyanopimelic acid was detected.

Based on the above morphological characteristics and containing LL-cyaminopimelic acid, strain 843405 is a strain belonging to the genus Strezotomyces.

The mycological properties of strain 843405 are summarized as follows. The tip of the aerial hyphae is straight or wavy, and its conidia consist of 10 or more spores linked together, and the surface of each spore is smooth.

The color tone of aerial mycelium is red color series, the color of the underside is yellow to brown or orange, and glycerin.
A slightly uniform color on the back side of the indicator is observed on nitrate agar medium. Soluble pigments are generally faint brown to red, but the pH changes on glycerin/nitrate agar medium.
A slightly changing soluble pigment is observed. Melanin-like pigments are produced frequently.

Based on the above properties, Scherling and Gottlieb's ISP report "International Schernal op.
Systematic Bacteriolozo (Interna)
tional Journal of Systemat
ic Bacteriology) J Vol. 18, pp. 69, 279, (1968), Vol. 19, 39
1 (1969), Vol. 22, p. 265 (1972) and [Bergey: s M
annual of Determinative Ba
As a result of searching from the 8th edition of 843405 strain, the following four species were found to be closely related to strain 843405.

Streptomyces vinaceus
Streptomyces rosa (Streptomyces rosa) Streptomycea moderatus
) Streptomyces californicus
US) Comparing strains 481 and 843405 above, Strenotomyces vinaceus reduces nitrate and liquefies gelatin, as well as L-arabinose, inositol,
It differs in that it does not use L-rhamnose or raffinose, and the back side exhibits a characteristic purple color. Streptomyces rosa has spiral formation, D-fructose,
They differ in that they use inositol and rynose. Streptomyces moderatus exhibits white aerial mycelium on nutrient agar, brown on malic acid/lime agar, and pink soluble pigment on nutrient agar, and also reduces nitrate and slightly reduces sucrose. Points of use,
The difference is that the layer surface does not show the characteristic reddish orange color like the 843405 strain. Streptomyces californicus is different in that it reduces nitrates, does not utilize L-arabinose, inositol, L-rhamnose, or raffinose, and has a characteristic purple color on its underside.

As described above, the 843405 strain does not match any species, so the present inventors investigated Strezotomyces S.b. 84340 in order to distinguish the 843405 strain from known strains.
5 (Strepto-myces sp, 8434
05) and was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7956 (FEBM P-7956).
Deposited as.

The physiologically active substance 5S43405E of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically. The physiologically active substance 5S43405E producing strain includes not only the above-mentioned 843405 strain, but also its artificial or natural mutant strains.
Any material capable of producing 5E can be used in the present invention. The above-mentioned artificial mutant strain 843405 can be easily obtained by, for example, ultraviolet irradiation, cobalt-60 irradiation, addition of a chemical mutagenic agent, etc., as in the case of other actinomycetes.

Next, culturing of the bacterial strain in the production of the physiologically active substance 5S43405E will be explained. That is, a normal actinomycete culture method is used to culture the strain producing the physiologically active substance 5S43405E belonging to the genus Strezotomyces.

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, etc.

As the carbon source, for example, glucose, fructose, mannitol, starch, molasses, etc. are 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, pezotone, meat extract, dried yeast, cottonseed meal. , proteose pezotone, casamino acid, corn-stizo liquor, etc. may be used alone or in combination. As the inorganic substance, for example, calcium carbonate, sodium chloride, steel sulfate, manganese chloride, zinc chloride, etc. are used alone or in combination. Helps the growth of 843405 other strains 5
A substance that promotes the production of S43405E or a general antifoaming agent such as silicone oil or γ-decanol (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.

Culture is carried out under aerobic conditions, and the appropriate temperature for culture is 2.
Although the temperature is 5 to 35°C, it is generally preferable to culture around 27°C. Physiologically active substance 5S43405E is cultured with shaking,
In any case of the deep agitation culture method, the production amount is 3 to 5.
Maximum reached after 1 day of incubation. Physiologically active substances in culture solution 5
When the accumulated amount of S43405E reaches the maximum, the culture is completely stopped and the target substance is separated and purified from the culture solution. Isolation of 5S43405E from the culture solution and production of Wt are 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 Examples below.

That is, since the physiologically active substance 5S43405F normally exists in the culture solution and the bacterial cells, the bacterial cells are separated from the culture by centrifugation or filtration, and the bacterial cells and the culture filtrate are subjected to a conventional separation method such as a solvent extraction method. ion exchange resin method,
The physiologically active substance 5S43405E is separated and purified by gel filtration, adsorption or distribution column chromatography, dialysis, precipitation, etc. alone or in appropriate combinations.

Examples of preferable separation and purification include the following methods. After fermentation, the culture is centrifuged and separated into a 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.
The filtrate is extracted with a suitable solvent such as ethyl acetate. The solvent of the extract was distilled off, and the residue was subjected to silica gel chromatography.

Next: After elution with an eluent such as chloroform, the active fractions are collected, concentrated under reduced pressure, and the residue is recrystallized with a suitable solvent such as a chloroform/methanol mixture to obtain yellow crystals of the physiologically active substance 8843405E. It will be done.

Physiologically active substance 5843405E obtained as above
has the following physical and chemical properties.

<Physical and chemical properties> ■ Elemental analysis CH Experimental value (%) 72.92 5.01 Theoretical value ←)
72.68 4.87 ■ Molecular formula % formula % [α] Bamboo = -14° (C = 0.50 methanol) ■
Ultraviolet absorption spectrum Figure 1 λMeOH nm (g) 239 (51800), 26
6ax (26800), 410 (9200) ■ Infrared absorption spectrum (KBr method) Figure 2 ■ IH-NMR spectrum (90 MHz) Figure 3 Measurements were made using TMS in heavy chloroform solution as a reference substance.

(2) Measurement was carried out using TMS in a solution of C-NMRx-e (22,5MH7) in deuterated chloroform as a reference substance.

δ (ppm), 187.1 (s), 181.8 (Touge 1
79.1 (s), 172.8 (s), 162.5 (s
), 156.7 (s), 149.6 (s), 136.2
(d), 135.9 (pass 132-3(s), 12
6.5(s), 125.4(d), 125.2(d
), 119.7(s), 119.1(d), 116.8
(s), ], ], 1.2 (dl, 40.4(d)
, 27.4(tl, 24.0(q), 17.8(q),
11.6(q) [Phase] Soluble Soluble in chloroform, dimethyl sulfoxide, ethyl acetate, methanol. n-Hexane, poorly soluble in water.

(■ Basic/acidic/neutral distinction Neutral 0 Color and properties of the substance Yellow crystals 0 Color reaction Positive in ferric chloride reaction Thin layer chromatography carrier Nijirica glucile) F's4 (manufactured by Merck & Co.) 0 Structural formula % Formula % From the above physical and chemical measurements and structural formula, the physiologically active substance 5S43405E of the present invention is a novel compound.

[For production]

The physiologically active substance 5S43405E of the present invention has the following biological properties.

■Carbon clearance effect Animal: ICR male mice, average weight 351, were used.

Carmen: Pelican Ink (all/ x4axa) at 300 Orpm.

After centrifugation for 15 minutes, supernatant diluted 5 to 6 times with physiological saline. I interpreted and used it.

Experimental method: Compound 5S43405E of the present invention was adjusted to various concentrations in 5% DMSO physiological saline (containing 1 drop of Tween 80%) and intraperitoneally administered to the above-mentioned group of 8 mice. After 24 hours, add 0.1 m7 of this diluted Pelican ink per 10 g of mouse! was administered into the tail vein,
After 4 minutes, pipet a 20μ micropipette into the orbital vein.
Blood was drawn.

After hemolyzing this with a 0.1% sodium carbonate solution of 3r111, the absorbance at a wavelength of 600 nm was measured using an absorbance meter, and the CK''J value was calculated using the following formula.
Shown in the table.

Tt - Tt 110gc1 Absorbance of blood after pcarbon administration (untreated group) 10gc2 Absorbance 4 minutes after zcarzene administration (untreated group and drug administration group) Tlp O (min) Tx ; 4 (min) Table 1 [ Effects of the Invention] Physiologically active substance 5S43405E has the effect of promoting the foreign body elimination function of the reticuloendothelial system, and is useful as a pharmaceutical.

〔Example〕

Next, an example will be given and explained.

Example A liquid medium having a composition of 2.0% soluble starch, 2.0% cottonseed meal, 1.0% corn stizo liquor, and 0.3% calcium carbonate was adjusted to pH 6.2, and 500-Yosaka Dispense into 100-ml flasks and sterilize. In addition to this, Strezotomyces S.B.
No. 6) and cultured with shaking at 27°C for 2 days to prepare a seed culture solution.

Next, glucose 1.0%, soluble starch 6. θ%, M actual phase 2.0%, corn stizo liquor 0.5 excellent,
A liquid medium having a composition of 0.5% yeast extract, 0.3% calcium carbonate, 0.0005% copper sulfate, 0.0005% mangon chloride, and 0.005% zinc sulfate was adjusted to pH 7.0 and placed in a 30 J volume Zoarfa. 18 seeds were prepared in a mentor, inoculated with 100 - of the above-mentioned seed culture solution, and 120 -
Incubate for hours. Note that the pH was adjusted to 7.2 during culturing using a 30% citric acid solution.

After the culture was completed, the culture solution was centrifuged, and the resulting filtrate was extracted twice with twice the amount of ethyl acetate.

On the other hand, methanol 5j was added to the bacterial cells, followed by stirring and filtration, and the resulting filtrate was concentrated under reduced pressure at 40° C. to about 1/1 volume. Next, an equivalent amount of water was added to this concentrated solution, and the mixture was extracted twice with 21 portions of ethyl acetate.

Combine with the previous extract and remove the solvent under reduced pressure to obtain crude extract 12-5.
I got r. This crude extract was collected using silica gel (Ki manufactured by Merck & Co., Ltd.).
esel Gel 60.23〇-400 mesh) column chromatography C4,0(yI) X 50c
The active fraction was obtained by elution with fluoroform.

The target substance thus obtained was concentrated to dryness, and then recrystallized from a chloroform/methanol mixture to obtain 5.
I got yellow crystal 125 lie of S43405F.

It was confirmed that this substance has the above-mentioned physical and chemical properties.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the physiologically active substance 8843405E of the present invention, Figure 2 shows the infrared absorption spectrum of the same,
Figure 3 is the H-NMR spectrum of the same.The above person: Miyuki Ariga, agent of Nisnis Pharmaceutical Co., Ltd., patent attorney;11..4 Procedural amendment (voluntary) April 8, 1985'1 """"""""'1""'1 Shiga 6] Description of the case 1985 Patent Application No. 31027 2 Title of the invention Novel physiologically active substance 5S43405E and its manufacturing method 3
Relationship with the case of the person making the amendment Applicant Address: 2-12-4 Nihonbashihama-cho, Chuo-ku, Tokyo
Name Nisnis Pharmaceutical Co., Ltd. Representative Nookata Yasumichi 4 Agent Sponsored 6 Column 7 of “Detailed Description of the Invention” of the specification to be amended Contents of the amendment (1) Page 7, line 9 of the specification “ ■ Correct the phrase "Penotonization of skimmed milk" to "-E!, +O)tonization of skim milk." (2) Same, page 7, line 1O, ``Generation of melanin-like pigment'' should be corrected to ``■ Generation of melanin-like pigment.'' (3) Same, page 9, lines 8 to 9, ``Gotslieb'' Tororo is corrected to ``Godlieb''. (4) Same, on the bottom line of page 9, the word ``Berzo's'' is corrected to ``Bersee's.'' (5) In the same article, page 10, lines 1 and 2, the words ``Bacteriorozoe'' are corrected to ``Bacteriorozoe''. (6) Same, page 14, line 14, "Steeg" and Ruru are corrected to "Steeg". (7) Same, page 16, line 10, "normal culture solution" is corrected to "normal culture filtrate". (8) Same, page 16, lines 10 to 11, and page 17, line 4, ``culture'' is corrected to ``culture solution.'' (9) Same, page 17, line 1 O [Silica gel chromatography J is corrected to read "silica gel column chromatography." QO same, page 23, lines 4-5, correct the word ``this dilution'' to ``dilution.'' 0]) Same, the bottom line of page 23 and the second line of page 24, the words "No treatment group" are corrected to "No treatment group." (13PJ, page 24, in the table, 2nd line "standard error" is corrected to ``standard deviation.'' Correct. (141, p. 25, lines 5 and 13 “Schizo
Correct ``stop'' to ``staap.'' (151, page 26, line 13, ``equivalent'' is corrected to ``equivalent''.

Claims (1)

[Claims] 1. A physiologically active substance SS43405E represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2. SS43, a physiologically active substance belonging to the genus Streptomyces
1. A method for producing physiologically active substance SS 43405E, which comprises culturing 405E-producing bacteria and collecting physiologically active substance SS 43405E from the culture. 3. The physiologically active substance SS43405E-producing bacterium is Streptomyces sp.
A method for producing physiologically active substance SS43405E according to claim 2, which is SS43405 (Feikoken Kyoiku No. 7956).