JPH02255655A - New physiologically active substance dc118 - Google Patents

Abstract

NEW MATERIAL:A physiologically active substance DC118 expressed by the formula. Appearance; light brown powder. Melting point; 149-152 deg.C. Solubility; soluble in methanol, benzene, etc., slightly soluble in n-hexane and insoluble in water. Rf value; 0.60 chloroform methanol (100:1). Elementary analysis; calculated value (%) C, 81.31; H, 8.53; N, 4.74; O, 5.42. Molecular formula; C20H25NO. USE:Useful for preventing and treating various disorders and diseases, such as angiopathy, inflammation or edema, having inhibitory action on peroxylipid formation. PREPARATION:A microorganism (FERM P-2275), belonging to the genus Stereptomyces and capable of producing the substance DC118 is cultured in a suitable liquid culture medium under aerobic conditions and the resultant culture is extracted with a solvent, such as ethyl acetate, or by adsorption, etc. The obtained extract is then purified to afford the substance DC118 expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] The present invention relates to a new substance, more specifically to a novel substance DC118, which can be produced by the strain Dc118 belonging to Streptomyces φchromofuscus and has an inhibitory effect on lipid peroxide production. .

Lipid peroxide generated in living organisms penetrates blood vessels. [Detailed Description of the Invention] Novel Substance DC118> 1) Chemical Structure The novel substance DC118 according to the present invention has a chemical structure represented by the above formula (I).

The chemical structure of DCllg was determined as described above by examining in detail the proton nuclear magnetic resonance spectrum, carbon-13 nuclear magnetic resonance spectrum, ultraviolet absorption spectrum, optical external absorption spectrum, and mass spectrometry spectrum.

2) Physicochemical properties (1) Appearance Light brown powder (2) Melting point 149-152°C (decomposed) (3) Solubility methanol, ethanol, chloroform, benzene,
Soluble in acetone, ethyl acetate, slightly soluble in n-hexane, insoluble in water (4) Rf value (Merck & Co., Ltd. “Silica Gel 60 F 25
4 J used) Chloroform-methanol (100:1) 0.6
0(5) FD-MS spectrum (m/z) 295 (
M'') (6) Ultraviolet absorption spectrum Figure 1 λsaxnm (g) 218 (15,29X10'
) (in methanol) 235 (14,05X10'
)254 (7,62xlO') 266 (6,03X104) 303 (8,29X10') 342 (2,01xlO') (7) Infrared absorption spectrum Figure 2 (KBr disk method) (8) Proton nuclear magnetic resonance Spectrum Figure 3 (50
0 MHz, in deuterochloroform) (9) Carbon-13 nuclear magnetic resonance spectrum Figure 4 (125 MHz, in deuterochloroform) (■0) Elemental analysis CHN O Analysis value C%) 81. 28 8. 55 4.74
5.43 Calculated value (1) 81. 31 8. 53
4. 74 5.42 (11) Molecular formula C20H25
NO<Manufacture of DCllg>Summary> DCllg can currently only be obtained by culturing microorganisms, but it can also be produced by synthetic chemical or microbiological modification of related compounds, or by total synthetic chemical method. It could also be manufactured.

When culturing a microorganism, a strain belonging to the genus Streptomyces and capable of producing DC118 is used. Specifically, the present inventors have revealed that the Streptomyces φriromofuscus strain DC118, which the present inventors isolated, produces DCllg;
As for other bacterial strains, suitable ones can be isolated from nature by conventional methods for isolating antibiotic-producing bacteria. In addition, Streptomyces chromofuscus DC11
There is also room to increase DCllg production ability by subjecting DCllg-producing bacteria, including the eight strains, to radiation or other mutation treatments. Furthermore, this DC118 strain DC1
It is also possible to use genetic engineering techniques using recombinants having genes encoding the production of 18 substances.

<Strain DC118> Strain DC118, which the present inventors have discovered as a Streptomyces strain capable of producing DC118, has the following content.

1. Origin and deposit number Strain DC118 was isolated from soil collected in Miyazaki City, Miyazaki Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 8, 1989. It has a number of FERM BP-2275 J (FERM BP-2275).

2. Mycological properties The mycological properties of the DC118 strain are as follows.

1) Morphology Strain DC118 grows poorly on sucrose/nitrate agar medium, but grows moderately on other media. Aerial mycelium grows moderately on starch/mineral salt agar, but poorly on yeast/malt agar, oatmeal agar, sucrose/nitrate agar, and nutrient agar, and is rarely observed on other media. It wasn't done. Aerial hyphae formed from substratum hyphae form simple branches and elongate, with chains of 10 to 50 spores. Although the spore chains are spiral, many are incompletely spiral, and many are loop-shaped.

The shape of the bottle is cylindrical to barrel-shaped, and the size is 0.5 to 0.7μ.
mXO is 19 to 1.1 μm, and its surface is thorn-like.

Special forms such as sporangia, flagellated spores, and sclerotia cannot be reviled.

2) Growth status on various media The results of culturing the DC118 strain on various media at 27°C for 3 weeks are shown in Table 1.

3) Physiological properties The physiological properties of the DC118 strain are as shown in Table 2.

4) Carbon source utilization The carbon source utilization of the DC118 strain (on Bridham-Gotlieb agar medium) is as shown in Table 3.

5) Analysis of diaminopimelic acid As a result of analysis of diaminopimelic acid, which is one of the amino acids constituting the cell wall, LL-diaminopimelic acid was detected.

Based on the above mycological properties, strain DC118 is judged to be a strain of the genus Streptomyces, and has the following characteristics.

(1) The spore chain is spiral to loop-shaped, and the surface of the spore is thorn-like.

(2) Aerial mycelial growth is generally poor on media other than starch/nitrate agar media, and the color is yellowish white to light olive gray, and the color of the underside is pale yellow to light yellowish brown to yellowish brown.

(3) Melanin-like pigment is produced on peptone yeast/iron agar medium, but no melanin-like pigment is observed on tyrosine agar medium or tryptone yeast liquid medium. Also, no soluble dyes are observed.

(4) Sucrose, rhamnose, and sorbitol are not assimilated.

From the above properties, I5P (International Streptomyces Project) records M (E, B).

Shirllng and D. Gottlieb:
Int, J, 5yst, Bact, 1869-189
．． 279-392 (1988); 19391-51
2 (1989); 22285-394 (1972)
) and Bergey's Manual of Determinative Bacteriology.
ual of' Determinatelve Bac
When searching for known related bacterial species using the 8th edition (1974) of Streptomyces chromofuscus (Streptomyces chromofuscus)
fuscus ) (Int, J.

5yst, Baet,: 1g, 307 (1968
)) is the closest approximation.

When strain DC118 is compared with Streptomyces chromofuscus, the morphological characteristics include spiral spore chains and spores with spiny surfaces, the fact that they produce melanin-like pigments on peptone, yeast, and iron agar media, and the fact that they produce aerial hyphae. The two match well in terms of color tone, etc. The difference is that DC
The 118 strain does not produce melanin-like pigment in tryptone yeast liquid medium and does not assimilate rhamnose.

As described above, although there are some differences, the DC118 strain closely matches those of Streptomyces chromofuscus in its basic characteristics, and therefore it is appropriate to identify it as Streptomyces chromofuscus. Therefore, D.C.
118 strains of Streptomyces chromofuscus (Str.
epton+yceschromofuscus) D
It is named strain C118.

Table 1 Table 2 Table 3 +: Utilization - Dual utilization culture/production of DC118> Compound DC118 is derived from DC belonging to the genus Streptomyces.
It can be produced by culturing 118-producing bacteria aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the DC118-producing bacteria. Specifically, for example, glucose, sucrose, MalF-su, glycerol, starch, fats and oils can be used as carbon sources, and soybean flour, fish meal, cottonseed meal, dried yeast, yeast extract, and corn can be used as nitrogen sources. Organic materials such as staple liquor and inorganic materials such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. In addition, if necessary, sodium chloride,
Inorganic salts such as potassium chloride, calcium carbonate, phosphates, heavy metal salts, etc. can be added. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 20 to 37°C, preferably 25 to 30°C. In this method, the production amount of DC118 reaches its maximum after 5 days of culture in both shaking culture and aerated agitation culture.

In this way, a culture enriched with D0118 is obtained. In the culture, a portion of DC118 exists in the culture filtrate, but most of it exists in the bacterial cells.

Any convenient method can be used to harvest DC118 from such cultures. One method is based on the principle of extraction, specifically, for DC118 in culture and solution, it is extracted from water-immiscible D
Solvent for C118 (see above) For example, extraction method with ethyl acetate, etc., or for DC118 in bacterial cells, method of recovering bacterial mass obtained by filtration, centrifugation, etc., by treating with methanol, ethanol, acetone, etc. be.

The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting DC118 from cultures is based on the principle of adsorption, in which D is already in liquid form.
For C118-containing substances, such as culture filtrate or extract obtained by performing the extraction operation as described above, an appropriate adsorbent such as silica gel, activated carbon, Diaion HP20J (manufactured by Mitsubishi Kasei Corporation), etc. DC118 can be obtained by adsorbing the target DC118 and then eluting it with an appropriate solvent.If the DC118 solution thus obtained is concentrated to dryness under reduced pressure, a crude sample of DC118 can be obtained. .

In order to further purify the crude sample of DC118 obtained in this way, the extraction method and adsorption method described above may be combined with gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary.

For example, adsorbents such as silica gel, gels such as Sephadex LH-20J (manufactured by Pharmacia), or column chromatography rMMC bags using general rules.
(manufactured by Yamamura Kagaku Co., Ltd.) or the like and a countercurrent distribution method can be carried out in an appropriate combination. Specifically, for example, a DC118 crude sample is dissolved in a small amount of methanol, and "Sephadex LH-20
Apply to J column, elute the active fraction with methanol, and concentrate to dryness to obtain a pure product of DC118.

<Applications of DC118> The compound DC118 according to the present invention is useful in that it has an excellent inhibitory effect on lipid peroxide production as described below, and is effective against various diseases, inflammation, and edema caused by circulation disorders in the brain, heart, and peripheral areas. It is expected to be a preventive/therapeutic agent for various pathological conditions such as.

The formulation and administration method for use as a drug is as follows:
Various conventionally known methods can be applied. That is, possible administration methods include injection, oral administration, and rectal administration.

The formulation may be in the form of injections, granules, tablets, powders, suppositories, or the like.

Furthermore, in the case of oral or rectal administration, it may be used as a sustained release preparation.

When making a drug, as long as it does not have a negative effect on DC118,
Various auxiliary agents used in medicine, ie, carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, emulsifiers, etc., may be used as necessary.

In the formulation, the content of DC118 can vary widely depending on the formulation.

The dosage of DC118 is 0.01 to 1 per adult per day.
The dosage is approximately 000 mg, but it is more preferable to increase or decrease the amount as appropriate depending on age, pathological condition, and symptoms.

Experimental example: Inhibition of lipid peroxide production
Homogenize with phosphate buffer (pH 7,4),
Brain homogenates were prepared. This rat brain homogenate was treated with ascorbic acid 100 μM 1F e S 0410.
a M1 and the test drug for 1 h at 37 °C, and the malondialdehyde (MDA) produced in the incubation mixture was analyzed by Yagi et al. (Anal, Bloch
em,,95.351. (1979)). During this time, the lipid peroxide production inhibition rate was calculated from the DA amount (a) and the control value (b) (MDA amount when no test drug was added) using the following formula.

Next, a regression linear equation is calculated from the lipid peroxide production inhibition rate at each concentration of the test drug, and the lipid peroxide production rate of the test drug is
The 0% inhibitory concentration (IC5o) was calculated.

Since the test substance is poorly soluble in water, it was dissolved and diluted with methanol before being subjected to the test.

(Results) From the above results, DC118 was found to have a strong inhibitory effect on lipid peroxide production.

Production> 1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.4.

Glycerol 30. Og fishmeal 20. Og Calcium carbonate 2.0g 100ml of the above medium was dispensed into a 500ml Erlenmeyer flask with warts, and after sterilization, one platinum loop of Streptomyces chromofuscus DC118 strain (FERM BP-2275) was inoculated from a slant and incubated at 27°C for 30 minutes. The seeds were cultured with shaking for several days and were used as seeds.

2) Add 100 ml of the above seed seed to a sterilized production medium having the same composition as that used in preparing the cultured seed mother by dispensing 25 liters into 50 liter jar fermenters, and heat to 27°C. for 4 days, 150 rpm, 0.6VVM
Aerated agitation culture was performed.

3) Collection of DC118 After culturing under the above conditions, the culture solution (50 liters) was filtered, the bacterial cells were extracted with 25 liters of acetone, and the extract was diluted with 50 liters of acetone.
After concentrating to liters, the pH is adjusted to 10 and extracted twice with equal volumes of ethyl acetate. After concentrating the extract, it was dissolved in 0.1 liter of chloroform, and the solution was poured into a column of silica gel (Wanogel C-200 manufactured by Wako Pure Chemical Industries, Ltd.)
01m) and eluted with chloroform. The active fraction is concentrated to dryness to obtain a crude sample of DC118.

This crude sample was dissolved in a small amount of methanol, subjected to gel filtration using a Sephadex LH-20 column (2 cmφ x 120 co+), and developed with methanol. DC118
The fractions were concentrated to dryness to obtain 26+1 g of purified DC118 product.

It is a copy of Tor.

Figure 4 shows the concentration of 12 in deuterated chloroform of DC118.
This is a reproduction of a 5 MHz carbon-13 nuclear magnetic resonance spectrum.

Claims (1)

[Claims] A novel physiologically active substance DC118 represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)