JPH03240496A - New physiologically active substance kdc 165 - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula having the following physical and chemical properties. Appearance: light yellowish powder. Melting point: 88-91 deg.C (decomposition). Solubility: soluble in methanol, ethanol, chloroform, benzene, acetone, ethyl acetate and n-hexane and insoluble in water. Elemental analysis: C 77.20, H 1069, O 12.11, etc. USE:An inhibitor of peroxide lipid formation. PREPARATION:Pseudomonas sp. DC165 strain (FERM P-2558) is cultured preferably by aerobic liquid culture at 20-27 deg.C for 5 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel substance, and more particularly to a novel substance KDC165 having an effect of inhibiting lipid peroxide production.

Prior art> Lipid peroxide generated in living organisms is known to damage blood vessels and other normal organs and tissues, causing secondary lesions such as vascular disorders, inflammation, and edema (Japanese Clinical ,
46 (I0), 64 (I988)).

Therefore, substances that suppress lipid peroxide production can be used to treat vascular disorders,
It can be used as a prophylactic and therapeutic agent for various disorders and diseases such as inflammation and edema, and it can be said that there is a constant desire for substances that have such effects.

[Summary of the invention]

Summary The present invention meets the above needs.

That is, the novel substance according to the present invention is a compound KDC165 represented by the following formula (I).

2 [Detailed Description of the Invention] [1) Novel Substance KDC165 1) Chemical Structure The novel substance KDC165 according to the present invention has the above formula (I
) has the chemical structure shown.

The chemical structure of KDC165 was determined as described above by examining in detail the proton nuclear magnetic resonance spectrum, carbon-13 nuclear magnetic resonance spectrum, ultraviolet absorption spectrum, infrared absorption spectrum, and mass spectrometry spectrum.

2) Physicochemical properties (I) Appearance Pale yellow powder (2>Melting point 88-91℃ (decomposed) (3>Solubility in methanol, ethanol, chloroform, benzene, acetone, ethyl acetate, n-hexane, Insoluble in water.

(4) Rf value (“Silica gel 60F254” manufactured by Merck & Co., Ltd.)
Used) Chloroform-methanol (I00:1) 0,71 (5) FD-MS spectrum (m/z) 264 (M) (6) Ultraviolet absorption spectrum as shown in FIG.

λmax nm (g) 273 (2200)
(in methanol) (7) Infrared absorption spectrum shown in Figure 2.

(KBr disk method) 8〉Proton nuclear magnetic resonance spectrum It is shown in FIG.

(500 MHz, in heavy dimethyl sulfoxide) (9) Carbon-13 nuclear magnetic resonance spectrum It is shown in FIG.

(I25 MHz, in heavy dimethyl sulfoxide) 3- (I0) Elemental analysis CHO Analysis value (%) 77.20 10.69 12
．． 11 Calculated value (%) 77.23 10.67
12.10(I1) Molecular formula C17H2802(
n) Overview of production of KDC165> KDC165 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 production. You could also do that. It may also be possible to use genetic engineering techniques. That is, compound KDC1
It would also be possible to integrate the gene involved in the production of 65 into a suitable microorganism, culture the resulting transformant, and obtain it from this culture.

In the case of culturing microorganisms, a strain having the ability to produce KDC165 belonging to the genus Pseudomonas is used. Specifically, the present inventors have revealed that the Pseudomonas sp strain DC165 isolated by the present inventors produces -KDC165; It is possible to separate suitable substances from nature by conventional methods. In addition, Pseudomonas sp, D
There is also room to increase the production ability of KDC185 by subjecting KDC165 producing bacteria, including the C165 strain, to irradiation or other mutation treatments. As mentioned above, genetic engineering techniques can also be used.

<Strain DC165> Strain DC165, which the present inventors have discovered as a Pseudomonas strain capable of producing KDC165, has the following content.

l) Origin and deposit number Strain DC165 was isolated from soil collected in Taketomi-cho, Shigeyama-gun, Okinawa Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on August 17, 1989, and was designated as a It has a number of Koken Joyori No. 2558 J (FERMBP-2558).

2) Mycological properties The mycological properties of the DC165 strain are as follows.

(I) Morphology Strain DC165 is a Durham-negative short rod-shaped bacterium with rounded ends, and the size is approximately 0.2-0.5 x 0.5-1.2μ.
It is m. They usually exist singly, but may also exist in series. It is motile and does not form spores.

(2) Growth status on various media The results of culturing the DC165 strain on various media at 27° C. for one week are shown in Table 1.

(3> Physiological properties The physiological properties of the DC165 strain are as shown in Table 2.

(4) Aerobic use of carbohydrates and production of acids and gases C1
The aerobic utilization of carbohydrates and production of acid and gas for the 65 strains are shown in Table 3.

(5) Anaerobic decomposition of carbohydrates Analysis of the aerobic decomposition of carbohydrates (Hugh-Leirson test method, 30°C, 7 days) by strain DC165 revealed that oxidation that produces acids aerobically (oxi
dation).

The above mycological properties are summarized as follows.

(I) Durham-negative short bacillus, motile and does not form spores.

(I1) Positive for catalase and oxidase.

(+, 11) It is aerobic and produces acids from carbohydrates, but no gas is produced. In addition, O by the Hugh-Leifson test method
In the F test, it is oxidation.

(Iv) Grows well on bouillon agar, potato dextrose agar and pine conch agar, forming shiny colonies.

Based on the above properties, Berges Manual of Systematic Bacteriology Volume 1 (Berge
y's Manual o(' Systematic
According to the identification from Bacteriology Vol. 1), this production bacterium strain DC165 can appropriately belong to the genus Pseudomonas in the family Psudmonadaceae. Therefore, I compared the properties of this strain with the relatively similar Pseudomonas species described in the Verseys◆ manual, but the results did not match any of the species.

Therefore, this DC165 strain was used as Pseudomonas (Pseud
omonas) sp, designated as DC165 strain.

Table 1 Table 2 Table 3 Table 1 2-Culture/Production of KDC165> Compound KDC165 is a compound of KDC165 that belongs to the genus Pseudomonas.
It can be produced by culturing C165-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 used by the KDC165-producing bacteria. Specifically, for example, glucose, sucrose, maltose, glycerol, starch, oils and fats can be used as carbon sources, and soybean flour, fish meal, cottonseed meal, dried yeast, yeast extract, and cornstarch can be used as nitrogen sources. Organics such as Epliquor and inorganics such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride are available. 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 20 to 27°C. With this method, the production amount of KDC165 reaches its maximum after 5 days of culture in both shaking culture and aeration stirring culture.

In this way an enriched culture of KDC165 is obtained. In the culture, a part of KDC165 exists in the culture filtrate, but most of it exists in the bacterial cells.

Any convenient method can be used to harvest KDC165 from such cultures.

One method is based on the principle of extraction, and specifically, for KDC165 in the culture filtrate, it is extracted with a water-immiscible KDC165 solvent (see the above section on physicochemical properties of KDC165), For example, there is a method of extraction with ethyl acetate or the like, or a method of recovering KDC165 in bacterial cells by treating a bacterial mass obtained by filtration, centrifugation, etc. with methanol, ethanol, acetone, etc.

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 KDC165 from cultures is based on the principle of adsorption, and is obtained by extracting KDC165-containing materials that are already in liquid form, such as culture filtrate, or by performing the extraction procedure as described above. The target KDC165 is adsorbed on the extract using a suitable adsorbent such as silica gel, activated carbon, Diaion HP20J (manufactured by Mitsubishi Kasei Corporation), and then eluted with an appropriate solvent to obtain KDC1, KDC1, 65 can be obtained.KDC165 obtained in this way
The solution is concentrated to dryness under reduced pressure to obtain a crude sample of KDC165.

In order to further purify the crude sample of KDC165 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, high-performance liquid chromatography and countercurrent distribution using adsorbents such as silica gel, column chromatography rYMC pack using gel filtration agents such as Sephadex LH-20J (manufactured by Pharmacia) (manufactured by Yamamura Kagaku Co., Ltd.), etc. Laws may be implemented in appropriate combinations. Specifically, for example, KDC165 crude sample is dissolved in a small amount of methanol, applied to a Sephadex L H-20J column, the active fraction is eluted with methanol, and concentrated to dryness.
65 pure products are obtained.

[:m] Use of KDC165 The compound KDC165 according to the present invention is useful in that it has an excellent effect of inhibiting lipid peroxide production as described below.

Physiological activity/inhibitory effect on lipid peroxide production> (Method) Wistar male rat brains were removed by decapitation, and 67mM
A brain homogenate was prepared by homogenizing with a phosphate buffer (pH 7.4). This rat brain homogenate was treated with ascorbic acid 100μM 5F e SO 410μ
M1 and test drug were incubated for 5-1 h at 37 °C, and the malondialdehyde (MDA) produced in the incubation mixture was analyzed by Yagi et al.
l, Biochem, 95,851. (I979)
) was measured by the thiobarbic acid method. The lipid peroxide production inhibition rate was determined from the MDA amount (a) and the control value (b) (MDAffi 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 (IC50) was calculated. Since the test substance is poorly soluble in water, it was dissolved in methanol and subjected to the test after dilution.

6- From the above results, KDC165 was found to have a strong inhibitory effect on lipid peroxide production.

Thus, it was revealed that the compound KDC165 according to the present invention exhibits an inhibitory effect on lipid peroxide production in animals.

Therefore, the compounds according to the present invention can be used as lipid peroxide production inhibitors or for treating diseases that can be caused by lipid peroxide production, such as in the brain.
Various diseases and inflammation based on circulation disorders in the heart and peripheral areas,
It can be used as a preventive/therapeutic agent for various pathological conditions such as edema.

When used as a pharmaceutical, various conventionally known methods can be used for formulation and administration. 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, various adjuvants used for pharmaceutical purposes, such as carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, emulsifiers, etc., may be used as long as they do not adversely affect KDC165. may be used as necessary. In the formulation, the content of KDC165 can vary widely depending on the formulation form.

When actually administering the compound of the present invention as a lipid peroxide production inhibitor, one typical method is to dissolve the compound in distilled water for injection or physiological saline and inject it. Specifically, in the case of animals, injection methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration, and in humans, intravascular injection or injection into a vein or artery. There are methods such as local administration.

The dosage of the compound of the present invention is determined in consideration of the results of animal tests and various situations so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage depends on the administration method, the situation of the patient or treated animal,
For example, it goes without saying that the appropriate amount and frequency of administration under certain conditions will vary depending on age, weight, sex, sensitivity, diet, administration time, concomitant drugs, patient and degree of disease. The appropriate dosage must be determined through a test conducted by a specialist based on the above guidelines. Specifically, it is about 0.01 to 1000 square meters per day for adults.

<Example>1> Culture The medium used was one in which the following components were dissolved in 1 liter of water and the pH was adjusted to 7.4.

Glycerol 30. CJg Fish Meal 20. 0g calcium carbonate 2. Og 100 ml of the above medium was dispensed into 500 ml Erlenmeyer flasks with warts, 20 flasks were prepared, and after sterilization, Pseudomonas sp, DC165 strain (FERM BP-2558) was inoculated from a slant with one platinum loop at a time, and the flasks were incubated at 27°C. 20 Orpm for 6 days at
Cultured with shaking.

9 2) Collection of KDC165 After culturing under the above conditions, filter the culture solution (2 liters),
Extract the bacterial cells with 2 liters of acetone and dilute the extract with 150 liters of acetone.
After concentration to milliliter, the pH is adjusted to 7 and extracted twice with equal volumes of ethyl acetate. After concentrating the extract, it is dissolved in 50 ml of chloroform, and 300 ml of hexane is added thereto to remove the precipitate. After concentrating the supernatant, 10 milliliters of hexane/ethyl acetate (5
0:1) and a column (3.5 cm φ x 30 cm
) and eluted with hexane/ethyl acetate (50:1). When the active fraction is concentrated to dryness, KDC1
Obtain 65 rough samples.

This crude sample was dissolved in a small amount of methanol, subjected to gel filtration using a Sephadex LH-20 column (2 cmφ x 40 cm), and developed with methanol.

The KDC165 fraction was concentrated to dryness to obtain KDC165 purified product 1.
94.7 mg was obtained.

0-

[Brief explanation of drawings]

FIG. 1 is a reproduction of the ultraviolet absorption spectrum of KDC165 in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of KDC165 obtained by the KBr disk method. FIG. 3 is a reproduction of the 500 MHz proton nuclear magnetic resonance spectrum of KDC165 in deuterated dimethyl sulfosite. FIG. 4 is a reproduction of the 125 MHz carbon-13 nuclear magnetic resonance spectrum of KDC165 in deuterated dimethyl sulfosite.

Claims (1)

[Claims] Compound KDC165 represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)