JPH0585998A - New compounds ca39-a and ca39-b, their use and production - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject new compounds useful as an antioxidant, etc., by aerobically culturing a strain, belonging to the genus Streptomyces and having the ability to produce CA39-A, CA39-B, etc., in a suitable culture medium and collecting the resultant product from the prepared culture. CONSTITUTION:A strain [e.g. Streptomyces-viridochromodenes 2220-SV2 (FERM P-3504)], belonging to the genus Streptomyces and having the ability to produce CA39-A and/or CA39-B is inoculated into a liquid culture medium, containing starch, soybean flour, a dried yeast and calcium carbonate, etc., and regulated to pH7.0 and subjected to roller tube culture at 27 deg.C for 3 days. The resultant culture solution is then centrifuged and the microbial cells are extracted with acetone. The extracted solution is concentrated, subsequently extracted with ethyl acetate and concentrated to dryness. The obtained solid substance is then dissolved in chloroform and n-hexane is added to collect formed precipitates by filtration. The collected precipitates are dissolved in chloroform and purified by adsorbing treatment using a silica gel column. Thereby, the objective compound expressed by the formula (R is CH2H or CHO) is obtained.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to novel substances, and more particularly to novel compounds CA39-A and B having antioxidant activity.

[0002]

2. Description of the Related Art Many antioxidants have already been put into practical use as pharmaceuticals. In general, the physiological activity of a chemical substance depends largely on its chemical structure,
It can be said that there is a constant desire for new compounds having antioxidant properties.

[Outline of the Invention]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel compound having the above-mentioned need, that is, a novel compound having antioxidant properties.

[0004]

The novel compounds according to the present invention include CA39-A and CA39 represented by the following formula (I).
-B, and its salts.

[Chemical 2] (In the formula, R is CH ₂ OH in the case of CA39-A, C is
In the case of A39-B, it is CHO. ) The invention also relates to the use of this compound. That is, the antioxidant according to the present invention contains at least one of the above compounds as an active ingredient. The invention also relates to the process for the preparation of this compound. That is, the compounds CA39-A and CA39- represented by the above formula (I) according to the present invention.
The production method of B belongs to the genus Streptomyces, CA39-
A strain capable of producing A and / or CA39-B is aerobically cultured in an appropriate medium, and CA3 is extracted from the culture.
9-A and / or CA39-B are obtained.

DETAILED DESCRIPTION OF THE INVENTION Novel Compounds CA39-A and B 1) Chemical Structure The novel compounds CA39-A and B according to the present invention have the chemical structure represented by the formula (I). And each compound has the following formula (Ia)
And (Ib). CA39-A:

[Chemical 3] CA39-B:

[Chemical 4] These chemical structures were determined as follows. The proton nuclear magnetic resonance spectrum and carbon-13 nuclear magnetic resonance spectrum of CA39-A and CA39 were measured using various solvents, but no signal was observed. Therefore, by adding methyl iodide in the presence of potassium carbonate in acetone, CA39-A is chemically converted to a dimethyl compound (hereinafter abbreviated as “AdiMe”) and CA39-B to a trimethyl compound (hereinafter abbreviated as “BtriMe”). Then
Structural analysis was performed using these. The molecular formula (AdiMe: C ₂₀ H ₁₇ NO ₅ , which was found by detailed examination of the proton nuclear magnetic resonance spectra of AdiMe and BtriMe and the carbon 13 nuclear magnetic resonance spectra (see FIGS. 9 to 12) and from the high-resolution mass spectrum (AdiMe: C ₂₀ H ₁₇ NO ₅ , Btri
Me: C ₂₁ H ₁₇ NO ₅ ) to give AdiMe and Btr
The structure of iMe was determined, and from these, the structures of CA39-A and B were determined as (Ia) and (Ib), respectively.

2) Physicochemical properties CA39-A CA39-B (1) Appearance Red powder Purple powder (2) Solubility Soluble Soluble Methanol, n-butanol, acetone Hardly soluble ethyl acetate Insoluble in ethyl acetate Chloroform, hexane, water ( 3) High-resolution FAB-mass measured value 324.0904 322.0756 spectrum calculated value 324.0872 322.0724 (M + H) ⁺ ( m / z ) (4) molecular formula C ₁₈ H ₁₃ NO ₅ C ₁₈ H ₁₁ NO ₅ (5) UV absorption spectrum Figure 1 3 shown in FIGS. 4 to 6 (6) Infrared absorption spectrum shown in FIG. 7 shown in FIG. 8 (KBr disk method) (7) Melting point 300 ° C. or higher 300 ° C. or higher Compound represented by formula (I) is OH There can be base addition salts and acid addition salts at the positions of the groups and NH ₂ groups, respectively. The compound of the present invention also includes these salts. Examples of the base addition salt include salts with alkali metal compounds (eg, sodium hydroxide, potassium hydroxide, etc.), salts with alkaline earth metal compounds (eg, calcium hydroxide, magnesium hydroxide, etc.), ammonium salts, organic bases. Examples thereof include salts with triethylamine, ethanolamine and the like. Examples of acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid,
Phosphoric acid or the like, or salts with organic acids such as acetic acid, propionic acid, maleic acid, oleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, laurylsulfonic acid, methanesulfonic acid. I can give you. Needless to say, when the base addition salt and the acid addition salt are used as a medicine, the base and the acid must be pharmaceutically acceptable.

Production of CA39-A and B 1) Overview Compounds CA39-A and B are currently obtained only by culturing microorganisms, but they can also be produced by synthetic chemical modification of related compounds, or total synthesis. It could be manufactured chemically. It could also be done by genetic engineering techniques. That is, compound CA39-A,
It would also be possible to introduce the gene involved in the production of B into a suitable microorganism, culture such a transformed microorganism and obtain it from this culture. Examples of strains in the case of culturing a microorganism include CA39- belonging to the genus Streptomyces.
Those having A and / or B producing ability are used. Specifically, the present inventors isolated Streptomyces viridochromo ( Streptomyces viridochromo
genes ) 2220-SV ₂ strain has been clarified by the present inventors to produce CA39-A and B, but other strains should be appropriately selected by a conventional method for isolating antibiotic-producing bacteria. It can be separated from the natural world. In addition, CA39-A and B producing bacteria including Streptomyces viridochromogenes 2220-SV ₂ strain were subjected to irradiation and other mutation treatments to produce CA39-
There is still room for increasing the productivity of A and B. As mentioned above, genetic engineering techniques are also possible. The CA39-A and B thus obtained can be converted into the base addition salt or acid addition salt as described above by a conventional method.

2) 2220-SV ₂ strain 2220-S found by the present inventors as a strain of the genus Streptomyces having CA39-A and B-producing ability.
The V ₂ strain has the following contents. (1) Origin and Deposit No. 2220-SV ₂ strain is an actinomycete isolated from soil, and was deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science on August 8, 1991. No. 3504 "(FER
MBP-3504). (2) Mycological properties of mycological properties 2220-SV ₂ is as follows. This strain 2220-SV ₂ is an actinomycete isolated from a soil sample. Characterization of this strain is based on the "Japan Patent Office Industry Examination Standards"
According to the method described. The basal hypha of this strain has a looped tip and does not divide. The aerial mycelium branches uniaxially from the long main axis and forms a spiral spore chain consisting of 50 or more at the tip. The spores are non-motile, oval-shaped, with a width of 0.4 to 0.5, a length of 0.7 to 0.8 μm, and a thorny surface. No sclerotium, sporangium, or other special morphology was observed.
The cell wall type is type I. The culture properties are shown in Table 1. The surface color of the colony is green, the back color is dark green to dark grayish blue, pH sensitive, and the diffusible pigments are glycerin / asparagine agar, which is light yellow green, and yeast / malt agar, light green, and pH. It is sensitive. The physiological properties are shown in Table 2.
This strain is mesophilic and its assimilation ability of carbon source utilizes all diagnostic sugars. This strain is located in the genus Streptomyces because of its morphological characteristics and cell wall type. As a result of searching for species of the genus Streptomyces listed in "Bacterial Name Approval List, 1980" and subsequent "Effective Name List" based on the above-mentioned properties, Streptomyces viridochromo
Selected genes . As shown in Table 3, this strain and Streptom
Morphological and physiological properties of yces viridochromogenes are in good agreement. However, although the color of the flora on the surface of the village, the hydrolyzing ability of starch and the availability of sucrose are different,
It is considered that they are the same species without judging that the species are different. Therefore, this strain 2220-SV ₂ was added to Streptomyces viridochromo.
It is named genes 2220-SV ₂ strain.

Table 1 Culture properties ^{Medium colony surface colony color Colony backside color Diffusible pigment}  Sucrose_{Green series (22cb)} Light yellow-green to gray-green_None ^{Nitrate agar (24 1 / 2dc ~ 24 1 / 2fe)}  Glucose and ass_{Green series (24fe)} Dark green_{Light yellow (1db)} ^{Paragin Agar (23nl ~ 23pn)}  Glycerin As_{Green series (22cb)} Dark ash blue light green grey ^{Paragin Agar (15po) (1 1 / 2lc)}  Inorganic salt / starch_{Green series (22ge)} Dark green_None ^{Agar (22 ml to 24 1/2 ml)} Tyrosine Agar Green series (23ig) Brownish gray (2ig) None Nutrient agar No aerial mycelium Pale yellow (2fb) None Yeast / malt agar Green series (22ih) Dark green (22pn) Light green (1 1 / 2gc) Oatmeal agar Green series (23ge) Dark grayish blue (15po) None Color Harmony Manual (container is in parentheses)
-Corporation of America, 1950)
Mark code.

Table 2 Physiological properties Growth temperature range 20-45 ℃ Optimal temperature 27-37 ℃ Melanin pigment Tyrosine agar ＋ Peptone yeast iron agar ＋ Tryptone yeast broth ＋ Starch hydrolysis ＋ Liquefaction of gelatin ＋ Peptonization of skim milk ＋ Coagulation of skim milk -Reduction of nitrate-Assimilation of carbon source D-glucose + L-arabinose + D-xylose + D-fructose + sucrose + L-rhamnose + raffinose + i -inositol + D-mannite +

Table 3 _{This strain 2220-SV S.viridochromogenes} 2 Spore chain morphology Looped − − Helical ＋ ＋ Spore surface Thorn ＋ ＋ Microbiota color Green ＋ ＋ Yellow － － Blue ＋ ＋ Back color Green ＋ ＋ Blue ＋ ＋ ＋ Diffusible pigment Green ++ pH sensitivity ++ Melanin pigment production Peptone yeast iron agar ++ Tyrosine agar ++ Proteolysis ++ Nitrate reduction ----- Starch hydrolysis ++ Growth temperature 45 ° C ++ Utilization of carbon source L-arabinose ++ Sucrose ＋ ± D-Xylose ＋ ＋ Inositol ＋ ＋ Mannitol ＋ ＋ D-Fructose ＋ ＋ L-rhamnose ＋ ＋ Raffinose ＋ ＋

Culture / Production of CA39-A and B Compounds CA39-A and B are produced by aerobically culturing CA39-A and B -producing bacteria belonging to the genus Streptomyces in an appropriate medium. It can be manufactured by collecting. The medium is CA39-A or B
It may contain any nutrient source available to the producing strain. Specifically, for example, glucose as a carbon source,
Sucrose, maltose, starch and fats and oils can be used, and organic substances such as soybean flour, cottonseed meal, dried yeast, yeast extract and corn steep liquor as nitrogen sources and ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and chloride. Inorganic substances such as ammonium can be used. Further, if necessary, inorganic salts such as sodium chloride, potassium chloride, phosphate, and heavy metal salt can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent such as silicone oil can be added according to a conventional method. As the culturing method, the aerobic liquid medium culturing method is most suitable as in the generally used antibiotic production method. Culture temperature is 20 to
37 ° C is suitable, but 25-30 ° C is preferable. With this method, the production amounts of CA39-A and CA39 reached the maximum in 3 to 4 days of culture in both shaking culture and aeration-agitation culture. In this way, an accumulated culture of CA39-A and B is obtained. In the culture, CA39-A and B are partially present in the culture filtrate, but most of them are present in the bacterial cells. Any purposeful method can be used to collect CA39-A or B from such a culture. One of the methods is based on the principle of extraction. Specifically, regarding CA39-A and B in the culture filtrate, this is a water-immiscible solvent for CA39-A and B (the above CA39- (Refer to the physicochemical properties section of A and B),
For example, there is a method of extracting with ethyl acetate or the like, or a method of collecting bacterial cells CA39-A and B in the bacterial cells by treating them with methanol, ethanol, acetone or the like, which is obtained by filtration or centrifugation. The culture itself may be subjected to the above extraction operation without separating the cells. Countercurrent partitioning with an appropriate solvent can also be included in the extraction category. Another method for collecting CA39-A or B from the culture is based on the principle of adsorption, which is the already liquid CA39-A, B-containing material, such as the culture filtrate or as described above. A suitable adsorbent such as silica gel, activated carbon, "Diaion HP-
20 "(manufactured by Mitsubishi Kasei Co., Ltd.)
CA39-A and B can be obtained by adsorbing A and B and then eluting with a suitable solvent. The CA39-A or B solution thus obtained is concentrated under reduced pressure to dryness to obtain a CA39-A or B crude product. In order to further purify the crude CA39-A or B sample thus obtained, the extraction method and the adsorption method described above may be combined with a gel filtration method, high performance liquid chromatography, etc., if necessary, to perform the required number of times. You can do it. For example,
Adsorbents such as silica gel, "Sephadex LH-2
Column chromatography using a gel filtration agent such as "0" (manufactured by Pharmacia), high performance liquid chromatography using "YMC Pack" (manufactured by Yamamura Scientific Co., Ltd.) and a countercurrent distribution method may be appropriately combined. it can. Specifically, for example, by applying the CA39-A, B crude sample to a "Sephadex LH-20" column, eluting the active fraction with a chloroform-methanol (1: 1) mixed solution, and concentrating to dryness A pure product of CA39-A or B is obtained.

Uses of CA39-A, B The compounds CA39-A and B according to the invention are useful in that they have antioxidant activity. 1) Antioxidant activity CA39-A and B showed lipid peroxidation inhibitory activity in rat liver microsomes. The produced lipid peroxide was detected by the thiobarbituric acid method. For example, 10% rat liver microsomes (0.5 ml) were added to various concentrations of CA3.
9-A or B (0.05 ml), and 0.0015M
After incubating with ascorbic acid (0.5 ml) at 37 ° C for 1 hour, 0.67% thiobarbituric acid (0.5
(ml)) and the extinction coefficient at a wavelength of 530 nm after heating at 100 ° C. for 20 minutes, the IC ₅₀ value is CA39-
0.04μg / ml for A, 0.07μ for CA39-B
g / ml of vitamin E (IC ₅₀ 10.8 μg / ml) as a positive control measured in the same manner.
2 times and 154 times the activity was shown. Further, CA39-A and B, 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) by strongly inhibited hemolysis of rat erythrocytes (IC _50: 0 in CA39-A.
93 μg / ml, CA39-B 1.60 μg / ml). As described above, it was revealed that CA39-A and B of the present invention exhibit antioxidant activity. Therefore, the CA3 of the present invention
9-A and B can be used as antioxidants.

2) Antioxidant As mentioned above, the compound of the present invention can be used as an antioxidant. That is, the antioxidant according to the present invention is
It contains at least one of CA39-A and B represented by the above formula (I) and salts thereof as an active ingredient. The compound of the present invention as an antioxidant can be administered by any purposeful administration route and in a dosage form determined by the administration route adopted. The drug is usually in a form diluted with a pharmaceutically acceptable carrier or diluent. When the compound of the present invention is actually administered as an antioxidant, one of the representative methods is to dissolve these in distilled water for injection or physiological saline and inject them. Specifically, there are methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into vein or artery, and local administration by injection.
The dose of the compound of the present invention is determined in consideration of the results of animal tests and various situations so that the total dose does not exceed a certain amount when administered continuously or intermittently. The specific dose depends on the method of administration, the situation of the patient or treated animal,
For example, age, weight, sex, sensitivity, diet, administration time,
Needless to say, it varies depending on the concomitant drug, the patient, or the degree of the disease, and the appropriate dose and the number of administrations under certain conditions must be determined by a dose determination test by a specialist based on the above guidelines. I won't. Specifically, it is about 0.01 to 1 g per day for an adult.

[0015]

【Example】

<Experimental example> (1) Culture The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 7.0. Starch 25 g Soybean flour 15 g Dry yeast 2 g Calcium carbonate 4 g 100 ml each of the above medium was dispensed into a 500 ml Erlenmeyer flask and sterilized, and Streptomyces viridochromogenes 2220-SV ₂ strain was inoculated to the sterilized mixture at 3 ° C at 27 ° C. Rotation culture was performed for a day. (2) Collection of CA39-A, B After culturing under the above conditions, the culture solution (60 liters) was centrifuged, and the bacterial cells were extracted with 5 liters of acetone. The extract was concentrated, extracted with 500 ml of ethyl acetate three times, and concentrated to dryness. This was dissolved in 50 ml of chloroform, and 30 ml of n-hexane was added to collect a precipitate, which was collected by filtration and washed three times with a mixed solution of chloroform-n-hexane (1: 6). The precipitate was dissolved in chloroform, adsorbed on a column of silica gel (“Wakogel C200” manufactured by Wako Pure Chemical Industries, Ltd.), and eluted with a chloroform-methanol (5: 1) mixed solution. The active fraction was concentrated to dryness and CA39-A
And CA39-B purified product 99 mg and 215 mg, respectively
Obtained.

[0016]

INDUSTRIAL APPLICABILITY The compound of formula (I) CA39-
A, CA39-B and their salts have excellent antioxidant activity. It can be said that such a property that the compound of the present invention has such an excellent antioxidant effect was unexpected to those skilled in the art.

[Brief description of drawings]

FIG. 1 is a copy of an ultraviolet absorption spectrum of CA39-A in methanol.

FIG. 2 is a copy of an ultraviolet absorption spectrum of CA39-A in 0.01N hydrochloric acid-methanol.

FIG. 3 is a copy of an ultraviolet absorption spectrum of CA39-A in 0.01N sodium hydroxide-methanol.

FIG. 4 is a copy of an ultraviolet absorption spectrum of CA39-B in methanol.

FIG. 5 is a copy of an ultraviolet absorption spectrum of CA39-B in 0.01N hydrochloric acid-methanol.

FIG. 6 is a copy of an ultraviolet absorption spectrum of CA39-B in 0.01N sodium hydroxide-methanol.

FIG. 7 is a copy of an infrared absorption spectrum of CA39-A by a KBr disk method.

FIG. 8 is a copy of an infrared absorption spectrum of CA39-B by a KBr disk method.

FIG. 9 is a reproduction of a 500 MHz proton nuclear magnetic resonance spectrum of AdiMe in deuterated acetone.

Figure 10: 5 of BtriMe in deuterated chloroform.
This is a copy of the 00 MHz proton nuclear magnetic resonance spectrum.

FIG. 11 is a reproduction of a 125 MHz carbon-13 nuclear magnetic resonance spectrum of AdiMe in deuterated acetone.

FIG. 12: 1 of BtriMe in deuterated chloroform.
It is a copy of a 25 MHz carbon-13 nuclear magnetic resonance spectrum.

Claims (3)

[Claims] 1. A novel compound CA39 represented by the following formula (I):
-A and CA39-B, or salts thereof. [Chemical 1] (In the formula, R is CH ₂ OH in the case of CA39-A, C is
In the case of A39-B, it is CHO. ) 2. An antioxidant containing at least one of the compound according to claim 1 and a salt thereof as an active ingredient. 3. A CA39-A belonging to the genus Streptomyces.
And / or CA39-B producing strain is aerobically cultured in an appropriate medium, and CA39 is extracted from the culture.
CA3 of formula (I) according to claim 1, characterized in that -A and / or CA39-B are obtained.
Method for producing 9-A and / or CA39-B.