JPH06277084A - New antibiotic ab4063-a and b, their production and use thereof - Google Patents

Abstract

PURPOSE:To obtain the subject new antibiotics for medicines and germicides, etc., for agriculture and horticulture, having activity capable of controlling bacteria and plant pathogenic fungi by culturing an antibiotic AB 4063s-producing bacterium belonging to the genus Phoma in a culture medium and then collecting a product from the cultured mixture. CONSTITUTION:An antibiotic AB4063-A and B-producing bacterium belonging to the genus Phoma [e.g. Phoma sp. AB4063 (FERM P-13358)] is inoculated into a culture medium and subjected to rotary shaking culture at 25 deg.C for 5 days using a rotary shaker, and then, the cultured mixture is filtered to collect the yeast cells. Then, acetone is added to the yeast cells and the cells are extracted with acetone at 55-60 deg.C for 30min and the extract is filtered to remove the yeast cells and the yeast extraction liquid is collected and concentrated under reduced pressure. Ethyl acetate is added to the residue and the residue is extracted and fractionated and the extracted liquid is concentrated under reduced pressure and the resultant brown oily substance is purified by a silica get column chromatography to provide the objective new antibiotics AB4063-A and -B expressed by formula I (R is formula II in the case of AB4063-A and formula III in the case of AB4063-B) or their salts.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to novel antibiotics AB4063-A and -B having antibacterial activity and antifungal activity (hereinafter, these are collectively referred to as AB4063 substances, but unless otherwise specified), The present invention relates to its manufacturing method and its use. More specifically, the present invention relates to AB40 belonging to the genus Houma.
The present invention relates to novel antibiotics AB4063-A and -B substances produced by culturing a 63-A or -B substance-producing bacterium, a method for producing them, and a novel microorganism useful as a bacterium producing the AB4063 substance. The AB4063 substance of the present invention has antibacterial activity and antifungal activity, and is expected as a medicinal fungicide useful for chemotherapy against bacterial infections, and as an agricultural / horticultural fungicide.

[0002]

2. Description of the Related Art Cephalosporins, erythromycin, etc. are known as antibacterial substances produced by microorganisms for use in medicine.
Moreover, kasugamycin, validamycin, etc. are known as fungicides for agriculture and horticulture. In addition, according to the present invention
As substances similar in chemical structure to those of AB4063, some substances are known as pharmaceuticals or veterinary drugs.
4-316578, JP-A-4-74163, JP-A-63-190
(See Japanese Patent No. 894).

However, the AB4063 substance of the present invention is inconsistent with the above-mentioned known substances in terms of various properties such as its physicochemical properties, target of biological activity and its effect.

[0004]

The compounds exemplified in the above-mentioned patent application publications are disclosed to have use as pharmaceuticals or veterinary medicines, but they are said to be useful as fungicides for agriculture and horticulture. There is no description. One object of the present invention is to
It is to provide a novel antibacterial substance having a novel chemical structure and having antibacterial activity against resistant bacteria and the like, which is useful as a drug or veterinary drug. It is intended to provide a novel antibiotic which can be used and is effective at low concentrations against phytopathogenic fungi, does not show phytotoxicity to plants, and has low toxicity to mammals.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted intensive studies on the isolation of microorganisms from various soils and on the antibiotics produced by the microorganisms for the purpose of solving the above-mentioned problems. As a result, it was discovered that a plurality of substances exhibiting antibacterial activity and antifungal activity were produced in the culture of a novel strain belonging to the genus Homa. Then, when these active substances were separated and purified from the culture of the microorganism and isolated, two types of substances were obtained, and they were found to exhibit the above-mentioned antibacterial activity and antifungal activity. We succeeded in clarifying the chemical structure of the substance. As a result, it was confirmed that the substance is a novel antibiotic and has antibacterial activity and antifungal activity.
We decided to call it 4063-B substance.

That is, in the first aspect of the present invention, the following general formula (I) [In the formula, R is the following formula for AB4063-A substance It is the base of AB4063-B substance A new antibiotic AB4063-A represented by
-B, or salts thereof.

Examples of the AB4063-A and -B substances of the present invention and salts thereof include organic salts such as quaternary ammonium salts and various metal salts such as alkali metal salts, especially sodium or potassium salts, and There are alkaline earth metal salts, especially calcium salts.

The AB4063-A substance according to the first invention is represented by the following structural formula (Ia) and has the following physicochemical properties.

[0009]

(1) Appearance: pale orange oily substance (2) Molecular formula: C ₂₆ H ₃₉ NO ₄ (3) Molecular weight: 429 (4) Specific optical rotation: [α] _D ²⁴ +28.6 (c = 1.0, Chloroform) (5) Ultraviolet absorption spectrum (in methanol solution): shown in FIG. 1 of the accompanying drawings. λmax, nm (ε): Acidity (0.01N HCl-in methanol): 227 (6,700) 292 (13,400) Neutral (in methanol): 228 (6,500) 292 (13,800) Alkaline (0.01N NaOH-in methanol) : 246 (11,200) 290 (14,600)

(6) Infrared absorption spectrum (KBr tablet): Shown in FIG. 2 of the accompanying drawings. (7) ¹ H-NMR spectrum (400 MHz, in deuterated chloroform): shown in FIG. 3 of the accompanying drawings. (8) ¹³ C-NMR spectrum (100 MHz, in deuterated chloroform): shown in FIG. 4 of the accompanying drawings. (9) Solubility: Soluble in chloroform, ethyl acetate and dimethylsulfoxide, but insoluble in water. (10) Thin-layer Chromatography: Rf value = 0.26 Adsorbent: Merck Kieselgel 60F ₂₅₄ Developing solvent: Chloroform-methanol-acetic acid (98: 2:
1) (11) Thin layer chromatography: Rf value = 0.21 adsorbent: Merck Kieselgel 60F ₂₅₄ developing solvent: hexane - ethyl acetate - acetic acid (50: 50: 1) (12) Color reaction: vanillin sulfuric acid, chloride Ferric and iodine reactions are positive. (13) Basic, acidic, neutral: acidic substance.

The AB4063-B substance according to the first invention is represented by the following structural formula (Ib) and has the following physicochemical properties.

[0013]

(1) Appearance: pale orange oily substance (2) Molecular formula: C ₂₆ H ₃₉ NO ₃ (3) Molecular weight: 413 (4) Specific optical rotation: [α] _D ²⁵ + 47.7 ° (c = 1.0) , Chloroform) (5) Ultraviolet absorption spectrum: λmax, nm (ε): Acidicity (0.01N HCl-in methanol): 204 (12,600) 226 (6,600) 290 (11,100) Neutrality (in methanol): 204 ( 18,000) 228 (6,200) 290 (12,800) Alkaline (in 0.01N NaOH-methanol): 209 (9,100) 248 (10,300) 290 (12,700) (6) Infrared absorption spectrum (KBr tablet): Figure 5 in the attached drawing
Shown in. (7) Solubility: Soluble in chloroform, ethyl acetate and dimethyl sulfoxide, but insoluble in water. (8) Thin-layer Chromatography: Rf value = 0.41 Adsorbent: Merck Kieselgel 60F ₂₅₄ Developing solvent: Hexane-ethyl acetate (7: 3) (9) Color reaction: vanillin sulfate, ferric chloride, iodine The reaction is positive. (10) Basic, acidic, neutral: acidic substance.

Furthermore, according to the second aspect of the present invention, AB4063-A substance-producing bacteria belonging to the genus Houma are cultured in a medium, and the antibiotic substance AB4063-A of the formula (Ia) is produced and accumulated in the culture,
There is then provided a process for the production of antibiotic AB4063-A of formula (Ia), characterized in that said AB4063-A substance is harvested from the culture.

Furthermore, according to the third aspect of the present invention, AB4063-B substance-producing bacteria belonging to the genus Houma are cultured in a medium, and the antibiotic AB4063-B of the formula (Ib) is produced and accumulated in the culture,
There is then provided a process for the preparation of the antibiotic AB4063-B of formula (Ib), characterized in that the AB4063-B material is harvested from the culture.

Further, according to the fourth aspect of the present invention, as a novel microorganism, the antibiotic AB40 represented by the above general formula (I) is used.
Provided is a strain of Homa sp. AB4063, which has the property of producing 63-A and -B substances and has the mycological characteristics described below.

Next, a method for producing the AB4063-A and -B substances will be described. The AB4063-A or -B substance-producing bacterium used in the method of the present invention may be any microorganism as long as it is capable of producing the AB4063-A or -B substance or a salt thereof. As a specific example thereof, there is strain AB4063 newly isolated by the present inventors from the soil of Yakushima, Kumage-gun, Kagoshima Prefecture. This strain belongs to the genus Houma and is an example of the AB4063 substance-producing bacterium most effectively used in the method of the present invention.

The bacteriological properties of this AB4063 strain are described below. (1) Growth condition in each medium Growth of this strain was rapid on oatmeal agar, and in culture in the dark at 25 ° C, the diameter of the colony was 46 to 48 mm on day 5 of culture and 65 to 48 on day 9 of culture. Reaches 67 mm. The community is fluffy, the surface of the community is grayish olive green and the back is light olive gray. Hyphae have septa and grow on and in the medium. The width of mycelium is 1.5-6.0 μm,
Colorless, smooth and branched.

The strain of the present invention grows rapidly on malt extract agar medium, and in culture in the dark at 25 ° C., the diameter of the colony reaches 38 to 40 mm on the 5th day of culture and 63 to 65 mm on the 7th day of culture. The community is fluffy and the surface of the community is grayish olive green. The back of the village is dark olive green.

The strain of this strain grows rapidly on corn meal agar, and when cultured in the dark at 25 ° C.
The diameter of the settlement reaches 42-43mm on the day 1 and reaches 74-76mm on the 9th day. The community is fluffy, the surface of the community is bright olive color, and the back of the community is dull yellow.

In culturing on oatmeal agar, the formation of anamorphs (features of incomplete generation) was
Formation of conidial shells (Pycnidium) was observed at week. Conidia husks appear dark olive green to olive black and are resident on the agar medium or embedded in the medium. Conidia shells are spherical to subspherical, or pearform
~ Ampulliform with a mesh surface,
The diameter is 120 to 250 μm, usually one hole mouth, sometimes 2 to
It has three ostium, and conidia are pushed out through the ostium. Conidia are unicellular, colorless, smooth and oval to oval in size.
It is 2.0 to 4.0 × 1.5 to 2.5 μm.

(2) Physiological properties Growth temperature Using malt extract agar medium, 5 ° C, 10 ° C, 15 ° C, 20
As a result of culturing at each temperature of ℃, 25 ℃, 30 ℃ and 37 ℃, AB
The 4063 strain grew at any temperature from 5 ° C to 30 ° C,
It did not grow at 37 ° C. The optimum growth temperature is 20 ° C to 25 ° C. Growth pH The malt extract agar medium adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 was used to culture at 25 ° C. As a result, AB4063 strain grew to pH 5 to 11. . The optimum growth pH is
pH is 8-10.

From the above mycological characteristics, the taxonomic position of the AB4063 strain is described by J.A. von Arcs, The Generala of Fanjay Spirating in Pure Culture, 3rd Edition, Jay Kramer, Badadz, 1981 (JAvon Arx, "The Genera of
Fungi Sporulating in pure Culture '', 3rd ed., J.Cr
amer, Vaduz, 1981) and Brian Sea Sutton, The Silomysetes Commonwealth Mycolological Institute, England, 1980 (Brian C. Sutton, "The Coelomycetes, Commonwealth
Mycological Institute, England ", 1980). As a result, it was confirmed that the present AB4063 strain belongs to the genus (Phoma), and the present inventors have decided to call the AB4063 strain the Homa SPB AB4063 strain.

The AB4063 strain was applied for deposit at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology (currently, the Institute of Biotechnology, Institute of Biotechnology of the Agency of Industrial Science and Technology) and was accepted as FERM P-13358 on December 25, 1992. There is.

AB40, which is an example of the AB4063 substance-producing bacterium, has been described above.
Although the 63 strains have been explained, generally, the mycological properties of filamentous fungi are extremely changeable, and ultraviolet irradiation, X-ray irradiation, and mutagenizing agents (for example, N-methyl- N-nitro-N-nitrosoguanidine and 2-aminopurine, etc.) or mutation by artificial mutation means using genetic recombination is a well-known fact. Thus, all strains that belong to the genus Homa and have the ability to produce the AB4063 substance, including natural mutants and artificial mutants, can be used in the method of the present invention.

In the method of the present invention, in order to produce the antibiotic AB4063, AB4063 substance-producing bacteria belonging to the genus Houma are cultured in a medium containing nutrients that can be used by ordinary microorganisms. In culturing the AB4063 substance-producing bacterium, a usual culturing method used for culturing a microorganism is applied. As a nutrient source, either a natural medium or a synthetic medium can be used as long as it is a medium containing a carbon source, a nitrogen source, an inorganic salt and the like that can be assimilated by microorganisms.

As carbon sources that can be assimilated, glucose,
Sucrose, galactose, dextrin, glycerol, starch, starch syrup, molasses, animal and vegetable oils, etc. can be used.
As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, ammonium nitrate, sodium nitrate, urea and the like can be used. In addition, if necessary,
It is useful to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions. Inorganic and organic substances that help the growth of the strain and promote the production of the AB4063 substance can be added appropriately. When a metal salt is present in the medium, the AB4063 substance may be obtained in the form of a salt with a corresponding metal cation.

The most suitable culture method is a culture method under aerobic conditions, particularly a deep liquid culture method. A suitable culturing temperature is 15 to 30 ° C, but in many cases, culturing is performed at 24 to 30 ° C. The production of AB4063 substance varies depending on the culture medium and culture conditions, but the accumulation reaches the maximum in 3 to 10 days in both shaking culture and tank culture.

The culture is preferably stopped when the accumulated amount of the AB4063 substance in the culture reaches the maximum. Then, according to the general method of collecting fermentation products from the culture, AB
It is recommended to collect and isolate AB4063-A or -B substance or both substances from the culture of the 4063 substance-producing bacterium. The culture conditions such as the composition of the medium, the liquidity of the medium, the culture temperature, the stirring speed and the aeration amount are appropriately adjusted or selected depending on the kind of the strain to be used and the external conditions so as to obtain preferable results. . If there is foaming in the liquid culture, defoaming agents such as silicone oil, vegetable oil and surfactant are used appropriately.

Since the AB4063-A or -B substance accumulated in the culture thus obtained has the physicochemical properties described above, it can be separated and purified from the culture according to the properties. In particular, it is preferable to efficiently separate and purify by the following method.

When the accumulated AB4063-A or -B substance is isolated and collected from the culture, a usual method for collecting a physiologically active substance from the culture medium is applied. That is, ethyl acetate from the culture, extraction with an organic solvent such as chloroform, AC partition using water or two or more organic solvent systems, ion exchange resin, adsorption resin, silica gel, silanized silica gel, alumina, cellulose, diatomaceous earth, gel The AB4063 substance can be isolated by adsorption / desorption treatment of the active substance by column chromatography or thin layer chromatography using a filtering agent or the like, or high performance liquid chromatography using a reverse layer column. The production examples of the AB4063-A and -B substances of the present invention are shown in Examples 1 to 3 below.

Further, the antibiotic AB4063 according to the present invention has antibacterial activity and phytopathogenic fungal growth inhibitory activity, as described below. Next, the fact that the AB4063 substance of the present invention has antibacterial activity and antifungal activity and is useful will be described by the test examples described below.

Test Example 1 Effect of AB4063 substance on bacteria Minimum growth inhibitory concentration (MIC) of AB4063 substance on various bacteria
(μg / ml) was measured by agar plate dilution method using Mueller Hinton agar medium (manufactured by Difco). The results are shown in Table 1 below.

[0035]

Test Example 2 Effect of AB4063 substance on phytopathogens Minimum growth inhibitory concentration (M
IC) (μg / ml) was measured by agar plate dilution method using potato-sucrose agar medium. The results are shown in Table 2 below.

[0037]

Therefore, according to the fifth aspect of the present invention, a pharmaceutical bactericidal agent comprising the antibiotic AB4063-A or -B represented by the above formula (I) or both of them as an active ingredient. An agricultural and horticultural fungicide is provided.

When the AB4063 substance of the present invention is used as a bactericidal agent for medicine, it is administered as an ointment, an injection, an oral preparation, a suppository or the like, either alone or mixed with an excipient. The excipient may be any pharmaceutically acceptable one, and its type and composition are appropriately selected depending on the administration route and administration method. The dose of the AB4063 substance varies depending on the age, body weight, etc., but usually about 1 to 1,000 mg is orally or parenterally (injected intravenously) to an adult.

When the AB4063 substance of the present invention is used as an agricultural and horticultural fungicide, it can be used alone depending on its intended use, but it can be used in admixture with a commonly used solid or liquid carrier. For example, in order to promote or stabilize the biological effect, as shown in Examples 4 to 6 described later, a mixture prepared by mixing with a suitable carrier and an auxiliary agent, and using the pharmaceutical composition directly Or, if necessary, dilute with water before use.

[0041]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples 1 to 3 which exemplify the fermentative production of the antibiotic AB4063 of the present invention, but this is merely an example, and The invention is not limited. Needless to say, it is possible to use many modified methods or modification means not illustrated here, and it can be an effective means. In the examples, “parts” means “parts by weight”.

Example 1 Production of substance AB4063-A As a production medium, glucose 1.0%, soluble starch 3.0
%, Polypeptone 0.5%, yeast extract 0.2%, malto extract 0.5%, calcium carbonate 0.5%, and a medium (pH 6.5) was used.

A total of 46 Erlenmeyer flasks with baffles having a capacity of 500 ml each containing 110 ml of the above-mentioned medium (for 5 liters of the medium) were used.
It was sterilized at 0 ° C. for 20 minutes, and 1-2 platinum loops of a slope agar culture of Houma sp. Strain AB4063 (FERM P-13358) were inoculated to this. Then, using a rotary shaker (180 rpm), the cells were subjected to rotary shaking culture at 25 ° C for 5 days. After the completion of the culture, the culture solution was filtered to obtain 5 liters of the culture filtrate and bacterial cells.

Acetone 1.5 liter was added to the above cells,
Extraction was carried out at 50-60 ° C for 30 minutes, and the cells were removed by filtration to obtain a cell extract. This operation was performed twice, and 3 liters of the obtained bacterial cell extract was concentrated under reduced pressure to remove acetone, and then twice the amount of ethyl acetate was added for extraction and liquid separation. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 1.08 g of a brown oil containing the active ingredient. This brown oil was adsorbed on a column (inner diameter 22 mm x length 200 mm) packed with silica gel (manufactured by Merck & Co., Inc.), chloroform, then a mixed solvent composed of chloroform-methanol (80: 1), and then chloroform-. Column chromatography was carried out by elution with a mixed solvent having a composition of methanol (40: 1). The active fractions were collected and concentrated to dryness under reduced pressure to obtain 319 mg of an oil containing the active ingredient.

An oil containing the active ingredient is then added to
The column was packed with Sephadex LH20 (manufactured by Pharmacia) in methanol (inner diameter 10 mm × length 450 mm), and column chromatography was performed with methanol. The active fractions were collected and concentrated to dryness under reduced pressure to obtain 146 mg of orange oily substance AB4063-A. Obtained ab
The 4063-A substance is dissolved in 50 ml of ethyl acetate, the solution is decolorized with activated carbon, washed with an equal amount of 0.01 N hydrochloric acid, then washed with water, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Thus, 109 mg of a pale orange oily substance AB4063-A was obtained.

Example 2 Production of AB4063-A substance As a production medium, glucose 1.0%, soluble starch 3.0
%, Polypeptone 0.5%, yeast extract 0.2%, malto extract 0.5%, calcium carbonate 0.5%, and a medium (pH 6.5) was used.

121 ml of 46 Erlenmeyer flasks with 500 ml capacity baffles (5 liters of medium) in which 110 ml of the above-mentioned medium was dispensed
Sterilize for 20 minutes at ℃, then add Homa sp.
One to two platinum loops of a slope agar culture of the strain (FERM P-13358) were inoculated. Then, using a rotary shaker (180 rpm), the cells were subjected to rotary shaking culture at 25 ° C for 5 days. After the completion of the culture, the culture solution was filtered to obtain 5 liters of the culture filtrate and bacterial cells.

To the above culture filtrate, twice the amount of ethyl acetate was added for extraction, liquid separation, and the ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a brown oily substance containing the active ingredient. 0.53 g was obtained. This brown oil was adsorbed on a column (internal diameter 18 mm x length 200 mm) packed with silica gel (Merck), and chloroform, then a mixed solvent having a composition of chloroform-methanol (80: 1), and then chloroform- Column chromatography was performed by elution with a mixed solvent having a composition of methanol (40: 1).
Active fractions (No. 54-64) are collected, concentrated to dryness under reduced pressure,
127 mg of an oil containing the active ingredient are obtained.

An oil containing the active ingredient is then added to
The column was packed with Sephadex LH20 (manufactured by Pharmacia) in methanol (inner diameter 10 mm × length 450 mm), and column chromatography was performed with methanol. The active fractions were collected and concentrated to dryness under reduced pressure to obtain 40 mg of AB4063-A substance as orange oil. Obtained AB40
The 63-A substance was dissolved in 50 ml of ethyl acetate, washed with an equal volume of 0.01 N hydrochloric acid, washed with water, dehydrated with anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure to give a pale orange oil AB40.
37.5 mg of 63 substances were obtained.

Example 3 Production of substances AB4063-A and -B As a production medium, glucose 1.0%, soluble starch 3.0
%, Polypeptone 0.5%, yeast extract 0.2%, malto extract 0.5%, calcium carbonate 0.5%, and a medium (pH 6.5) was used.

121 500 ml capacity Erlenmeyer flasks with baffles (2 liters of medium) into which 110 ml of the above medium was dispensed
It was sterilized at 0 ° C. for 20 minutes, and 1-2 platinum loops of slant agar culture of Houma SPB strain AB4063 (FERMP-13358) were inoculated to this. Then, using a rotary shaker (180 rpm), it was subjected to rotary shaking culture at 25 ° C. for 2 days to give an inoculum. 900 ml of the inoculum prepared above was transplanted to two 65 liter fermenters containing 45 liter of the above composition,
Aeration of 45 liters per minute, 25 ° C with stirring at 300 revolutions per minute
The cells were cultured for 5 days. After the culture was completed, the culture solution was filtered to obtain cultured bacterial cells.

The above-mentioned bacterial cells were extracted with 36 liters of acetone to remove the bacterial cells to obtain a bacterial cell extract. This operation was repeated twice to obtain 72 liters of cell extract. The resulting cell extract is 3 l
The mixture was concentrated to an iter under reduced pressure, acetone was removed, and then twice the amount of ethyl acetate was added, and the mixture was shake-extracted and then separated. The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a brown oil containing active ingredients AB4063-A and -B.

This brown oil was adsorbed on a column (inner diameter 45 mm × length 300 mm) packed with silica gel (manufactured by Merck & Co., Inc.) and eluted with various developing solvents, and the eluate was fractionated by 9 g. Fraction Nos. 1 to 50 are hexane-ethyl acetate (2:
1), Fraction Nos. 51 to 100 are hexane-ethyl acetate-
Fraction No. 101, mixed solvent composed of formic acid (400: 200: 3)
To 150 is hexane-ethyl acetate-formic acid (100: 100: 1)
Chromatography was performed by eluting with a mixed solvent having the composition described above. Active fraction containing only AB4063-A substance (No. 1
15-133) were collected and concentrated to dryness under reduced pressure to obtain 2.19 g of an oily substance I.

Fractions containing active ingredients AB4063-A and -B substance (Nos. 103 to 114) were collected, concentrated to dryness under reduced pressure, and 2.
01 g of oil II was obtained. Next, the oily substance I containing only the active ingredient AB4063-A substance was placed on a column (inner diameter 28 mm x length 450 mm) filled with Sephadex LH20 (Pharmacia) with chloroform-methanol (1: 1). , Developed with methanol and subjected to chromatography. The active fractions were collected and concentrated to dryness under reduced pressure to obtain 0.96 g of pale orange oily substance AB4063-A.

Similarly, for oil II containing active ingredients AB4063-A and -B, Sephadex LH20 (manufactured by Pharmacia) was packed in a column (inner diameter 28 mm) with chloroform-methanol (1: 1). × length 450 mm), developed with methanol and chromatographed.
Fractions containing active ingredients AB4063-A and -B were collected and concentrated to dryness under reduced pressure to give 1.32 g of a pale orange oily active fraction.

Separation of the active ingredients AB4063-A and -B substance was carried out by thin layer chromatography. That is, the active fraction was analyzed by thin layer chromatography (Gieselgel 60F manufactured by Merck).
₂₅₄ , 20 cm x 20 cm x 0.5 cm, developed with chloroform-methanol = 40: 1), scrape off the UV absorption band at Rf = 0.66, elute with a mixed solvent consisting of chloroform-methanol (1: 1), and then concentrate. , Pale yellow oil AB4063-A substance 0.7
g was obtained. Further, the UV absorption band at Rf = 0.86 was scraped off, eluted with a mixed solvent of chloroform-methanol (1: 1) and then concentrated to obtain 56 mg of AB4063-B substance as a pale orange oil.

Next, the present invention will be explained concretely with reference to Examples 4 to 6 which exemplify composition examples of the bactericide according to the fifth present invention.

Example 4 (Wettable powder) 20 parts of AB4063-A or -B substance, 3 parts of potassium alkylbenzene sulfonate, 5 parts of polyoxyethylene nonylphenyl ether and 72 parts of clay were uniformly mixed and pulverized. A wettable powder containing 20% of the active ingredient was obtained.

Example 5 (powder) AB4063-A or -B substance 2 parts, PAP (physical property improver) 1
Parts and 97 parts of clay were uniformly mixed and ground to obtain a dust containing 2% of active ingredient.

Example 6 (Emulsion) 30 parts of AB4063-A or -B substance, 40 parts of methyl ethyl ketone and 30 parts of polyoxyethylene nonylphenyl ether were mixed and dissolved to obtain an emulsion containing 30% of active ingredient. .

Furthermore, the control effects of the AB4063 substance used in the agricultural and horticultural fungicide according to the fifth aspect of the present invention against plant pathogens will be illustrated by Test Examples 3 to 5.

Test Example 3 (Cucumber downy mildew control effect test) The above-mentioned test was carried out on the second leaf stage cucumber seedlings (cultivar: Sagamihanjiro) which were cultivated in a greenhouse in a clay pot with a diameter of 9 cm. 20 ml of a dilute solution of the AB4063-A substance wettable powder having a predetermined concentration prepared according to Example 4 was sprayed per pot. On the other hand, with a moistened brush, spores are scraped from diseased leaves of cucumber and fungus ( Pseudoperonospora cubensis ), suspended in a 50 ppm aqueous solution of a spreading agent (polyoxyethylene alkyl ether), and then spores are removed. Concentration 5 × 10
^The spore suspension was prepared by adjusting the number of spores (6 / ml).

The spore suspension of the cucumber beetle and the disease fungus was spray-inoculated on a cucumber seedling one day after spraying the drug. Then, the cucumber seedlings were allowed to stand in a moist chamber at 20 ° C. and 100% humidity for 2 days, and then moved to the sick room to cause cucumber bean and disease. Six days after the inoculation, the cucumber bean per leaf and the lesion area ratio (%) were investigated, the average lesion area ratio was calculated, and the control value (%) was calculated by the following formula. The test results are shown in Table 3 below.

[0064]

Test Example 4 (Wheat Red Rust Control Effect Test) No. 1 cultivated in a greenhouse in a clay pot with a diameter of 9 cm
20 ml per 3 pots of a diluted dilute solution of the AB4063-A substance wettable powder prepared according to Example 4 were sprayed on wheat seedlings (variety: Norin 61) in the true leaf stage. One day later, the leaf rust of wheat ( Puccinia recon) was previously formed on the leaves of wheat.
dita : Pucinia recondita) was added with Tween 20 [polyoxyethylene sorbitan monolaurate trade name manufactured by Kao Co., Ltd.] so that the concentration of spores per field of view was about 50 with a microscope of 150 times. A spore suspension was prepared by suspending in sterilized water. The leaves to be treated were then spray inoculated with the spore suspension. Then, the wheat seedlings were transferred to a greenhouse at 20 ° C and 100% humidity to promote the disease.
Ten days after the inoculation, the number was taken out, and the number of summer spore stacks infected per leaf was investigated, and the control value was calculated by the following formula. This test was carried out with 3 pots per test, and the average control value (%) was determined. The test results are shown in Table 4 below.

[0066]

Test Example 5 (Pear black spot disease control effect test) New development of pear tree (cultivar: twentieth century) New shoots containing 4 to 5 leaves are cut and dipped in 300 ml of Kolben for testing. I went to 20 ml of a dilute solution of a wettable powder of AB4063-A substance prepared according to Example 4 at a predetermined concentration was sprayed onto the shoots. Alternaria kikuchiana pest for inoculation ( Alternaria kikuchiana) is 24 ℃ on a plate of apricot decoction agar.
Were cultured for 10 days. Then, the conidia formed on the plate were scraped off and dispersed in a 50 ppm solution of Tween 20 [trade name of Kao Corporation]. Microscopic spore concentration
(150 times) A spore suspension was prepared by adjusting the number to about 20 per visual field.

Then, the suspension was inoculated one day after spraying the drug with a spray gun. Pear shoots after inoculation (300 ml
(Kolben watered) was kept in a humid chamber at 24 ℃ for 24 hours, and then kept in a growth cabinet at 24-26 ℃ and 85-95% humidity. Five days after disease inoculation, the lesion area ratio (%) per leaf was investigated, and the control value (%) was calculated by the following calculation formula. The test was carried out under the control of 1 branch and 3 branches, and the average control value (%) was determined. The test results are shown in Table 5 below.

[0069]

[0070]

The novel antibiotic AB4063-A of the present invention and
As described above, the substance-B has an antibacterial activity and a strong control activity against plant pathogens, and thus is useful as a novel pharmaceutical antibacterial agent and agricultural / horticultural fungicide.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum diagram of AB4063-A substance in methanol.

FIG. 2 is an infrared absorption spectrum diagram of AB4063-A substance in a potassium bromide tablet.

FIG. 3 is a ¹ H nuclear magnetic resonance spectrum of AB4063-A substance in deuterated chloroform.

FIG. 4 is a ¹³ C nuclear magnetic resonance spectrum of AB4063-A substance in deuterated chloroform.

FIG. 5 is an infrared absorption spectrum diagram of AB4063-B substance in a potassium bromide tablet.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C07D 405/08 207 7602-4C C12P 17/16 7432-4B // (C12P 17/10 C12R 1: (01) (C12P 17/16 C12R 1:01) (72) Inventor Atsushi Takahashi 4-25-18 Nishikita, Tama-ku, Kawasaki City, Kanagawa Prefecture (72) Yasuyuki Tezuka 2-24 Morinosato, Atsugi City, Kanagawa Prefecture No. 7 (72) Saint Sato, 200, Higashidawara, Hadano City, Kanagawa Prefecture 96 (72) Inventor Tomio Takeuchi 5-11, Higashigotanda, Shinagawa-ku, Tokyo Nyufuji Mansion 701 (72) Inventor, Masaru Hamada Tokyo 26, No.405 Hidewa Residence, 26 Naito-cho, Shinjuku-ku, Tokyo (72) Inventor Hiroshi Naganawa 3-17, Denenchofuhonmachi, Ota-ku, Tokyo

Claims (5)

[Claims] 1. The following general formula (I): [In the formula, R is the following formula for AB4063-A substance It is the base of AB4063-B substance A new antibiotic AB4063-A represented by
-B, or their salts. 2. An antibiotic AB4063-A-producing bacterium belonging to the genus Houma is cultivated in a medium, and the antibiotic AB4063 is contained in the culture.
Antibiotic AB according to claim 1, characterized in that -A is produced and accumulated, and then the antibiotic is collected from the culture.
4063-A manufacturing method. 3. An antibiotic AB4063-B producing bacterium belonging to the genus Houma is cultured in a medium, and the antibiotic AB4063 is contained in the culture.
Antibiotic AB according to claim 1, characterized in that it produces and accumulates -B and then collects the antibiotic from the culture.
4063-B manufacturing method. 4. A bacterium belonging to the genus Homa having the characteristic of being capable of producing the antibiotics AB4063-A and -B represented by the general formula (I) according to claim 1 and having the following mycological characteristics, AB40.
63 shares. 5. A pharmaceutical bactericidal agent or agricultural / horticultural bactericidal agent comprising the antibiotics AB4063-A or -B according to claim 1 or both of them as active ingredients.