WO1997026367A1

WO1997026367A1 - Antibiotics tkr 400-a and tkr 400-b and processes for producing these

Info

Publication number: WO1997026367A1
Application number: PCT/JP1997/000094
Authority: WO
Inventors: Kazutoh Takesako; Katsushige Ikai; Tomoko Iwasaki; Masanobu Kuroda; Ikunoshin Kato
Original assignee: Takara Shuzo Co., Ltd.
Priority date: 1996-01-19
Filing date: 1997-01-20
Publication date: 1997-07-24
Also published as: AU1399697A

Abstract

Novel antibiotics useful as a remedy for fungal infections; and a process for producing antibiotic TKR 400-A having the following physicochemical properties or pharmacologically acceptable salts thereof: (1) it gives a mass spectrum having a peak of m/z 1,273 [M+H]+ according to FAB-MS; (2) it gives in methanol an ultraviolet absorption spectrum having major peaks at wavelengths (nm) of 250 sh and 294, whose values of E1%1cm are 96 and 20, respectively; (3) it gives an infrared absorption spectrum by the KBr method which has major peaks at wavenumbers of 3,450, 2,970, 1,680, 1,640, 1,530, 1,410, 1,220 and 1,150 cm-1; (4) threonine, alanine, valine, and isoleucine are detected in the analysis for amino acids by the ninhydrin reaction; and (5) it is soluble in methanol but sparingly soluble in chloroform, water, and hexane.

Description

Description Antibiotics TKR 400-A and TKR 400-B and their manufacturing methods

The present invention relates to antibiotics TKR 400-A and TKR 400-B useful as therapeutic agents for fungal infections, a method for producing them, and microorganisms producing them. Background art

Fungi are known to infect humans, animals, plants and the like and cause various diseases. For example, it causes superficial mycosis in the skin and oral cavity of humans, systemic mycosis in the internal organs, brain, etc., and the same infectious disease in dead animals such as pets and livestock. In addition, it causes various diseases on plants such as fruit trees and vegetables.

Among these, the main fungi that cause human infection and cause systemic mycosis are Candida and Cryptococcus> Aspergillicus. It is known that superficial mycosis is mainly caused by Candida, which infects the skin, oral cavity, vagina, etc., and Bacillus bacillus, which infects the skin of hands and feet. Various other fungi exist in the living environment and are thought to cause animal and plant contamination.

Only a very small number of antifungal agents are currently known that can be used to treat and protect against such fungal infections and contamination. Of these, especially for systemic infections in animals, including humans Examples of the therapeutic agent include amphotericin B, flucytosine, miconazole, and fluconazole. However, these have problems in terms of efficacy, toxicity, antibacterial spectrum, etc., and have not been sufficient as therapeutic agents. Summary of the Invention

An object of the present invention is to provide a novel antibiotic which is useful as a therapeutic agent for fungal infections in view of the above situation.

The present inventors, for the purpose of searching for new antibiotics, isolated a large number of microorganisms from nature, isolated the antibiotics produced by them, and examined their biological properties. Aureobasidium ( Aureo b_a sidium) has been found to contain antibiotics that exhibit antibacterial activity against pathogenic fungi such as Candida albicans and Cryptococcus neoformans in cultures of microorganisms belonging to the genus Aureo b_a sidium. Subsequently, the present inventors isolated this antibiotic and examined its physicochemical properties.As a result, the present inventors confirmed that the two novel substances had unique physicochemical properties and were not described in the literature. The antibiotics were named TKR400-A and TKR400-B. The present invention provides the above antibiotics TKR400-A and TKR400-B, and methods for producing them. Brief description of the drawing

FIG. 1 is a diagram showing an ultraviolet absorption spectrum of the antifungal substance TKR 400-A. The horizontal axis shows the wavelength (nm).

FIG. 2 is a diagram showing an infrared absorption spectrum of the antifungal substance TKR 400-A. The horizontal axis shows the wave number (cm- ¹ ).

Figure 3 shows the 'H-NMR spectrum of the antifungal TKR 400—A. FIG. The horizontal axis shows the chemical shift value (ppm).

FIG. 4 is a diagram showing a ' ³ C-NMR spectrum of the antifungal substance TKR 400-A. The horizontal axis shows the chemical shift value (ppm).

FIG. 5 is a diagram showing the elution positions of the antifungal substance TKR 400-A in HPLC. The vertical axis indicates the relative ultraviolet absorption intensity, and the horizontal axis indicates the retention time (minutes).

FIG. 6 is a diagram showing an ultraviolet absorption spectrum of the antifungal substance TKR400-B. The horizontal axis shows the wavelength (nm).

FIG. 7 is a diagram showing an infrared absorption vector of a fungal substance TKR 400-1B. The horizontal axis shows the wave number (cm- ¹ ).

FIG. 8 is a diagram showing a 'Η-NMR spectrum of the antifungal substance KR400-B. The horizontal axis shows the chemical shift value (ppm).

FIG. 9 is a diagram showing a ' ³ C-NMR spectrum of the antifungal substance TKR 400 -B. The horizontal axis shows the chemical shift value (ppm).

FIG. 10 is a diagram showing the elution position of the antifungal substance TKR 400-B in HPLC. The vertical axis indicates the relative ultraviolet absorption intensity, and the horizontal axis indicates the retention time (minutes). Detailed Disclosure of the Invention

The antibiotic TKR400-A has the following physicochemical properties (1), (2), (3), (4) and (5).

(1) Mass spectrum by FAB-MS method has a peak of mZz 127 3 [M + H] ⁺

(2) The major absorption wavelength (nm) of the ultraviolet absorption spectrum in methanol is 250 s sh 294, and their E ' _c 9 are 96 and 20. (3) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3450 cm- ', 2970 cm-', 1680 cm- ', 1640 c nT ^!, 1 5 3 0 cm - ', 1 4 1 0 cm one', is 1 2 2 0 cm- 1 1 5 0 cm _ l

(4) Threonine, alanine, phosphoric acid, and isoleucine are detected in aminoacid analysis by ninhydrin reaction

(5) Soluble in methanol, poorly soluble in black form, water and hexane

The antibiotic TKR400-B has the following physicochemical properties (6), (7), (8), (9) and (10).

(6) Mass spectrum by FA B-MS method has a peak of mZz 1101 [M + H] +

(7) Main absorption wave length of the ultraviolet absorber scan Bae-vector in METHANOL in Le (nm) force, a 2 5 0 sh, 2 9 4 , their E _'cm Chikaraku, 1 0 7. 2 4

(8) The main absorption wave numbers of the infrared absorption spectrum according to the KBr method are 3450 cm- ', 3350 cm-', 297 cm-168 cm- ' , 1640 cm ^_ \ 1530 cm " ¹ .1470 cm-', 1 4 1 0 c πΤ', 1 2 1 0 cm-', 1 1 4 0 cm-'

(9) In the amino acid analysis by the ninhydrin reaction, valin and iso-isocyanate are detected.

(10) Soluble in methanol, slightly soluble in chloroform, water and hexane

The antibiotic TKR 4 0 0 - A is the show 'H- NMR-spectrum Le 3 has a ^{1 3} C-NMR spectrum shown in Figure 4, the reversed-phase high performance liquid chroma preparative chromatography, It has the property of being eluted at the position shown in Fig. 5. O

Furthermore, the antibiotic TKR 4 0 0- B is shown in FIG. 8 'H- NMR scan Bae-vector, shown in Figure 9' has a ³ C-NMR spectrum, reversed-phase high performance liquid body chromatography It has the characteristic of being eluted at the position S shown in FIG.

The TKR 400-A belongs to the genus Aureobasidium, and the strain producing the TKR 400-A is cultured, and then isolated from the culture of the strain. O

The strain used in the present invention is not particularly limited as long as it belongs to the genus Aureobasidium and produces the TKR400-A. For example, the strain Aureobasidium SP (Aureobasidium sp.) TKR 400 strain (hereinafter referred to as “TKR 400 strain”) and the like.

The TKR400-B belongs to the genus Aureobasidium and is used to culture the strain that produces the TKR400-B. Thereafter, the TKR400-B is isolated from a culture of the strain. Can be manufactured by 0

The strain to be used in the present invention is not particularly limited as long as it is a strain belonging to the genus Au-reobasidium and produces the TKR400-B. TKR 400 strains and the like can be mentioned.

The strain producing TKR400-A and the strain producing TKR400-B produce only one of TKR400-A and TKR400-B. It may be one that produces both TKR 400 -A and TKR 400 -B. Using the latter strain If used, both TKR 400-A and TKR 400-B can be manufactured. The TKR400 strain is one of strains capable of producing both TKR400-A and TKR400-B.

The above-mentioned TKR 400 strain is a new strain not described in the literature, and was first isolated and identified by the present inventors from a sample collected from Crystal Beach, Fukui Prefecture. It has the property of producing 1 ^ 400-B in an advantageous manner. Hereinafter, the mycological properties of the TKR400 strain will be described in detail.

The TKR400 strain has the color tone of colonies (hereinafter also referred to as “populations”) in various media as shown in Table 1. In addition, the color tone in the table is based on the color name according to the Japanese Industrial Standard JISZ 8102 (1989) .After inoculating the culture medium, culture at 25 ° C, and after 4 days, 7 days and This is shown by the results observed 14 days later. The diameter of the settlement was measured 14 days later.

table 1

The TKR400 strain grows moderately on a malt extract agar medium, a YpSS agar medium, etc., and the center of the colony is glossy and usually exhibits a viscosity or a paste. However, it may become leathery as the culture days elapse. The periphery of the colony shows a remarkable root-like shape. The color of the colony is white in the early stage of the culture, and then gradually changes from light grayish yellow to peach to dark grayish yellow, but gradually changes from dull reddish yellow to brownish brown. As the days elapse, the color of the colony changes from brown to dark brown. Dyes are insoluble.

Hyphae are 2-4 m 柽 and develop well, but do not form aerial hyphae and extend into the agar. From the tip or side of the hypha, budding conidia of 2 to 4 × 4 to 8 / m are often formed in the shape of a fingertip, and some are grown to a ball-like mass. Young vegetative cells are yeast-like, measuring 3-7 X 6-14 / m, their shape is oval or lemon-shaped, multipolar Propagate by budding. It forms arthropod spores with a size of 4 to 6 x 8 to 18 m, thick membrane spores with a size of 4 to 8 x 8 to 16 um, and no ascospores.

Among the mycological properties of the TKR400 strain, the physiological properties are as shown below.

Growth temperature range: The growth temperature range is 10 to 25, and the optimum growth temperature is around 20 ° C.

Growth pH range: The pH range at which growth is possible is pH 3 to pH 9, and the optimal growth pH is pH 4 to pH 8.

Species having the above-mentioned mycological properties were identified by WB Cook, WB Cook, My copathologiaet My co1 ogia A pplicata. Vol. 7, pp. I-43 (1962); written by JA von Arx, the 'Jienera' ob. Fanzine * Sport rating in 'Pure '' Culture (The Generaof F ungi S porulatingin

Pure Culture); J. Cramer Lehre; E. J. Hermanides—Nijhoff, Study's • It is described in literature such as Mycology (Studies in My cology), No. 15, pp. 141-166, CPS Baarn (1977). By searching for strains of the genus Aureobasidium, the above TKR400 strain can be identified as a strain belonging to Aureobasidium.

However, it is a strain belonging to Aureum basidium 厲, which has the ability to produce TKR400-A, and has the ability to produce TKR400-B. Have not been reported before. Therefore, the present inventors designated this strain as a new strain and named it Aureobasidium 'SP TKR 400 strain (A ureobasidium sp. TKR 400), and named it um sp. The International Deposit No. FE RM was registered at the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (address: 1-3-1 Higashi, Tsukuba, Ibaraki, Japan).

BP-5780 (May 17, 1995) (deposited in the present invention, in addition to the above-mentioned TKR400 strain, the TKR400 natural strain) Alternatively, an artificial mutant, a strain belonging to Aureobasidium, and a strain (microorganism) capable of producing TKR400-A can be used.

In addition, in the present invention, in addition to the above-mentioned TKR400 strain, natural or artificial mutant strains of TKR400 strain, other strains belonging to the genus Aureobasidium, etc. A strain (microorganism) capable of producing TKR400-B can be used.

In the present invention, TKR 400-A and TKR 400-B are the above-mentioned TKR 400-A producing strain and TKR 400-B producing strain in a nutrient source-containing medium. Among the above nutrient sources that can be produced by inoculation and culturing, examples of carbon sources include glucose, fructose, saccharose, starch, dextrin, glycerin, molasses, starch syrup, oils and fats, and the like. Organic acids and the like can be mentioned.

Among the above nutrient sources, nitrogen sources include, for example, organic nitrogen compounds such as soybean flour, cottonseed flour, corn steep liquor, casein, peptone, yeast extract, meat extract, germ, urea, amino acids, ammonium salts, and inorganic salts. And nitrogen compounds. Among the above nutrient sources, salts include, for example, sodium salt, potassium salt, calcium salt, magnesium salt, and phosphate salt. And the like inorganic salts. Each of these may be used alone or in combination as appropriate.

The nutrient-containing medium may further contain, if necessary, heavy metals such as iron salts, copper salts, zinc salts, and cobalt salts; vitamins such as biotin and vitamin B; Organic substances, inorganic substances, and the like that promote the production of TKR400-A and TKR400-B can be appropriately added.

In addition to the above-mentioned nutrient sources, an antifoaming agent such as silicone oil or polyalkylene glycol ether, a surfactant, or the like can be added to the above-mentioned medium containing the nutrients, if necessary.

When culturing the above-mentioned strain producing TKR400-A and the strain producing TKR400-B in the above-mentioned nutrient-containing medium, it is generally necessary to produce antibiotics by culturing microorganisms. The method used can be employed, but a liquid culture method, in particular, a shaking or deep aeration stirring culture method can be suitably used.

The above culture is preferably performed at 15 to 25 ° C., and the pH of the medium is usually pH 3 to 8, but is preferably around pH 5. A sufficient production amount can be obtained usually in a culture period of 3 to 15 days.

By the above-described culture method, TKR400-A and TKR400-B are contained in the culture solution and the cells and accumulate in the culture. In the present invention, TKR 400-A and TKR 400-B accumulated in the culture are separated from the culture using the physicochemical properties of these antifungal substances, and It can be further purified and obtained as needed.

The above separation can be performed by extracting the whole culture with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, chloroform, butanol, and methyl isobutyl ketone. Separate the culture into culture solution and cells by filtration or centrifugation, and then separate from the culture solution and cells. You can also.

To separate TKR 400-A and TKR 400-B from the above culture solution. The method of extracting with the above non-hydrophilic organic solvent can also be adopted. Alternatively, a method may be employed in which TKR400-A and TKR400-B in the culture solution are adsorbed on a carrier and then eluted with a solvent. Examples of the carrier include activated carbon, powdered cellulose, and adsorbent resin. The solvent may be used singly or in combination of two or more depending on the type and properties of the carrier.For example, an aqueous solution of a water-soluble organic solvent such as aqueous acetate or aqueous alcohol may be used. Appropriate combinations can be given. To separate T KR 400 -A and T KR 400 -B from the above cells, a method of extracting with a hydrophilic organic solvent such as acetone can be adopted.

In the present invention, the crude extracts of TKR400-A and TKR400-B thus separated from the culture may be subjected to a step of further purification, if necessary. it can. The above-mentioned purification can be carried out by a method usually used for separation and purification of fat-soluble antibiotics. Examples of such a method include silica gel, activated alumina, activated carbon, and adsorbent resin. Column chromatography using a solid, high-performance liquid mouth chromatography, and the like. When column chromatography using silica gel as a carrier is adopted, examples of the eluting solvent include chromatographic form, ethyl acetate, methanol, acetone, and water. Can be used together.

When the high performance liquid chromatography is employed, examples of the carrier include a chemically bonded silica gel having an octadecyl group, an octyl group, a fuunyl group, etc. bonded thereto; a polystyrene-based porous polymer gel, and the like. As the mobile phase, for example, hydrated methanol, hydrated acetonitrile For example, an aqueous solution of a water-soluble organic solvent such as toluene can be used.

The TKR 400 -A and TKR 400 -B of the present invention can be used in medicine as they are or as pharmacologically acceptable salts thereof. The salt is not particularly limited as long as it is pharmacologically acceptable. For example, salts of mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, and hydrobromic acid; Salts of organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, etc. : Alkali metals such as sodium, potassium and calcium or salts of alkaline earth metals and the like.

When the TKR400-A, TKR400-B of the present invention or a pharmacologically acceptable salt thereof is administered as a medicament, the TKR400-A, TKR400-B of the present invention is administered. Or a pharmaceutically acceptable salt thereof, as it is, or in a pharmaceutically acceptable non-toxic and inert carrier, for example, 0.1 to 99.5%, preferably 0.5 to It is administered to animals containing humans as a pharmaceutical composition containing 90%.

Examples of the carrier include solid, semi-solid or liquid diluents, fillers, and other prescription auxiliaries. One or more of these can be used.

The pharmaceutical composition is preferably administered in the form of a dosage unit, and can be administered orally, intrathecally, topically (eg, transdermally), or rectally. Naturally, the above pharmaceutical composition is administered in a dosage form suitable for these administration methods.

When TKR400-A, TKR400-B or a pharmacologically acceptable salt thereof of the present invention is administered as a medicament, the dose as an antifungal agent depends on the condition of the patient such as age and weight. , Administration route, the nature and extent of the disease, etc. It is desirable to adjust the above, but usually, for humans, the amount of the active ingredient of the present invention for adults is in the range of 1 to 200 mg per day. Dosages below the above range may be sufficient, while conversely, doses above the above range may be required. When administering large amounts, it is preferable to divide the dose into several doses. In addition, depending on the formulation, it is desirable to administer it once a week or once a week.

The above-mentioned oral administration can be carried out in solid, powder or liquid dosage units, for example, powders, powders, tablets, dragees, capsules, drops, sublinguals, other dosage forms, etc. .

Parenteral administration can be accomplished, for example, by using liquid dosage unit forms for subcutaneous, intramuscular, or intravenous injection, such as solutions and suspensions. These can be used for the TKR400-A, TKR400-B or a pharmacologically acceptable salt thereof according to the present invention, for example, in an aqueous or oily medium, for example, mineral oil ( It is manufactured by suspending or dissolving in a non-toxic liquid carrier suitable for the purpose of injection, such as incomplete Freund's adjuvant.

The above-mentioned topical administration (such as transdermal administration) can be carried out, for example, by using external preparations such as liquids, creams, powders, bases, gels, and ointments. These can be used as the TKR400-A, TKR400-B of the present invention, or a certain amount of their pharmacologically acceptable salts, in a fragrance, colorant, or filling suitable for the purpose of the external preparation. It is manufactured by combining with one or more of agents, surfactants, humectants, emollients, gelling agents, carriers, preservatives, stabilizers and the like.

Rectal administration is performed by treating a certain amount of TKR 400-A, TKR 400-B or a pharmacologically acceptable salt thereof of the present invention with, for example, higher esters such as myristyl palmitate, Polyethylene glycol, cocoa butter, It can be carried out using a suppository mixed in a low melting point solid such as a mixture thereof. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1

One platinum loop was obtained from the slant culture of TKR400 strain (FERMP-149927), and 100 ml of a liquid medium (Difcoyce Tonitogen base 0.67% (W / V), glucose 2.0% (W / V)) was inoculated into a 500 ml Erlenmeyer flask, and shaken at 25 ° C for 3 days to obtain a seed culture solution. 1.0 ml of this seed culture solution was inoculated into one 500 ml Erlenmeyer flask containing 100 ml of the above liquid medium, shaken for 25 days, and cultured for 8 days. r pm). The culture solution thus obtained was centrifuged and separated into a supernatant and cells. The obtained supernatant was adsorbed on a Diaion HP20 (manufactured by Mitsubishi Chemical Corporation) column (1 L) which was made into a bite with water, and washed with 50% methanol. This was eluted with 3 L of methanol to obtain an active fraction.

In addition, 1 L of methanol was added to the cells, mixed well, and extracted. 1 L of water was added to the obtained residue and mixed well. This was adsorbed on an HP20 column that had been washed with 50% methanol. After washing this column with 50% methanol, it was eluted with 3 L of methanol to obtain an active fraction. The culture supernatant and the active fraction derived from the bacterial cells were collected and concentrated under reduced pressure to obtain 467 mg of a residue.

To this, add 300 ml of water and ethyl acetate, mix well, and add An ethyl washing operation was performed. The water eyebrows were concentrated under reduced pressure, 100 ml of methanol was added to the residue, mixed well, and an extraction operation was performed. The extract was concentrated under reduced pressure to obtain 100 mg of a residue.

This was dissolved in 0.8 ml of methanol, and applied to a Sephadex LH-20 (manufactured by Pharmacia) gel equilibrated with methanol. ¾S Supercolumn (1.85 cm x 78 cm) was added to methanol. The active fraction was obtained by eluting with. This fraction was concentrated under reduced pressure to obtain 87 mg of a residue.

This was dissolved in 0.8 ml of methanol and subjected to high performance liquid chromatography to obtain two active fractions. These fractions were concentrated under reduced pressure to obtain 40 mg of partially purified TKR400-A and 10 mg of partially purified TKR400-B. The conditions for high performance liquid chromatography are as follows: ad ^ «k

Equipment: L C 8 A (manufactured by Shimadzu Corporation)

Column: YMC pc c C 18 [2.0 cm x 25 cm] (manufactured by Iemushi Corporation)

Mobile phase: 0.05% (V / V) containing 5% trifluoroacetic acid

Each of the above partially purified products was dissolved in 0.4 ml of methanol, and subjected to high performance liquid chromatography again to obtain an active fraction. The fractions were concentrated under reduced pressure to give purified products of TKR 400-A and TKR 400-B as 18 mg and 2 mg of white powder, respectively. The conditions of the high-performance liquid chromatography were as follows.

Equipment: L C 8 A (manufactured by Shimadzu Corporation)

Column: YMC pccC18 (2.0 cm x 25 cm) (manufactured by Iemushi)

Mobile phase: 40% (VZV) acetate containing 0.05% trifluoroacetic acid Nitrilno water

Physicochemical properties

For mass spectrometry, a JMS-DX302 mass spectrometer (manufactured by JEOL Ltd.) was used. For UV absorption spectrum analysis (in methanol), UV-2

A 50-type self-recording spectrophotometer (manufactured by Shimadzu Corporation) was used. For the infrared absorption spectrum analysis (KBr method), a 270-30 type infrared spectrophotometer (manufactured by Hitachi, Ltd.) was used. Amino acid was detected by a ninhydrin reaction using L-850, manufactured by Hitachi, Ltd. 'H- NMR (heavy methanol in one le, standard: Te Torame Chirushira down), and,' ^S C-NMR (in deutero meta Nord, standard: heavy methanol) the measurement of, J NM A 5 A nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.) was used. The physicochemical properties of TKR 400 -A and TKR 400 -B are described below.

(1) Mass spectrometry

Purified white powders of TKR 400-A and TKR 400-B obtained by high-performance liquid chromatography and concentrating the obtained active fraction under reduced pressure were analyzed by mass spectrometry. FAB—MS measurement revealed that m 2 z 273 [M + H] +, 1101 [M + H] ⁺ .

(2) UV absorption spectrum

In high-performance liquid chromatography, the active fraction obtained was concentrated under reduced pressure, and the resulting white powder of TKR 400-A and TKR 400-B in methanol was purified. The results of the UV absorption spectrum measurement at the above were as follows.

For TKR 400 — A, UV (nm) (E ^l¾ _{l cm} ): ²⁵⁰ sh (9

6). 2 9 4 (2 0)

TKR 4 0 0 - For B, UV (nm) (E l9i l cm): 2 5 0 sh (1 0 7), 2 9 4 (2 4) The ultraviolet absorption spectra of the obtained TKR 400-A and TKR 400-B are shown in FIGS. 1 and 6, respectively.

(3) Infrared absorption spectrum

Infrared absorption of the purified white powder of TKR 400-A and TKR 400-B obtained by high-performance liquid chromatography and concentration of the obtained active fraction under reduced pressure by the KBr method The spectrum measurement results were as follows.

In the case of TKR 400 1 A, IR (cm-R: 3450, 2970, 1680, 1640, 1550 30.14.0.120.20 , 1 1 5 0

For TKR 400—B, IR (cnT: 3450, 3350, 2970, 1680, 1640, 1530, 1470, 1 4 1 0, 1 2 1 0, 1 1 4 0

The infrared absorption spectra of the obtained TKR400-A and TKR400-B are shown in FIGS. 2 and 7, respectively.

(4) 'Η-NMR and ^13C- NMR spectra

The purified TKR400- し and TKR400-B white powders, which were obtained by high-performance liquid chromatography and concentrating the obtained active fractions under reduced pressure, were subjected to 'H-NMR It was subjected to a vector measurement and a ¹³ C-NMR spectrum measurement. The 'H-NMR spectra of the obtained TKR 400 -8 and 1 ^ 1 ^ 400 —8 are shown in FIGS. 3 and 8, respectively, and the' ³ C-NMR spectrum is shown in FIG. Figures 4 and 9 show the results.

(5) Amino acid analysis

The purified fractions of TKR 400-A and TKR 400-B obtained by high-performance liquid chromatography and concentrating the obtained active fractions under reduced pressure were treated with 6N hydrochloric acid. , I 10, for 24 hours. After decomposition, the product was analyzed by an amino acid analyzer. Lanin, balin, and isoleucine were detected in TKR400-B, and parin and isoleucine were detected.

The solubility of the obtained TKR 400-A and TKR 400-B substances in various solvents is both soluble in methanol, but slightly soluble in chloroform, water and hexane. Met.

According to the above analysis results, the purified white powder obtained by subjecting the obtained active fraction to high-performance liquid chromatography under reduced pressure is TKR400-A and TKR400-B. It has been found.

The above TKR400-A and TKR400-B were converted to reversed-phase high-performance liquid chromatograph (HPLC) using LC-10A type high-performance liquid chromatograph (manufactured by Shimadzu Corporation). Analysis. The conditions for high performance liquid chromatography were as follows.

Column: CAPCELLP AK C _{I 8} (6 mm 150 mm) (manufactured by Shiseido)

Mobile phase: 40% (V / V) acetate containing 0.05% trifluoric acid

Force ram temperature: 40. C

Detection UV wavelength: 220 nm

As a result, it was revealed that the above TKR 400 -A and TKR 400 -B were eluted at the positions shown in FIGS. 5 and 10, respectively.

Biological properties

1) Using the obtained TKR400-A and TKR400-B, antibacterial spectrum against various microorganisms was examined. For the measurement, the concentration that almost completely inhibited the growth of the bacteria was determined as the minimum growth inhibitory concentration (/ z gZml) by the liquid culture medium dilution method. Table 2 shows the results. In addition, the minimum concentration that partially inhibits bacterial growth was determined as the half-inhibitory concentration (gZml). In parentheses. In the table, YNBG represents a medium containing 0.67% of yeast tonite trogen base (manufactured by Difco) and 1.0% of glucose. Table 2

From the results in Table 2, the antifungal substances TKR 400-A and TKR 400-B of the present invention are antibacterial against pathogenic fungi such as Candida albicans, Candida kefir, and Cryptococcus neoformans. It was found to have activity.

2) Effect on mouse Aspergillus systemic infection model

Aspergillus' spores of Fumigatus TI MM 177 6 strain 2 X 10 ICR mice (female, 5 weeks old, 5 mice per group, but 8 mice in the control group only) were inoculated individually from the tail vein. Four hours after the inoculation, and once a day for four days after the inoculation, once a total of five times, a total of 10 times the 10% ethanol solutions of TKR 400--8 and 1 ^ 1¾ 400--8 in 0.2% were added. Each ml was intraperitoneally administered. The control group received only 0.2 ml of the solvent. Table 3 shows the results of observing life and death for 30 days. Table 3

From the results in Table 3, it was revealed that TKR 400-A has a therapeutic effect on Aspergillus infection.

3) Efficacy in a mouse Candida systemic infection model

A 10% solution of TKR 400-B in ethanol was thoroughly mixed with an equal volume of incomplete adjuvant adjuvant to give a water-in-oil formulation of 3.715 mg MgZkg. C57BLZ6 mice (female, 7-week-old, 5 mice per group, but only 6 mice in the control group) were subcutaneously administered as needed. One week later, the same amount was subcutaneously administered again. Incomplete volume equivalent to saline as control The mixture with Freund's adjuvant was similarly administered subcutaneously. One week later, 1 ^× 10 ^s cells obtained by culturing Candida albicans TIMM1768 strain in a Sabouraud. Dextros liquid medium were inoculated into the tail vein of each mouse. Seven days after the inoculation, the mice were dissected on the 7th day, both of the kidneys were aseptically removed, homogenized, and the viable cell count was determined using Sabouraud's dextrose agar medium. Table 4 shows the results. Table 4

') Logarithmic (log). "Significant difference 0.005 From the results in Table 4, it was revealed that TKR-400B had a protective effect against Candida infection. It was suggested that the above effects could be exhibited by the action.

4) Acute toxicity

The obtained TKR-400 OA and TKR-400B were administered intraperitoneally to ICR mice at 5 O mg / kg, respectively, but no toxicity was observed. Industrial applicability

According to the present invention, it is possible to provide antifungal substances TKR400-A and TKR400-B which are useful as clinical drugs such as therapeutic agents for mycosis, and methods for producing them.

Claims

The scope of the claims

1. An antibiotic TKR400-A characterized by having the following physicochemical properties (1), (2), (3), (4) and (5) or a pharmacologically acceptable antibiotic: salt.

(1) The mass spectrum by the FAB-MS method has a peak of mZz 1273 3 [M + H] +

(2) main main absorption wavelength of the ultraviolet absorber scan Bae click Bokuru during methanol (nm) Chikaraku, 2 5 0 sh, 2 9 4 Deari their £ ^'9 ₁ ^ Kaka', 9 6, 2 0

(3) major absorption wave of KB infrared absorption spectrum by r method, 3 4 5 0 cm one ^{1, 2 9 7 0 cm "} \ 1 6 8 0 cm"'> 1 6 4 0 cm-', 1 5 3 0 cm—1 4 1 0 cm— ', 1 2 2 0 cm—1 1 5 0 cm—'

(4) Threonine, alanine, parin and isoleucine are detected in aminoacid analysis by ninhydrin reaction

(5) Soluble in methanol, poorly soluble in chloroform, water and hexane

2. Antibiotic TKR 400-1B characterized by having the following physicochemical properties (6), (7), (8), (9) and (10) or a pharmacologically acceptable antibiotic thereof. Salt.

(6) Mass spectrum by FAB-MS method has a peak of mZz 1101 [M + H] ⁺

(7) The main absorption wavelength (nm) of the UV absorption spectrum in methanol is 250 sh, 294, and their E _{cm is} 10 _cm. 7, 2 4

(8) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3450 cm- ', 3350 cm-', 297 cm "1680 cm- ', 16 4 0 cm— ', 1 5 3 0 cm—', 1 4 7 0 cm— ', 1 4 1 0 cm—', 1 2 1 0 cm— ', 1 1 4 0 cm— ¹

(9) In the amino acid analysis by ninhydrin reaction, valin and iso-isocyanate are detected.

(10) Soluble in methanol, slightly soluble in chloroform, water and hexane

3. A strain belonging to Aureobasidium 厲, which is a strain that produces the antibiotic TKR 400-A, is cultured, and then the target substance is isolated from the culture of the strain. A method for producing the antibiotic TKR 400-A, which is characterized in that it is released.

4. Culture of a strain belonging to Aureobasidium that produces the antibiotic TKR400-B, and then isolating the target substance from the culture of the strain A method for producing the antibiotic TKR400-B.

5. A microorganism belonging to the genus Aureobasidium (Aureobasidium), which produces the antibiotic TKR400-A.

6. A microorganism belonging to the genus Aureobasidium (Aurebobasidum) and producing the antibiotic TKR400-B.