JP3530563B2 - KO-8119 substance and process for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a KO-8119A substance, a KO-8119B substance, a KO-8119C substance and / or a KO- substance which are useful as therapeutic and preventive agents for coccidiosis, which is an important disease in the poultry industry. 811
For more details on the 9D substance and its manufacturing method, see KO.
-8119A substance, KO-8119B substance, KO-81
The 19C substance and / or KO-8119D substance has an antibacterial activity and an antiviral activity, and is useful as a therapeutic drug or preventive drug for an antibiotic or a viral disease.

[0002]

2. Description of the Related Art Conventionally, as anticoccidial agents, sulfa agents, quinoline agents, antithiamine agents, antibiotics, etc. have been put into practical use, and recently, polyether antibiotics such as monensin, salinomycin, lasalocid, etc. Widely used.

[0003]

However, coccidium resistant to these drugs has appeared and its effect is weakened, and an anticoccidial agent to replace them has been demanded. Therefore, from the above viewpoint, the object of the present invention is to provide a drug which is effective not only against drug-resistant coccidium but also against coccidium which has acquired resistance to polyether compounds.

[0004]

In general, drug cross-resistance is established when the structures are similar or the mechanism of action is similar, and compounds that differ structurally or mechanism of action from conventional coccidial agents are It is useful as a therapeutic or preventive agent for coccidiosis. The present inventors continued various studies to find out a novel drug effective against coccidial parasites that have acquired resistance to conventional drugs from the natural world, using monensin-resistant coccidial protozoa as a test organism, metabolism produced by microorganisms. As a result of searching among the products, it was found that a substance effective against coccidium is produced in the culture solution of the KO-8119 strain newly isolated from the soil in Nagoya City, Aichi Prefecture.

Then, as a result of separating and purifying the anticoccidial active substance from the culture, each substance having the physicochemical properties described below was obtained. Since these substances have not been known at all in the past, this substance is referred to as KO-8119A substance or KO-
8119B substance, KO-8119C substance and KO-8
119D substance (hereinafter, sometimes collectively referred to as KO-8119 substance). The present invention has been completed based on such findings, and the following formula

[0006]

[Chemical 8] The KO-8119 substance represented by or a non-toxic salt thereof is provided.

Furthermore, the present invention comprises culturing in a medium a microorganism belonging to the genus Streptomyces and having an ability to produce a KO-8119 substance represented by the following formula, and KO-8 in the culture.
119 substance was accumulated and KO-8119 was collected from the culture.
The present invention provides a method for producing a KO-8119 substance, which comprises collecting a substance and, if desired, producing a non-toxic salt thereof.

[0008]

[Chemical 9]

KO-8119A substance of the present invention, KO-8
119B substance, KO-8119C substance and KO-81
The physicochemical properties of 19D substance are as follows. [1] KO-8119A substance (1) Elemental analysis value: C50.61%, H6.96%, N
10.75%, S6.08% (2) Estimated molecular formula: C ₂₂ H ₃₄ N ₄ O ₈ S (according to high resolution mass spectrum) (3) Molecular weight: 514 The measurement value according to high resolution FAB mass spectrum is as follows. is there. Calculated value (M + H): 515.2176 Measured value (M + H): 515.2190 (4) Specific rotation: [α] _D ²⁴ + 103 ° (C = 0.0)
7, in methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum near 215, 256 and 309 nm. (6) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr method is as shown in FIG.
2930, 1660, 1580, 1480, 1330,
It has absorption bands at 1130, 1060, and 1040 cm ^-1 . (7) Color reaction: Positive in Dragendorff reaction (8) Solubility in solvent: methanol, ethanol,
Soluble in ethyl acetate, chloroform and dimethylsulfoxide, sparingly soluble in water, insoluble in n-hexane

(9) Distinction between basic, acidic and neutral: basic (10) substance property: pale yellow powder (11) nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a deuterated chloroform solution using an NMR spectrometer at 0 and 400 MHz are as shown in FIGS. 3 and 4, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 1 below.

[0012]

[Table 1]

[2] KO-8119B substance (1) Elemental analysis value: C56.84%, H6.86%, N
12.72% (2) Estimated molecular _{formula: C 26 H 37 N 5 O} 8 ( by high resolution mass spectrum) (3) Molecular weight: 547 measurements by high-resolution FAB mass spectrum are as follows. Calculated value (M + H): 548.2720 Measured value (M + H): 548.2816 (4) Specific rotation: [α] _D ²⁵ + 122 ° (C = 0.1,
(In methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum near 204, 255 and 339 nm. (6) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr method is as shown in FIG.
2930, 1660, 1600, 1560, 1480,
1330, 1250, 1180, 1070, 1040c
It has an absorption band at m ^-1 . (7) Color reaction: Positive in Dragendorff reaction (8) Solubility in solvent: methanol, ethanol,
Acetic acid ethyl ester, soluble in dimethyl sulfoxide,
Insoluble in water and chloroform, insoluble in n-hexane

(9) Distinction between basic, acidic and neutral: basic (10) substance property: pale yellow crystal (11) nuclear magnetic resonance spectrum: Varian XL-40
¹ H measured at 0,400 MHz using a NMR spectrometer in a mixed solution of deuterated chloroform and deuterated methanol
-NMR spectrum and ¹³ C-NMR spectrum are
This is as shown in FIGS. 7 and 8, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 2 below.

[0016]

[Table 2]

[3] KO-8119C substance (1) Elemental analysis value: C55.14%, H7.36%, N
11.09% (2) Estimated molecular formula: C ₂₃ H ₃₆ N ₄ O ₈ (according to high resolution mass spectrum) (3) Molecular weight: 496 Measured values according to high resolution FAB mass spectrum are as follows. Calculated value (M + H): 497.2611 Actual value (M + H): 497.2601 (4) Specific rotation: [α] _D ²⁵ + 105 ° (C = 0.1,
(In methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum near 210, 262 and 300 nm. (6) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr method is as shown in FIG.
2930, 1640, 1560, 1490, 1330,
It has absorption bands at 1130, 1070 and 1040 cm ^-1 . (7) Color reaction: Positive in Dragendorff reaction (8) Solubility in solvent: methanol, ethanol,
Soluble in ethyl acetate, chloroform and dimethylsulfoxide, sparingly soluble in water, insoluble in n-hexane

(9) Distinction between basic, acidic and neutral: basic (10) substance property: white powder (11) nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a deuterated chloroform solution using an NMR spectrometer at 0 and 400 MHz are as shown in FIGS. 10 and 11, respectively. The chemical shifts of hydrogen and carbon are shown in Table 3 below.

[0020]

[Table 3]

[4] KO-8119D substance (1) Elemental analysis value: C55.13%, H7.37%, N
11.09% (2) Estimated molecular formula: C ₂₃ H ₃₆ N ₄ O ₈ (according to high resolution mass spectrum) (3) Molecular weight: 496 Measured values according to high resolution FAB mass spectrum are as follows. Calculated value (M + H): 497.2611 Measured value (M + H): 497.2611 (4) Specific rotation: [α] _D ²⁴ + 144 ° (C = 0.1 in methanol)

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 2, and shows a characteristic absorption maximum near 201, 258 and 300 nm. (6) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr method is as shown in FIG.
0, 2910, 1640, 1560, 1480, 138
It has absorption bands at ⁰ , 1330, 1250, 1070, and 1040 cm ⁻¹ . (7) Color reaction: Positive in Dragendorff reaction (8) Solubility in solvent: methanol, ethanol,
Soluble in ethyl acetate, chloroform and dimethylsulfoxide, sparingly soluble in water, insoluble in n-hexane

(9) Distinction between basic, acidic and neutral: basic (10) substance property: white powder (11) nuclear magnetic resonance spectrum: Varian XL-40
The ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a deuterated chloroform solution using an NMR spectrometer at 0 and 400 MHz are as shown in FIGS. 13 and 14, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 4.

[0024]

[Table 4]

The above-mentioned KO-8119A substance, KO-81
19B substance, KO-8119C substance and / or KO
A microorganism having the ability to produce the −8119D substance (hereinafter referred to as a KO-8119 substance-producing bacterium) belongs to the genus Streptomyces. For example, the Streptomyces sp. KO-8119 strain isolated by the present inventors is This is an example of the most effectively used strain of the present invention, and the bacteriological characteristics of this strain are as follows.

I. Morphological properties Vegetative hyphae develop well on various agar media and no fragmentation is observed. Aerial aerial hyphae grow abundantly on starch, inorganic salt agar, etc., and show white to gray. When observed under a microscope, the aerial mycelium has a spiral shape, and a chain of 20 or more spores is observed. The size of spores is 0.58 x 0.81 μm
It has a cylindrical shape. The surface of spores is smooth. No sclerotia, sporangia and zoospores are found.

II. Properties on various media EB Shirrin
g) and the method of D. Gottlieb (International Journal of Systematic Bacteriology, Vol. 16, 313, 1966), the culture properties of the productive bacteria are shown in Table 5 below. . The color tone was determined using the Color Harmony Manual Fourth Edition (Container Corporation of America Chicago, 1958) as the standard color, and the code is also shown in brackets along with the color chart name. The following are the results of observation in each medium at 27 ° C. for 2 weeks unless otherwise specified.

[0028]

[Table 5]

[0029] III. Physiological properties   (1) Generation of melanin pigment     (A) Tyrosine agar negative     (B) Peptone, yeast, iron agar No growth     (C) Glucose / peptone / gelatin medium positive     (D) Tryptone yeast solution positive   (2) Tyrosinase reaction negative   (3) Hydrogen sulfide production Negative   (4) Nitrate reduction Positive

[0030]   (5) Liquefaction of gelatin (21 ℃ -23 ℃) Positive         (Glucose / peptone / gelatin medium)   (6) Starch hydrolysis positive   (7) Coagulation of skim milk (27 ° C) Negative   (8) Peptonization of skim milk (27 ° C) Positive   (9) Growth temperature range 9 ° C to 32 ° C   (10) Availability of carbon source         (Pleedam Gotrive Agar)       Glucose, arabinose, xylose, raffinose, melibiose,       Mannitol, fructose, rhamnose, inositol, sucrose       Not use   (11) Decomposition of cellulose             negative

IV. Cell wall composition Cell wall diaminopimelic acid is of the LL type. The following is a summary of the mycological properties of this bacterium. Diaminopimelic acid in the cell wall is LL type. The aerial hyphal morphology is spiral and forms long spore chains. The surface of spores is smooth. As for various properties in culture, vegetative hyphae have a colorless or brown color tone, and aerial hyphae have a white or gray pigment. It does not produce soluble pigments, but tryptone
A slight amount of melanin-like pigment is produced in yeast solution medium and glucose / peptone / gelatin medium.

From these results, this strain belongs to the genus Streptomyces and is classified into Pridham and Tresner (Virgin's Manual of Determinant Bacteriology, 8th Edition, pp. 748-829). It is considered to be a bacterial species belonging to the Gray series by. This strain is Streptomyces sp. KO
-8119 (Streptomyces sp. KO-
8119) has been deposited with the Institute of Biotechnology, Industrial Technology Institute. Deposit number is FERM P-1339
8. The deposit date is February 1, 1993.

Above, KO-8119A substance, KO-81
19B substance, KO-8119C substance and KO-811
Although the 9D substance-producing bacterium has been described, as a general property of the bacterium, the bacteriological properties are very liable to vary and are not constant, and the UV irradiation or a mutant derivative such as N-methyl-naturally or commonly performed is used. N'-nitro-N
-It is a well-known fact that mutation is carried out by an artificial mutagenesis method using nitrosoguanidine, ethyl methanesulfonate or the like.

KO-8119 belongs to the genus Streptomyces, including natural mutants as well as artificial mutants.
Any strain capable of producing substance A, substance KO-8119B, substance KO-8119C and / or substance KO-8119D can be used in the present invention.
In addition, strains mutated by cell engineering such as cell fusion and gene manipulation are also included in the KO-8119 substance-producing bacterium of the present invention.

In the present invention, KO-8119A substance, KO-8119B substance belonging to the genus Streptomyces,
KO-8119C substance and / or KO-8119D
The substance-producing bacteria are cultured in the medium. In culturing the bacterium, a usual culturing method for actinomycetes is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be assimilated, and, if necessary, an inorganic acid salt is used. As the assimilable carbon source, glucose, sucrose, sugar concentrate, dextrin, starch, cellulose and the like are used alone or in combination.

Examples of the carbon source used in the present invention include glucose, glycerol, fructose,
Carbohydrates such as maltose, mannitol, xylose, galactose, ribose, starch or hydrolysates thereof can be used. Its concentration is 0.1% with respect to normal medium
~ 5% is desirable. Further, various organic acids such as gluconic acid, pyruvic acid, lactic acid, acetic acid, various amino acids such as glycine, glutamic acid, and alanine, further alcohols such as methanol and ethanol, and non-aromatic hydrocarbons such as normal paraffin, or a plant or Various animal fats and oils can also be used.

Examples of the nitrogen source used in the present invention include ammonium salts of various inorganic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate and ammonium nitrate, urea, peptone and NZ-.
Nitrogen-containing organic substances such as amine, meat extract, yeast extract, dry yeast, corn steep liquor, casein hydrolyzate, fish meal or its digest, soybean flour or its digest, defatted soybean or its digest, hydrolyzate Furthermore, various amino acids such as glycine, glutamic acid, and alanine can be used.

As the inorganic substance, for example, various phosphates, magnesium sulfate, sodium chloride, and trace amounts of heavy metal salts are added. Also, when using a mutant strain that exhibits an auxotrophy, it is naturally necessary to add a substance that satisfies the auxotrophy to the medium, but this type of nutrient is not available when using a medium containing a natural product. , In some cases, no addition is required.

Culturing is usually carried out under aerobic conditions such as shaking or aeration stirring culture. From the industrial point of view, deep aeration stirring culture is preferred. The pH of the culture is, for example, 5.0-8.
Although it is 0, it is preferable to carry out the culture in the vicinity of neutrality. The culture temperature may be 20 to 37 ° C., but usually 26 to 32 ° C.
It is preferable to keep the temperature (preferably around 27 ° C.). When the culture time is liquid, the KO-8 is usually obtained by culturing for 2 to 5 days.
119A substance, KO-8119B substance , KO-8119
Since the C substances it and KO-8119D substance is accumulated,
The culture may be terminated when the accumulated amount in the culture reaches the maximum.

The culture conditions such as the medium composition, the liquidity of the medium, the culture temperature, the stirring speed, and the aeration rate are appropriately adjusted and selected so as to obtain preferable results depending on the kind of the strain to be used and external conditions. It goes without saying that it will be done. In liquid culture, when foaming occurs, a defoaming agent such as silicone oil, vegetable oil, or surfactant can be used as appropriate.

The KO-8119A substance, KO-8119B substance and KO accumulated in the culture thus obtained
Since the -8119C substance and the KO-8119D substance are contained in the microbial cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the microbial cells.
119A substance, KO-8119B substance, KO-8119
It is advantageous to collect material C and KO-8119D material. In order to collect the KO-8119A substance, the KO-8119B substance, the KO-8119C substance and the KO-8119D substance, the means usually used for collecting the metabolites from the culture of the microorganism is used alone or in combination,
Or it is used repeatedly.

That is, for example, extraction filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, precipitation, crystallization, recrystallization, phase transfer, countercurrent distribution method utilizing the difference in solubility between various solvents. Means such as chromatography are used. KO-8119A substance, KO-811 from culture solution
9B substance, KO-8119C substance and KO-8119
To collect the substance D, first, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, benzene, or the culture filtrate or cell extract is activated carbon, alumina, porous synthetic polymer resin. It may be adsorbed on an ion exchange resin or the like, eluted with an elution solvent such as methanol, the obtained extract is concentrated under reduced pressure, and then extracted with an organic solvent such as ethyl acetate.

The obtained crude substance is further subjected to a known method usually used for the purification of fat-soluble substances, for example, column chromatography using a carrier such as silica gel, alumina or reverse phase chromatography using an ODS carrier, Further, it can be purified by a method such as high performance liquid chromatography.

KO-8119A thus obtained
The substance, KO-8119B substance, KO-8119C substance, and KO-8119D substance are basic substances and are hardly soluble in water, but can be converted into a salt with an acid by a known method. Examples of such pharmaceutically acceptable salts include hydrochloride, sulfate, nitrate, phosphate, tartrate, citrate, glutamate, aspartate and the like.

The anticoccidial activity and other biological activities of the above-mentioned KO-8119A substance, KO-8119B substance, KO-8119C substance and KO-8119D substance of the present invention on chickens are as follows.

Chicken Coccidiosis Growth Inhibitory Activity Primary cultured cells (chicken primary cells) were prepared from fertilized egg embryos at 10 days of hatching. Chicken coccidia, resistant to monensin
The method of inhibiting the growth of coccidium on the primary cells of chick host cells by using the oocysts of Eimeria tenella (Oonaga, Ishii: Journal of the Japan Veterinary Association, 3
1, 592-596 (1978)), the growth inhibitory action concentration of each of the KO-8119A substance, KO-8119B substance, KO-8119C substance and KO-8119D substance of the present invention was 0.3 μg / ml. , 0.6
μg / ml, 0.6 μg / ml and 2.5 μg / ml
Met. No cytotoxicity was observed at a concentration of 0.3 to 2.5 μg / ml, which shows the activity of inhibiting the growth of Eimeria tenella, on the primary culture cells of the chick, which is the host.

On the other hand, the same administration experiment was carried out for the known chicken coccidial growth inhibitor monnesin. As a result, monensin showed no coccidial growth inhibitory activity, and toxicity was observed in host cells at 0.03 μg / ml administration. Present Invention KO-8119A Substance, KO-8119B
The substances, KO-8119C substance and KO-8119D substance, were found to be less toxic than the known chicken coccidiosis growth inhibitor monensin.

Antibacterial activity KO-811 was placed on an agar plate overlaid with various test bacteria.
9A substance, KO-8119B substance, KO-8119C substance and KO-8119D substance 5 μg in diameter impregnated 6
The antibacterial activity was investigated by placing a mm-mm pulp disk and measuring the size of the diameter of the growth inhibition circle produced.

KO-8119A substance, KO-8119B
Both the substance and the KO-8119C substance exhibited antibacterial activity against bacteria such as Staphylococcus aureus, Bacillus subtilis, Sarsina lutea, Mycobacterium smegmatis, Escherichia coli, and Zanthomonas oryzae. In addition, antibacterial activity was observed against the anaerobic bacterium Bacteroides fragilis, and also against Mycoplasma acholeplasma ledrowii. The KO-8119D substance was found to have antibacterial activity against Micrococcus luteus and Acoreplasma ledrowie.

Antiviral activity MDBK cells (cell line derived from bovine kidney) were used as a host, infected with influenza virus (A / WSN / 33), and then cultured overnight, and cytopathic effect by virus proliferation (C
PE) was observed under a microscope. The KO-8119A substance and the KO-8119C substance inhibited cytopathic change due to virus growth at concentrations of 10 μg / ml and 20 μg / ml, respectively. No cytotoxicity to host cells was observed at these concentrations.

[0051]

As described above, the present invention KO-8119A
Since the substance, the KO-8119B substance, the KO-8119C substance and the KO-8119D substance have the activity of inhibiting the growth of coccidial parasites resistant to the polyether antibiotic monensin, they can lead to the amelioration of coccidiosis. In addition, it can be used as a concomitant drug that can prolong the effect of a known anticoccidial drug remarkably. Further, KO-8119A substance, KO-8119B substance, KO-8119C substance and / or KO-811 substance
9D substance can be used as an antibiotic because it shows antibacterial activity against bacteria including anaerobic bacteria or mycoplasma. Furthermore, KO-8119A substance and KO-8119C substance inhibit influenza virus infection. It has activity and is effective as an antiviral agent.

[0052]

【Example】

Example 1 In a 500 ml Erlenmeyer flask, glucose 0.1%, starch 2.4%, peptone 0.3%, yeast extract 0.5%,
Dispense 100 ml of liquid medium (pH 7.0) containing 0.3% of meat extract and 0.4% of calcium carbonate and
Steam sterilized for 0 minutes. Streptomyces sp. KO-8119 strain (F
The bacterial cells of ERM P-13398) were aseptically inoculated with a platinum loop and cultured at 27 ° C. for 3 days to obtain a seed culture solution. Then, 2% maltose 30L jar fermenter, dried 1.5% yeast, brewer's yeast (trade name Ebios) 2.5%, 0.3% _{saline, KH 2 PO 4 0.05%,} MgSO 4 · 7
20 L of liquid medium (pH 7.0) containing 0.05% H ₂ O
Was sterilized and steam sterilized at 121 ° C. for 20 minutes. 400 ml of the above-mentioned seed culture was inoculated into this and cultured at 27 ° C for 3 days with aeration and stirring.

After adjusting the pH of 18 L of the culture broth to 8.5, 18 L of ethyl acetate was added and well stirred, followed by centrifugation to separate the ethyl acetate layer. The ethyl acetate extract was concentrated to dryness to obtain 8.58 g of a crude extract. The crude extract was washed with n-hexane, the substance insoluble in methanol was removed, and the crude product was subjected to silica gel chromatography (developing solvent: chloroform, methanol).
9 mg was obtained. Next, it is subjected to reverse phase chromatography (developing solvent: methanol, water) using an ODS carrier, and reverse phase (ODS) high performance liquid chromatography (developing solvent: 60% methanol, 10 mM phosphate buffer pH 7.
8) is carried out, and 8.0 mg of KO-8119A substance, KO-
11.6 mg of 8119B substance and 7.1 mg of KO-8119C substance were respectively obtained.

Example 2 As in Example 1, Streptomyces sp. KO
An inoculum culture of strain-8119 was obtained. Soluble starch 2%, dry yeast 1%, brewer's yeast (trade name Ebios) 2%, peptone 0.3%, yeast extract 0.
5%, salt 0.3%, KH ₂ PO ₄ 0.05%, MgS
O ₄ · 7H ₂ O0.05%, was charged CaCO ₃ liquid medium (pH 7.0) 20L containing 0.4%, 121 ℃, 2
Steam sterilized for 0 minutes. 400 ml of the above seed culture
Was inoculated and cultured with aeration and stirring at 27 ° C. for 2 days. KO-8119A substance, KO-8119B substance, K in culture supernatant
The accumulated amounts of O-8119C substance and KO-8119D substance were 12.0 μg / ml and 33.3 μg / m, respectively.
1, 15.3 μg / ml and 11.5 μg / ml.

18 L of this culture solution was centrifuged to separate it into a supernatant and cells, and the pH of the supernatant was adjusted to 8.5.
After L ethyl acetate was added and well stirred, the mixture was centrifuged to separate the ethyl acetate layer. The ethyl acetate extract was concentrated under reduced pressure and dried to obtain 10.25 g of a crude extract. This crude extract was washed with n-hexane, the insoluble material was removed from methanol, and the residue was subjected to silica gel chromatography (developing solvent: chloroform, methanol), followed by reverse phase chromatography with an ODS carrier (developing solvent: methanol,
Water) to give 60.8 mg of KO-8119A substance, KO
-8119B substance 151 mg, KO-8119C substance 7
2.1 mg and 52.4 mg of KO-8119D substance were obtained respectively.

Example 3 KO-8119A substance, KO-8119B substance, KO-
Growth inhibition activity of the 8911C substance and the KO-8119D substance for the chicken coccidia: Primary cultured cells (chicken primary cells) were prepared from fertilized egg embryos at 10 days of hatching. The method of inhibiting the growth of coccidium on host chick primary cells using the oocysts of chicken coccidium and Eimeria tenella, which are resistant to monensin, is the method of Onaga et al. (Oonaga, Ishii: Journal of the Japan Veterinary Association, 31, Table 6 shows the results of measurement according to 592-596 (1978)).

KO-8119A substance, KO-811
9B substance, KO-8119C substance and KO-8119
The concentration of growth inhibitory effect of substance D on chicken coccidium is
0.3 μg / ml, 0.6 μg / ml, 0.6 μg respectively
/ Ml and 2.5 μg / ml. In addition, the concentration of this substance is 0.3-
No cytotoxicity was observed at 2.5 μg / ml.

On the other hand, a similar administration experiment was carried out on monensin, which is a known chicken coccidiosis growth inhibitor.
No coccidial growth inhibitory activity was observed with monensin, and toxicity was observed with host cells at 0.03 μg / ml administration.

[0059]

[Table 6]

Antibacterial activity KO-811 was placed on an agar plate overlaid with various test bacteria.
9A substance, KO-8119B substance, KO-8119C substance and KO-8119D substance 5 μg in diameter impregnated 6
The antibacterial activity was investigated by placing a mm-mm pulp disk and measuring the size of the diameter of the growth inhibition circle produced. KO-8
119A substance, KO-8119B substance or KO-8
All the 119C substances exhibited antibacterial activity against bacteria such as Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Mycobacterium smegmatis, Escherichia coli, and Zanthomonas oryzae.

In addition, antibacterial activity was also recognized against the anaerobic bacterium Bacteroides fragilis, and further against Mycoplasma acholeplasma ledrowii. The KO-8119D substance was found to have antibacterial activity against Micrococcus luteus and Acoreplasma ledrowie. The results are shown in Table 7. still,*
t: A slight growth inhibition circle diameter is observed.

[0062]

[Table 7]

Anti-virus activity MDBK cells (cell line derived from bovine kidney) were used as hosts, infected with influenza virus (A / WSN / 33), and cultured overnight, and cytopathic effect by virus growth (C
PE) was observed under a microscope. The KO-8119A substance inhibited about 50% of cell degeneration due to virus growth at a concentration of 10 μg / ml, and almost 100% at a concentration of 20 μg / ml. The KO-8119C substance inhibited the cytopathic effect due to virus growth by about 50% at a concentration of 20 μg / ml. It should be noted that neither the KO-8119A substance nor the KO-8119C substance was found to be cytotoxic to host cells at these concentrations.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of a KO-8119A substance, a KO-8119B substance and a KO-8119C substance (measured as a methanol solution). The solid line in the figure is the KO-8119A substance, and the broken line is KO-8119B.
Substance, dashed line shows KO-8119C substance.

FIG. 2 is an ultraviolet absorption spectrum of KO-8119D substance.

FIG. 3 is an infrared absorption spectrum of a KO-8119A substance (KBr method).

FIG. 4 is a proton nuclear magnetic resonance spectrum of KO-8119A substance (deuterated chloroform solution).

FIG. 5 is a ¹³ C nuclear magnetic resonance spectrum of KO-8119A substance (deuterated chloroform solution).

FIG. 6 is an infrared absorption spectrum of a KO-8119B substance (KBr method).

FIG. 7 is a proton nuclear magnetic resonance spectrum of KO-8119B substance (deuterated chloroform / methanol mixed solution).

FIG. 8 is a ¹³ C nuclear magnetic resonance spectrum of KO-8119B substance (deuterated chloroform / methanol mixed solution).

FIG. 9 is an infrared absorption spectrum of a KO-8119C substance (KBr method).

FIG. 10 is a proton nuclear magnetic resonance spectrum of KO-8119C substance (deuterated chloroform solution).

FIG. 11 is a ¹³ C nuclear magnetic resonance spectrum of KO-8119C substance (deuterated chloroform solution).

FIG. 12 is an infrared absorption spectrum of KO-8119D substance (KBr method).

FIG. 13 is a proton nuclear magnetic resonance spectrum of KO-8119D (deuterated chloroform solution).

FIG. 14 is a ¹³ C nuclear magnetic resonance spectrum of KO-8119D substance (deuterated chloroform solution).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12P 19/28 C12R 1: 465 C12R 1: 465) (72) Inventor Yuzuru Iwai 5-9-1 Shirokane, Minato-ku, Tokyo Corporate Hojin Kitasato Institute (56) References J. Antibiot. , July 1994, Vol. 47, No. 7, p. 774-781 J. Antibiot. , Vol. 47, No. 7, p. 782-786 Kangsengsu, 1985, Vol. 10, p. 285-295 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12P 19/00-19/64 JSTPlus (JOIS) MEDLINE (STN) REGISTRY (STN) CA (STN) PubMed

Claims (9)

(57) [Claims] 1. The following formula: KO-8119 substance represented by or a non-toxic salt thereof. 2. The following formula: KO-8119A substance represented by or a non-toxic salt thereof. 3. The following formula: KO-8119B substance represented by or a non-toxic salt thereof. 4. The following formula: KO-8119C substance represented by or a non-toxic salt thereof. 5. The following formula: KO-8119D substance represented by or a non-toxic salt thereof. 6. A microorganism belonging to the genus Streptomyces and having an ability to produce a KO-8119 substance represented by the following formula is cultured in a medium to accumulate the KO-8119 substance in the culture, and KO is obtained from the culture. -8119 substance was collected,
K, characterized by producing a non-toxic salt thereof if desired
Manufacturing method of O-8119 substance. [Chemical 6] 7. A KO-8119 substance, a KO-8119A substance, a KO-8119B substance, and a KO-indicated by the following formula:
8119C substance and KO-8119D substance and KO-8119 selected from the group consisting of non-toxic salts thereof
The KO-8 according to claim 6, which is a substance or a non-toxic salt thereof.
A method for producing 119 substances or their non-toxic salts. [Chemical 7] 8. KO-81 belonging to the genus Streptomyces
Microorganisms capable of producing 19 substances are Streptomyces sp. KO-8119 (Streptom
yces sp. KO-8119) FERM P-13
The production method according to claim 6 or 7, which is 398. 9. KO-81 belonging to the genus Streptomyces
Streptomyces spp. Capable of producing 19 substances
Speedy KO-8119 (Streptomyces)
sp. KO-8119) FERM P-13398.
That microorganisms.