JPH07233185A - Fo-2030 substance and its production - Google Patents

Abstract

PURPOSE:To obtain a new FO-2030 substance useful for treating coccidium of chicken, effective even against coccidium resistant to a polyether-based compound by culturing a fungus which belongs to the genus Penicillium, capable of producing FO-2030 substance in a medium. CONSTITUTION:A fungus [e.g. Penicillium sp. FO-2030 (FERM P-14,000)] belonging to the genus Penicillium, capable of producing FO-2030 substance, is cultured in a medium and FO-2030 substance is accumulated in a culture mixture. A product is collected from the culture mixture to give objective FO-2030 substance having the following chemical and physical properties. Elemental analysis value: C 72.0% H 8.82%. Molecular formula: C15H22O3 (by mass spectrometry having high resolving power). Molecular weight: 250. Melting point: decomposing at >=300 deg.C. Specific rotatory power: [alpha]<28>D+4.4 deg. (C=0.1, in methanol). Coloring reaction: coloring in 50% H2SO4. Negative in ninhydrin. Solubility: soluble in methanol, ethanol, ethyl acetate and chloroform and insoluble in water. Properties of substance: neutral color powder.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a FO-2030 substance which is useful as a therapeutic drug and a preventive drug for coccidiosis, which is an important disease in the poultry industry, and a method for producing the same.

[0002]

Conventionally, as anticoccidial agents, sulfa agents, quinoline agents, antithiamine agents, antibiotics, etc. have been put to practical use, and recently, polyether antibiotics such as monensin, salinomycin, lasalocid are widely used. Has been done.

[0003]

However, coccidium resistant to these drugs has emerged and the results have weakened, and there is a demand for anticoccidial agents to replace them. Therefore, an object of the present invention is to provide a drug effective not only against drug-resistant coccidium but also against coccidium that has acquired resistance to a polyether compound, from the above viewpoint. .

[0004]

Means for Solving the Problems In general, drug cross-resistance is established when the substances acting have a similar structure or a similar mechanism of action, and are structurally or mechanism of action similar to conventional anticoccidial agents. Compounds differing in are useful as therapeutic or preventive agents 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 was produced in the culture solution of the FO-2030 strain newly isolated from soil.

Then, as a result of separating and purifying the anticoccidial active substance from the culture, it was found that the FO-2030 substance having the physicochemical properties described below is a substance that has never been known. The present invention has been completed based on such findings, and provides an FO-2030 substance having the physicochemical properties described below or a pharmaceutically acceptable salt thereof. Furthermore, the present invention cultivates a microorganism that belongs to the genus Penicillium and has the ability to produce the FO-2030 substance in a medium to accumulate the FO-2030 substance in the culture, and collect the FO-2030 substance from the culture. The present invention provides a method for producing the FO-2030 substance or a pharmaceutically acceptable salt thereof.

The physicochemical properties of the FO-2030 substance of the present invention are as follows. (1) Elemental analysis: C72.01%, H8.82% (2 ) Molecular formula: C ₁₅ H ₂₂ O ₃ (by high resolution mass spectrum) (3) Molecular weight: 250 (measured by high-resolution EI mass spectrum Calculated value: 250.1570 Measured value: 250.1572 (4) Melting point: Decomposition above 300 ° C

(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum near 203 and 228 nm. (6) Infrared absorption spectrum: KBr The infrared absorption spectrum measured by the method is as shown in FIG.
2902, 2891, 1700, 1674, 1473,
It has absorption bands near 1380, 1277, 1075, and 1010 cm ⁻¹ (7) Color reaction: Color is developed with 50% H ₂ SO ₄ . Ninhydrin negative (8) Solubility in solvents: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water (9) Basic, acidic, neutral: neutral

(10) Material properties: colorless powder (11) Nuclear magnetic resonance spectrum: Varian XL-40
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 shown in FIGS. 3 and 4, respectively.
As shown in. The chemical shifts of hydrogen and carbon are as shown in Table 1 below.

[0009]

[Table 1]

The above-mentioned FO-2030 used in the present invention.
Microorganisms that produce substances (hereinafter referred to as FO-2030 substance-producing bacteria) belong to the genus Penicillium. Among them, for example, Penicillium sp.
p. ) The FO-2030 strain is an example of the most effectively used strain of the present invention, and the mycological properties of this strain are as follows.

I. Morphological Properties This strain grows relatively well on potato / glucose agar medium, YpSs agar medium, wort agar medium and the like, and conidia are in a moderate degree. When the colonies grown on the potato-glucose agar medium are observed under a microscope, the hyphae are transparent and have septa, and the conidia peduncle is directly growing from the basal hyphae. Penicillus is a single-wheel student, and the conidia peduncle rarely branches,
Conidia stalks have partition walls, and those that are long and those that are short are mixed. The infarcts are 2 to 4 in a pen-tip type.
The size is 7.5 to 10.0 × 2 to 3 μm. Initially, one Fiaro-conidia settles on the apical end of the infarct and becomes chain-like with the lapse of culture time, and finally this chain reaches around 100 μm. Conidia are spherical to subspherical, have a size of 2.5 to 3.0 μm, and have a smooth surface.

II. Various Properties on Various Media Table 2 shows the results of macroscopic observation when the cells were cultured on various media at 25 ° C. for 14 days. In each medium, formation of secretory fluid and sclerotia associated with the growth of the fungus was not observed.

[0013]

[Table 2]

III. Physiological Properties 1) Optimal Growth Conditions The optimum growth conditions for this strain are pH 4 to 4 in YpSs medium.
8 and the growth temperature is 15 to 30 ° C. 2) Range of growth The range of growth of this strain is pH 2-1 in YpSs medium.
0, the growth temperature is 8 to 37 ° C. 3) Distinction between aerobic and anaerobic: aerobic

Based on the above-mentioned morphological characteristics, culture properties and physiological properties, an attempt was made to compare it with known bacterial strains. As a result, this strain is considered to belong to the genus Penicillium, and Penicillium sp. FO
It was named -2030. This strain is Penicillium sp. FO-2030 (Penicillium sp.
FO-2030) has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology. Deposit number is FERM P-1
The contract date is December 6, 1993.

The FO-2030 substance-producing bacterium has been described above. However, as a general property of the bacterium, the bacteriological properties are extremely variable and are not constant. It is a well-known fact that mutation is carried out by an artificial mutagenesis method using radiation or a mutant derivative such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc. Of course, all strains including the natural mutant strains that belong to the genus Penicillium and have the ability to produce the FO-2030 substance can be used in the present invention. In addition, strains mutated by cell engineering such as cell fusion and gene manipulation are also included as FO-2030 substance-producing bacteria.

In the present invention, first, a production bacterium having an ability to produce the FO-2030 substance belonging to the genus Penicillium is cultured in a medium. In culturing the bacterium, a fungal culture method is generally used. As the medium, a synthetic medium or a natural medium that appropriately contains a carbon source that can be assimilated by microorganisms, a nitrogen source that can be assimilated, and an inorganic substance and, if necessary, other nutrients can be used. The carbon source and nitrogen source used in the medium may be any type as long as the strain used can be used.

Examples of assimilable carbon sources are glucose, sucrose, glycerol, fructose,
Various carbohydrates such as maltose, mannitol, xylose, galactose, ribose, dextrin, sugar condensate, starch or hydrolysates thereof can be used. Usually, its concentration is preferably 0.1 to 5% with respect to the medium. Further, various organic acids such as gluconic acid, pyruvic acid, lactic acid and acetic acid, various amino acids such as glycine, glutamic acid and alanic acid, alcohols such as methanol and ethanol, non-aromatic hydrocarbons such as normal paraffin, or plants. Alternatively, various animal fats and oils can be used.

Examples of the assimilable nitrogen source include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, and the like. Nitrogen-containing organic substances such as dry yeast, corn steep liquor, cottonseed flour, peanut flour, soybean flour or its digest, casein or its hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine are used. It is possible.

As the inorganic substance, for example, various phosphates, sulfates, salt, calcium carbonate, magnesium sulfate and the like can be used. Further, if necessary, trace amounts of heavy metal salts, animals as defoaming agents, plants, mineral oils and the like can be added. Further, when using a mutant strain showing auxotrophy,
Naturally, a substance satisfying the auxotrophy must be added to the medium, but this kind of nutrient may not be particularly required when using a medium containing a natural product.

The culture 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 at a pH of around 6.5. The culturing temperature may be, for example, 20 to 37 ° C, but it is usually preferable to keep it at 26 to 32 ° C (preferably around 27 ° C). When the culture time is liquid, the FO-2030 substance of the present invention is usually accumulated when the culture is performed for 2 to 5 days. Therefore, the culture may be terminated when the accumulated amount during 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 FO-2 of the present invention accumulated in the culture thus obtained
Since the 030 substance is contained in the cells and the culture filtrate, the culture is centrifuged to separate the culture filtrate and the cells.
It is advantageous to collect the FO-2030 material of the invention from each.

In order to collect the FO-2030 substance from the culture filtrate, the means generally used for collecting metabolites from the culture of microorganisms can be used alone or in any combination or repeatedly. That is, for example, extraction filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, utilizing the difference in solubility in various solvents such as precipitation, crystallization, recrystallization, phase transfer, countercurrent distribution method, chromatography. Etc. are used.

The FO-2030 substance can be collected from the culture broth by collecting it from the culture filtrate from which the bacterial cells have been removed. For example, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate or benzene, or the culture filtrate or cell extract is adsorbed on activated carbon, alumina, porous synthetic polymer resin, ion exchange resin, etc. Then, the extract is 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 purification of a fat-soluble substance, for example, column chromatography using a carrier such as silica gel, alumina or reverse phase chromatography using an ODS carrier. It can be purified. Further, the FO of the present invention
As the pharmaceutically acceptable salt of −2030 substance, for example, sodium salt, potassium salt, ammonium salt and the like can be produced by a conventional method.

[0026]

The anticoccidial activity of the FO-2030 substance of the present invention on chickens will be described below. Chicken coccidiosis growth inhibitory activity: Using chicken coccidium, Eimeria tenella oocysts resistant to monensin,
Hamster kidney-derived cells (BHK-21 cells) that are the host
The growth inhibitory activity on the above was determined by the method of Tabata et al. (Journal of Antibiotex, 46, 749-755 (19
93)). The measurement results are shown in Table 3.

[0027]

[Table 3]

The concentration of the FO-2030 substance of the present invention on the growth inhibitory effect on chicken coccidium was 4.0 μg / ml. Cytotoxicity was observed at a concentration of 40 μg / ml of the FO-2030 substance, but this substance is a cell at a concentration showing a growth inhibitory activity of Eimeria tenella on hamster kidney-derived cells (BHK-21 cells) as a host. No toxicity was observed. On the other hand, the same growth inhibition experiment was conducted on monensin, which is a known chicken coccidial growth inhibitory activity. As a result, monensin showed no coccidial growth inhibitory activity, and cytotoxicity to host cells was observed at 0.02 μg / ml administration.

As described above, the FO-2030 substance of the present invention has the activity of inhibiting the growth of the coccidial parasite that is resistant to the polyether antibiotic monensin, and therefore can lead to the amelioration of coccidiosis. It can also be used as a concomitant drug that remarkably prolongs the effect of a known anticoccidial drug.

[0030]

EXAMPLES The present invention will be described in detail below, but the present invention is by no means limited thereto. Example 1 In a 500 ml Erlenmeyer flask, glucose 2.0%, polypeptone 0.5%, yeast extract (manufactured by Oriental Yeast Co., Ltd.) 0.2%, magnesium sulfate heptahydrate 0.0
Dispense 100 ml of a liquid medium (pH 6.0) consisting of 5%, potassium dihydrogen phosphate 0.1% and agar 0.1%,
Sterilized by autoclaving at 121 ° C for 15 minutes and then grown on an agar slant medium. Penicillium SP (Penici)
Illium sp. ) FO-2030 strain (FERM P
Aseptically inoculate the fungus body of (14000) with a platinum loop, and
The seed culture was obtained by culturing at 7 ° C. for 3 days.

Next, in a 30 L jar culture tank, soluble starch 3%, glycerol 1%, soybean powder 2%, dry yeast (fermipan, sold by Asahi Kasei Kogyo Co., Ltd.) 0.3
%, Potassium chloride 0.3%, potassium dihydrogen phosphate 0.
20 L of a liquid medium (pH 6.5) containing 5%, magnesium sulfate heptahydrate 0.05% and calcium carbonate 0.2% was charged, and steam sterilized at 121 ° C. for 20 minutes. 200 ml of the above seed culture solution was inoculated into this, and the stirring speed was 250 rp.
The culture was carried out at 27 ° C. for 2 days at a flow rate of 10 L / min.

18 L of ethyl acetate was added to 18 L of this culture solution, stirred well, and then centrifuged to separate the ethyl acetate layer. The ethyl acetate extract was concentrated under reduced pressure to dryness to obtain 23.4 g of a crude extract. The crude extract was washed with 6 L of silica gel (6 L of chloroform and then the active substance was eluted with 6 L of chloroform: methanol (99: 1) to obtain 2.6 g of a crude product. Then, the crude product was dissolved in methanol and sonicated well. The insoluble matter was removed by centrifugation to obtain 646 mg of a crudely purified product, and reverse phase (ODS) high performance liquid chromatography (developing solvent: 0.05%).
3.9 mg of FO-2030 substance was obtained by phosphoric acid-containing 40% acetonitrile to 100% acetonitrile).

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of a FO-2030 substance.

FIG. 2 is an infrared absorption spectrum of a FO-2030 substance.

FIG. 3 is a proton nuclear magnetic resonance spectrum of a FO-2030 substance.

FIG. 4 is a ¹³ C nuclear magnetic resonance spectrum of a FO-2030 substance.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:80) (72) Inventor Yuzuru Iwai 5-9-1 Shirokane, Minato-ku, Tokyo Kitasato Institute (72) Inventor, Katsuji Haneda 5-9-1 Shirokane, Minato-ku, Tokyo Kitasato Institute

Claims (5)

[Claims] 1. FO-20 having the following physicochemical properties:
30 substances or pharmaceutically acceptable salts thereof. (1) Elemental analysis: C72.01%, H8.82% (2 ) Molecular formula: C ₁₅ H ₂₂ O ₃ (by high resolution mass spectrum) (3) Molecular weight: 250 (4) Melting point: decomposed at 300 ° C. or higher (5) Specific optical rotation: [α] _D ²⁸ + 4.4 ° (C = 0.1,
(In methanol) (6) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG.
It shows a characteristic absorption maximum around 3,228 nm. (7) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr method is as shown in FIG.
2902, 2891, 1700, 1674, 1473,
It has absorption bands near 1380, 1277, 1075, and 1010 cm ^-1 (8) Color reaction: Color is developed with 50% H ₂ SO ₄ . Ninhydrin negative (9) Solubility in solvents: methanol, ethanol,
Acetic acid ethyl ester, soluble in chloroform, insoluble in water (10) Basic, acidic, neutral: neutral (11) substance property: colorless powder (12) 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. 2. FO-2030 belonging to the genus Penicillium
A FO characterized by culturing a microorganism having the ability to produce a substance in a medium to accumulate the FO-2030 substance in the culture, and collecting the FO-2030 substance from the culture.
-A method for producing 2030 substances. 3. FO-2030 belonging to the genus Penicillium
Penicillium is a microorganism that has the ability to produce substances.
SP FO-2030 (FERM P-1400
0) The production method according to claim 2. 4. A microorganism belonging to the genus Penicillium and capable of producing a FO-2030 substance. 5. The microorganism is Penicillium sp. FO.
The microorganism according to claim 4, which is -2030 (FERM P-14000).