JP3592740B2 - FO-2030 substance and method for producing the same - Google Patents

Description

【００１０】
本発明で使用される上記のＦＯ−２０３０物質を生産する微生物（以下、ＦＯ−２０３０物質生産菌と称する）は、ペニシリウム属に属するが、その中で例えば本発明者らが分離したペニシリウム属に属するペニシリウム エスピー（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）ＦＯ−２０３０菌株は、本発明の最も有効に使用される菌株の一例であって、本菌株の菌学的性状を示すと次の通りである。
【００１１】
Ｉ．形態的性質
本菌株はバレイショ・ブドウ糖寒天培地、ＹｐＳｓ寒天培地、麦芽汁寒天培地等で比較的良好に生育し、分生子の着生は中程度である。バレイショ・ブドウ糖寒天培地に生育したコロニーを顕微鏡で観察すると菌糸は透明で隔壁を有しており、分生子柄は基底菌糸より直生している。ペニシラスは単輪生で、分生子柄はまれに分岐し、分生子柄が隔壁を有し長くなるものと、短いものが混在する。梗子はペン先型〜とっくり型で２〜４個群生し、大きさは７．５〜１０．０×２〜３μｍである。はじめはフィアロ型分生子が、梗子の頂端に一個着生し、培養時間の経過と共に連鎖状となり、最終的にはこの連鎖は１００μｍ前後に達する。分生子は球形〜亜球形で、大きさは２．５〜３．０μｍであり、その表面は平滑である。
【００１２】
ＩＩ．各種培地上での諸性状
各種培地上で２５℃、１４日間培養した場合の肉眼的に観察した結果を表２に示した。各培地において、菌の生育に伴う分泌液および菌核の形成は観察されなかった。
【００１３】
【表２】

【００１４】
ＩＩＩ．生理的性状
１）最適生育条件
本菌株の最適生育条件はＹｐＳｓ培地においてｐＨ４〜８であり、生育温度は１５〜３０℃である。
２）生育の範囲
本菌株の生育範囲はＹｐＳｓ培地においてｐＨ２〜１０、生育温度は８〜３７℃である。
３）好気性、嫌気性の区別：好気性
【００１５】
以上の形態的特徴、培養性状および生理的性状に基づき、既知菌種との比較を試みた結果、本菌株はペニシリウム（Ｐｅｎｉｃｉｌｌｉｕｍ）属に属する菌種であると考えられ、ペニシリウム エスピー・ＦＯ−２０３０と命名した。本菌株はペニシリウム エスピー・ＦＯ−２０３０（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ・ＦＯ−２０３０）として、工業技術院生命工学工業技術研究所に寄託されている。受託番号はＦＥＲＭ Ｐ−１４０００であり、受託日は平成５年１２月６日である。
【００１６】
以上、ＦＯ−２０３０物質生産菌について説明したが、菌の一般的性状として菌学上の性状はきわめて変異し易く、一定したものではなく、自然的にあるいは通常行われる紫外線照射またはＸ線照射または変異誘導体、例えばＮ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニジン、エチルメタンスルホネートなどを用いる人工的変異手段により変異することは周知の事実であり、このような人工的変異株は勿論、自然変異株も含め、ペニシリウム属に属し、ＦＯ−２０３０物質を生産する能力を有する菌株はすべて本発明に使用することができる。又、細胞融合、遺伝子操作などの細胞工学的に変異させた菌株もＦＯ−２０３０物質生産菌として包含される。
【００１７】
本発明においては、先ずペニシリウム属に属するＦＯ−２０３０物質を生産する能力を有する生産菌が培地に培養される。本菌の培養においては、通常真菌類の培養法が一般に用いられる。培地としては、微生物が同化し得る炭素源、資化し得る窒素源および無機物、更に必要に応じてその他の栄養物をほどよく含有する合成培地または天然培地を使用することができる。培地に使用される炭素源および窒素源としては、使用菌株の利用可能なものならばいずれの種類でもよい。
【００１８】
同化し得る炭素源としては、例えばグルコース、シュークロース、グリセロール、フラクトース、マルトース、マンニトール、キシロース、ガラクトース、リボース、デキストリン、糖密、澱粉またはその加水分解物等の種々の炭水化物が利用できる。その濃度は通常、培地に対して０．１〜５％が好ましい。又、グルコン酸、ピルビン酸、乳酸、酢酸等の各種有機酸、グリシン、グルタミン酸、アラニン酸等の各種アミノ酸、さらにはメタノール、エタノール等のアルコール類やノルマルパラフィン等の非芳香属炭化水素、あるいは植物もしくは動物性の各種油脂等も使用可能である。
【００１９】
資化し得る窒素源としては、例えばアンモニア、塩化アンモニウム、リン酸アンモニウム、硫酸アンモニウム、硝酸アンモニウム等の各種無機酸あるいは有機酸のアンモニウム塩類、尿素、ペプトン、ＮＺ−アミン、肉エキス、酵母エキス、乾燥酵母、コーン・ステイープ・リカー、綿実粉、落花生粉、大豆粉あるいはその消化物、カゼインあるいはその水分解物などの含窒素有機物質、さらには、グリシン、グルタミン酸、アラニン等の各種アミノ酸が使用可能である。
【００２０】
無機物としては、例えば各種リン酸塩、硫酸塩、食塩、炭酸カルシウム、硫酸マグネシウム等を使用できる。さらには必要に応じて微量の重金属塩、消泡剤としての動物、植物、鉱物油等を添加することもできる。また、栄養要求性を示す変異株を用いる場合には、当然その栄養要求性を満足させる物質を培地に加えなければならないが、この種の栄養素は、天然物を含む培地を使用する場合は、とくに添加を必要としない場合がある。
【００２１】
培養は通常振とうまたは通気攪拌培養などの好気的条件下で行うのがよい。工業的には深部通気攪拌培養が好ましい。培養のｐＨはたとえば５．０〜８．０であるが、ｐＨ６．５付近で培養を行うのが好ましい。培養温度は例えば２０〜３７℃で行い得るが、通常は２６〜３２℃（好ましくは２７℃付近）に保つのがよい。培養時間は液体の場合、通常２〜５日間培養を行うと、本発明のＦＯ−２０３０物質が蓄積されるので、培養中の蓄積量が最大に達した時に、培養を終了すればよい。
【００２２】
これらの培地組成、培地の液性、培養温度、攪拌速度、通気量などの培養条件は使用する菌株の種類や外部の条件などに応じて好ましい結果が得られるように適宜調節、選択されることはいうまでもない。液体培養において、発泡があるときは、シリコン油、植物油、界面活性剤などの消泡剤を適宜使用できる。このようにして得られた培養物に蓄積される本発明のＦＯ−２０３０物質は菌体内および培養濾液中に含有されるので、培養物を遠心分離して培養濾液と菌体とに分離し、各々から本発明のＦＯ−２０３０物質を採取するのが有利である。
【００２３】
培養濾液からＦＯ−２０３０物質を採取するには、通常の微生物の培養物から代謝物を採取するのに用いられる手段を単独あるいは任意の順序に組み合わせ、または反復して用いられる。すなわち、例えば抽出濾過、遠心分離、透析、濃縮、乾燥、凍結、吸着、脱着、各種溶媒に対する溶解度の差を利用する例えば沈澱、結晶化、再結晶、転溶、向流分配法、クロマトグラフイー等の手段が用いられる。
【００２４】
培養液からＦＯ−２０３０物質を採取するには、菌体を除去した培養濾液から採取すればよい。例えば、培養濾液を酢酸エチル、酢酸ブチル、ベンゼンなどの非親水性有機溶媒で抽出するか、あるいは培養濾液あるいは菌体抽出液を活性炭、アルミナ、多孔性合成高分子樹脂、イオン交換樹脂等に吸着させ、メタノール等の溶出溶媒で溶出し、得られた抽出液を減圧濃縮後、酢酸エチル等の有機溶媒で抽出すればよい。
【００２５】
得られた粗物質は、さらに脂溶性物質の精製において通常用いられている公知の方法、例えばシリカゲル、アルミナ等の担体を用いるカラムクロマトグラフイーあるいはＯＤＳ担体を用いる逆相クロマトグラフイーにより精製することができる。また、本発明のＦＯ−２０３０物質の薬学的に許容し得る塩としては、例えばナトリウム塩、カリウム塩、アンモニウム塩等を常法により製造することができる。
【００２６】
【発明の効果】
次に、本発明のＦＯ−２０３０物質の鶏に対する抗コクシジウム活性について述べる。
鶏コクシジウム生育阻害活性：
モネンシンに耐性の鶏コクシジウム、エイメリア・テネラのオーシストを用い、宿主であるハムスター腎由来細胞（ＢＨＫ−２１細胞）上での生育阻害活性を田畠らの方法（ジャーナル・オブ・アンチバイオテックス、４６、７４９−７５５（１９９３））に準じて測定した。その測定結果を表３に示した。
【００２７】
【表３】

【００２８】
本発明のＦＯ−２０３０物質の鶏コクシジウムに対する生育阻害作用濃度は、４．０μｇ／ｍｌであった。なお、ＦＯ−２０３０物質４０μｇ／ｍｌ濃度で細胞毒性が認められたが、本物質は宿主であるハムスター腎由来細胞（ＢＨＫ−２１細胞）に対してエイメリア・テネラの生育阻害活性を示す濃度において細胞毒性は認められなかった。一方、既知鶏コクシジウム生育阻害活性モネンシンについても同様の生育阻害実験を行った。その結果、モネンシンでは、コクシジウム生育阻害活性は認められず、０．０２μｇ／ｍｌ投与で宿主細胞に対する細胞毒性が認められた。
【００２９】
以上のように、本発明のＦＯ−２０３０物質は、ポリエーテル系抗生物質モネンシンに耐性のコクシジウム原虫の生育阻害活性を有することから、コクシジウム病の寛解に導くことが可能となる。また、公知の抗コクシジウム剤の薬物の効果を著しく持続せしめる併用剤として用いることもできる。
【００３０】
【実施例】
以下に本発明を具体的に説明するが、本発明はけっしてこれのみに限定されるものではない。
実施例１
５００ｍｌ容三角フラスコに、グルコース２．０％、ポリペプトン０．５％、酵母エキス（オリエンタル酵母工業（株）製）０．２％、硫酸マグネシウム７水塩０．０５％、リン酸２水素カリウム０．１％、寒天０．１％からなる液体培地（ｐＨ６．０）を１００ｍｌを分注し、１２１℃で１５分間高圧蒸気滅菌し、これに寒天斜面培地に生育させたペニシリウム エスピー（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）ＦＯ−２０３０株（ＦＥＲＭ Ｐ−１４０００）の菌体を白金耳にて無菌的に接種し、２７℃、３日間培養して種培養液を得た。
【００３１】
次いで３０Ｌ容ジャー培養槽に可溶性澱粉３％、グリセロール１％、大豆粉２％、ドライイースト（ｆｅｒｍｉｐａｎ、旭化成工業（株）販売）０．３％、塩化カリウム０．３％、リン酸２水素カリウム０．５％、硫酸マグネシウム７水塩０．０５％、炭酸カルシウム０．２％を含む液体培地（ｐＨ６．５）を２０Ｌを仕込み、１２１℃で２０分間蒸気滅菌した。これに上記の種培養液２００ｍｌを接種し、攪拌速度２５０ｒｐｍ、通気量１０Ｌ／分で２７℃で２日間培養した。
【００３２】
この培養液１８Ｌに１８Ｌの酢酸エチルを加えてよく攪拌した後、遠心分離して酢酸エチル層を分離した。この酢酸エチル抽出液を減圧濃縮乾固して粗抽出物２３．４ｇを得た。この粗抽出物をシリカゲルクロマトグラフイー（クロロホルム６Ｌで洗浄後クロロホルム：メタノール（９９：１）６Ｌで活性物質を溶出させ粗精製物２．６ｇを得た。次いでメタノールに溶解させ、よくソニック後、遠心分離の操作で不溶物を除去し、粗精製物６４６ｍｇを得た。更に逆相（ＯＤＳ）系の高速液体クロマトグラフイー（展開溶媒：０．０５％リン酸含有４０％アセトニトリル〜１００％アセトニトリル）により、ＦＯ−２０３０物質３．９ｍｇを得た。
【図面の簡単な説明】
【図１】ＦＯ−２０３０物質の紫外部吸収スペクトルである。
【図２】ＦＯ−２０３０物質の赤外部吸収スペクトルである。
【図３】ＦＯ−２０３０物質のプロトン核磁気共鳴スペクトルである。
【図４】ＦＯ−２０３０物質の^１３Ｃ核磁気共鳴スペクトルである。[0001]
[Industrial applications]
The present invention relates to a FO-2030 substance useful as a therapeutic or preventive agent for coccidiosis, which is an important disease in the poultry industry, and a method for producing the same.
[0002]
[Prior art]
Conventionally, as anticoccidial agents, sulfa agents, quinoline agents, antithiamine agents, antibiotics and the like have been put to practical use, and recently, polyether antibiotics such as monensin, salinomycin, lasalocid and the like have been widely used.
[0003]
[Problems to be solved by the invention]
However, coccidia resistant to these drugs have appeared and their effects have been weakened, and anticoccidium agents replacing them have been demanded.
Accordingly, an object of the present invention is to provide a drug effective against not only drug-resistant coccidium but also coccidium which has acquired resistance to a polyether compound in view of the above viewpoints. .
[0004]
[Means for Solving the Problems]
In general, drug cross-resistance is established when the structures of the acting substances are similar or the mechanisms of action are similar, and compounds having a different structural or action mechanism from conventional anticoccidial agents are used for the treatment of coccidiosis. It is useful as an agent or prophylactic agent. The present inventors have continued various studies in order to find a new drug effective against coccidia protozoa that has acquired resistance to conventional drugs, from the natural world. As a result of searching from among the products, it was found that a substance effective for coccidium was produced in a culture solution of the FO-2030 strain newly isolated from soil.
[0005]
Next, as a result of separating and purifying the anticoccidial active substance from the culture, it was found that the FO-2030 substance having the following physicochemical properties was a substance which was not known at all. The present invention has been completed based on such findings, and provides a FO-2030 substance having the following physicochemical properties or a pharmaceutically acceptable salt thereof. Further, the present invention provides a method for culturing a microorganism belonging to the genus Penicillium and having the ability to produce a FO-2030 substance in a culture medium to accumulate the FO-2030 substance in a culture, and collecting the FO-2030 substance from the culture. And a method for producing a FO-2030 substance or a pharmaceutically acceptable salt thereof.
[0006]
The physicochemical properties of the FO-2030 substance of the present invention are as follows.
(1) Elemental analysis value: C 72.01%, H 8.82%
(2) Molecular formula: C ₁₅ H ₂₂ O ₃ (by high-resolution mass spectrum)
(3) Molecular weight: 250 (measured values by high-resolution EI mass spectrum are as follows)
Calculated value: 250.1570
Obtained value: 250.1572
(4) Melting point: decomposed at 300 ° C. or higher
(5) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1 and shows a characteristic absorption maximum around 203 and 228 nm. (6) Infrared absorption spectrum: measured by KBr method. The infrared absorption spectrum obtained is as shown in FIG. 2, and has absorption bands around 3412, 2902, 2891, 1700, 1674, 1473, 1380, 1277, 1075, and 1010 cm ^−1. (7) Color reaction: 50% Color develops with H ₂ SO ₄ . Negative for ninhydrin (8) Solubility in solvent: soluble in methanol, ethanol, ethyl acetate, chloroform, insoluble in water (9) Basic, acidic, neutral distinction: neutral
(10) Material properties: colorless powder (11) Nuclear magnetic resonance spectrum: ¹ H-NMR spectrum and ¹³ C-NMR spectrum measured in a heavy chloroform solution using a Varian XL-400, 400 MHz, NMR spectrometer, respectively This is as shown in FIGS. The chemical shifts of hydrogen and carbon are as shown in Table 1 below.
[0009]
[Table 1]

[0010]
Microorganisms that produce the above-mentioned FO-2030 substance used in the present invention (hereinafter referred to as FO-2030 substance-producing bacteria) belong to the genus Penicillium. Among them, for example, those belonging to the genus Penicillium isolated by the present inventors. The Penicillium sp. FO-2030 strain to which the present invention belongs is one of the most effectively used strains of the present invention, and the bacterial properties of the strain are as follows.
[0011]
I. Morphological properties The strain grows relatively well on potato-glucose agar medium, YpSs agar medium, wort agar medium, and the like, and conidium formation is moderate. When the colonies grown on the potato-glucose agar medium are observed under a microscope, the hyphae are transparent and have septum, and the conidiophores grow directly from the basal hyphae. Penicillus is monocyclic, and the conidiophores rarely diverge, and the conidiophores have a partition and are long and short. Strokes are pen nib type to sharp type and grow in groups of 2 to 4 and are 7.5 to 10.0 × 2 to 3 μm in size. Initially, a single phialoconidium attaches to the apex of the infarct and becomes a chain as the culture time elapses, and finally this chain reaches about 100 μm. Conidia are spherical to subspherical in size, 2.5 to 3.0 μm in size, and their surfaces are smooth.
[0012]
II. Various properties on various media Table 2 shows the results of macroscopic observation when cultured on various media at 25 ° C for 14 days. In each medium, the formation of secretions and sclerotium was not observed with the growth of the bacteria.
[0013]
[Table 2]

[0014]
III. Physiological properties 1) Optimum growth conditions The optimum growth conditions of this strain are pH 4-8 in YpSs medium, and the growth temperature is 15-30 ° C.
2) Range of growth The growth range of this strain is 2 to 10 in the YpSs medium, and the growth temperature is 8 to 37 ° C.
3) distinction between aerobic and anaerobic: aerobic
Based on the above morphological characteristics, culture characteristics and physiological characteristics, an attempt was made to compare with known bacterial species. As a result, this strain is considered to be a bacterial species belonging to the genus Penicillium, and Penicillium sp. FO-2030 is considered. It was named. This strain has been deposited as Penicillium sp. FO-2030 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. The deposit number is FERM P-14000 and the deposit date is December 6, 1993.
[0016]
As described above, the FO-2030 substance-producing bacterium has been described. However, as a general property of the bacterium, the mycological properties are extremely susceptible to variation, are not constant, and are naturally or normally performed by ultraviolet irradiation or X-ray irradiation or It is a well-known fact that mutation is performed by an artificial mutation means using a mutant derivative, for example, N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc. All strains belonging to the genus Penicillium and having the ability to produce FO-2030 substances, including mutant strains, can be used in the present invention. Strains mutated by cell engineering such as cell fusion and genetic manipulation are also included as FO-2030 substance producing bacteria.
[0017]
In the present invention, first, a production bacterium capable of producing a FO-2030 substance belonging to the genus Penicillium is cultured in a medium. In culturing the fungus, a fungal culture method is generally used. As the medium, a synthetic medium or a natural medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source and inorganic substances that can be assimilated, and, if necessary, other nutrients can be used. As the carbon source and the nitrogen source used in the medium, any type can be used as long as the strain to be used can be used.
[0018]
As the assimilable carbon source, various carbohydrates such as glucose, sucrose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, molasses, starch or a hydrolyzate thereof can be used. Usually, the concentration is preferably 0.1 to 5% based on the medium. Also, 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; Alternatively, various animal fats and oils can be used.
[0019]
Examples of assimilable nitrogen sources include, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium salts of various inorganic or organic acids such as ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, Nitrogen-containing organic substances such as 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 can be used. .
[0020]
As the inorganic substance, for example, various phosphates, sulfates, salt, calcium carbonate, magnesium sulfate and the like can be used. Furthermore, if necessary, trace amounts of heavy metal salts, animals, plants, mineral oils and the like as antifoaming agents can be added. Also, when using a mutant strain that exhibits auxotrophy, a substance that satisfies the auxotrophy must of course be added to the medium, but this type of nutrient is used when a medium containing natural products is used. In some cases, no addition is required.
[0021]
Culture is preferably performed under aerobic conditions such as shaking or aeration and stirring culture. Industrially, deep aeration stirring culture is preferred. The pH of the culture is, for example, 5.0 to 8.0, but it is preferable to perform the culture at around pH 6.5. The cultivation temperature can be, for example, 20 to 37 ° C, but it is usually good 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 normally accumulated when culturing is performed for 2 to 5 days. Therefore, the cultivation may be terminated when the accumulated amount during the cultivation reaches the maximum.
[0022]
The culture conditions, such as the culture medium composition, the liquid properties of the culture medium, the culture temperature, the stirring speed, and the aeration rate, are appropriately adjusted and selected so as to obtain preferable results according to the type of the strain used, external conditions, and the like. Needless to say. If there is foaming in the liquid culture, an antifoaming agent such as silicone oil, vegetable oil, or a surfactant can be used as appropriate. Since the FO-2030 substance of the present invention accumulated in the culture thus obtained is contained in the cells and in the culture filtrate, the culture is centrifuged to separate the culture filtrate and the cells, It is advantageous to collect the FO-2030 substance of the invention from each.
[0023]
In order to collect the FO-2030 substance from the culture filtrate, the means used for collecting metabolites from the culture of ordinary microorganisms may be used alone, combined in any order, or used repeatedly. That is, for example, extraction filtration, centrifugation, dialysis, concentration, drying, freezing, adsorption, desorption, utilizing differences in solubility in various solvents, such as precipitation, crystallization, recrystallization, phase transfer, countercurrent distribution, and chromatography. Is used.
[0024]
To collect the FO-2030 substance from the culture solution, the FO-2030 substance may be collected from the culture filtrate from which the cells have been removed. For example, the culture filtrate is extracted with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, benzene, etc., or the culture filtrate or cell extract is adsorbed on activated carbon, alumina, porous synthetic polymer resin, ion exchange resin, etc. Then, the mixture is eluted with an elution solvent such as methanol, and the obtained extract is concentrated under reduced pressure and then extracted with an organic solvent such as ethyl acetate.
[0025]
The obtained crude substance is further purified by a known method generally used in the purification of fat-soluble substances, for example, column chromatography using a carrier such as silica gel or alumina or reverse phase chromatography using an ODS carrier. Can be. In addition, as a pharmaceutically acceptable salt of the FO-2030 substance of the present invention, for example, a sodium salt, a potassium salt, an ammonium salt and the like can be produced by a conventional method.
[0026]
【The invention's effect】
Next, the anticoccidial activity of the FO-2030 substance of the present invention on chickens will be described.
Chicken coccidium growth inhibitory activity:
Using oocysts of chicken coccidium and Eimeria tenella resistant to monensin, the growth inhibitory activity on the host hamster kidney-derived cells (BHK-21 cells) was determined by the method of Tabata et al. (Journal of Antibiotics, 46, 749-755 (1993)). Table 3 shows the measurement results.
[0027]
[Table 3]

[0028]
The growth inhibitory concentration of the FO-2030 substance of the present invention against chicken coccidium was 4.0 μg / ml. Although cytotoxicity was observed at a concentration of 40 μg / ml of the FO-2030 substance, the substance was found to be cytotoxic at a concentration showing the growth-inhibiting activity of Eimeria tenella on hamster kidney-derived cells (BHK-21 cells) as a host. No toxicity was observed. On the other hand, a similar growth inhibition experiment was carried out for known chicken coccidial growth inhibitory activity monensin. As a result, monensin did not show coccidial growth inhibitory activity, and showed cytotoxicity to host cells when administered at 0.02 μg / ml.
[0029]
As described above, the FO-2030 substance of the present invention has an activity of inhibiting the growth of coccidial protozoa resistant to the polyether antibiotic monensin, and thus can lead to the remission of coccidial disease. In addition, it can be used as a concomitant drug that significantly prolongs the effect of a known anticoccidial drug.
[0030]
【Example】
Hereinafter, the present invention will be described in detail, but the present invention is not 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 0.05%, potassium dihydrogen phosphate 0 100 ml of a liquid medium (pH 6.0) consisting of 0.1% and 0.1% agar, sterilized by high-pressure steam at 121 ° C. for 15 minutes, and penicillium sp. (Penicillium sp.) Grown on an agar slant medium. ) Cells of the FO-2030 strain (FERM P-14000) were aseptically inoculated with a platinum loop and cultured at 27 ° C for 3 days to obtain a seed culture.
[0031]
Then, soluble starch 3%, glycerol 1%, soybean flour 2%, dry yeast (fermipan, sold by Asahi Kasei Kogyo Co., Ltd.) 0.3%, potassium chloride 0.3%, potassium dihydrogen phosphate are placed in a 30 L jar culture tank. 20 L of a liquid medium (pH 6.5) containing 0.5%, magnesium sulfate heptahydrate 0.05%, and calcium carbonate 0.2% was charged, and steam-sterilized at 121 ° C. for 20 minutes. This was inoculated with 200 ml of the above seed culture solution, and cultured at 27 ° C. for 2 days at a stirring speed of 250 rpm and an aeration rate of 10 L / min.
[0032]
After 18 L of ethyl acetate was added to 18 L of the culture and stirred well, the mixture was centrifuged to separate an 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 chloroform and eluted with 6 L of chloroform: methanol (99: 1) to elute the active substance to obtain 2.6 g of a crude product. The insoluble matter was removed by centrifugation to obtain 646 mg of a crude product, which was further subjected to high-performance liquid chromatography of an inverse phase (ODS) system (developing solvent: 40% acetonitrile containing 0.05% phosphoric acid to 100% acetonitrile). ), 3.9 mg of FO-2030 substance was obtained.
[Brief description of the 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 the FO-2030 substance.

Claims (4)

FO-2030 substance having the following physicochemical properties or a pharmaceutically acceptable salt thereof.
(1) Elemental analysis value: C 72.01%, H 8.82%
(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 rotation: [α] _D ²⁸ + 4.4 ° (C = 0.1 in methanol)
(6) Ultraviolet absorption spectrum: ultraviolet absorption spectrum measured in methanol exhibits a characteristic absorption maximum around 203,228Nm (7) Infrared absorption spectrum: The infrared absorption spectrum measured by KBr method, 3412, 2902, 2891, 1700, 1674, 1473, 1380, 1277, 1075, 1010 having an absorption band in the vicinity of 1010 cm ^-1. (8) Color reaction: Color is developed with 50% H ₂ SO ₄ . Negative to ninhydrin (9) Solubility in solvent: soluble in methanol, ethanol, ethyl acetate, chloroform, insoluble in water (10) Basic, acidic, neutral: neutral (11) Material: colorless powder 2. A microorganism having the ability to produce the FO-2030 substance according to claim 1, which belongs to the genus Penicillium, is cultured in a medium, the FO-2030 substance is accumulated in the culture, and the FO-2030 substance is collected from the culture. A method for producing a FO-2030 substance. 3. The method according to claim 2, wherein the microorganism capable of producing the FO-2030 substance according to claim 1 belonging to the genus Penicillium is Penicillium SP FO-2030 (FERM P-14000). Microorganism having an ability to produce FO-2030 substance according to claim 1 belonging to the Penicillium genus, Bae Nishiriumu sp · FO-2030 (FERM P- 14000).

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