JP4105805B2 - Novel antifungal active substances PF1163A and PF1163B substances, processes for producing them, and antifungal agents containing them as active ingredients - Google Patents
Novel antifungal active substances PF1163A and PF1163B substances, processes for producing them, and antifungal agents containing them as active ingredients Download PDFInfo
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【０００１】
【発明の属する技術分野】
本発明は新規抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質、それらの製造法ならびにそれらを有効成分とする抗真菌剤に関するものである。
【０００２】
【従来の技術】
１９５０年代以降の抗生物質に関する研究開発の急速な進歩およびその広範な普及により、細菌性の感染症に対する多くの治療薬が開発されてきた。その一方で、平素は無害な弱病原性微生物による感染症、いわゆる日和見感染症が近年大きな問題となりつつある。この日和見感染症は免疫不全症や悪性腫瘍等の疾病、または免疫抑制剤や抗炎症剤等の投与によって免疫機能が低下した場合、また抗生物質の投与による共生菌の抑制から生じる菌交代、などが原因とされている。このような日和見感染症の中で真菌が原因である症例が数多く報告されている。
【０００３】
【発明が解決しようとする課題】
現在使用されている主な抗真菌剤としては、ポリエンマクロライド系、アゾール系およびフルシトシン等がある。浅在性真菌症の治療には、主に外用剤が使用され、それらには多種のイミダゾール系薬剤を始め、フルシトシン、ポリエンマクロライドのナイスタチン等が用いられている。一方、深在性真菌症の治療においては、アゾール系薬剤であるフルコナゾールが、その高い安全性から多用されているが、これは抗菌スペクトルが狭いことが難点とされている。また、ポリエンマクロライド系薬剤であるアムホテリシンＢは、抗菌スペクトルが広く、有効性は高いが、毒性の問題が残されている。
【０００４】
近年、真菌症が増加傾向にある状況下で、より有効かつ安全な新規抗真菌剤の開発が望まれている。
【０００５】
【課題を解決するための手段】
本発明者らは、より有効かつ安全な新規抗真菌活性物質を見い出すべく、幅広く微生物の探索を行った結果、ペニシリウム属に属する菌を培養することによって、抗真菌活性を示す物質が培養物中に生産、蓄積されることを見い出した。
【０００６】
次いで、本発明者らは培養物中から式（Ｉ）で表される有効物質ＰＦ１１６３Ａ物質および式（ＩＩ）で表わされるＰＦ１１６３Ｂ物質を単離、精製し、その理化学的性状を明らかにした。したがって、本発明は、新規抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を提供するものである。
【０００７】
更に、本発明は、ペニシリウム属に属し、ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を生産する能力を有する微生物を培地に培養し、培養物中にＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を蓄積させ、該培養物からＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を採取することを特徴とするＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の製造法を提供するものである。
【０００８】
本発明の第１の要旨とするところは、前述式（Ｉ）で表され、下記の理化学的性状を有する新規抗真菌活性物質ＰＦ１１６３Ａ物質にある。
【０００９】
本発明の第２の要旨とするところは、式（ＩＩ）で表わされるＰＦ１１６３Ｂ物質にある。
本発明の第３の要旨とするところは、ペニシリウム属に属する抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質生産菌を培養し、その培養物から抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を採取することを特徴とする、抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の製造法にある。本発明に使用される新規抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質生産菌の一例としては、新たに分離され、工業技術院生命工学工業技術研究所に生命研菌寄第１５４７３号（ＦＥＲＭ Ｐ−１５４７３）として寄託されているペニシリウム属ＰＦ１１６３株（以下「本菌株」と略記することもある）が挙げられる。本菌株の菌学的性状は以下のとおりである。
【００１２】
３．ＰＦ１１６３株の菌学的性状
（１）コロニーの性状
ツァペック酵母エキス寒天培地上で生育良く、２５℃、７日間の培養でコロニーの直径は２５〜３０ｍｍに達する。淡茶色、ベルベット状、しわ状のコロニーとなり、分生子をまばらに形成する。裏面は茶色となる。
麦芽エキス寒天培地上で生育はやや抑制的で、２５℃、７日間の培養でコロニーの直径は２０〜２５ｍｍに達する。淡橙色、ベルベット状、平坦もしくは、わずかなしわ状のコロニーとなり、分生子はほとんど形成しない。裏面は黄土色となる。３７℃の培養では、どの培地上でも生育しない。
【００１３】
（２）形態的性状
分生子柄は、コロニー表面、もしくは気菌糸より生じ、滑面、大きさ１５０〜３００×２〜２．５μｍである。ペニシリは、複輪生となる。メトレは８〜１３×２〜２．５μｍ、３〜５本の輪生体となる。フィアライドはアンプル型、５〜８×１．８〜２μｍ、５〜８本の輪生体となる。分生子は球形〜楕円形、滑面、大きさ２〜２．５μｍ、連鎖状に生じる。
【００１４】
以上の菌学的性状より、本菌株を、ペニシリウム属に属するカビと同定した。
【００１５】
本菌株は他のカビに見られるようにその性状が変化し易い。例えば、本菌株に由来する突然変異株（自然発生または誘発性）、形質接合体または遺伝子組換え体であっても、ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を生産するものはすべて本発明に使用できる。
【００１６】
【発明の実施の形態】
１．ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質生産菌の培養法
本発明の方法では、ペニシリウム属に属するＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質生産菌を通常の微生物が利用し得る栄養物を含有する培地で培養する。栄養源としては、従来カビの培養に利用されている公知のものが使用できる。例えば、炭素源としては、グルコース、シュクロース、水飴、デキストリン、澱粉、グリセロール、糖蜜、動植物油等を使用し得る。また、窒素源としては、大豆粉、小麦胚芽、コーン・スティープ・リカー、綿実粕、肉エキス、ペプトン、酵母エキス、硫酸アンモニウム、硝酸ナトリウム、尿素等を使用し得る。その他必要に応じてナトリウム、カリウム、カルシウム、マグネシウム、コバルト、塩素、燐酸、硫酸およびその他のイオンを生成することができる無機塩類を添加することは有効である。また、菌の発育を助け、ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の生産を促進するような有機物および無機物を適当に添加することができる。
【００１７】
培養法としては、好気的条件での培養法、特に静置培養法が最も適している。培養に適当な温度は２５〜３０℃であるが、多くの場合２６℃付近で培養する。ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の生産は、培地や培養条件によって異なるが、静置培養、振とう培養、タンク培養のいずれにおいても、通常２〜１４日間でその蓄積量が最高に達する。培養物中のＰＦ１１６３Ａ物質あるいはＰＦ１１６３Ｂ物質の蓄積量が最高になった時に培養を停止し、培養物から目的物質を単離、精製する。
【００１８】
２．ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の精製法
本発明によって得られるＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質は、前記する理化学的性状を有するので、その性状に従って培養物から精製することが可能である。例えば、有機溶媒を用いて培養物よりＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を抽出した後、吸着剤を用いた吸脱着法、ゲル濾過剤を用いた分子分配法等を用いて精製することが可能である。例えば、有効成分を含む培養物を酢酸エチルにより抽出する。抽出液を減圧濃縮し、この抽出物を少量のクロロホルムに溶解し、クロロホルムで平衡化したシリカゲルカラムを用いて、クロロホルム／メタノールの溶媒でクロマトグラフィーを行う。活性物質を含む溶出液を減圧濃縮し、これをメタノールに再溶解して、メタノールで平衡化したセファデックスＬＨ−２０（ファルマシアファインケミカルズ社製）によるゲル濾過、あるいはヘキサン／酢酸エチルの溶媒で平衡化したシリカゲルカラムを用いて、ヘキサン／酢酸エチルの溶媒でクロマトグラフィー、を行うなどにより目的とするＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を得ることができる。
【００１９】
本発明に用いられるＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質を抗真菌剤として使用するには、種々の投与形態あるいは使用形態に合わせて、公知の担体および必要に応じて公知の補助剤とを組み合わせて製剤化すればよい。
【００２０】
抗真菌剤における投与形態としては、皮下注射、静脈内注射、筋肉内注射、座薬等による非経口投与あるいは錠剤、カプセル剤、散剤、顆粒剤等による経口投与の全身投与のほか、軟膏剤、ローション剤、膣座薬等の局所投与の形態を例示することができる。
【００２１】
【実施例】
以下に本発明の実施例を示すが、これは単なる一例であって本発明を限定するものではなく、ここに例示しなかった多くの変法あるいは修飾手段のすべてを包括するものである。
【００２２】
実施例１
種培地として、澱粉２．０％、グルコース１．０％、ポリペプトン０．５％、小麦胚芽０．６％、酵母エキス０．３％、大豆粕０．２％および炭酸カルシウム０．２％の組成からなる培地（殺菌前ｐＨ７．０）を用いた。
また、生産培地として、十分に水を吸収させた米に、大豆粕２．５％を添加した固形培地を用いた。
前記の種培地２０ｍｌを分注した１００ｍｌ容三角フラスコを１２０℃で１５分間殺菌し、これにＰＦ１１６３株（ＦＥＲＭ Ｐ−１５４７３）の斜面寒天培養の１白金耳を植菌後、２５℃で２日間振とう培養した。次いで、生産培地１００ｇを分注した５００ｍｌ容三角フラスコを１２０℃で１５分間殺菌し、これに上記種培養物を４ｍｌずつ植菌し、よく撹拌後、２８℃で１４日間静置培養した。
【００２３】
こうして得られた培養物７．５ｋｇを酢酸エチル１０ｌで抽出し、酢酸エチル層を減圧濃縮して約２７ｇの油状物質を得た。得られた油状物質を６０ｍｌのヘキサンで３回洗浄し、残査約１５ｇを得た。得られた残査に少量の酢酸エチルを加えて溶解させ、これに２０ｇのシリカゲルを加えた後、減圧下で酢酸エチルを留去し、残査をシリカゲルに均一に吸着させた。これをクロロホルムにて平衡化したシリカゲルカラム（シリカゲル１５０ｇ）を用いて、クロマトグラフィーを行った。溶離はメタノール濃度を順次増加させたクロロホルム／メタノール溶液（メタノール濃度が０％を２５００ｍｌ、１％を２０００ｍｌ、２％を５００ｍｌ）で行った。有効成分を含む分画を集めて減圧濃縮し、ＰＦ１１６３Ａ物質を主成分として含む乾固物を３．８ｇと、またＰＦ１１６３Ｂ物質を主成分として含む乾固物を６００ｍｇ得た。
【００２４】
ＰＦ１１６３Ａ物質を主成分として含む乾固物３．８ｇを、少量のクロロホルムに溶解させ、生じた沈澱物を除去した後、クロロホルムにて平衡化したシリカゲルカラム（シリカゲル１７０ｇ）を用いて、クロマトグラフィーを行った。溶離はメタノール濃度を順次増加させたクロロホルム／メタノール溶液（メタノール濃度が０％を７００ｍｌ、１％を１０００ｍｌ）で行った。有効成分を含む分画を集めて減圧濃縮し、ＰＦ１１６３Ａ物質を含む乾固物１．２ｇとＰＦ１１６３Ｂ物質を含む乾固物１００ｍｇを得た。
【００２５】
ＰＦ１１６３Ａ物質を含む乾固物１．２ｇを、５０％の酢酸エチルを含むヘキサンの少量に溶解させ、これを５０％の酢酸エチルを含むヘキサンにて平衡化したシリカゲルカラム（シリカゲル４０ｇ）を用いて、クロマトグラフィーを行った。溶離は酢酸エチル濃度を順次増加させたヘキサン／酢酸エチル溶液（酢酸エチル濃度が５０％を５００ｍｌ、６０％を２００ｍｌ、６７％を２４０ｍｌ）で行った。ＰＦ１１６３Ａ物質を含む分画を集め、減圧濃縮し、ＰＦ１１６３Ａ物質を、ほぼ純粋な油状物質として８３０ｍｇ得た。
【００２６】
ＰＦ１１６３Ｂ物質を主成分として含む乾固物を合わせて約７００ｍｇとし、３０％の酢酸エチルを含むヘキサンの少量に溶解させ、これを３０％の酢酸エチルを含むヘキサンにて平衡化したシリカゲルカラム（シリカゲル６５ｇ）を用いて、クロマトグラフィーを行った。溶離は酢酸エチル濃度を順次増加させたヘキサン／酢酸エチル溶液（酢酸エチル濃度が３０％を３８０ｍｌ、５０％を３８０ｍｌ、６７％を１９００ｍｌ）で行った。ＰＦ１１６３Ｂ物質を含む分画を集めて減圧濃縮し、これを少量のメタノールに溶解させ、あらかじめメタノールで充填したセファデックスＬＨ−２０（ファルマシアファインケミカルズ社製、３００ｍｌ）カラムを用いて、メタノールにて展開して、クロマトグラフィーを行った。ＰＦ１１６３Ｂ物質を含む分画を集め、減圧濃縮し、ＰＦ１１６３Ｂ物質を、ほぼ純粋な油状物質として３００ｍｇ得た。
【００２７】
試験例１
ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質の抗真菌活性を液体培養希釈法により、各種菌株に対する最小阻止濃度（ＭＩＣ）として測定した。培地としてＲＰＭＩ１６４０（日水製薬社製）を用い、３７℃で２０時間（Ｃ．ａｌｂｉｃａｎｓおよびＣ．ｐａｒａｐｓｉｌｏｓｉｓについては１５時間）培養した。その測定結果を表１に示す。
【００２８】
【表１】
【発明の効果】
本発明の新規抗真菌活性物質ＰＦ１１６３Ａ物質およびＰＦ１１６３Ｂ物質は、表１に示したように、医療上問題となっているＣ．ａｌｂｉｃａｎｓに対し抗真菌活性を有しており、これらを有効成分とする抗真菌剤は、Ｃ．ａｌｂｉｃａｎｓを起因菌とする真菌症の治療に有用である。
【図面の簡単な説明】
【図１】本発明の新規抗真菌活性物質ＰＦ１１６３Ａ物質の重クロロホルム中での４００ＭＨｚ 1Ｈ−ＮＭＲスペクトルである。
【図２】本発明の新規抗真菌活性物質ＰＦ１１６３Ａ物質の重クロロホルム中での１００ＭＨｚ 13Ｃ−ＮＭＲスペクトルである。
【図３】本発明の新規抗真菌活性物質ＰＦ１１６３Ｂ物質の重クロロホルム中での４００ＭＨｚ 1Ｈ−ＮＭＲスペクトルである。
【図４】本発明の新規抗真菌活性物質ＰＦ１１６３Ｂ物質の重クロロホルム中での１００ＭＨｚ 13Ｃ−ＮＭＲスペクトルである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel antifungal active substance PF1163A substance and PF1163B substance, a process for producing them, and an antifungal agent containing them as an active ingredient.
[0002]
[Prior art]
Due to the rapid progress in research and development of antibiotics since the 1950s and their widespread use, many therapeutic agents for bacterial infections have been developed. On the other hand, infectious diseases caused by weakly pathogenic microorganisms, so-called opportunistic infections, are becoming a major problem in recent years. This opportunistic infection is caused by diseases such as immunodeficiency and malignant tumors, or when the immune function is reduced by the administration of immunosuppressants or anti-inflammatory agents, or the bacterial turnover resulting from suppression of commensal bacteria by administration of antibiotics, etc. Has been the cause. There have been many reports of such opportunistic infections caused by fungi.
[0003]
[Problems to be solved by the invention]
The main antifungal agents currently used include polyene macrolides, azoles and flucytosine. For the treatment of superficial mycosis, external preparations are mainly used, including various imidazole drugs, flucytosine, polyene macrolide nystatin and the like. On the other hand, in the treatment of deep mycosis, fluconazole, which is an azole drug, is frequently used due to its high safety, but this has a difficulty in having a narrow antibacterial spectrum. In addition, amphotericin B, which is a polyene macrolide, has a wide antibacterial spectrum and high effectiveness, but a problem of toxicity remains.
[0004]
In recent years, development of new antifungal agents that are more effective and safer under the circumstances where mycosis is increasing has been desired.
[0005]
[Means for Solving the Problems]
The present inventors have extensively searched for microorganisms in order to find a more effective and safe novel antifungal active substance. As a result, by culturing a bacterium belonging to the genus Penicillium, a substance exhibiting antifungal activity is found in the culture. Found to be produced and accumulated.
[0006]
Next, the present inventors isolated and purified the active substance PF1163A substance represented by the formula (I) and the PF1163B substance represented by the formula (II) from the culture, and clarified their physicochemical properties. Accordingly, the present invention provides novel antifungal active substances PF1163A and PF1163B.
[0007]
Further, the present invention relates to a microorganism belonging to the genus Penicillium and having a capability of producing PF1163A substance and PF1163B substance in a medium, accumulating the PF1163A substance and PF1163B substance in the culture, and PF1163A substance and PF1163B from the culture. The present invention provides a method for producing a PF1163A substance and a PF1163B substance, characterized by collecting the substance.
[0008]
The first gist of the present invention resides in a novel antifungal active substance PF1163A substance represented by the aforementioned formula (I) and having the following physicochemical properties.
[0009]
The second gist of the present invention resides in the PF1163B substance represented by the formula (II).
The third gist of the present invention is characterized by culturing an antifungal active substance PF1163A substance and a PF1163B substance-producing bacterium belonging to the genus Penicillium, and collecting the antifungal active substance PF1163A substance and the PF1163B substance from the culture. And a method for producing an antifungal active substance PF1163A substance and a PF1163B substance. As an example of the novel antifungal active substance PF1163A substance and PF1163B substance-producing bacterium used in the present invention, it was newly isolated, and Biotechnological Institute No. 15473 (FERM P-15473) ) And Penicillium PF1163 strain (hereinafter may be abbreviated as “the present strain”). The bacteriological properties of this strain are as follows.
[0012]
3. Mycological properties of PF1163 strain (1) Colony properties Good growth on zapek yeast extract agar medium, colony diameter reaches 25-30 mm after 7 days of culture at 25 ° C. Light brown, velvet-like, and wrinkle-like colonies that form conidia sparsely. The back side is brown.
Growth on the malt extract agar medium is somewhat suppressive, and the colony diameter reaches 20 to 25 mm after culturing at 25 ° C. for 7 days. Pale orange, velvet-like, flat or slightly wrinkled colony with few conidia. The back is ocher. In the culture at 37 ° C., it does not grow on any medium.
[0013]
(2) Morphological properties Conidial pattern is generated from the colony surface or aerial hyphae, and has a smooth surface and a size of 150 to 300 × 2 to 2.5 μm. Penisiri is a bicyclist. Metre becomes 8 to 13 × 2 to 2.5 μm and 3 to 5 ring organisms. The phialide is an ampoule type, 5 to 8 × 1.8 to 2 μm, and 5 to 8 rings. Conidia are spherical to elliptical, have a smooth surface, a size of 2 to 2.5 μm, and are chained.
[0014]
From the above bacteriological properties, this strain was identified as mold belonging to the genus Penicillium.
[0015]
This strain is likely to change its properties as seen in other molds. For example, any mutant strain (naturally occurring or inducible), zygote or gene recombinant derived from this strain that produces PF1163A substance and PF1163B substance can be used in the present invention.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
1. In the method of the present invention, the PF1163A substance and the PF1163B substance-producing bacteria belonging to the genus Penicillium are cultured in a medium containing nutrients that can be used by normal microorganisms. As the nutrient source, known ones conventionally used for mold cultivation can be used. For example, glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal and vegetable oils and the like can be used as the carbon source. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, and the like can be used. In addition, it is effective to add inorganic salts capable of generating sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions as required. In addition, organic substances and inorganic substances that assist the growth of bacteria and promote the production of the PF1163A substance and the PF1163B substance can be appropriately added.
[0017]
As the culture method, a culture method under aerobic conditions, particularly a stationary culture method is most suitable. A suitable temperature for culturing is 25 to 30 ° C., but in many cases, culturing is performed at around 26 ° C. Production of the PF1163A substance and the PF1163B substance varies depending on the medium and culture conditions, but the accumulation amount reaches the maximum in 2 to 14 days in any of stationary culture, shaking culture, and tank culture. When the accumulated amount of the PF1163A substance or PF1163B substance in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture.
[0018]
2. Purification method of PF1163A substance and PF1163B substance The PF1163A substance and PF1163B substance obtained by the present invention have the physicochemical properties described above, and can be purified from the culture according to their properties. For example, the PF1163A substance and the PF1163B substance can be extracted from the culture using an organic solvent, and then purified using an adsorption / desorption method using an adsorbent, a molecular partition method using a gel filtration agent, or the like. For example, a culture containing active ingredients is extracted with ethyl acetate. The extract is concentrated under reduced pressure, and the extract is dissolved in a small amount of chloroform and chromatographed with a chloroform / methanol solvent using a silica gel column equilibrated with chloroform. Concentrate the eluate containing the active substance under reduced pressure, redissolve it in methanol, gel filtration with Sephadex LH-20 (Pharmacia Fine Chemicals) equilibrated with methanol, or equilibrate with hexane / ethyl acetate solvent The target PF1163A substance and PF1163B substance can be obtained by performing chromatography with a hexane / ethyl acetate solvent using the silica gel column prepared.
[0019]
In order to use the PF1163A substance and the PF1163B substance used in the present invention as an antifungal agent, a known carrier and, if necessary, a known adjuvant may be combined in accordance with various dosage forms or usage forms. That's fine.
[0020]
Administration forms for antifungal agents include subcutaneous injections, intravenous injections, intramuscular injections, parenteral administration such as suppositories, or oral administration such as tablets, capsules, powders, granules, ointments, lotions Examples of topical administration forms such as an agent and a vaginal suppository can be exemplified.
[0021]
【Example】
Examples of the present invention will be described below, but this is merely an example and is not intended to limit the present invention, and encompasses all of the many variations or modification means not exemplified here.
[0022]
Example 1
As a seed medium, starch 2.0%, glucose 1.0%, polypeptone 0.5%, wheat germ 0.6%, yeast extract 0.3%, soybean meal 0.2% and calcium carbonate 0.2% A medium having a composition (pH 7.0 before sterilization) was used.
Moreover, the solid culture medium which added 2.5% of soybean meal to the rice which fully absorbed water was used as a production medium.
The 100 ml Erlenmeyer flask into which 20 ml of the seed medium was dispensed was sterilized at 120 ° C. for 15 minutes, and after inoculating one platinum loop of slant agar culture of PF1163 strain (FERM P-15473) at 25 ° C. for 2 days. Cultured with shaking. Next, a 500 ml Erlenmeyer flask into which 100 g of production medium had been dispensed was sterilized at 120 ° C. for 15 minutes, 4 ml of the above seed culture was inoculated thereto, and after sufficient stirring, static culture was performed at 28 ° C. for 14 days.
[0023]
7.5 kg of the thus obtained culture was extracted with 10 l of ethyl acetate, and the ethyl acetate layer was concentrated under reduced pressure to obtain about 27 g of an oily substance. The obtained oily substance was washed with 60 ml of hexane three times to obtain a residue of about 15 g. A small amount of ethyl acetate was added to the obtained residue to dissolve it, and 20 g of silica gel was added thereto, and then ethyl acetate was distilled off under reduced pressure, and the residue was uniformly adsorbed on silica gel. This was chromatographed using a silica gel column (silica gel 150 g) equilibrated with chloroform. Elution was carried out with a chloroform / methanol solution (methanol concentration was 2500 ml at 0%, 2000 ml at 1%, 500 ml at 2%, 500 ml) with increasing methanol concentration. Fractions containing the active ingredient were collected and concentrated under reduced pressure to obtain 3.8 g of a dry solid containing the PF1163A substance as the main component and 600 mg of a dry solid containing the PF1163B substance as the main component.
[0024]
3.8 g of the dried product containing the PF1163A substance as a main component was dissolved in a small amount of chloroform, and the resulting precipitate was removed, followed by chromatography using a silica gel column (silica gel 170 g) equilibrated with chloroform. went. Elution was performed with a chloroform / methanol solution (methanol concentration 0% 700 ml, 1% 1000 ml) with increasing methanol concentration. Fractions containing the active ingredient were collected and concentrated under reduced pressure to obtain 1.2 g of dried product containing PF1163A substance and 100 mg of dried product containing PF1163B substance.
[0025]
Using a silica gel column (silica gel 40 g) in which 1.2 g of the dried product containing PF1163A substance was dissolved in a small amount of hexane containing 50% ethyl acetate and equilibrated with hexane containing 50% ethyl acetate. Chromatography was performed. Elution was carried out with a hexane / ethyl acetate solution (ethyl acetate concentration: 50%, 500 ml, 60%, 200 ml, 67%, 240 ml) with increasing ethyl acetate concentration. Fractions containing PF1163A material were collected and concentrated in vacuo to give 830 mg of PF1163A material as an almost pure oil.
[0026]
A total of about 700 mg of the dried product containing the PF1163B substance as a main component was dissolved in a small amount of hexane containing 30% ethyl acetate, and this was equilibrated with hexane containing 30% ethyl acetate (silica gel). 65g) was used for chromatography. Elution was performed with a hexane / ethyl acetate solution (ethyl acetate concentration of 380 ml for 30%, 380 ml for 50%, 1900 ml for 67%) with increasing ethyl acetate concentration. Fractions containing the PF1163B substance were collected, concentrated under reduced pressure, dissolved in a small amount of methanol, and developed with methanol using a Sephadex LH-20 (Pharmacia Fine Chemicals, 300 ml) column previously filled with methanol. And chromatographed. Fractions containing the PF1163B material were collected and concentrated in vacuo to yield 300 mg of the PF1163B material as an almost pure oil.
[0027]
Test example 1
The antifungal activity of the PF1163A substance and the PF1163B substance was measured as the minimum inhibitory concentration (MIC) for various strains by the liquid culture dilution method. RPMI1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) was used as the medium and cultured at 37 ° C. for 20 hours (15 hours for C. albicans and C. parapsilosis ). The measurement results are shown in Table 1.
[0028]
[Table 1]
【The invention's effect】
As shown in Table 1, the novel antifungal active substance PF1163A substance and PF1163B substance of the present invention are C.I. Antifungal agents having antifungal activity against albicans and containing these as active ingredients are C.I. It is useful for the treatment of mycosis caused by albicans .
[Brief description of the drawings]
FIG. 1 is a 400 MHz 1 H-NMR spectrum of de novo antifungal active substance PF1163A substance of the present invention in deuterated chloroform.
FIG. 2 is a 100 MHz 13 C-NMR spectrum in deuterated chloroform of a novel antifungal active substance PF1163A substance of the present invention.
FIG. 3 is a 400 MHz 1 H-NMR spectrum of de novo antifungal active substance PF1163B substance of the present invention in deuterated chloroform.
FIG. 4 is a 100 MHz 13 C-NMR spectrum in deuterated chloroform of the novel antifungal active substance PF1163B substance of the present invention.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22424298A JP4105805B2 (en) | 1998-08-07 | 1998-08-07 | Novel antifungal active substances PF1163A and PF1163B substances, processes for producing them, and antifungal agents containing them as active ingredients |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22424298A JP4105805B2 (en) | 1998-08-07 | 1998-08-07 | Novel antifungal active substances PF1163A and PF1163B substances, processes for producing them, and antifungal agents containing them as active ingredients |
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| Publication Number | Publication Date |
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| JP2000053663A JP2000053663A (en) | 2000-02-22 |
| JP4105805B2 true JP4105805B2 (en) | 2008-06-25 |
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| CN114652714B (en) * | 2022-04-18 | 2023-09-15 | 中国医学科学院医药生物技术研究所 | Application of macrolide in pancreatic cancer resistance |
| CN115281217A (en) * | 2022-08-29 | 2022-11-04 | 云南大学 | Preparation method and application of organic extract for promoting lateral root development of plant |
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