JP4404710B2 - ステロールの5−エン−3−オン体の製造方法、及び分析方法 - Google Patents
ステロールの5−エン−3−オン体の製造方法、及び分析方法 Download PDFInfo
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- JP4404710B2 JP4404710B2 JP2004207885A JP2004207885A JP4404710B2 JP 4404710 B2 JP4404710 B2 JP 4404710B2 JP 2004207885 A JP2004207885 A JP 2004207885A JP 2004207885 A JP2004207885 A JP 2004207885A JP 4404710 B2 JP4404710 B2 JP 4404710B2
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- Prior art keywords
- sterol
- cholesterol oxidase
- dione
- hydrocarbon solvent
- ene
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/12—Acting on D ring
- C12P33/16—Acting at 17 position
Landscapes
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Description
〔1〕コレステロールオキシダーゼ含有液からなる水層と、炭化水素系溶媒からなる炭化水素系溶媒層とで構成される二層溶液に、ステロールを含有させて反応させ、
上記コレステロールオキシダーゼ含有液は、コレステロールオキシダーゼ活性を示す微生物の培養液であり、
上記微生物は、アルスロバクター(Arthrobacter)属であることを特徴とするステロールの5−エン−3−オン体の製造方法。
〔2〕上記炭化水素系溶媒が、n−ブタン、n−ヘキサン、ヘプタン及びシクロアルカンから選ばれる少なくとも1種である上記〔1〕記載のステロールの5−エン−3−オン体の製造方法。
〔3〕上記反応の終了後に、アルコールを反応液に添加する上記〔1〕又は〔2〕記載のステロールの5−エン−3−オン体の製造方法。
〔4〕上記ステロールが、植物ステロールである上記〔1〕乃至〔3〕のいずれかに記載のステロールの5−エン−3−オン体の製造方法。
〔5〕アセトニトリル及びイソプロピルアルコールを体積基準で(3〜5)/(5〜7)の割合で混合して得られた混合液を移動相として、
高速液体クロマトグラフィ(HPLC)法にてステロールの5−エン−3−オン体及び3,6−ジオン体を同時に分離定量するステロールの5−エン−3−オン体及び3,6−ジオン体の分析方法であって、
上記高速液体クロマトグラフィ(HPLC)法におけるカラムの温度を10〜30℃とすることを特徴とするステロールの5−エン−3−オン体及び3,6−ジオン体の分析方法。
更に、本発明のステロールの5−エン−3−オン体及び3,6−ジオン体の分析方法によれば、従来は困難であったHPLC法によるステロールの5−エン−3−オン体及び3,6−ジオン体の同時分離定量を行うことができる。
本発明の上記「二層溶液」を構成する上記「コレステロールオキシダーゼ含有液からなる水層」は、水系溶媒中にコレステロールオキシダーゼを含有する層である。該水系溶媒の種類は、上記炭化水素系溶媒と混合せずに二層に分離する性質を有する限り、特に限定はない。上記水系溶媒として、通常は水が用いられる。
前培養培地として、以下の表1(A)に記載の組成の水系液体培地(pH;7.0±0.1)を調製した。また、本培養培地として、以下の表1(B)に記載の組成の水系液体培地(pH;7.0±0.1)を調製した。更に、使用菌体として、グラム陽性桿菌である市販のアルスロバクター シンプレックス(Arthrobacter Simplex)を用いた。
アセトニトリル及びイソプロピルアルコールを4/6(体積基準)の割合で混合し、アスピレーターを用いて減圧脱気することにより、移動相を調製した。次いで、該移動相をカラムボリュームの10倍量通液し、カラムの平衡化を行った。カラムの平衡化は、ベースラインをモニターし、ベースラインが安定したら完了した。
そして、上記生成物を正確に秤量し、上記移動相に0.2%(v/v)となるように溶解した。溶解後、該溶液をフィルター処理(0.42μm)し、HPLC試料とした。そして、以下の条件でHPLCを行った。
〔HPLC条件〕
カラム;「Cadenza CD C−18」(4.6×500mm;インタクト社製)
流量;0.55ml/min
検出器及び検出波長;「UV detecter」 210nm
カラム温度;18℃
試料溶解溶媒;移動相と同じ
注入量;20μl
得られたピーク面積の値と検量線から試料中の各成分の含量を求め、以下の式により各成分濃度を定量した。
各成分濃度(%、v/v)=検量線より求めた試料中の各成分の含量×100/40
一方、コレステロールオキシダーゼ含有液からなる水層と、炭化水素系溶媒であるヘキサン層で構成される二層溶液に基質であるコレステロールを添加し、反応を行った実施例1では、比較例1と異なり、5−エン体を得ることができることが分かる。また、植物ステロールを基質として用いた場合、実施例2に示すように、5−エン体を得ると共に、3,6−ジオン体をも得ることができることが分かる。
Claims (5)
- コレステロールオキシダーゼ含有液からなる水層と、炭化水素系溶媒からなる炭化水素系溶媒層とで構成される二層溶液に、ステロールを含有させて反応させ、
上記コレステロールオキシダーゼ含有液は、コレステロールオキシダーゼ活性を示す微生物の培養液であり、
上記微生物は、アルスロバクター(Arthrobacter)属であることを特徴とするステロールの5−エン−3−オン体の製造方法。 - 上記炭化水素系溶媒が、n−ブタン、n−ヘキサン、ヘプタン及びシクロアルカンから選ばれる少なくとも1種である請求項1記載のステロールの5−エン−3−オン体の製造方法。
- 上記反応の終了後に、アルコールを反応液に添加する請求項1又は2記載のステロールの5−エン−3−オン体の製造方法。
- 上記ステロールが、植物ステロールである請求項1乃至3のいずれかに記載のステロールの5−エン−3−オン体の製造方法。
- アセトニトリル及びイソプロピルアルコールを体積基準で(3〜5)/(5〜7)の割合で混合して得られた混合液を移動相として、
高速液体クロマトグラフィ(HPLC)法にてステロールの5−エン−3−オン体及び3,6−ジオン体を同時に分離定量するステロールの5−エン−3−オン体及び3,6−ジオン体の分析方法であって、
上記高速液体クロマトグラフィ(HPLC)法におけるカラムの温度を10〜30℃とすることを特徴とするステロールの5−エン−3−オン体及び3,6−ジオン体の分析方法。
Priority Applications (6)
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JP2004207885A JP4404710B2 (ja) | 2004-07-14 | 2004-07-14 | ステロールの5−エン−3−オン体の製造方法、及び分析方法 |
PCT/JP2005/012869 WO2006006608A1 (ja) | 2004-07-14 | 2005-07-12 | ステロールの5-エン-3-オン体又は3,6-ジオン体の製造方法、脂質代謝改善剤、飲食品及び動物用飼料の製造方法、並びに分析方法 |
KR1020067026761A KR101040099B1 (ko) | 2004-07-14 | 2005-07-12 | 스테롤의 5-엔-3-온체 또는 3,6-디온체의 제조방법, 및 분석방법 |
US11/632,072 US8008040B2 (en) | 2004-07-14 | 2005-07-12 | Process for production of 5-ene-3-one or 3,6-dione derivatives of sterols, processes for production of lipid metabolism improvers, foods, drinks, and animal feeds, and analytical method |
CN2005800231293A CN101006184B (zh) | 2004-07-14 | 2005-07-12 | 甾醇的5-烯-3-酮衍生物或3,6-二酮衍生物的制造方法,脂质代谢改善剂、饮食品以及动物饲料的制造方法,和分析方法 |
TW094123948A TWI421256B (zh) | 2004-07-14 | 2005-07-14 | Method for producing 5-ene-3-keto or 3,6-diketone of fatty alcohol, method for producing lipid metabolism improving agent, food and animal and animal feed, and analysis method |
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US8008040B2 (en) | 2011-08-30 |
TW200606173A (en) | 2006-02-16 |
TWI421256B (zh) | 2014-01-01 |
CN101006184A (zh) | 2007-07-25 |
WO2006006608A1 (ja) | 2006-01-19 |
US20080248520A1 (en) | 2008-10-09 |
JP2007284348A (ja) | 2007-11-01 |
KR20070041450A (ko) | 2007-04-18 |
CN101006184B (zh) | 2011-08-03 |
KR101040099B1 (ko) | 2011-06-09 |
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