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WO2002086142A1 - Procede de production de soyasapogenol b - Google Patents

Procede de production de soyasapogenol b Download PDF

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Publication number
WO2002086142A1
WO2002086142A1 PCT/JP2002/003612 JP0203612W WO02086142A1 WO 2002086142 A1 WO2002086142 A1 WO 2002086142A1 JP 0203612 W JP0203612 W JP 0203612W WO 02086142 A1 WO02086142 A1 WO 02086142A1
Authority
WO
WIPO (PCT)
Prior art keywords
soyasapogenol
plant
producing
hydroxylase
derived
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2002/003612
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English (en)
Japanese (ja)
Inventor
Hiroaki Hayashi
Kenichiro Inoue
Masato Tani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Meiji Seika Kaisha Ltd
Original Assignee
Meiji Seika Kaisha Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Meiji Seika Kaisha Ltd filed Critical Meiji Seika Kaisha Ltd
Publication of WO2002086142A1 publication Critical patent/WO2002086142A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms

Definitions

  • the present invention relates to a method for producing soyasapogenol B, and more particularly to a method for producing soyasapogenol B from sophoradiol using a plant hydroxylase.
  • Sosasapogenol B (12-oleanane-3,22,24-triol) is a triterpene (Chem.
  • Soyasapogenol B has been reported to have anti-complement activity, platelet aggregation inhibitory effect (JP-A-61-37749), antitumor activity (JP-A-10-234396), and hepatoprotective effect (Bioorg. Med. Chem. Lett. 7, ⁇ -88, 1997), etc., and their usefulness as pharmaceuticals or pharmaceutical raw materials is expected.
  • soyasapogenol B As a method for producing soyasapogenol B, there is known a method of hydrolyzing the sugar chain of savonin contained in soybean seeds and then purifying the soyasapogenol B. However, this method is not an effective method and practical because the ratio of saponin contained in soybean seeds is as low as about 0.2% (Pharmaceutical Journal, 104, P162-168, 1984).
  • an object of the present invention is to establish an efficient method for producing sasa sapogenol B. It is to stand. Disclosure of the invention
  • sophoradole (12-oleanene-3, 22-diol) which is structurally similar to soshapogenol B. This compound has been reported as a component contained in Sophora elegans (Pharmaceutical Journal, 78, pl090-1094, 1958).
  • the present inventors prepared an enzyme fraction from cultured Glycyrrhiza glabra cells, and examined conditions for hydroxylating the 24-position of sophoradiol using this enzyme. As a result, it is possible to add NADPH and sophoradiol to the microsomal fraction of the cultured cells of Kanzo, and incubate the mixture. As a result, Sosasapogenol B was successfully produced, and the present invention was completed.
  • the present invention is a method for producing sosapogenol B, which comprises producing sosapogenol B from sophoradyl using a plant-derived hydroxylase.
  • a plant-derived hydroxylase is used, but any plant may be used as long as it contains a hydroxylase for biosynthesizing sojasapogenol B. Since this enzyme is thought to have a function of hydroxylating the 24th position of the oleanan skeleton, the plants are not limited to Kanzo, but are soybeans and soybeans, which are leguminous plants containing soyasapogenol B. Lara and Alf Alpha are also available. These have the same biosynthetic pathway, and it is considered that the same enzymes exist in classification.
  • the form of the plant containing the enzyme is not particularly limited, and may be any of a young plant that has just germinated from a seed, a cultivated plant, or a cultured cell such as a callus.
  • a method for extracting and purifying a hydroxylase from a plant will be described.
  • a plant or cells of the plant are crushed by an appropriate method, and this is stirred in a buffer containing an antioxidant, an enzyme stabilizer, a polyphenol adsorbent, a metal ligand, and the like.
  • an operation such as filtration or centrifugal sedimentation is applied to remove insolubles and extract the enzyme.
  • a method of preparing a microsomal fraction from a crude extract by ultracentrifugation or the like is effective. It is also possible to further increase the specific activity of the enzyme by a conventional method such as gel filtration or ion exchange resin.
  • a conventional method such as gel filtration or ion exchange resin.
  • an enzyme fraction is prepared.
  • the sosapogenol B according to the present invention can be manufactured by the following method. NADPH and Sophoradio are added to the enzyme fraction suspended in a suitable buffer. And incubate.
  • the enzyme fraction is suspended by adding a buffer of a smaller amount than the fresh weight of the original plant, preferably a buffer of a fresh weight of 15 to 150, and suspending NADPH to a final concentration of 0.1 to 1 OmM. And sophoradiol as a starting material are added to a final concentration of 100 to 1000 M, respectively, and then incubated.
  • a buffer that does not exert a biological effect can be used, and a buffer having a buffering capacity in a neutral region (pH 7.0 to 7.5), such as Tris-HCl buffer / phosphate buffer, is preferred. No.
  • the incubation is performed at 15 to 37 ° C for 10 minutes to several hours, preferably 30 to 120 minutes.
  • the suspension is extracted several times, usually twice, using an organic solvent, for example, ether, ethyl acetate, or chloroform, to obtain a soyasapogenol B extract.
  • an organic solvent for example, ether, ethyl acetate, or chloroform
  • soshapogenol B extract can be treated with silica gel octadedecyl (ODS) -silica gel to purify soshapogenol B.
  • ODS silica gel octadedecyl
  • the organic solvent extract was subjected to thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography. It can be detected and quantified by analytical means such as Z-mass spectrometry (GC-MS) and liquid chromatography / mass spectrometry (LC-MS).
  • the cultured cells of Kanzo II were derived from calli derived from young plants of Glycyrrhiza glabra by 100 M naphthalene acetic acid and 1 uM 6-benzyl. Subcultured in Linsmaier-Skoog liquid medium containing aminopurine (Phytochemi stry, 29, p3127-3129, 1990) was used.
  • the cells were harvested on day 10 (30 g Fresh weight). The cells were then frozen in liquid nitrogen and ground in a mortar, after which polyvinylpolypyrrolidone (PVPP) 38, 0.2 60 ml of 0.1 M Tris-HCl buffer (pH 7.5) containing sucrose, 2 mM EDTA and 20 mM 2-mercaptoethanol was added and homogenized.
  • PVPP polyvinylpolypyrrolidone
  • NADPH final concentration: 2 mM
  • sophora diol final concentration: 50 ju U Bioorg. Med. Chem. Lett., 7, P85-88, 1997) were added to the microsomal surface area lm1, respectively. Incubation was performed at 20 ° C for 30 minutes.
  • the extract was analyzed by LC-MS (HP1090, manufactured by Hewlett-Packard), and the retention time and mass fragment were compared with those of soyasapogenol B (manufactured by Wako Pure Chemical Industries). As a result, production of soyasapogenol B could be confirmed (conversion rate from sofuoradiol: 7.6%).
  • LC-MS HP1090, manufactured by Hewlett-Packard
  • soyasapogenol B manufactured by Wako Pure Chemical Industries
  • the present invention provides an efficient method for producing soyasapogenol B using a plant hydroxylase. Sosapogenol B is useful as a pharmaceutical or a raw material for the synthesis of pharmaceuticals.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de production de soyasapogénol B qui est utile en tant que médicament ou que matière pour la production de médicaments. Le sophoradiol utilisé comme matière de base est traité avec une hydroxylase issue d'une plante afin d'hydroxyler le composé sur sa position 24. On peut ainsi produire efficacement le soyasapogénol B cible.
PCT/JP2002/003612 2001-04-16 2002-04-11 Procede de production de soyasapogenol b Ceased WO2002086142A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-117449 2001-04-16
JP2001117449A JP2005137201A (ja) 2001-04-16 2001-04-16 ソヤサポゲノールｂの製造法

Publications (1)

Publication Number Publication Date
WO2002086142A1 true WO2002086142A1 (fr) 2002-10-31

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/003612 Ceased WO2002086142A1 (fr) 2001-04-16 2002-04-11 Procede de production de soyasapogenol b

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JP (1) JP2005137201A (fr)
WO (1) WO2002086142A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080572A1 (fr) 2004-02-25 2005-09-01 Meiji Seika Kaisha, Ltd. Triterpène hydroxylase

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103635184B (zh) 2011-06-28 2016-02-10 J-制油株式会社 大豆皂醇组合物
EP2878295A4 (fr) 2012-07-24 2015-12-23 J Oil Mills Inc Composition

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57163331A (en) * 1981-03-31 1982-10-07 Kikkoman Corp Preparation of soya sapogenol b
WO1997003088A1 (fr) * 1995-07-07 1997-01-30 Meiji Seika Kabushiki Kaisha Derives triterpenes et medicament pour les maladies du foie

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57163331A (en) * 1981-03-31 1982-10-07 Kikkoman Corp Preparation of soya sapogenol b
WO1997003088A1 (fr) * 1995-07-07 1997-01-30 Meiji Seika Kabushiki Kaisha Derives triterpenes et medicament pour les maladies du foie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIROAKI HATASHI ET AL.: "Glycyrrhetinic acid 24-hydroxylase activity in microsomes of cultured licrice cells", PHYTOCHEMISTRY, vol. 34, no. 5, 1993, pages 1303 - 1307, XP002954151 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080572A1 (fr) 2004-02-25 2005-09-01 Meiji Seika Kaisha, Ltd. Triterpène hydroxylase
JPWO2005080572A1 (ja) * 2004-02-25 2007-10-25 明治製菓株式会社 トリテルペン水酸化酵素
US7575892B2 (en) 2004-02-25 2009-08-18 Meiji Seika Kaisha, Ltd. Expression vector encoding a triterpene hydroxylase polypeptide
EP2145959A3 (fr) * 2004-02-25 2010-08-25 Meiji Seika Kaisha, Ltd. Triterpene Hydroxylase
JP4668176B2 (ja) * 2004-02-25 2011-04-13 明治製菓株式会社 トリテルペン水酸化酵素

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