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WO2003040383A1 - Obtention de 20-$g(b)-d-pyranoglucosyl-protopanaxadiol par hydrolyse enzymatique du ginsenoside - Google Patents

Obtention de 20-$g(b)-d-pyranoglucosyl-protopanaxadiol par hydrolyse enzymatique du ginsenoside Download PDF

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Publication number
WO2003040383A1
WO2003040383A1 PCT/CN2002/000786 CN0200786W WO03040383A1 WO 2003040383 A1 WO2003040383 A1 WO 2003040383A1 CN 0200786 W CN0200786 W CN 0200786W WO 03040383 A1 WO03040383 A1 WO 03040383A1
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WO
WIPO (PCT)
Prior art keywords
preparing
ginsenodiol
enzymatic hydrolysis
type
hydrolysis
Prior art date
Application number
PCT/CN2002/000786
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English (en)
Chinese (zh)
Inventor
Ling Yany
Ke-Jiang He
Yi Yang
Hong-Wei Luan
Original Assignee
Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences
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 Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences filed Critical Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences
Publication of WO2003040383A1 publication Critical patent/WO2003040383A1/fr

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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • 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

Definitions

  • the present invention relates to the preparation of 20- ⁇ -D-glucopyranosylprotopanaxadiol from ginsenosides.
  • C-K Compound-K
  • Ginsenoside c- ⁇ is a metabolite of diol-type ginsenosides in the intestine after oral administration. Studies have shown that c- ⁇ has antitumor and immunomodulatory effects, improves microcirculation, regulates digestive functions, soothe the nerves, anti-aging, anti-aging Various biological activities such as stress, prevention of gastrointestinal ulcers, improvement of quality of life, enhancement of memory and learning ability. Therefore, c- ⁇ production methods have attracted much attention. Kyobashi Kobashi (Chem Pharm Bull 21 (3): 245-249, 1988) dissolved 500 mg of ginsenoside Rbl and 250 mg of naringinase in 32 ml of phosphate buffer solution (pH 4.0, containing 10% ethanol).
  • reaction solution was centrifuged into a precipitate and a mother liquor, and the precipitate was dissolved in 3.5 ml of ethanol, and added to the mother liquor with 32 ml of a buffer solution and 250 mg of naringinase. Hydrolyzed for 7 days.
  • the reaction solution was extracted with ether, and the ether phase was evaporated to dryness to obtain ginsenoside C-K.
  • the mother liquor was extracted with n-butanol, and the n-butanol phase was washed with water, dried, and evaporated to obtain a residue; the residue was separated by preparative TLC to separate C-K; and combined C-K to obtain 253 mg.
  • This method has a high yield (86%), but has the following disadvantages: long hydrolysis time; large amount of enzyme, and the source of the enzyme is inconvenient and uneconomical; it requires extraction with explosive ether, so it is not suitable for mass production .
  • Technical content of the invention :
  • the present invention provides a method for preparing CK by enzymatic hydrolysis of ginsenosides, including hydrolysis and collection steps, and is characterized in that the diol is hydrolyzed by a snail enzyme in a buffer solution.
  • Type ginsenoside the weight ratio of diol type ginsenoside to snail enzyme is 1: 1 ⁇ 10: 1, pH value is between 4.0 ⁇ 5.0, hydrolysis temperature is 30 ⁇ 45 ° C, and hydrolysis time is 2 ⁇ 3 days.
  • the collecting process is as follows: the reaction precipitate is washed with water and then repeatedly washed with ethanol. After the combined ethanol solutions were evaporated to dryness, the collected residue was C-K.
  • the washing liquid can be combined with the mother liquor and concentrated for reuse in enzymatic hydrolysis.
  • the snail enzyme used may be a crude snail enzyme.
  • the optimum temperature for hydrolyzing diol-type ginsenosides by crude snail enzyme is 40 ° C, and the optimum pH is 4.5.
  • the type of buffer solution has little effect on the enzyme activity.
  • the pH of the invention is 4.5. Phosphoric acid-citrate buffer with a strength of 0.01.
  • a certain amount of organic solvent should be added to the reaction solution.
  • the organic solvent assists the intermediate product to facilitate the normal progress of the enzymatic hydrolysis reaction.
  • the type and amount of organic solvents are based on those that do not affect enzyme activity. After screening, 5 ⁇ 15% ethanol is the best.
  • the ratio of diol saponin to enzyme is 6: 1; generally speaking, after 8 hours of hydrolysis, the hydrolysis time is prolonged, and the yield of CK is extremely increased. Less, so it is not necessary to lengthen the hydrolysis time.
  • the invention provides a convenient and low-cost enzyme for preparing ginsenoside C-K by enzymatic hydrolysis of ginseng glycol saponin.
  • the process for preparing C-K using this enzyme is simple, convenient, low cost and high recovery rate.
  • the C-K content prepared by this method is 95%, and the aglycon yield is 91%.
  • FIG. 1 shows the effect of pH on C-K production
  • Figure 2 shows the effect of material ratio on C-K output
  • FIG. 3 shows the effect of temperature on C-K production
  • Figure 4 shows the effect of ionic strength on C-K production
  • FIG. 5 shows the change of C-K output over time
  • Example 1 '500 mg of diol-type ginsenoside, 84 mg of snail enzyme was dissolved in 32 ml of phosphate-citrate buffer (pH 4.5, ionic strength 0.01, containing 10% ethanol), and hydrolyzed in a water bath at 40 ° C for 1 day The reaction solution was collected by centrifugation. The precipitate was repeatedly washed with water (10ml X 3). The washing solution was combined with the mother liquor and concentrated to 32ml for reuse. The precipitate was repeatedly washed with ethanol (10ml X 5). The ethanol solution was combined, evaporated to dryness, and collected. The residue gave 106 mg of CK.
  • phosphate-citrate buffer pH 4.5, ionic strength 0.01, containing 10% ethanol
  • Diol ginsenoside and snail enzyme were dissolved in 3.2ml phosphate-citrate buffer (pH4.5, ionic strength 0.01, containing 10% ethanol) in different proportions, and hydrolyzed in a 40 ° C water bath for 1 day; reaction The precipitate was collected by centrifugation. The precipitate was repeatedly washed with water (lml X 3). The washing solution was combined with the mother liquor and concentrated to 3.2 ml for reuse. The precipitate was repeatedly washed with ethanol (lml X 5). The ethanol solution was combined, evaporated to dryness, and the residue was collected. The CK yield is shown in Figure 2.

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

Abstract

La présente invention concerne un procédé d'obtention de 20-β-D-pyranoglucosyl-protopanaxadiol par hydrolyse enzymatique de ginsenoside suivie d'une opération de recueil. La caractéristique du procédé est que le diol se détache du glycosyle moléculaire du ginsenoside et migre vers un site sous l'effet de l'hélice d'enzyme, produisant ainsi l'hélice d'enzyme du composé attendu, en l'occurrence le 20-β-D-pyranoglucosyl-protopanaxadiol. L'hélice d'enzyme utilisé dans l'invention est facilement disponible et bon marché. Le procédé d'obtention du C-K est simple, commode, bon marché, et d'un bon rendement. Il peut se prêter à la préparation d'un médicament anti-tumoral.
PCT/CN2002/000786 2001-11-06 2002-11-05 Obtention de 20-$g(b)-d-pyranoglucosyl-protopanaxadiol par hydrolyse enzymatique du ginsenoside WO2003040383A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN01133410.X 2001-11-06
CNB01133410XA CN1167805C (zh) 2001-11-06 2001-11-06 用酶水解人参皂甙制备20－β-D-吡喃葡萄糖基原人参二醇

Publications (1)

Publication Number Publication Date
WO2003040383A1 true WO2003040383A1 (fr) 2003-05-15

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Country Status (2)

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CN (1) CN1167805C (fr)
WO (1) WO2003040383A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1508146A (zh) * 2002-12-13 2004-06-30 中国科学院大连化学物理研究所 一种新型抗肿瘤人参皂苷
CN1305479C (zh) * 2003-09-28 2007-03-21 中国科学院大连化学物理研究所 具有抗肿瘤活性的低极性人参皂苷组合物
US8541035B2 (en) 2009-05-14 2013-09-24 Il Hwa Co., Ltd. Methods for preparing a fermented ginseng concentrate or powder
JP6170674B2 (ja) * 2009-05-19 2017-07-26 イル・ファ・カンパニー・リミテッドＩｌ Ｈｗａ Ｃｏ．，Ｌｔｄ． 発酵高麗人参濃縮液または粉末の製造方法
CN101768619A (zh) * 2010-02-10 2010-07-07 华侨大学 一种以Rd为底物制备稀有人参皂甙IH-901的方法
CN102154417A (zh) * 2010-12-13 2011-08-17 天津中医药大学 一种杠柳次苷的制备方法
CN102251009A (zh) * 2011-06-09 2011-11-23 华侨大学 一种稀有人参皂甙ih-901结晶的简易生产方法
CN103360441B (zh) * 2012-04-01 2016-08-10 浙江海正药业股份有限公司 人参皂苷c-k化合物多晶型及其制备方法
CN105949264A (zh) * 2012-04-01 2016-09-21 浙江海正药业股份有限公司 人参皂苷c-k的两种晶型及其制备方法
CN103073611B (zh) * 2013-01-10 2015-10-28 天津大学 一种人参皂苷c-k半水合物晶体及制备方法
CN104622928A (zh) * 2015-01-04 2015-05-20 中国药科大学 人参总皂苷通过激活IL-1β/IL-18活化分泌发挥免疫调节作用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0648946A (ja) * 1991-06-21 1994-02-22 Takuo Kosuge サポニン誘導体を含有する補気薬
CN1184851A (zh) * 1996-11-29 1998-06-17 株式会社一和 人参皂草苷代谢物的制备方法
CN1190098A (zh) * 1998-01-22 1998-08-12 白求恩医科大学基础医学院 人参分组皂甙的制备方法,其药物组合物及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0648946A (ja) * 1991-06-21 1994-02-22 Takuo Kosuge サポニン誘導体を含有する補気薬
CN1184851A (zh) * 1996-11-29 1998-06-17 株式会社一和 人参皂草苷代谢物的制备方法
CN1190098A (zh) * 1998-01-22 1998-08-12 白求恩医科大学基础医学院 人参分组皂甙的制备方法,其药物组合物及应用

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CN1417345A (zh) 2003-05-14
CN1167805C (zh) 2004-09-22

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