CN101768619A - Method for preparing rare ginsenoside IH-901 with Rd as substrate - Google Patents
Method for preparing rare ginsenoside IH-901 with Rd as substrate Download PDFInfo
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- CN101768619A CN101768619A CN 201010113762 CN201010113762A CN101768619A CN 101768619 A CN101768619 A CN 101768619A CN 201010113762 CN201010113762 CN 201010113762 CN 201010113762 A CN201010113762 A CN 201010113762A CN 101768619 A CN101768619 A CN 101768619A
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Abstract
The invention discloses a method for preparing rare ginsenoside IH-901 with Rd as a substrate. The scheme of the method is as follows: 1 unit mass of monomer ginsenoside Rd extracted from fiveleaf gynostemma herb, 0.5 to 1 unit mass of domestic snail enzyme and 1 unit mass of sterile water are uniformly mixed; after heat preservation is carried out for 72 to 120 hours under the temperature of 30 to 45 DEG C, the monomer ginsenoside Rd can be converted into the rare ginsenoside IH-901. The method for preparing the rare ginsenoside IH-901 adopts the monomer ginsenoside Rd extracted from the fiveleaf gynostemma herb as the reaction substrate, and adopts the cheap domestic snail enzyme as the reaction enzyme; compositions in a reacted system are simple, and the extraction of subsequent products is pretty simple. The content of the monomer ginsenoside Rd in the fiveleaf gynostemma herb is 8 times higher than that in ginseng. Compared with the prior art, the preparation method in the invention has the following advantages: the high-content monomer ginsenoside Rd in the cheap fiveleaf gynostemma herb is adopted as raw materials, the cheap domestic snail enzyme is adopted, and the sterile water is adopted as a reaction medium; therefore, the cost is low, the compositions in the system are simple after the reaction is finished so as to be beneficial for product extracting, and the like.
Description
Technical field
The present invention relates to a kind of method for preparing rare ginsenoside IH-901.
Background technology
Modern pharmacological research shows, the final active substance of genseng performance antitumor action is not the natural ginseng saponin as the Ginsenoside Rb
1, Rb
2, Rb
3, Rc, Rd etc., but the metabolite that the effect of its a series of intestinal microfloras is produced down, promptly the natural ginseng saponin is antitumor natural prodrug, ginsenoside entero-bacte meta-bolites is only anti-tumor activity essence or active entity.The metabolism of ginsenoside discovers that wherein, ginsenoside entero-bacte metabolite: 20-O-β-D-glucopyranoside-20 (S)-protopanoxadiol (being called for short rare ginsenoside IH-901, M1 or Compound K) is natural glycol group ginsenoside such as Rb
1, Rb
2Etc. the final main metabolites of oral back, be one of final main activity form of ginsenoside antitumor action by the secreted Glycosylase selective hydrolysis C-3 position carbohydrate ligands gained of intestinal bacteria.And in a large number about the constantly confirmation of metabolism relation research of natural ginseng saponin and IH-901, the glycol group saponin that some content are higher, polarity is bigger can be that IH-901 is absorbed by body by entero-bacte metabolism eventual degradation all.
Rare ginsenoside IH-901 belongs to glycol group saponin, does not exist in natural genseng, is the catabolites of other glycol group ginsenosides in people's enteron aisle.Many in recent years many biologic activity of genseng that studies show that are caused by this compound that all particularly antitumor, anti-ageing, aspects such as anti-inflammatory all embody good drug effect.The focus of its research at present embodies a concentrated reflection of mass preparation and two aspects of biological activity further investigation of IH-901 compound, and China still is in the starting stage to the research of this compound, does not see the systematic study report.Because IH-901 is an interior metabolism product, thus nontoxic, studies show that to have anti-tumor activity widely, so far, the clinical value of rare ginsenoside IH-901 has caused that various countries pay much attention to, and becomes the focus of each big drugmaker research.
IH-901 will be a up-and-coming antitumor drug, following will the listing successively with healthcare products and national new drug form in succession, the scarce ginsenoside project is in 21 century biological medicine and biological healthy product are forward-looking, scarcity and the huge property of demand, and domestic and international market has great potential to its demand.Rare ginsenoside IH-901 costs an arm and a leg owing to it at present, content reaches 1,000 ten thousand yuan/kilogram in 99% above price on the market, and only 800 yuan/kilogram of ginsenosides be difficult to be used by tumour patient, so the production method of rare ginsenoside IH-901 receive much concern.
Because the specificity of 3 and 20 glycosidic links on the ginsenoside tetracyclic triterpene mother nucleus structure determined IH-901 to be obtained by the bioconversion method of gentleness, and acid or basic hydrolysis method commonly used is impracticable.At present, bioconversion method generally includes microbial fermentation and two kinds of main method of enzymatic conversion.
If microbe fermentation method prepares used microbial host enteron aisle anerobe, and the research of other microorganisms is fewer.The enteron aisle anaerobic bacterial fermentation is to ferment with the total fungus in people's movement at first, the single entero-bacte of research screening afterwards transforms, as adopt enteron aisle lactic acid bacteria tranformation diol type saponin(e to prepare IH-901, effect is best when finding to use Bifidobacterium minimum KK-1 and B.choerinum KK-2 to ferment conversion Rb1 altogether, transformation efficiency reaches about 41%, but entero-bacte mostly is anerobe, culture condition harshness, culture medium cost height.Other has the research screening primary soil microorganism ginsenoside of degrading, but the enzymolysis ability is low, and the purpose product yield is not high, must improve conversion capability by methods such as optimization of fermentation conditions and induction mutation of bacterium.Dalian Chemiclophysics Inst., Chinese Academy of Sciences's application patent of invention prepares the method for rare low polarity ginsenoside with aspergillus niger enzymolysis ginsenoside, used black-koji mould is that the black-koji mould with various sources changes in the substratum that contains ginsenoside, the bacterial strain that can glycolysis produces scarce ginsenoside and glucoside unit that obtains through the cultivation of going down to posterity, domestication, screening.Be not suitable at present the sophisticated method or the technology of mass preparation aspect preparing at microbial fermentation.
And with reference to bacterium metabolism principle in the intestines, the enzyme transforming process preparation is to utilize enzyme to realize the process of chemical conversion as catalyzer, be that zymin is carried out the chemical reaction that structural modification took place to exogenous substrate, enzyme and enzyme system can be converted into many natural compoundss has higher bioactive material, and the advantage of enzymatic conversion method method is that flow process is short, specificity is strong, the easily separated purifying of product.The enzyme major part that is used to prepare is an industrial enzyme preparation, and the general enzyme with enzymolysis glycosidic link that adopts is as naringinase, polygalacturonase, cellulase, helicase and Sumylact L etc.Domestic scholars utilizes beta-glucosidase that notoginseng stem and leaf total saponin is transformed, and obtains the better conversion effect.Also have and separate the stronger saccharase of specificity from the bacterial strain with activity of conversion, the thick enzyme as extracting from Edible microorganismus Bifidobacterium sp.Int57 and Bifidobacterium sp.SJ32 can transform Rb
2Become Rd and F with Rc
2, and then changing into end product ginsenoside IH-901, the glucuroide from people's entero-bacte Fusodobacterium K-60 separation and purification also can directly transform Rb
1Generate rare ginsenoside IH-901.The consumption of enzyme is big, the source is difficult, the more high shortcoming of substrate composition but these methods also exist, so aforesaid method also still can't be applied to suitability for industrialized production.Strong, the general product of enzyme transforming process selectivity also is convenient to extraction separation, and technical study control is convenient, and this preparation method more helps scale preparation, and its research is crucial to be to obtain cheapness and efficient single-minded industrial enzyme preparation arranged.
Along with going deep into of natural ginseng oside compound enteron aisle metabolism research, what studies show that its effect of entero-bacte metabolic degradation ginsenoside is not thalline itself, but entero-bacte excretory biological enzyme, metabolic essence is that enzyme has carried out bio-modification or transformation to the structure of natural ginseng saponin, directly embodies bioactive meta-bolites and generate.With reference to the entero-bacte metabolic mechanism, the IH-901 preparation method mainly contains microbe fermentation method and two kinds of methods of enzymatic conversion method method at present.Microbe fermentation method is because the culture condition harshness of fermentation is cultivated the cost height, and fermentation also needs to search out tool thalline efficiently, still can't satisfy the suitability for industrialized production needs.Biological method enzymolysis transforms preparation, because enzyme digestion reaction gentleness, condition is easy to control, the selectivity of enzyme, specificity in addition only act on the fixed position of special construction, to other structure and not influence of sterie configuration, and the stability of the glycosidic link of ginsenoside 20 position in acidic medium is lower than 3 glycosidic links, and the steric restriction effect makes that the glycosidic link vigor of 20 of enzymic hydrolysiss is very low, and therefore, enzyme transforming process becomes the main preparation methods of present employing.It is to screen wide material sources, low, the efficient narrow spectrum biological enzyme of cost that the external enzymatic conversion method of accomplishing scale production prepares key, and utilizes cheap enzymatic conversion starting material, sets up applicable to industrialization process.At present according to research reports; there are enzymatic conversion method China genseng, Radix Ginseng, Radix Panacis Quinquefolii or pseudo-ginseng etc. to be the technical study of rare ginsenoside IH-901 and bibliographical information both at home and abroad; Shang Weijian is the report of raw material with the gynostemma pentaphylla of cheapness; and there are shortcomings such as the source difficulty, raw materials cost height of enzyme mostly in these methods, still can't be applied to scale preparation or suitability for industrialized production.
Gynostemma pentaphylla [Gynostemma pentaphyllum (Thunb.) Makino] has another name called Rhizome or herb of Fiveleaf Gynostemma, Pentapanax leschenaultii, Herb Gynostemmae Pentaphylli etc., is the herbaceous perennial vine plant of Curcurbitaceae gynostemma pentaphyllum genus.Gynostemma total saponin content is 3 times of genseng, and wherein the content of monomer ginsenoside Rd is in the genseng 8 times.Having not yet to see with the extremely abundant monomer ginsenoside Rd of content in gynostemma pentaphylla is substrate, is converted into the technical study and the bibliographical information of rare ginsenoside IH-901 through homemade helicase.
Summary of the invention
The purpose of this invention is to provide a kind of is substrate with monomer ginsenoside Rd cheap, that be easy to get, through the enzyme catalysis of cheapness, prepares the method for expensive rare ginsenoside IH-901.
Technical scheme of the present invention is such: a kind of is the method that substrate prepares rare ginsenoside IH-901 with Rd, be achieved by the following scheme: monomer ginsenoside Rd, 0.5-1 unit mass home-made helicase and 1 unit volume sterilized water that 1 unit mass is extracted from gynostemma pentaphylla mix, after 30-45 ℃ is incubated 72-120 hour down, just the monomer ginsenoside Rd can be converted into rare ginsenoside IH-901.
After adopting such scheme, the method for preparing rare ginsenoside IH-901 of the present invention is a reaction substrate with the single ginsenoside Rd that extracts from gynostemma pentaphylla, and the enzyme of reaction is cheap homemade helicase, composition is simple in the reacted system, and follow-up product extracts very simple.Because the content of monomer ginsenoside Rd is in the genseng 8 times in the gynostemma pentaphylla, compared with prior art, preparation method of the present invention, the raw material that adopts is the monomer ginsenoside Rd that content is very high in the gynostemma pentaphylla of cheapness, the enzyme that adopts is cheap homemade helicase, the reaction medium that adopts is a sterilized water, and it is low to have a cost, and reaction finishes, and composition simply helps advantages such as product extraction in the system of back.
Embodiment
Embodiment one:
Of the present invention a kind of be the method that substrate prepares rare ginsenoside IH-901 with Rd, be achieved by the following scheme:
1. get 1 milligram of extraction from the monomer ginsenoside Rd of gynostemma pentaphylla in aseptic reaction tubes, add 0.5 milligram of home-made helicase and 1 ml sterile water;
2. monomer ginsenoside Rd, helicase and sterilized water are mixed, in 30 ℃ of waters bath with thermostatic control 120 hours, reaction substrate monomer ginsenoside Rd was proved through thin-layer chromatography and liquid-phase chromatographic analysis and is converted into the product rare ginsenoside IH-901.
Embodiment two:
Of the present invention a kind of be the method that substrate prepares rare ginsenoside IH-901 with Rd, be achieved by the following scheme:
1. get 1 milligram of extraction from the monomer ginsenoside Rd of gynostemma pentaphylla in aseptic reaction tubes, add 0.75 milligram of home-made helicase and 1 ml sterile water;
2. monomer ginsenoside Rd, helicase and sterilized water are mixed, in 40 ℃ of waters bath with thermostatic control 100 hours, reaction substrate monomer ginsenoside Rd was proved through thin-layer chromatography and liquid-phase chromatographic analysis and is converted into the product rare ginsenoside IH-901.
Embodiment three:
Of the present invention a kind of be the method that substrate prepares rare ginsenoside IH-901 with Rd, be achieved by the following scheme:
1. get 1 milligram of extraction from the monomer ginsenoside Rd of gynostemma pentaphylla in aseptic reaction tubes, add 1 milligram of home-made helicase and 1 ml sterile water;
2. monomer ginsenoside Rd, helicase and sterilized water are mixed, in 45 ℃ of waters bath with thermostatic control 72 hours, reaction substrate monomer ginsenoside Rd was proved through thin-layer chromatography and liquid-phase chromatographic analysis and is converted into the product rare ginsenoside IH-901.
Claims (1)
1. one kind is the method that substrate prepares rare ginsenoside IH-901 with Rd, it is characterized in that: be achieved by the following scheme: monomer ginsenoside Rd, 0.5-1 unit mass home-made helicase and 1 unit volume sterilized water that 1 unit mass is extracted from gynostemma pentaphylla mix, after 30-45 ℃ is incubated 72-120 hour down, just the monomer ginsenoside Rd can be converted into rare ginsenoside IH-901.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251009A (en) * | 2011-06-09 | 2011-11-23 | 华侨大学 | Simple production method of rare ginsenoside IH-901 |
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| CN86105166A (en) * | 1985-07-22 | 1987-03-04 | 武田药品工业株式会社 | Produce a kind of method of ginsenoside-rd |
| CN1417345A (en) * | 2001-11-06 | 2003-05-14 | 中国科学院大连化学物理研究所 | Ginsenoside enzymatically hydrolyzing process to prepare 2o-beta-D-pyranoglucose protopanoxadiol |
| CN1417342A (en) * | 2001-11-06 | 2003-05-14 | 中国科学院大连化学物理研究所 | Glusulase degrading process of natural product and polysaccharide |
| CN1508260A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Anti-tumour ginseng saponin preparing method |
| CN1508144A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Method for preparing true aglycone of ginseng saponin |
| CN1623554A (en) * | 2003-12-06 | 2005-06-08 | 山东绿叶天然药物研究开发有限公司 | Anticancer auxiliary medicine using C-K as effective component and its application |
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2010
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Patent Citations (6)
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| CN86105166A (en) * | 1985-07-22 | 1987-03-04 | 武田药品工业株式会社 | Produce a kind of method of ginsenoside-rd |
| CN1417345A (en) * | 2001-11-06 | 2003-05-14 | 中国科学院大连化学物理研究所 | Ginsenoside enzymatically hydrolyzing process to prepare 2o-beta-D-pyranoglucose protopanoxadiol |
| CN1417342A (en) * | 2001-11-06 | 2003-05-14 | 中国科学院大连化学物理研究所 | Glusulase degrading process of natural product and polysaccharide |
| CN1508260A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Anti-tumour ginseng saponin preparing method |
| CN1508144A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Method for preparing true aglycone of ginseng saponin |
| CN1623554A (en) * | 2003-12-06 | 2005-06-08 | 山东绿叶天然药物研究开发有限公司 | Anticancer auxiliary medicine using C-K as effective component and its application |
Non-Patent Citations (1)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102251009A (en) * | 2011-06-09 | 2011-11-23 | 华侨大学 | Simple production method of rare ginsenoside IH-901 |
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Open date: 20100707 |