CN101671714A - Method for preparing rare ginsenoside IH-901 - Google Patents
Method for preparing rare ginsenoside IH-901 Download PDFInfo
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- CN101671714A CN101671714A CN200910193156A CN200910193156A CN101671714A CN 101671714 A CN101671714 A CN 101671714A CN 200910193156 A CN200910193156 A CN 200910193156A CN 200910193156 A CN200910193156 A CN 200910193156A CN 101671714 A CN101671714 A CN 101671714A
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Abstract
The invention discloses a method for preparing rare ginsenoside IH-901, which is realized by the solution as below: 1 unit mass of monomer ginsenoside Rb1, 0.5-1 unit mass of domestic helicase and 1 unit volume of buffer solution with 3-5 of pH are mixed uniformly and are subject to heat preservation for 8 to 14 hours at 30 to 45 DEG C, and then the monomer ginsenoside Rb1 can be completely converted into the product of rare ginsenoside IH-901. According to the invention, the monomer ginsenoside Rb1 is used as reaction substrate and the cheap domestic helicase is used as enzyme for the reaction, so in the system after the reaction, the monomer ginsenoside Rb1 as reaction substrate, except for the ingredients of the buffer solution, is completely converted into the product of rare ginsenoside IH-901, and subsequent products can be extracted quite easily. Compared with the prior art, as single substrate and cheap domestic helicase are adopted, the preparation method has the advantages ofabundant sources of enzyme, low cost of raw materials and the fact that the ingredients in the system after the reaction are so simple as to be favorable for the extraction of the products.
Description
Technical field
The present invention relates to a kind of preparation method of 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 CK, 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 scarce ginsenoside CK.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; but 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.
Summary of the invention
The purpose of this invention is to provide a kind of is substrate with single saponin, the preparation method of the rare ginsenoside IH-901 that the source of enzyme is abundant, raw materials cost is low.
Technical scheme of the present invention is such: the preparation method of rare ginsenoside IH-901, be achieved by the following scheme: the buffered soln of 1 unit mass monomer ginsenoside Rb1,0.5-1 unit mass home-made helicase and 1 unit volume pH=3-5 is mixed, after 30-45 ℃ is incubated 8-14 hour down, just the monomer ginsenoside Rb1 can be converted into the product rare ginsenoside IH-901 fully.
Above-mentioned buffered soln is phosphoric acid citric acid solution or aqueous acetic acid.
After adopting such scheme, the preparation method of rare ginsenoside IH-901 of the present invention, with the monomer ginsenoside Rb1 is reaction substrate, the enzyme of reaction is cheap homemade helicase, in the reacted system except that the composition of buffer system, reaction substrate monomer ginsenoside Rb1 is converted into the product rare ginsenoside IH-901 fully, and follow-up product extracts very simple.Compared with prior art, preparation method of the present invention, owing to adopt single substrate and cheap homemade helicase, enrich in the source with enzyme, raw materials cost is low, and reaction finishes, and composition simply helps advantages such as product extraction in the system of back.
Embodiment
Embodiment one:
The preparation method of rare ginsenoside IH-901 of the present invention is achieved by the following scheme:
1. accurately draw 100 microlitre monomer ginsenoside Rb1s (containing 1 milligram of monomer ginsenoside Rb1) in aseptic reaction tubes, add the phosphoric acid citric acid solution of 1 milligram of home-made helicase and 1 milliliter of pH=3;
2. monomer ginsenoside Rb1, helicase and phosphoric acid citric acid solution are mixed, in 30 ℃ of waters bath with thermostatic control 14 hours, reaction substrate monomer ginsenoside Rb1 is converted into the product rare ginsenoside IH-901 fully, in the system after reaction finishes except that the composition of buffer system, only remaining product rare ginsenoside IH-901.
Embodiment two:
The preparation method of rare ginsenoside IH-901 of the present invention is achieved by the following scheme:
1. accurately draw 1000 microlitre monomer ginsenoside Rb1s (containing 10 milligrams of monomer ginsenoside Rb1s) in aseptic reaction tubes, add the aqueous acetic acid of 5 milligrams of home-made helicases and 10 milliliters of pH=4;
2. monomer ginsenoside Rb1, helicase and aqueous acetic acid are mixed, in 37 ℃ of waters bath with thermostatic control 11 hours, reaction substrate monomer ginsenoside Rb1 is converted into the product rare ginsenoside IH-901 fully, in the system after reaction finishes except that the composition of buffer system, only remaining product rare ginsenoside IH-901.
Embodiment three:
The preparation method of rare ginsenoside IH-901 of the present invention is achieved by the following scheme:
1. accurately draw 100 microlitre monomer ginsenoside Rb1s (containing 1 milligram of monomer ginsenoside Rb1) in aseptic reaction tubes, add the phosphoric acid citric acid solution of 0.6 milligram of home-made helicase and 1 milliliter of pH=5;
2. monomer ginsenoside Rb1, helicase and phosphoric acid citric acid solution are mixed, in 45 ℃ of waters bath with thermostatic control 8 hours, reaction substrate monomer ginsenoside Rb1 is converted into the product rare ginsenoside IH-901 fully, in the system after reaction finishes except that the composition of buffer system, only remaining product rare ginsenoside IH-901.
Extract and qualification process:
1. termination reaction adds the n-butanol extraction twice of reactant 1/2 volume, merges n-butanol layer;
2. draw on a small quantity with kapillary, point sample (activates 90 minutes down at 110 ℃ before using) on silica-gel plate, and cold wind dries up;
3. placing in the beaker that fills developping agent, is contrast with IH-901 standard substance and Rb1 standard substance, and with chloroform: methyl alcohol: water=65: 35: 10 is that developping agent launches;
Take out in about 1 centimetre away from silica-gel plate top be in the forward position of 4. treating developping agent, volatilizes solvent, evenly sprays the developer of 10% sulfuric acid, one ethanolic soln;
5. identify in 105 ℃ of heating colour developing in 10 minutes.
The compound method of related reagent:
1, developping agent
Chloroform: methyl alcohol: water=65: 35: 10 (lower floor)
Propyl carbinol: ethyl acetate: water=4: 1: 5 (upper strata)
Chloroform: ethyl acetate: methyl alcohol: water=15: 40: 20: 10 (lower floors)
Upper strata, lower floor be meant that solution preparation mixes and leave standstill the back layering, and what developping agent used is upper strata or lower floor, and remaining discards.
2, sulfuric acid one ethanol developer
Sulfuric acid: ethanol=1: 10.
3, Rb1 standard solution
20 milligrams of Rb1 add 10 microlitre DMSO (dimethyl sulfoxide (DMSO)), add ethanol to 2 milliliter ,-20 ℃ of freezing preservations.
4, IH-901 standard solution
20 milligrams of IH-901 add 10 microlitre DMSO (dimethyl sulfoxide (DMSO)), add ethanol to 2 milliliter, freezing preservation under-20 ℃ temperature.
5, phosphoric acid citrate buffer solution
| ??pH | ??0.2mol/L ??Na 2HPO 4(milliliter) | 0.1mol/L citric acid (milliliter) | ??pH | ??0.2mol/L ??Na 2HPO 4(milliliter) | 0.1mol/L citric acid (milliliter) |
| ??2.2 ??2.4 ??2.6 ??2.8 ??3.0 ??3.2 ??3.4 ??3.6 ??3.8 ??4.0 ??4.2 ??4.4 ??4.6 ??4.8 ??5.0 | ??0.40 ??1.24 ??2.18 ??3.17 ??4.11 ??4.94 ??5.70 ??6.44 ??7.10 ??7.71 ??8.28 ??8.82 ??9.35 ??9.86 ??10.30 | ??10.60 ??18.76 ??17.82 ??16.83 ??15.89 ??15.06 ??14.30 ??13.56 ??12.90 ??12.29 ??11.72 ??11.18 ??10.65 ??10.14 ??9.70 | ??5.2 ??5.4 ??5.6 ??5.8 ??6.0 ??6.2 ??6.4 ??6.6 ??6.8 ??7.0 ??7.2 ??7.4 ??7.6 ??7.8 ??8.0 | ??10.72 ??11.15 ??11.60 ??12.09 ??12.63 ??13.22 ??13.85 ??14.55 ??15.45 ??16.47 ??17.39 ??18.17 ??18.73 ??19.15 ??19.45 | ??9.28 ??8.85 ??8.40 ??7.91 ??7.37 ??6.78 ??6.15 ??5.45 ??4.55 ??3.53 ??2.61 ??1.83 ??1.27 ??0.85 ??0.55 |
Na
2HPO
4Molecular weight=14.98,0.2mol/L solution are 28.40 grams per liters.
Na
2HPO
4-2H
2O molecular weight=178.05,0.2mol/L solution contains 35.01 grams per liters.
C
4H
2O
7H
2O molecular weight=210.14,0.1mol/L solution are 21.01 grams per liters.
Claims (2)
1, the preparation method of rare ginsenoside IH-901, it is characterized in that: be achieved by the following scheme: the buffered soln of 1 unit mass monomer ginsenoside Rb1,0.5-1 unit mass home-made helicase and 1 unit volume pH=3-5 is mixed, after 30-45 ℃ is incubated 8-14 hour down, just the monomer ginsenoside Rb1 can be converted into the product rare ginsenoside IH-901 fully.
2, the preparation method of rare ginsenoside IH-901 according to claim 1 is characterized in that: above-mentioned buffered soln is phosphoric acid citric acid solution or aqueous acetic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN102251009A (en) * | 2011-06-09 | 2011-11-23 | 华侨大学 | Simple production method of rare ginsenoside IH-901 |
| CN102973623A (en) * | 2012-12-20 | 2013-03-20 | 刘淑莹 | Separation method of ginseng rare saponins in ginseng fibrils |
| CN116355879A (en) * | 2023-03-31 | 2023-06-30 | 吉林农业大学 | Preparation of compound oleanolic acid glucosyl ester with anti-tumor activity |
-
2009
- 2009-10-13 CN CN200910193156A patent/CN101671714A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN102251009A (en) * | 2011-06-09 | 2011-11-23 | 华侨大学 | Simple production method of rare ginsenoside IH-901 |
| CN102973623A (en) * | 2012-12-20 | 2013-03-20 | 刘淑莹 | Separation method of ginseng rare saponins in ginseng fibrils |
| CN116355879A (en) * | 2023-03-31 | 2023-06-30 | 吉林农业大学 | Preparation of compound oleanolic acid glucosyl ester with anti-tumor activity |
| CN116355879B (en) * | 2023-03-31 | 2024-06-07 | 吉林农业大学 | Preparation of compound oleanolic acid glucosyl ester with anti-tumor activity |
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Application publication date: 20100317 |