CN106282270B - Method for glycosidation of polydatin - Google Patents
Method for glycosidation of polydatin Download PDFInfo
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- CN106282270B CN106282270B CN201610596643.6A CN201610596643A CN106282270B CN 106282270 B CN106282270 B CN 106282270B CN 201610596643 A CN201610596643 A CN 201610596643A CN 106282270 B CN106282270 B CN 106282270B
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- polydatin
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- glycosyl donor
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
Abstract
The invention discloses a method for glycosidation of polydatin, which comprises the steps of taking polydatin as a substrate, carrying out stirring reaction in an aqueous solution with pH of 5-7 and temperature of 40-70 ℃ in the presence of cyclodextrin glycosyltransferase and glycosyl donor, finishing the reaction when the polydatin glucoside is not increased in the reaction process detected by HPLC (high performance liquid chromatography), then carrying out post-treatment operation of product and substrate separation, specifically, removing redundant glycosyl donor in the reaction liquid, and then separating the product and the substrate by adopting an extraction method or a column chromatography. The method solves the separation problem of polydatin glucoside and polydatin, and the obtained polydatin glucoside has high conversion rate and good purity.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a method for glycosidation of polydatin.
Background
The polydatin is a mono-glucoside of polyphenol compound resveratrol, has a chemical name of 3, 4' -5-trihydroxy stilbene 3-O-D-glucoside, and is mainly derived from plants such as peanut, grape (red wine), giant knotweed rhizome, mulberry and the like. The polydatin is a natural polyphenol substance with strong biological property, is an effective active ingredient of the traditional Chinese medicine, has the effects of resisting infection, preventing tumor, reducing platelet aggregation, preventing and treating atherosclerosis, cardiovascular and cerebrovascular diseases and the like, and has wide application prospect.
The method disclosed at present has the problems of low conversion rate (<80%), expensive auxiliary materials (α -cyclodextrin), low conversion rate by adopting low-cost auxiliary materials (such as starch and the like), and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for glycosidation of polydatin.
In order to achieve the purpose, the invention adopts the technical scheme that: a polydatin glycosidation method comprises the steps of taking polydatin as a substrate, carrying out stirring reaction in an aqueous solution with pH of 5-7 and temperature of 40-70 ℃ in the presence of cyclodextrin glycosyltransferase and glycosyl donor, finishing the reaction when the polydatin glucoside is not increased in the reaction process detected by HPLC (high performance liquid chromatography), carrying out post-treatment operation of product and substrate separation, specifically, removing redundant glycosyl donor in a reaction liquid, and separating the product and the substrate by an extraction method or a column chromatography.
Preferably, when the extraction method is used for post-treatment operation of separating the product from the substrate, the specific process is as follows: soaking the macroporous resin I in 95% ethanol overnight, packing the macroporous resin I into a column by a wet method, and washing the column by distilled water until no ethanol flows out; and filtering and concentrating the reaction solution, loading the reaction solution, washing the reaction solution by using distilled water until a sugar-free donor flows out, then washing the reaction solution by using 40% ethanol until all products flow out, finally combining the product samples, concentrating the product samples, extracting the product samples for multiple times by using ethyl acetate with the same volume, collecting a water layer, and freeze-drying the product samples to obtain a pure product.
Further preferably, the macroporous resin I is DM18 macroporous adsorption resin.
Further preferably, the glycosyl donor used during the reaction is α -cyclodextrin, corn starch or a combination thereof.
Preferably, when the post-treatment operation of separating the product from the substrate is performed by column chromatography, the specific process is as follows: loading macroporous resin II into a chromatographic column, soaking the macroporous resin II in absolute ethyl alcohol overnight, and sequentially washing the macroporous resin II by adopting absolute ethyl alcohol and deionized water with the volume being three times that of the chromatographic column for later use; and (3) centrifuging the reaction solution, collecting the liquid, loading the sample, washing the sample with deionized water, eluting the sample with 30% ethanol solution, collecting the eluent, performing HPLC (high performance liquid chromatography) detection analysis, combining the solutions with the substrate content of less than 1%, replacing the solvent with water, and freeze-drying to obtain a pure product.
Further preferably, the macroporous resin II is a type LXT-J420 macroporous adsorption resin which is available from New science and technology materials, Inc. of Xian blue, West.
Further preferably, the glycosyl donor used is corn starch.
Preferably, in the reaction process, the charging mass ratio of the cyclodextrin glycosyltransferase, the glycosyl donor and the polydatin is as follows: 0.5-1: 60-90:6.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: compared with the prior art, the method for glycosidating the polydatin adopts the extraction or column chromatography method to separate the product and the substrate after the polydatin is glycosidated, can obtain the product with the purity of more than 98 percent and the content of more than 90 percent, saves the production cost, improves the product yield, solves the problem that the substrate and the product are difficult to separate after the polydatin is glycosidated in the prior art, can carry out scale-up production, and has strong practicability.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.
Example 1 (reaction with α -Cyclodextrin as glycosyl Donor)
Adding cyclodextrin glycosyltransferase (purchased from Tianye enzyme preparation) 3 mL, glycosyl donor α -cyclodextrin 270 g and deionized water 2L into substrate polydatin 18 g, adjusting pH to 5.5, heating to 60 deg.C, stirring for 6 hr, detecting conversion rate by HPLC to 90%, stopping reaction, and performing post-treatment.
Example 2 (reaction using corn starch as glycosyl donor)
Adding cyclodextrin glycosyltransferase (purchased from Tianye enzyme preparation) 7.5 mL, glycosyl donor corn starch 450 g and deionized water 2L into substrate polydatin 45 g, adjusting pH to 5.5, heating to 60 deg.C, stirring for 6 hr, detecting conversion rate by HPLC to 70%, stopping reaction, and performing post-treatment.
Example 3 (post-extraction treatment)
The reaction solution in example 1 was filtered through filter paper, concentrated by one time, and passed through DM18 macroporous resin (purchased from santong, lu, anti-lithology chemical limited) to remove glycosyl donor, which specifically includes steps of soaking 100 g macroporous resin with 95% ethanol overnight, packing with wet method, washing with distilled water until no ethanol flows out, washing with distilled water until no glycosyl donor flows out after loading, washing with 40% ethanol until all products flow out, combining product samples, concentrating to 0.5L, extracting with equal volume of ethyl acetate for 10 times, collecting water layer, and freeze-drying to obtain 36 g product (yield 90%), HPLC purity 98%, content 90%, and content of polydatin 0.1% by HPLC analysis.
Example 4 (column chromatography post-treatment)
The reaction solution in example 2 was centrifuged at 4000 r/m for 10 min, and the solution was collected. 9L of macroporous adsorption resin LXT-J420 (from New science and technology materials Co., Ltd., Xian blue) was prepackaged in a chromatographic column, soaked overnight with 9L of absolute ethanol, washed with 3X 9L of absolute ethanol, and then washed with 3X 9L of deionized water for further use. The above centrifuged liquid was loaded and washed with 2 x 9L deionized water, then eluted with 3 x 9L 30% aqueous ethanol, and the eluate from the last 3 x 9L ethanol washing of the column was collected at 4.5L/vial. And (3) sending the collecting bottles to HPLC analysis, combining the collecting bottles with the polydatin content of less than 1%, replacing the solvent with water, and freeze-drying to obtain 54 g of a product (yield is 70%). Purity of HPLC 99%, product content 99%, and polydatin content 0.2% by HPLC analysis.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (2)
1. A polydatin glycosylation method is characterized in that polydatin is used as a substrate, the polydatin is stirred and reacted in an aqueous solution with pH of 5-7 and temperature of 40-70 ℃ in the presence of cyclodextrin glycosyltransferase and glycosyl donor, HPLC (high performance liquid chromatography) detects that the reaction progresses until the polydatin glucoside is not increased any more, the reaction is ended, and then post-treatment operation of product and substrate separation is carried out;
when the post-treatment operation of separating the product and the substrate is carried out on the reaction liquid of which the glycosyl donor is α -cyclodextrin by adopting an extraction method, the specific process comprises the following steps of soaking macroporous resin I by ethanol with the concentration of 95% overnight, carrying out wet column packing, washing by distilled water until no ethanol flows out, filtering and concentrating the reaction liquid, loading the reaction liquid, washing by distilled water until no glycosyl donor flows out, washing by ethanol with the concentration of 40% until all products flow out, finally combining the product samples, concentrating, extracting for multiple times by ethyl acetate with the same volume, collecting a water layer, and freeze-drying to obtain a pure product, wherein the macroporous resin I is DM18 macroporous adsorption resin;
when the post-treatment operation of separating the product from the substrate is carried out on the reaction solution of which the glycosyl donor is the corn starch by adopting a column chromatography, the specific process is as follows: loading macroporous resin II into a chromatographic column, soaking the macroporous resin II in absolute ethyl alcohol overnight, and sequentially washing the macroporous resin II by adopting absolute ethyl alcohol and deionized water with the volume being three times that of the chromatographic column for later use; centrifuging the reaction solution, collecting the liquid, loading the sample, washing the sample with deionized water, eluting with 30% ethanol solution, collecting the eluate, performing HPLC detection and analysis, mixing the solutions with the substrate content of less than 1%, replacing the solvent with water, and lyophilizing to obtain pure product; the macroporous resin II is a type LXT-J420 macroporous adsorption resin which is purchased from New science and technology materials Co.
2. The method of claim 1, wherein the cyclodextrin glycosyltransferase, the glycosyl donor, and the polydatin are fed in a mass ratio of: 0.5-1: 60-90:6.
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| CN106282270B true CN106282270B (en) | 2020-06-16 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243140A (en) * | 2013-04-19 | 2013-08-14 | 江南大学 | Preparation method of composite cyclodextrin |
| CN103757074A (en) * | 2014-01-16 | 2014-04-30 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside M through enzyme method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1269831C (en) * | 2003-12-12 | 2006-08-16 | 深圳海王药业有限公司 | Method for preparing polygonin and resveratrol |
| CN102344472A (en) * | 2010-08-06 | 2012-02-08 | 苏州瑞蓝博中药技术开发有限公司 | Piceid extraction technology |
| CN105641219A (en) * | 2016-01-25 | 2016-06-08 | 济南星懿医药技术有限公司 | Pharmaceutical composition for treating depression |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243140A (en) * | 2013-04-19 | 2013-08-14 | 江南大学 | Preparation method of composite cyclodextrin |
| CN103757074A (en) * | 2014-01-16 | 2014-04-30 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside M through enzyme method |
Non-Patent Citations (5)
| Title |
|---|
| Bioconversion of Piceid to Piceid Glucoside Using Amylosucrase from Alteromonas macleodii Deep Ecotype;Park Hyunsu et al;《J. Microbiol. Biotechnol》;20120907;第22卷(第12期);第1698-1704页 * |
| Enzymatic Synthesis of Piceid Glucosides Using Maltosyltransferase from Caldicellulosiruptor bescii DSM 6725;Hyunsu Park et al;《J. Agric. Food Chem.》;20120723(第60期);第8183-8189页 * |
| Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase;Sindhu Mathew et al;《Process Biochemistry》;20111122(第47期);摘要、第第2.2、2.4、2.6及第3节、表1 * |
| Sindhu Mathew et al.Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase.《Process Biochemistry》.2011,(第47期), * |
| α-环糊精葡萄糖基转移酶催化合成α-熊果苷;赵如奎，等;《生物加工过程》;20140731;第13卷(第4期);摘要、第1.2-1.5及第2节 * |
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