CN106282270A - A kind of method that Polydatin is glycosylation - Google Patents
A kind of method that Polydatin is glycosylation Download PDFInfo
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- CN106282270A CN106282270A CN201610596643.6A CN201610596643A CN106282270A CN 106282270 A CN106282270 A CN 106282270A CN 201610596643 A CN201610596643 A CN 201610596643A CN 106282270 A CN106282270 A CN 106282270A
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- polydatin
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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
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Abstract
The invention discloses a kind of method that Polydatin is glycosylation, with Polydatin as substrate, in the presence of cyclodextrin glycosyl transferases and glycosyl donor, pH be 5 ~ 7, temperature be the aqueous solution of 40 ~ 70 DEG C in stirring reaction, HPLC detection reaction process is not further added by product Polydatin glucosides, terminates reaction, then carry out the post-processing operation that product separates with substrate, concrete, first remove glycosyl donor unnecessary in reactant liquor, then use extraction or column chromatography product and substrate to be separated.The method using the present invention, solves the separation problem of Polydatin glucosides and Polydatin, and the conversion ratio of obtained Polydatin glucosides is high, and purity is good.
Description
Technical field
The invention belongs to biological chemical field, be specifically related to a kind of method that Polydatin is glycosylation.
Background technology
Polydatin is single glucoside of polyphenol compound resveratrol, and chemical name is 3,4 '-5-trihydroxies two
Styrene 3-O-D-glucoside, is mainly derived from the plants such as Semen arachidis hypogaeae, Fructus Vitis viniferae (red wine), Rhizoma Polygoni Cuspidati, Fructus Mori.Polydatin is
A kind of the strongest biological natural polyphenol class material, is the effective active composition of Chinese medicine, has an infection, prophylaxis of tumours,
Reduce platelet aggregation, prevent and treat atherosclerosis, cardiovascular and cerebrovascular disease etc., being with a wide range of applications.
Polydatin dissolubility in water is the highest, it is difficult to be absorbed by the body, and is therefore subject in food, medicine and cosmetic field
To limiting.Polydatin is carried out glycosylation after can preferably solve this problem.Cyclodextrin glycosyl is utilized to shift at present
Enzyme carries out the report that Polydatin is glycosylation, such as Process Biochemistry 47 (2012): 528 532, J. Agric.
Food Chem. 2012,60,8183 8189 etc..There is conversion ratio low (< 80%) in presently disclosed method, adjuvant is expensive
(alpha-cyclodextrin), uses the problems such as low cost adjuvant (such as starch etc.) conversion ratio is low.The most key, at present, due to cannot
Thoroughly being converted completely by substrate Polydatin, how presently disclosed method carries out the separation of product and substrate if not being reported for work, therefore difficult
With problems such as amplifications, it is difficult to practical.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of method that Polydatin is glycosylation.
For reaching above-mentioned purpose, the technical solution used in the present invention is: a kind of method that Polydatin is glycosylation, with Polydatin
For substrate, in the presence of cyclodextrin glycosyl transferases and glycosyl donor, pH be 5 ~ 7, during temperature is the aqueous solution of 40 ~ 70 DEG C
Stirring reaction, HPLC detection reaction process is not further added by product Polydatin glucosides, terminates reaction, then carries out product and substrate
The post-processing operation separated, concrete, first remove glycosyl donor unnecessary in reactant liquor, then use extraction or column chromatography to incite somebody to action
Product and substrate separate.
Preferably, when using extraction to carry out the post-processing operation that product separates with substrate, its detailed process is as follows: will be big
Hole resin I, by the soak with ethanol overnight rear wet method dress post that concentration is 95%, then flows out to without ethanol with distilled water flushing；Will
Loading after reacting liquid filtering, concentration, uses distilled water flushing to flow out to without glycosyl donor, the second using concentration to be 40% the most again
Alcohol rinses and flows out to all products, finally merges outturn sample, concentrates and use isopyknic ethyl acetate repeatedly to extract,
Collect water layer, lyophilizing obtains product sterling.
It is further preferred that described macroporous resin I is DM18 macroporous adsorbent resin.
It is further preferred that the glycosyl donor employed in described course of reaction is alpha-cyclodextrin, corn starch or its group
Close.
Preferably, when using column chromatography to carry out the post-processing operation that product separates with substrate, its detailed process is as follows:
Chromatographic column loads macroporous resin II by soaked in absolute ethyl alcohol overnight, use successively three times of chromatographic column volumes dehydrated alcohol and
Described macroporous resin II is rinsed by deionized water, stand-by；It is centrifuged reactant liquor processing, collects liquid, and loading, on
Using deionized water rinsing, the ethanol solution finally using concentration to be 30% to carry out eluting, collect eluent after sample, HPLC detects
Analyzing, merge the substrate content solution less than 1%, solvent is replaced as water, lyophilizing obtains product sterling.
It is further preferred that described macroporous resin II is to produce purchased from Xi'an Sunresin New Materials Co., Ltd.
Model be LXT-J420 macroporous adsorbent resin.
It is further preferred that the glycosyl donor used is corn starch.
Preferably, in course of reaction, the mass ratio that feeds intake of described cyclodextrin glycosyl transferases, glycosyl donor and Polydatin
For: 0.5-1:60-90:6.
Due to the utilization of technique scheme, the present invention compared with prior art has the advantage that the Rhizoma Polygoni Cuspidati of the present invention
The method that glycoside is glycosylation, compared with prior art, after glycosylation to Polydatin, then use extraction or column chromatography by product and
Substrate separates, it is possible to obtain purity reach more than 98%, content reach more than 90% product, saved production cost, improve
Product yield, solves substrate and product after Polydatin glucosides 1 is changed in prior art and is difficult to the problem separated, can be amplified raw
Produce, practical.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following enforcement
Example.The implementation condition used in embodiment can do further adjustment, not marked enforcement according to specifically used different requirement
Condition is the condition in normal experiment.
Embodiment 1(uses alpha-cyclodextrin to react as glycosyl donor)
Substrate Polydatin 18 g, adds cyclodextrin glycosyl transferases (purchased from amano enzyme preparation) 3 mL, glycosyl donor alpha-cyclodextrin
270 g, deionized water 2 L, regulate pH to 5.5, be heated to 60 DEG C, and after stirring 6 hours, HPLC detection conversion ratio is 90%, stops
Reaction, carries out post processing.
Embodiment 2(uses corn starch to react as glycosyl donor)
Substrate Polydatin 45 g, adds cyclodextrin glycosyl transferases (purchased from amano enzyme preparation) 7.5 mL, and glycosyl donor Semen Maydis forms sediment
Powder 450 g, deionized water 2 L, regulate pH to 5.5, be heated to 60 DEG C, and after stirring 6 hours, HPLC detection conversion ratio is 70%, stops
Only reaction, carries out post processing.
Embodiment 3(extraction post processing)
By the reactant liquor in embodiment 1 after filter paper filtering, after concentrating one times, cross DM18 macroporous resin (anti-vertical purchased from Shandong, Shandong
Chemical Co., Ltd. of section) to remove glycosyl donor, concretely comprise the following steps and first the ethanol that 100 g macroporous resin concentration are 95% is soaked
Steep overnight rewetting method dress post, use distilled water flushing to flow out to without ethanol, with distilled water flushing to without glycosyl donor stream after loading
Going out, the alcohol flushing using concentration to be 40% flows out to all products, merges outturn sample, is concentrated into 0.5 L, with isopyknic
Ethyl acetate extracts 10 times, collects water layer lyophilizing and obtains product 36 g(yield 90%), HPLC purity 98%, content 90%, HPLC divides
Analysis Polydatin content is 0.1%.
Embodiment 4 (column chromatography post processing)
Reactant liquor in embodiment 2 is centrifuged 10 min through 4000 r/m, collects liquid.Pre-install 9 L macroporous adsorbent resin LXT-
J420(is purchased from Xi'an Sunresin New Materials Co., Ltd.) in chromatographic column, use the most afterwards by 9 L soaked in absolute ethyl alcohol
3*9 L dehydrated alcohol rinses, stand-by with 3*9 L deionized water rinsing the most again.By above-mentioned centrifugal liquid loading and go with 2*9 L
Ionized water rinses, and then with 3*9 L 30% ethanol water eluting, collects last 3*9 L alcohol flushing post by 4.5 L/ bottles
The eluent that son obtains.Send HPLC to analyze above-mentioned receiving flask, merge the wherein Polydatin content receiving flask less than 1%, and by molten
Agent is replaced as water, and lyophilizing obtains product 54 g (yield 70%).HPLC purity 99%, product content 99%, HPLC analyzes Polydatin content
It is 0.2%.
Above-described embodiment, only for technology design and the feature of the explanation present invention, its object is to allow person skilled in the art
Scholar will appreciate that present disclosure and implements according to this, can not limit the scope of the invention with this.All according to the present invention
The equivalence that spirit is made changes or modifies, and all should contain within protection scope of the present invention.
Claims (8)
1. the method that a Polydatin is glycosylation, it is characterised in that with Polydatin as substrate, at cyclodextrin glycosyl transferases and sugar
In the presence of base donor, pH be 5 ~ 7, temperature be the aqueous solution of 40 ~ 70 DEG C in stirring reaction, HPLC detection reaction process is to producing
Product Polydatin glucosides is not further added by, and terminates reaction, then carries out the post-processing operation that product separates with substrate, concrete, first removes
Glycosyl donor unnecessary in dereaction liquid, then use extraction or column chromatography product and substrate to be separated.
The method that Polydatin the most according to claim 1 is glycosylation, it is characterised in that use extraction to carry out product and the end
During the post-processing operation that thing separates, its detailed process is as follows: by macroporous resin I by soak with ethanol that concentration is 95% overnight after
Wet method dress post, then flows out to without ethanol with distilled water flushing；By loading after reacting liquid filtering, concentration, use distilled water flushing
Flowing out to without glycosyl donor, the alcohol flushing using concentration to be 40% the most again to all products flows out, and finally merges product sample
Product, concentrate and use isopyknic ethyl acetate repeatedly to extract, and collection water layer, lyophilizing obtain product sterling.
The method that Polydatin the most according to claim 2 is glycosylation, it is characterised in that described macroporous resin I is DM18
Macroporous adsorbent resin.
The method that Polydatin the most according to claim 2 is glycosylation, it is characterised in that employed in described course of reaction
Glycosyl donor is alpha-cyclodextrin, corn starch or a combination thereof.
The method that Polydatin the most according to claim 1 is glycosylation, it is characterised in that use column chromatography carry out product and
During the post-processing operation that substrate separates, its detailed process is as follows: loads macroporous resin II in chromatographic column and soaks with dehydrated alcohol
Bubble overnight, uses the dehydrated alcohol of three times of chromatographic column volumes and deionized water to be rinsed described macroporous resin II successively, treats
With；Being centrifuged reactant liquor processing, collect liquid, and loading, use deionized water rinsing after loading, finally employing concentration is
The ethanol solution of 30% carries out eluting, collects eluent, and HPLC detects analysis, merges the substrate content solution less than 1%, by solvent
Being replaced as water, lyophilizing obtains product sterling.
The method that Polydatin the most according to claim 5 is glycosylation, it is characterised in that described macroporous resin II is for being purchased from
The model that Xi'an Sunresin New Materials Co., Ltd. produces is LXT-J420 macroporous adsorbent resin.
The method that Polydatin the most according to claim 5 is glycosylation, it is characterised in that the glycosyl donor used is Semen Maydis
Starch.
The method that Polydatin the most according to claim 1 is glycosylation, it is characterised in that in course of reaction, described ring is stuck with paste
Glycosyltransferase, glycosyl donor with the mass ratio that feeds intake of Polydatin be: 0.5-1:60-90:6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610596643.6A CN106282270B (en) | 2016-07-27 | 2016-07-27 | Method for glycosidation of polydatin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610596643.6A CN106282270B (en) | 2016-07-27 | 2016-07-27 | Method for glycosidation of polydatin |
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| CN106282270A true CN106282270A (en) | 2017-01-04 |
| CN106282270B CN106282270B (en) | 2020-06-16 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546503A (en) * | 2003-12-12 | 2004-11-17 | 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 |
| 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 |
| 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 |
-
2016
- 2016-07-27 CN CN201610596643.6A patent/CN106282270B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546503A (en) * | 2003-12-12 | 2004-11-17 | 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 |
| 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 |
| 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 |
Non-Patent Citations (6)
| Title |
|---|
| HYUNSU PARK ET AL: "Enzymatic Synthesis of Piceid Glucosides Using Maltosyltransferase from Caldicellulosiruptor bescii DSM 6725", 《J. AGRIC. FOOD CHEM.》 * |
| PARK HYUNSU ET AL: "Bioconversion of Piceid to Piceid Glucoside Using Amylosucrase from Alteromonas macleodii Deep Ecotype", 《J. MICROBIOL. BIOTECHNOL》 * |
| SINDHU MATHEW ET AL: "Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase", 《PROCESS BIOCHEMISTRY》 * |
| 罗永明，等: "《中药化学》", 30 September 2013 * |
| 苏奥: ""莱鲍迪苷A分离提纯方法的研究"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 赵如奎，等: "α-环糊精葡萄糖基转移酶催化合成α-熊果苷", 《生物加工过程》 * |
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