CN1259428C - Synthesis process for enzymatic of sedopeptose natural product - Google Patents
Synthesis process for enzymatic of sedopeptose natural product Download PDFInfo
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- CN1259428C CN1259428C CN 200410016652 CN200410016652A CN1259428C CN 1259428 C CN1259428 C CN 1259428C CN 200410016652 CN200410016652 CN 200410016652 CN 200410016652 A CN200410016652 A CN 200410016652A CN 1259428 C CN1259428 C CN 1259428C
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- glucose
- tyrosol
- rhodioside
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Abstract
The present invention an enzymatic synthesis process for a natural product of salidroside, which has the technical scheme that home-made apple seed powder is used as a cheap catalytic agent; glucose or/and P-Hydroxyphenylethanol are used as substrate and are reacted in a single-phase organic solvent which is water by one step, and the salidroside is compounded; the yield is 15.8%. The present invention has the advantages that the separating and extracting process of products are simple, unreacted tyrosol can be completely recovered and reused, and production cost is relatively low. The present invention is a new method for preparing the natural medicine of pure salidroside products and has good prospect of industrial application and development.
Description
Technical field
The invention belongs to biological chemical field, relate to the preparation method of rhodioside, be specifically related to the synthetic and purifying process of rhodioside enzyme process.
Rhodioside, or be called rhodioloside, be the main effective constituent that is studied maximum in the rhodiola plant, have pharmacological actions such as anti-hypoxia, antifatigue, resisting microwave radiation, stimulating central nervous system, therefore as dietary supplements and medicine, rhodioside has good application prospects (environmental adaptation medicine).At present, mainly be to separate from the Rhodida plant rhizome by the method for extracting to obtain rhodioside, general process is: rhizome immersion, alcohol reflux extraction, organic solvent extraction, polymeric adsorbent and silica gel column chromatography.The step complexity is generally extracted productive rate 0.4%～0.8%.The process of chemosynthesis rhodioside mainly contains: pass through into glucoside, deacetylation with excessive tyrosol and synthesized rhodioside; With the 4-hydroxyphenylacetic acid ethyl ether is that raw material has synthesized rhodioside through steps such as benzylization, reduction, glucosideization and debenzylations; Be that raw material is through allylation, epoxidation, glucosideization with take off steps such as allyl group and synthesized rhodioside with the p bromophenol; With the p-hydroxyphenylaceticacid is raw material, through the synthetic rhodioside of steps such as benzylization, esterification, reduction, glucosideization.In a word, the chemosynthesis step is more, must be through hydroxyl protection and deprotection steps.People begin to utilize microorganism or enzyme to carry out the synthetic or modification of biocatalysis of natural product in recent years.Because the biocatalytic reaction process is simple, mild condition, easy to operate, environmental pollution is little, can obtain the compound of single steric configuration and becomes the problem that many scientific and technical personnel pay close attention to.At present, synthesize directly related open report seldom with the rhodioside enzymatic, have only one piece of document to relate to the technology of this respect: Tetrahedron Asymmetry 1999,10:2429-2439 discloses the reaction of a kind of employing almond beta-glucosidase (β-D-glucosidase from almond, Sigma G-0395) catalysis commentaries on classics glucose glycosidation and has synthesized the method that a series of natural beta-glucosides comprise rhodioside.But this method adopts expensive glucokinase activity glycosides (as the p-nitrophenyl glucoside) as the glucosyl group donor, and most glucokinase activity glycosides generation hydrolysis reaction, can not reclaim, and synthetic cost is very high, is not suitable for industrial applications.
In sum, the technology of the synthetic rhodioside of existing enzymatic adopts expensive commercial enzyme and glycosyl donor, and cost is very high, has therefore limited this method in industrial applying.
The technical issues that need to address of the present invention are the effective enzymatic synthesis methods that disclose a kind of natural product rhodioside, to overcome the above-mentioned defective of prior art.
Design of the present invention is such:
The contriver finds, apple seed powder is as a kind of glucuroide, on catalytic performance with almond beta-glucosidase (β-D-glucosidase from almond, Sigma G-0395) suitable, and prepare easy, cheap, stability high (because of in fact apple seed itself has served as immobilization material).Therefore as the catalyzer of enzymatic glycosylation reaction production cost is significantly reduced with self-control apple seed powder.
Because the activity of enzyme is subjected to the influence of buffered soln kind and pH in the system, so the contriver selects suitable buffered soln and pH can improve the speed of reacting in reaction system;
Because the substrate (tyrosol) of enzyme reaction is a solid, solvability is limited in water, therefore is necessary to add the dispersion situation that a kind of solubility promoter improves substrate in reaction system, improves its solubleness in reaction system, to accelerate the speed of enzyme reaction;
Because glucose and tyrosol are condensed into the control that the reaction (claiming glucosides to separate reaction against the current again) of glucosides is subjected to thermodynamic(al)equilibrium, therefore the contriver is by the water-content in the regulation system, make to be reflected in the low water content and carry out, and suitably improve the concentration of substrate tyrosol, can significantly improve the equilibrium yield of rhodioside.
According to above design, the contriver has proposed realization as described below technical scheme of the present invention:
(1) method of the present invention comprises the steps:
Apple seed powder, substrate glucose and tyrosol (p-hydroxyphenylethanol) are reacted in buffered soln and organic solvent, and the processing condition of reaction are such:
Said apple seed powder is for adopting the product after general apple seed is removed the peel, ground, and the add-on of apple seed powder is 1～100g/L;
Said organic cosolvent is through preferably adopting a kind of in dioxane, the trimethyl carbinol, acetonitrile, acetone, dimethyl sulfoxide (DMSO) or the dimethyl formamide;
Temperature of reaction is 20～70 ℃, and optimum temps is 50～60 ℃;
Reaction times is 1～5 day, specifically can decide according to the actual process of reaction;
The pH of buffered soln is 4～8, and best pH is 5.5～6.5;
Glucose concn: 0.01～0.50M, optimum concn is 0.2～0.3M;
The mol ratio of glucose and tyrosol is: glucose: tyrosol=1: (1～15), optimum proportion 1: (5-10);
Volume of organic solvent content is 0～99% (v/v) in the system, and optimum content is 80～99% (v/v).
(2) adopt conventional separation method,, can from reaction solution, collect the product that the present invention mentioned--rhodioside and unreacted tyrosol as extraction and column chromatography etc.
Adopt enzymatic process disclosed in this invention, can not only obtain highly purified rhodioside simply and easily, and production cost is more much lower than existing technology, it is a kind of production method with wide application prospect, can satisfy the needs of the medicine industry that develops rapidly, below will be further described technology contents of the present invention by embodiment.
Embodiment
Embodiment 1
The preparation of apple seed powder: the apple seed is removed the peel, ground, wash 3 times with ethyl acetate earlier, wash 2 grease removal with acetone again and dewater, place the vacuum drying oven drying.Can obtain 8g apple seed powder from 28kg apple total, it is stand-by to put 4 ℃ of refrigerator cold-storages.
Embodiment 2
In 50ml ground triangular flask, add 250mg apple seed powder successively, 0.272g (1.37mmol) glucose, 0.5ml phosphoric acid buffer (0.07M Na
2HPO
4-KH
2PO
4, pH 6.0), 2.28g tyrosol (16.5mmol), 5.0ml dioxane, jam-pack ground bottle stopper, and seal, oscillatory reaction 120h on 50 ℃, the rotary shaking table of 160r/min with raw material band.
Reaction finishes back adding methyl alcohol 10ml and removes by filter enzyme, and the enzyme that leaches is used methanol wash (20ml * 3) again, adds anhydrous sodium sulfate drying in filtrate, rotary evaporation removes and desolvates, rapid column chromatography (ethyl acetate/methanol=13/1) gets rhodioside solid 66mg, productive rate 15.8%.
Embodiment 3
Add 2.16g (10mmol) glucose, 4ml damping fluid (0.07MNa in the 250ml ground triangular flask
2HPO
4-KH
2PO
4, pH6.0) and 16.7g (0.11mol) p-hydroxyphenylethanol, adding 2g apple seed powder and 40ml dioxane again, jam-pack ground bottle stopper, and seal with raw material band was put on 50 ℃, the rotary shaking table of 160r/min reaction 5 days.Measure the concentration of product rhodioside in the reaction process with the HPLC method: sample liquid 0.04ml is answered in negate, and the methanol solution and the 0.86ml methanol mixed that add 0.10ml p-NP (0.5mM) are even, use the reversed-phase HPLC post then, the concentration of assay products Root of Kirilow Rhodiola.Chromatographic column YWS C-18 (Merck, 200 * 5.0mm, 10 μ); Moving phase be methanol (40/60, v/v, 0.8ml/min); UV-detector wavelength 275nm.The appearance time of glucosides, interior mark and substrate is respectively 4.0,5.7 and 17.6min.
Reaction finishes back adding methyl alcohol 20ml and removes by filter enzyme, and the enzyme that leaches is used methanol wash three times (20ml * 3) again, and the filtrate rotary evaporation removes and desolvates.Remaining solid adds 200ml ethyl acetate and 100ml deionized water dissolving, pours the separating funnel vibration into, standing demix, and ethyl acetate is used 100ml deionized water back extraction one time mutually again.Merge water, and with the 100ml ethyl acetate extraction once, the combined ethyl acetate phase, rotary evaporation removes to desolvate and promptly obtains unreacted most of substrate tyrosol.The water rotary evaporation except that anhydrating, is carried out rapid column chromatography (ethyl acetate/methanol=12/1) after the drying, get rhodioside solid 0.51g, productive rate 15.5%.
Embodiment 4
Adopt the identical method of embodiment 2, and to adopt the 5.0ml trimethyl carbinol be organic solvent, the result gets rhodioside solid 58mg, productive rate 13.9%.
Embodiment 5
Adopt the identical method of embodiment 2, but do not add organic solvent, the result obtains rhodioside 18mg, productive rate 4.3%.
Claims (10)
1. the enzymatic synthesis method of rhodioside is characterized in that, comprises the steps:
Apple seed powder, substrate glucose and tyrosol are reacted in buffered soln and organic solvent, adopt conventional separation method to collect product-rhodioside then from reaction solution, said organic solvent adopts a kind of in dioxane, the trimethyl carbinol, acetonitrile, acetone, dimethyl sulfoxide (DMSO) or the dimethyl formamide.
2. method according to claim 1 is characterized in that, the add-on of apple seed powder is 1～100g/L.
3. method according to claim 1 is characterized in that, the pH of buffered soln is 4～8.
4. method according to claim 1 is characterized in that, volume of organic solvent content is 0～99% (v/v) in the system.
5. method according to claim 4 is characterized in that, volume of organic solvent content is 80～99% (v/v) in the system.
6. method according to claim 1 is characterized in that, temperature of reaction is 20～70 ℃.
7. method according to claim 6 is characterized in that, temperature of reaction is 50～60 ℃.
8. method according to claim 1 is characterized in that, the reaction times is 1～5 day.
9. according to each described method of claim 1～8, it is characterized in that glucose concn is: 0.01～0.5M, the mol ratio of glucose and tyrosol is: glucose: tyrosol=1: (1～15).
10. method according to claim 1 is characterized in that, glucose concn is: 0.2～0.3M, the mol ratio of glucose and tyrosol is: glucose: tyrosol=1: (5～10).
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| CN 200410016652 CN1259428C (en) | 2004-03-01 | 2004-03-01 | Synthesis process for enzymatic of sedopeptose natural product |
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| CN 200410016652 CN1259428C (en) | 2004-03-01 | 2004-03-01 | Synthesis process for enzymatic of sedopeptose natural product |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100489113C (en) * | 2006-02-22 | 2009-05-20 | 华东理工大学 | Method for enzymatic synthesis of gadol glycoside through separation and coupling |
| CN102676478A (en) * | 2012-04-20 | 2012-09-19 | 华东理工大学 | Novel beta-glucosidase and genes thereof and application of beta-glucosidase to glucoside synthesis |
| CN103999648B (en) * | 2013-02-27 | 2015-09-09 | 湖南中医药大学 | A kind of cultivation method containing salidroside plant and plant application |
| CN103999747B (en) * | 2013-02-27 | 2016-05-04 | 湖南中医药大学 | Make its plant cultivation method that contains rhodioside and plant application |
| CN103642880B (en) * | 2013-12-11 | 2015-09-09 | 福州大学 | A kind of beta-glucosidase cross-linked aggregates catalyzes and synthesizes the technique of rhodioside |
| CN104404013B (en) * | 2014-10-14 | 2017-04-12 | 莆田市汇龙海产有限公司 | Abalone viscera [beta]-glucosidase and method for synthesizing rhodioloside thereby |
| CN104585374A (en) * | 2015-01-06 | 2015-05-06 | 湖南中医药大学 | Preparation method and application of tea containing salidroside |
| CN104542036A (en) * | 2015-01-06 | 2015-04-29 | 湖南中医药大学 | Cultivation method of vegetable containing salidroside and application |
| CN104911237B (en) * | 2015-06-10 | 2017-09-29 | 淮阴工学院 | A kind of method that recessed soil load black cloth woods seed β glucuroides cross-linked polymer synthesizes rhodioside |
| CN111172738B (en) * | 2020-02-11 | 2021-11-19 | 南通大学 | Antibacterial cotton fabric and preparation method thereof |
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