CN102311985A - Preparation method of baohuoside I - Google Patents
Preparation method of baohuoside I Download PDFInfo
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- CN102311985A CN102311985A CN201110186242A CN201110186242A CN102311985A CN 102311985 A CN102311985 A CN 102311985A CN 201110186242 A CN201110186242 A CN 201110186242A CN 201110186242 A CN201110186242 A CN 201110186242A CN 102311985 A CN102311985 A CN 102311985A
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Abstract
The invention provides a preparation method of baohuoside I, which has a high efficiency and a high conversion rate. The method is characterized in that baohuoside I is obtained by treating a certain concentration of ethanol as a reaction medium, and carrying out enzyme hydrolysis on icariin. The method which adopts a low concentration of an ethanol solution as the reaction medium has the advantages of increase of the specific surface area of a contact reaction of icariin with an enzyme, shortening of the reaction time, improvement of the conversion efficiency and the conversion rate, simple and feasible technology, good purposiveness, no pollution, and realization of industrial production, and overcomes disadvantages of complex operation, much time consumption, low conversion rate, bad purposiveness, difficult separation and purification, and the like existing in the prior art.
Description
Technical field
The present invention relates to the preparation method of a kind of precious leaves of pulse plants glycosides I, belong to medical technical field.
Background technology
Herba Epimedii, medicinal history is long, and determined curative effect is clinical conventional Chinese medicine.Its traditional function is a kidney-replenishing, strengthening the bones and tendons, wind-damp dispelling.Be used for impotence and seminal emission, the muscles and bones impotence is soft, rheumatic arthralgia, numbness contracture.The modern pharmacology experimental study shows; Herba Epimedii can strengthen the cardiovascular and cerebrovascular blood flow, promotes hemopoietic function, regulates bone metabolism, improve immunologic function; Also have multiple efficacies such as anti-ageing, antitumor and anti-AIDS, clinically be used to treat diseases such as osteoporosis, involution syndrome, hypertension, coronary heart disease, poliomyelitis, chronic bronchitis.Herba Epimedii is because its abundant resources of medicinal plant, various pharmacological action and definite clinical efficacy, is the focus of research both at home and abroad always, has huge research and development potentiality.
Precious leaves of pulse plants glycosides I, chemical name: 3,5,7-trihydroxy--4 '-methoxyl group-8-isoamylene radical chromocor-3-O-α-L-pyrans rhamnoside, be a kind of poly-hydroxy flavonoid monomer component.Pharmacological research shows that precious leaves of pulse plants glycosides I has effects such as antitumor, osteoporosis.But, be difficult to the direct separation purifying and obtain, so can't carry out preparation of industrialization because of its content in epimedium herb is lower.Shown in the chemical structure (I).
Icarin, chemical name: 3,5,7-trihydroxy--4 '-methoxyl group-8-isoamylene radical chromocor-3-O-α-L-pyrans rhamnosyl-7-O-β-D-glucopyranoside.Its structural formula is shown in (II).
Abundant because of naturally occurring icarin content in plant, a kind ofly prepare the method for precious leaves of pulse plants glycosides I so the invention provides from icarin, prepare precious leaves of pulse plants glycosides I through the glucoside bond in the hydrolysis icarin.The method of the hydrolysis sugar glycosidic bond of having reported has: (1) chemical process, and like acid, alkali hydrolysis method.Acid-hydrolysis method is used hydrochloric acid, sulfuric acid or nitric acid more, and alkali hydrolysis method is many with sodium hydroxide or Pottasium Hydroxide.This method purpose is not strong, and reaction is violent, and product is destroyed easily, and environmental pollution is bigger.(2) biotransformation method can contain enzymatic conversion liquid with bacterium, mould, yeast or the preparation of animal vegetable tissue cell cultures, icarin is put into conversion fluid react, and perhaps directly adds suitable enzymatic conversion icarin.This method conversion capability is not strong, and complicated operation is consuming time many, and transformation efficiency is low, and separation and purification is difficult, is unfavorable for suitability for industrialized production.
The present invention adopts icarin and the enzyme precious leaves of pulse plants glycosides of prepared in reaction I in certain density ethanol, and this method has increased icarin and the catalytic chance of enzyme, has shortened the reaction times; Improved transformation efficiency, technology is simple and feasible, and purpose is strong; Pollution-free; Post-reaction treatment is convenient, and purifying is simple, can realize suitability for industrialized production.
Summary of the invention
The method that the purpose of this invention is to provide the precious leaves of pulse plants glycosides of a kind of efficient production I.
To the foregoing invention purpose, the present invention provides following technical scheme:
The preparation method of a kind of precious leaves of pulse plants glycosides I is characterized in that:, obtained through enzyme digestion reaction and separation and purification by icarin as reaction medium with certain density ethanol.
Described preparation method, the purity of icarin are greater than 50%, and the purity of preferred icarin is greater than 95%.
Described preparation method, the enzyme that said enzyme digestion reaction uses is beta-glucosidase, cellulase.
Described preparation method, said reaction concrete steps are: icarin is dissolved in the ethanol of 40-100%, with the aqueous solution of enzyme, makes alcohol dilution to 5-50%, control reaction temperature is carried out enzyme digestion reaction, separation and purification at 0-60 ℃.
Described preparation method, preferably: icarin is dissolved in 50% the ethanol, with the aqueous solution of enzyme, makes alcohol dilution to 20-30%, control reaction temperature is carried out enzyme digestion reaction, separation and purification at 30-60 ℃.
Described preparation method also comprises the separation and purification of precious leaves of pulse plants glycosides I, and said separation and purification concrete steps are to add ethanol sedimentation, cross leaching filtrating, fling to solvent, adopt organic solvent recrystallization or column chromatography or preparative hplc repeatedly, promptly get precious leaves of pulse plants glycosides I.
Alcoholic acid per-cent described in the present invention means the ratio of 20 ℃ of capacity; The ethanol of various concentration is formed by the second alcohol and water.
Beneficial effect of the present invention mainly is:
1, compares with the method that icarin enzyme digestion reaction in water prepares precious leaves of pulse plants glycosides I; With certain density ethanol as reaction medium; The solubleness of icarin in reaction medium increases, and the icarin molecule that can contact with enzyme is many, and reaction efficiency increases substantially.
2, in the time of will being dissolved with the aqueous solution of ethanol and enzyme of icarin; The part icarin can be separated out; But because the existence of enzyme molecule; Can the icarin of separating out be stabilized in the smaller particle size scope, the icarin dissolution rate is fast in enzymolysis process, and enzyme digestion reaction efficient is significantly higher than directly icarin and enzyme are dispersed in the enzyme digestion reaction that carries out in the certain density ethanol.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but should notice that scope of the present invention does not receive any restriction of these instances.
Embodiment 1
Get icarin (purity 95.7%) 2g and be dissolved in the ethanol of 500mL50%, other gets beta-glucosidase 0.5g and is dissolved in the 330mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme; Make alcohol concn be diluted to 30%; In 37 ℃ of stirrings 6 hours,, remove solvent with the reaction solution rotary evaporation in vacuo; High-efficient liquid phase technique detects the transformation efficiency of icarin, and the result has 98.5% icarin to be converted into precious leaves of pulse plants glycosides I.
Get icarin (purity 95.7%) 2g, beta-glucosidase 0.5g, be scattered in the 830mL water, stirred 6 hours in 37 ℃; With the reaction solution rotary evaporation in vacuo; Remove solvent, high-efficient liquid phase technique detects the transformation efficiency of icarin, and the result has 36.3% icarin to be converted into precious leaves of pulse plants glycosides I.
Get icarin (purity 95.7%) 2g, beta-glucosidase 0.5g; Disperse in the ethanol of 830mL30%; In 37 ℃ of stirrings 6 hours,, remove solvent with the reaction solution rotary evaporation in vacuo; High-efficient liquid phase technique detects the transformation efficiency of icarin, and the result has 67.9% icarin to be converted into precious leaves of pulse plants glycosides I.
Embodiment 2
Get icarin (purity 95.7%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, other gets beta-glucosidase 0.5g and is dissolved in the 330mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme; Make alcohol concn be diluted to 30%, in 37 ℃ of stirrings 6 hours, after reaction finishes; Add equivalent ethanol sedimentation beta-glucosidase; Cross leaching filtrating, volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.52g.Again with absolute ethyl alcohol recrystallization 3 times repeatedly, the precious leaves of pulse plants glycosides I 1.08g of purity 99.5%.
Get icarin (purity 95.7%) 2g, beta-glucosidase 0.5g, be scattered in the 830mL water,, after reaction finishes, add equivalent ethanol sedimentation beta-glucosidase, cross leaching and filtrate, volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.72g in 37 ℃ of stirrings 6 hours.Again with absolute ethyl alcohol recrystallization 3 times repeatedly, the precious leaves of pulse plants glycosides I 0.31g of purity 92.3%.
Get icarin (purity 95.7%) 2g, beta-glucosidase 0.5g, disperse to stir 6 hours in 37 ℃ in the ethanol of 830mL30%; After reaction finishes, add equivalent ethanol sedimentation beta-glucosidase, cross leaching filtrating; Volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.61g.Again with absolute ethyl alcohol recrystallization 3 times repeatedly, the precious leaves of pulse plants glycosides I 0.65g of purity 95.7%.
Embodiment 2
Get icarin (purity 65.7%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, in addition extracting cellulose enzyme 1g is dissolved in the 330mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme; Make alcohol concn be diluted to 30%, in 50 ℃ of stirrings 10 hours, after reaction finishes; Add equivalent ethanol sedimentation cellulase; Cross leaching filtrating, volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.57g.Again with absolute ethyl alcohol recrystallization 5 times repeatedly, the precious leaves of pulse plants glycosides I 0.68g of purity 96.5%.
Embodiment 3
Get icarin (purity 65.7%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, in addition extracting cellulose enzyme 1g is dissolved in the 330mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme, makes alcohol concn be diluted to 30%; In 40 ℃ of stirrings 12 hours, after reaction finishes, add equivalent ethanol sedimentation cellulase, cross leaching and filtrate; Volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.55g, use the D101 macroporous resin adsorption; Water, 30% ethanol, 60% ethanol elution are collected 60% ethanol position, rotary evaporation in vacuo respectively; Remove solvent, absolute ethyl alcohol is recrystallization 2 times repeatedly, the precious leaves of pulse plants glycosides I 0.55g of purity 99.2%.
Embodiment 4
Get icarin (purity 65.7%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, in addition extracting cellulose enzyme 2g is dissolved in the 750mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme, makes alcohol concn be diluted to 20%; In 40 ℃ of stirrings 8 hours, after reaction finishes, add equivalent ethanol sedimentation cellulase; Cross leaching filtrating, volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.50g; Separate with silicagel column, the absolute ethyl alcohol recrystallization, the precious leaves of pulse plants glycosides I 0.61g of purity 98.7%.
Embodiment 5
Get icarin (purity 80.3%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, in addition extracting cellulose enzyme 2g is dissolved in the 750mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme, makes alcohol concn be diluted to 20%; In 50 ℃ of stirrings 6 hours, after reaction finishes, add equivalent ethanol sedimentation cellulase, cross leaching and filtrate; Volatilize solvent, promptly get precious leaves of pulse plants glycosides I bullion 1.63g, use the AB-8 macroporous resin adsorption; Water, 30% ethanol, 60% ethanol elution are collected 60% ethanol position, rotary evaporation in vacuo respectively; Remove solvent, absolute ethyl alcohol is recrystallization 2 times repeatedly, the precious leaves of pulse plants glycosides I 0.82g of purity 98.8%.
Final product is carried out nuclear magnetic resonance spectroscopy,
1H NMR,
13C NMR data are following:
1H?NMR(DMSO，400MHz，30℃)，δ1.2(3H，s，CH3-4″)，1.6(3H，s，CH3-5″)，2.5(2H，s，OH)，3.4(2H，d，J＝6.8Hz，H-11)，3.8(3H，s，4′-OCH
3)，5.1(1H，t，J＝6.8Hz，H-2″)，6.3(1H，s，H-6)，7.1(2H，d，J＝8.4Hz，H-3′，H-5′)，8.1(2H，d，J＝8.4Hz，H-2′，H-6′)，9.4(1H，s，OH-3)，10.7(1H，s，OH-7)，12.3(1H，s，OH-5)；
13C?NMR(DMSO，100MHz，30℃)，δ153.9(C-2)，136.3(C-3)，176.7(C-4)，160.9(C-5)，98.3(C-6)，161.7(C-7)，103.5(C-8)，146.6(C-9)，106.0(C-10) 122.9(C-1′)，129.6(C-2′，6′)，114.5(C-3′，5′)，158.7(C-4′)，21.6(C-1″)，124.0(C-2″)，131.4(C-3″)，25.9(C-4″)，18.3(C-5″)，55.8(4′-OCH3)。
The precious leaves of pulse plants glycosides I data consistent that provides with document.
Embodiment 6
Get icarin (purity 80.3%) 2g and be dissolved in the Diluted Alcohol of 500mL50%, in addition extracting cellulose enzyme 2g is dissolved in the 750mL water, and the Diluted Alcohol solution of icarin is slowly added in the aqueous solution of enzyme; Make alcohol concn be diluted to 20%, in 50 ℃ of stirrings 6 hours, after reaction finishes; Add equivalent ethanol sedimentation cellulase, cross leaching filtrating, volatilize solvent; Promptly get precious leaves of pulse plants glycosides I bullion 1.67g, separate precious leaves of pulse plants glycosides I, get the precious leaves of pulse plants glycosides I 0.87g of purity 99.6% with preparative hplc.
Claims (6)
1. the preparation method of a precious leaves of pulse plants glycosides I is characterized in that:, obtained through enzyme digestion reaction and separation and purification by icarin as reaction medium with certain density ethanol.
2. preparation method according to claim 1, the purity of icarin are greater than 50%, and the purity of preferred icarin is greater than 95%.
3. preparation method according to claim 1, the enzyme that said enzyme digestion reaction uses is beta-glucosidase, cellulase.
4. preparation method according to claim 1, said reaction concrete steps are: icarin is dissolved in the ethanol of 40-100%, with the aqueous solution of enzyme; Make alcohol dilution to 5-50%; Control reaction temperature is carried out enzyme digestion reaction, separation and purification at 0-60 ℃.
5. according to the described preparation method of claim 1-4, said reaction concrete steps preferably: icarin is dissolved in 50% the ethanol, with the aqueous solution of enzyme; Make alcohol dilution to 20-30%; Control reaction temperature is carried out enzyme digestion reaction, separation and purification at 30-60 ℃.
6. according to the described preparation method of claim 1-5, also comprise the separation and purification of precious leaves of pulse plants glycosides I, said separation and purification concrete steps are to add ethanol sedimentation; Cross leaching filtrating; Fling to solvent, adopt organic solvent recrystallization or column chromatography or preparative hplc repeatedly, promptly get precious leaves of pulse plants glycosides I.
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| CN201110186242A CN102311985A (en) | 2011-07-05 | 2011-07-05 | Preparation method of baohuoside I |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305572A (en) * | 2013-05-31 | 2013-09-18 | 西昌丹阳生物科技有限责任公司 | Method for producing baohuoside I through herba epimdii |
| CN106148454A (en) * | 2015-03-24 | 2016-11-23 | 北京珅奥基医药科技有限公司 | A kind of preparation method of baohuoside Ⅰ |
| CN106755214A (en) * | 2016-12-08 | 2017-05-31 | 江苏大学 | A kind of method that two-phase enzyme hydrolysis obtains precious leaves of pulse plants glycosides I |
| CN107059035A (en) * | 2017-03-08 | 2017-08-18 | 安徽省伟业净化设备有限公司 | A kind of Medical vessel scale remover and preparation method thereof |
| CN107164436A (en) * | 2017-05-12 | 2017-09-15 | 南京林业大学 | Application of the β glucuroides in conversion barren wort total chromocor prepares precious glycosides I suddenly |
| WO2019205026A1 (en) * | 2018-04-25 | 2019-10-31 | 邦泰生物工程(深圳)有限公司 | USAGE OF β-GLUCOSIDASE AND METHOD FOR PREPARING BAOHUOSIDE I BY USING SAME |
| CN112176008A (en) * | 2020-10-13 | 2021-01-05 | 烟台大学 | Enzymolysis method for efficiently and quickly preparing icariside II |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101316573A (en) * | 2005-11-30 | 2008-12-03 | 株式会社太平洋 | Cosmetic composition containing hydrolysates of icariin |
| CN101516325A (en) * | 2006-09-19 | 2009-08-26 | 株式会社太平洋 | Method for preparing icariside II, cosmetic composition containing the same and the use thereof for skin whitening |
-
2011
- 2011-07-05 CN CN201110186242A patent/CN102311985A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101316573A (en) * | 2005-11-30 | 2008-12-03 | 株式会社太平洋 | Cosmetic composition containing hydrolysates of icariin |
| CN101516325A (en) * | 2006-09-19 | 2009-08-26 | 株式会社太平洋 | Method for preparing icariside II, cosmetic composition containing the same and the use thereof for skin whitening |
Non-Patent Citations (1)
| Title |
|---|
| 贾东升等: "纤维素酶转化淫羊藿苷制备宝藿苷I的研究", 《中草药》, vol. 41, no. 6, 30 June 2010 (2010-06-30), pages 888 - 892 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305572A (en) * | 2013-05-31 | 2013-09-18 | 西昌丹阳生物科技有限责任公司 | Method for producing baohuoside I through herba epimdii |
| CN103305572B (en) * | 2013-05-31 | 2014-12-24 | 西昌丹阳生物科技有限责任公司 | Method for producing baohuoside I through herba epimdii |
| CN106148454A (en) * | 2015-03-24 | 2016-11-23 | 北京珅奥基医药科技有限公司 | A kind of preparation method of baohuoside Ⅰ |
| CN106755214A (en) * | 2016-12-08 | 2017-05-31 | 江苏大学 | A kind of method that two-phase enzyme hydrolysis obtains precious leaves of pulse plants glycosides I |
| CN106755214B (en) * | 2016-12-08 | 2020-06-26 | 江苏大学 | Method for obtaining baohuoside I by means of two-phase enzymatic hydrolysis |
| CN107059035A (en) * | 2017-03-08 | 2017-08-18 | 安徽省伟业净化设备有限公司 | A kind of Medical vessel scale remover and preparation method thereof |
| CN107059035B (en) * | 2017-03-08 | 2019-04-23 | 安徽省伟业净化设备有限公司 | A kind of Medical vessel scale remover and preparation method thereof |
| CN107164436A (en) * | 2017-05-12 | 2017-09-15 | 南京林业大学 | Application of the β glucuroides in conversion barren wort total chromocor prepares precious glycosides I suddenly |
| WO2019205026A1 (en) * | 2018-04-25 | 2019-10-31 | 邦泰生物工程(深圳)有限公司 | USAGE OF β-GLUCOSIDASE AND METHOD FOR PREPARING BAOHUOSIDE I BY USING SAME |
| CN110770351A (en) * | 2018-04-25 | 2020-02-07 | 邦泰生物工程(深圳)有限公司 | β -glucosidase application and baohuoside I preparation method using same |
| CN112176008A (en) * | 2020-10-13 | 2021-01-05 | 烟台大学 | Enzymolysis method for efficiently and quickly preparing icariside II |
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Application publication date: 20120111 |