CN102617670A - Preparation method of icariin monomer - Google Patents
Preparation method of icariin monomer Download PDFInfo
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- CN102617670A CN102617670A CN201110030373XA CN201110030373A CN102617670A CN 102617670 A CN102617670 A CN 102617670A CN 201110030373X A CN201110030373X A CN 201110030373XA CN 201110030373 A CN201110030373 A CN 201110030373A CN 102617670 A CN102617670 A CN 102617670A
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Abstract
The invention provides a preparation method of an icariin monomer. The preparation method adopts a semipreparative high-speed countercurrent chromatographic instrument. The preparation method comprises the following steps of dissolving an icariin crude product in a lower phase of a chloroform-methanol-water ternary solvent, filling a stationary phase which is an upper phase of the chloroform-methanol-water ternary solvent into a high-speed countercurrent chromatographic column, turning a main engine, pumping a mobile phase which is the lower phase of the chloroform-methanol-water ternary solvent into the high-speed countercurrent chromatographic column, feeding the icariin crude product dissolved by the lower phase into the high-speed countercurrent chromatographic column from a feed valve, receiving object components according to a detector map, concentrating a solution containing the object components, and carrying out freeze drying. The preparation method is suitable for preparing the icariin monomer having high purity from crude icariin extract having different purity. The preparation method of the icariin monomer has an advanced technology, high separation efficiency, a high recovery rate, high product purity and a large preparation amount.
Description
Technical field
The present invention relates to a kind of preparation method of icarin, design a kind of preparation high purity icarin monomer methods especially.
Background technology
The modern Chinese herbal medicine pharmaceutical research shows; Herba Epimedii have good invigorating the liver and kidney, strengthening the bones and tendons, wind-damp dispelling, anti-ageing, improve immunizing power, suppress pharmacological actions such as tumour; Therefore received the extensive attention of Chinese scholars, and deep research has been carried out in resource kind, regional distribution, chemical ingredients and the pharmacological action of barrenwort.The compound of in barrenwort, finding in recent years is main with flavones, it is generally acknowledged flavones with 8-isopentene group and glycoside thereof be Herba Epimedii to the cardiovascular and active staple of immunological enhancement, and have anti-tumor activity.Wherein icarin is a characteristic component, has the content that increases Theelin,dihydro-, and stimulates the secretion of Adrenalone, sexual function improving; Reduce the emission levels of hepatocellular gpt of infective virus and SDH, have immune effect such as anti-hepatotoxin and antitumor action etc.
In order to further investigate the pharmacologically active of icarin, the research of icarin monomer component preparation has also obtained corresponding attention.Adopt solvent to extract repeatedly like patent CN101607976A, the isolating method of silicagel column and polyamide column chromatography has made purity greater than 98% icarin; Patent CN101747391A separates with the alcohol precipitation removal of impurities through preliminary post and decolours, and adopts preparative high performance liquid chromatography to obtain purity greater than 98% icarin chemical reference substance at last.
In the preparation separation method research of icarin monomer component, adopt the separation and purification means of column chromatography mostly, it is used very extensively, has advantages such as applied sample amount is big, and its shortcoming is a complex operation step, and sample recovery rate is low.Also have correlative study to adopt performance liquid chromatography separation etc. to obtain the very high icarin monomer component of purity, but instrument cost is expensive, solvent-oil ratio greatly also restricted should technology development.
Yet; The liquid luquid partition chromatography stripping technique of a kind of continuous high-efficient that high speed adverse current chromatogram (HSCCC) grows up between the seventies and eighties as twentieth century; In conjunction with the advantage of liquid-liquid extraction and distribution chromatography, be a kind of liquid luquid partition chromatography technology that need not any solid-state carrier or support.Through the cf-that produces in the planetary operation process; Make the unidirectional distribution in the spiral tube of high speed rotating of two kinds of immiscible solvents; Phase that fixes wherein carries the moving phase that is loaded with sample to pass stationary phase by constant flow pump, utilize sample two mutually in the difference of partition ratio realize separation.HSCCC has no irreversible adsorption as a kind of separating and purifying technology commonly used, and the recovery is high, and simple and quick, sample size is big, plurality of advantages such as separation efficiency height.The separation and the purifying that very are suitable for natural plant extracts, and the preparation of high purity standard model.
Summary of the invention
The purpose of this invention is to provide a kind of icarin method for preparing monomer.
For the purposes of the present invention, the present invention provides the icarin shown in a kind of formula I monomeric preparation method, adopts half countercurrent chromatography appearance, comprises the steps:
1) ternary solvent of preparation chloroform, first alcohol and water, getting on it is stationary phase mutually, following to moving phase, and the icarin bullion is dissolved in phase down;
2), rotate main frame with the stationary phase of filling with the step 1) preparation in the high-speed counter-current chromatograph pillar;
3) moving phase with the step 1) preparation pumps into chromatographic column, gets into the icarin bullion of phased soln down by sampling valve again, according to detector collection of illustrative plates receiving target composition, will contain the solution concentration of target compound composition, lyophilize.
Formula I
Wherein, the volume ratio of chloroform, first alcohol and water is 2～5: 2～5 in the ternary solvent of described chloroform, first alcohol and water: 2, be preferably volume ratio 3.5: 2.5: 2.
Wherein, the detection wavelength that is used to detect icarin of the present invention is 280nm.
Wherein, the flow velocity of described high-speed counter-current chromatograph is 2～4mL/min, is preferably 3mL/min.
Wherein, the rotating speed of described high-speed counter-current chromatograph is 700～1000rpm, is preferably 900rpm.
Wherein, described separation temperature is 20～30 ℃.
According to the present invention, the Herba Epimedii bullion of separable 200～400mg, the purity of preferred Herba Epimedii bullion is 30～70%.
Wherein, described Herba Epimedii bullion is a solid, can prepare by means commonly known in the art, as preparing according to the CN200510061251.1 disclosed method; Also can commercially availablely obtain, as available from Sanjiang Biologica Engineering Co., Ltd., Xi-an City.
According to the present invention, the usable highly effective liquid chromatography detects the sample that separation obtains, and chromatographic condition is WondaSil C18 chromatographic column (5 μ m; 250 * 4.6mm i.d.); Acetonitrile: water=27: 73 is as moving phase, flow velocity 1mL/min, column temperature: 30 ℃; Detect wavelength: 270nm, analysis time: 30min.
According to the present invention, adopt Bruker 500MHz superconduction nuclear magnetic resonance spectrometer to carry out
1H-NMR reaches
13C-NMR analyzes, and deuterated solvent is a DMSO 99.8MIN..
Icarin monomeric compound purity with this law preparation can reach more than 98%, and preparation amount is in the gram magnitude.This law is suitable for the highly purified icarin monomeric compound that different icarin bullions is a feedstock production.This law characteristics such as technology advanced person, separation efficiency are high, the recovery is high, product purity is high, preparation amount is bigger that possess skills.Compare with prepare the icarin monomer methods with performance liquid chromatography, method provided by the invention more can be saved solvent, and preparation amount is big, and sample purity is high.
Description of drawings
Fig. 1 is the color atlas of the high speed adverse current chromatogram of the embodiment of the invention 1 icarin bullion.
Fig. 2 is that the performance liquid chromatography of icarin bullion before the embodiment of the invention 1 purifying detects collection of illustrative plates.
Fig. 3 is that the monomeric performance liquid chromatography of the icarin behind the embodiment of the invention 1 purifying detects collection of illustrative plates.
Fig. 4 is the color atlas of the high speed adverse current chromatogram of the embodiment of the invention 2 icarin bullions.
Fig. 5 is the color atlas of the high speed adverse current chromatogram of the embodiment of the invention 3 icarin bullions.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1
(1) adopt chloroform, methyl alcohol and aqueous systems are that 50% icarin bullion is available from Sanjiang Biologica Engineering Co., Ltd., Xi-an City with purity) be feedstock production icarin monomer.
(2) adopt half countercurrent chromatography, solvent systems is a chloroform, the first alcohol and water, and its volume ratio is 3.5: 2.5: 2.Above-mentioned solvent system is disposed in the separating funnel, static layering behind the shake well, getting is stationary phase mutually, is moving phase mutually down.Get 50% icarin bullion 250mg and be dissolved in phase solution 10mL down,, regulate rotating speed 900rpm then main frame is rotated being full of stationary phase in the high-speed counter-current chromatograph chromatographic column; The temperature of water bath with thermostatic control is set at 30 ℃; It is 280nm that UV-detector detects wavelength set, again moving phase is pumped into chromatographic column with 2.5mL/min, waits for that the system balance finishes; Get into crude samples by sampling valve again; According to detector collection of illustrative plates receiving target composition (see figure 1), will contain the solution concentration of target compound composition, lyophilize.
(3) to above-mentioned icarin bullion and the samples using efficient liquid phase chromatographic analysis behind the purifying (seeing Fig. 2 and Fig. 3); Its condition is following: WondaSil C18 chromatographic column (5 μ m, 250 * 4.6mm i.d.), and acetonitrile: water=27: 73 is as moving phase; Flow velocity 1mL/min; Column temperature: 30 ℃, detect wavelength: 270nm, analysis time: 30min.Through present embodiment, can be purified into purity and be 99.0% icarin monomer 105mg.
Embodiment 2
(1) adopt chloroform, methyl alcohol and aqueous systems are that 30% icarin bullion is available from Sanjiang Biologica Engineering Co., Ltd., Xi-an City with purity) be feedstock production icarin monomer.
(2) adopt half countercurrent chromatography, solvent systems is a chloroform, the first alcohol and water, and its volume ratio is 3: 2: 2.Above-mentioned solvent system is disposed in the separating funnel, static layering behind the shake well, getting is stationary phase mutually, is moving phase mutually down.Get 30% icarin bullion 200mg and be dissolved in phase solution 8mL down,, regulate rotating speed 700rpm then main frame is rotated being full of stationary phase in the high-speed counter-current chromatograph chromatographic column; The temperature of water bath with thermostatic control is set at 25 ℃; It is 280nm that UV-detector detects wavelength set, again moving phase is pumped into chromatographic column with 3.5mL/min, waits for that the system balance finishes; Get into crude samples by sampling valve again; According to detector collection of illustrative plates receiving target composition (see figure 4), will contain the solution concentration of target compound composition, lyophilize.
(3) the samples using efficient liquid phase chromatographic analysis to receiving, its condition is following: WondaSil C18 chromatographic column (5 μ m, 250 * 4.6mm i.d.); Acetonitrile: water=27: 73 is as moving phase, flow velocity 1mL/min, column temperature: 30 ℃; Detect wavelength: 270nm, analysis time: 30min.Through present embodiment, can be purified into purity and be 98.7% icarin monomer 50mg.
Embodiment 3
(1) adopt chloroform, methyl alcohol and aqueous systems are that 70% icarin bullion (available from Sanjiang Biologica Engineering Co., Ltd., Xi-an City) is a feedstock production icarin monomer with purity.
(2) adopt half countercurrent chromatography, solvent systems is a chloroform, the first alcohol and water, and its volume ratio is 5: 4: 2.Above-mentioned solvent system is disposed in the separating funnel, static layering behind the shake well, getting is stationary phase mutually, is moving phase mutually down.Get 70% icarin bullion 300mg and be dissolved in phase solution 12mL down,, regulate rotating speed 1000rpm then main frame is rotated being full of stationary phase in the high-speed counter-current chromatograph chromatographic column; The temperature of water bath with thermostatic control is set at 20 ℃; It is 280nm that UV-detector detects wavelength set, again moving phase is pumped into chromatographic column with 2mL/min, waits for that the system balance finishes; Get into crude samples by sampling valve again; According to detector collection of illustrative plates receiving target composition (see figure 5), will contain the solution concentration of target compound composition, lyophilize.
(3) the samples using efficient liquid phase chromatographic analysis to receiving, its condition is following: WondaSil C18 chromatographic column (5 μ m, 250 * 4.6mm i.d.); Acetonitrile: water=27: 73 is as moving phase, flow velocity 1mL/min, column temperature: 30 ℃; Detect wavelength: 270nm, analysis time: 30min.Through present embodiment, can be purified into purity and be 98.9% icarin monomer 180mg.
Embodiment 4
Adopt Bruker 500MHz superconduction nuclear magnetic resonance spectrometer to carry out the icarin monomer of embodiment 1 preparation is carried out
1H-NMR reaches
13C-NMR analyzes, and deuterated solvent is a DMSO 99.8MIN..
The result is as shown in table 1, shows that the product that obtains by the present invention is the icarin monomer.
Table 1 NMR detected result
Product to embodiment 2 and embodiment 3 preparations carries out magnetic resonance detection, and what the result also all showed preparation is the icarin monomer.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (9)
1. the monomeric preparation method of icarin is characterized in that, adopts half countercurrent chromatography appearance that the icarin bullion is carried out purifying, comprises the steps:
1) ternary solvent of preparation chloroform, first alcohol and water, getting on it is stationary phase mutually, following to moving phase, and the icarin bullion is dissolved in phase down;
2), rotate main frame with the stationary phase of filling with the step 1) preparation in the high-speed counter-current chromatograph pillar;
3) moving phase with the step 1) preparation pumps into chromatographic column, gets into the icarin bullion of phased soln down by sampling valve again, according to detector collection of illustrative plates receiving target composition, will contain the solution concentration of target compound composition, lyophilize.
2. method according to claim 1 is characterized in that, the detection wavelength of the UV-detector of said high-speed counter-current chromatograph is 280nm.
3. method according to claim 1 is characterized in that, the volume ratio of described chloroform, first alcohol and water is 2～5: 2～5: 2.
4. method according to claim 3 is characterized in that, the volume ratio of said chloroform, first alcohol and water 3.5: 2.5: 2.
5. method according to claim 1 is characterized in that, the flow velocity of said high-speed counter-current chromatograph is 2～4mL/min.
6. method according to claim 1 is characterized in that, the rotating speed of described high-speed counter-current chromatograph is 700～1000rpm.
7. method according to claim 1 is characterized in that, described separation temperature is 20～30 ℃.
8. method according to claim 1 is characterized in that, the applied sample amount of described icarin bullion is 200～400mg.
9. according to each described method of claim 1～8, it is characterized in that the purity of icarin is 30～70% in the described icarin bullion.
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| CN201110030373XA CN102617670A (en) | 2011-01-27 | 2011-01-27 | Preparation method of icariin monomer |
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| CN201110030373XA CN102617670A (en) | 2011-01-27 | 2011-01-27 | Preparation method of icariin monomer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019241947A1 (en) * | 2018-06-21 | 2019-12-26 | 邦泰生物工程(深圳)有限公司 | Method of separating and purifying icariin from epimedium extract |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1394866A (en) * | 2001-07-05 | 2003-02-05 | 北京天纯维通生物技术有限公司 | Method for preparing high-purity icariin |
| WO2008035918A1 (en) * | 2006-09-19 | 2008-03-27 | Amorepacific Corporation | Method for preparing icariside ii, cosmetic composition containing the same and the use thereof for skin whitening |
| CN101607976A (en) * | 2008-06-19 | 2009-12-23 | 贵州省中国科学院天然产物化学重点实验室 | A kind of preparation method of icarin |
-
2011
- 2011-01-27 CN CN201110030373XA patent/CN102617670A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1394866A (en) * | 2001-07-05 | 2003-02-05 | 北京天纯维通生物技术有限公司 | Method for preparing high-purity icariin |
| WO2008035918A1 (en) * | 2006-09-19 | 2008-03-27 | Amorepacific Corporation | Method for preparing icariside ii, cosmetic composition containing the same and the use thereof for skin whitening |
| CN101607976A (en) * | 2008-06-19 | 2009-12-23 | 贵州省中国科学院天然产物化学重点实验室 | A kind of preparation method of icarin |
Non-Patent Citations (1)
| Title |
|---|
| RENMIN LIU，等: "Preparative isolation and purification of three flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai by high-speed counter-current chromatography", 《JOURNAL OF CHROMATOGRAPHY A》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019241947A1 (en) * | 2018-06-21 | 2019-12-26 | 邦泰生物工程(深圳)有限公司 | Method of separating and purifying icariin from epimedium extract |
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Application publication date: 20120801 |