CN1277068A - Highspeed counter-current chromatographic separation process for preparing high purity catechin - Google Patents
Highspeed counter-current chromatographic separation process for preparing high purity catechin Download PDFInfo
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Abstract
The chromatographic separation process includes preparing solvent system constituting fixed phase and mobile phase; filling the countercurrent chromatographic column with the fixed phase; rotating the main unit and pumping the mobile phase into the column; introducing sample via sample valve; and receiving target component according to detector charts. The solvent system is a two-phase one including the upper fixed phase and the lower mobile phase, and is constituted either by fatty ester, fatty alcohol or fatty ketone, and water, or by alkane, fatty ester and water. The process can obtain high-purity catechin monomer with EGCG, GCG and ECG purity being as high as 98%.
Description
The present invention relates to the adverse current chromatogram method for separating and preparing of a kind of highspeed counter-current chromatographic separation process for preparing, particularly high purity catechin (EGCG, GCG, ECG).
Contain multiple polyphenols in the tealeaves, be referred to as Tea Polyphenols (tea ployphenols, TP).Wherein of paramount importance is catechin compounds (catechins).They mainly contain catechin (catechin, C), epicatechin (epicatechin, EC), nutgall catechin (gallocatechin, GC), epigallocatechin (epigallocatechin, EGC), catechin and gallate (catechin-3-O-gallate, CG), L-Epicatechin gallate (epicatechin-3-O-gallate, ECG), nutgall catechin gallic acid ester (gallocatechin-3-O-gallate, GCG), Epigallo-catechin gallate (EGCG) (epigallocatechin-3-O-gallate, EGCG).
Its structure is as follows:
I II R R
C EC H H
GC EGC H OH
CG ECG G H
GCG EGCG G OH
Pharmacological research shows, Tea Polyphenols (mainly being catechin) has tangible removing interior free yl, the formation of blocking-up N-nitroso compound suppresses lipoxygenase activity and lipid peroxidation, and anti-sudden change, anti-cancer, radiation proof and pharmacologically active such as anti-ageing, antibiotic, antiviral are arranged.Research finds that also two kinds of catechins of ECG and EGCG not only have very strong inhibitory action to human body HIV (HIV) reverse transcriptase, and are the polymerization proteases inhibitors of AIDS virus DNA and RNA.
Though Tea Polyphenols has been widely used in numerous areas such as food, medicine, light industry at present, what the overwhelming majority adopted is the Tea Polyphenols crude product.Further the especially pharmacology and the activity of catechin compounds of all kinds of materials in the research Tea Polyphenols is significant for the tea resources of development and utilization China's abundant better, like this method for separating and preparing of classes of compounds is had higher requirement.
The difficulty that catechin compounds separates is mainly from the characteristic of its structure, because the existence of a plurality of phenolic hydroxyl groups, make it be easy to by Irreversible Adsorption on general column chromatography filler and lose, and because such material is very unstable, on solid packing, also can degrade, thereby the rate of recovery is relatively poor, and traditional column chromatography separative efficiency is low, operates loaded down with trivial details.
The purpose of this invention is to provide a kind of preparation high purity catechin [epi-nutgall theine nutgall acid fat (epigallocatechin-3-O-gallate that from the Tea Polyphenols crude product, separates, EGCG), nutgall catechin gallic acid ester (gallocatechin-3-O-gallate, GCG), L-Epicatechin gallate (epicatechin-3-O-gallate, ECG)] method, this method separative efficiency height, easy and simple to handle.
For achieving the above object, the present invention takes following design: adopt highspeed counter-current chromatographic separation process for preparing, it comprises that preparation constitutes the fixedly dicyandiamide solution of phase, the phase that flows; Make and be full of fixedly phase in the counter-current chromatograph pillar; Its main frame is rotated, and will flow pumps in the post mutually again; By the sampling valve sample introduction; According to detector spectrogram receiving target composition.Described dicyandiamide solution mainly contains two: No. one dicyandiamide solution is made of three components, the A component can be selected from fatty ester classes such as ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate, the B component can be selected from fatty alcohol or aliphatic ketones such as methyl alcohol, ethanol, propyl alcohol, acetone, and the C component is a water.Ethyl acetate-ethanol-water system; No. two dicyandiamide solution also is made of three components, and the A component can be selected from alkane such as normal hexane, benzinum, thiacyclohexane, and the B component can be selected from fatty ester classes such as ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate, and the C component is a water.Preferred normal hexane-ethyl acetate-aqueous systems.
In the ethyl acetate, alcohol and water system, their consumption volume ratio can be at (1-10): (0-1): preparation in 10 the scope, for example: 10: 1: 10,20: 1: 20,20: 1: 50,25: 1: 100 etc.; In normal hexane-ethyl acetate-aqueous systems, their consumption volume ratio can be at (0-1): 10: preparation in the scope (10-100), for example: 1: 10: 10,1: 10: 50,1: 20: 20,1: 20: 100 etc.When the ratio of wherein ethanol and normal hexane became 0, two individual system just all became ethyl acetate-aqueous systems.More than the above-mentioned system mutually for fixing phase, be mobile phase mutually down, all can be used for from the Tea Polyphenols crude product, separating EGCG, and can obtain the EGCG pure component, also can obtain GCG and two pure components of ECG simultaneously through first separation, it is longer that but latter two component goes out the time at peak, the peak shape broad that becomes.
For separating of GCG and two components of ECG, when adopting the ethyl acetate, alcohol and water system, can be after isolating EGCG, on the basis of former setting proportioning, increase the following volume ratio of middle ethanol/water mutually gradually, [this moment, the volume ratio of ethanol/water can be at (0-1): 10 scope in] promptly adopts the gradient elution mode, the shortening disengaging time; Also can be after isolating EGCG, directly change to a suitable solvent burden ratio, also be to improve ethanol/water volume ratio wherein [this moment, the volume ratio of ethanol/water can be at (0-1): in 10 the scope], perhaps reelect normal hexane-ethyl acetate-aqueous systems, the component that wash-out is not gone out is as new sample, carry out secondary separation after concentrating, back two kinds are called the mode that substep separates, can obtain GCG and ECG pure component in the short period of time equally.
When adopting normal hexane-ethyl acetate-aqueous systems to separate GCG and two components of ECG, the mode that can only adopt substep to separate shortens disengaging time, promptly after isolating EGCG, in the raising mutually in the volume ratio of normal hexane/ethyl acetate, as bring up to 1: 3,1: 4 etc., the component that wash-out is not gone out was as new sample, after concentrating, carry out secondary separation, obtain GCG, ECG pure component.
Room temperature has certain influence to separating degree, and above separation condition is fit to room temperature to be used in the time of 16-30 ℃, for the ethyl acetate, alcohol and water system, when room temperature is higher, can reduce the ethanol/water volume ratio, and with the raising separating degree, but disengaging time can prolong; Vice versa.For normal hexane-ethyl acetate-aqueous systems, when room temperature is higher, can reduce normal hexane/ethyl acetate volume ratio, can improve separating degree, but disengaging time can correspondingly prolong also; Vice versa.
To adopt ethyl acetate-n-butanol-water system for the separation of other non-ester catechin.
Because the unstability of Tea Polyphenols preferably adopts the N2 gas shiled in the process of separation and collection target component.The product of collecting will carry out freeze drying immediately, places the closed container low-temperature dark to preserve then.
Advantage of the present invention: adopted the high speed adverse current chromatogram isolation technics in this method, because high speed adverse current chromatogram is a kind of continuous liquid liquid partition chromatography technology without solid support or carrier, it has overcome the Irreversible Adsorption that is caused by solid support, make separated object rate of recovery height, again because adopted preferred dicyandiamide solution, and corresponding process conditions, can obtain highly purified catechin (Epigallo-catechin gallate (EGCG) (EGCG), nutgall catechin gallic acid ester (GCG), L-Epicatechin gallate (ECG)) efficiently.This method is applicable to from preparing above three kinds of catechin monomers by separating the various Tea Polyphenols crude products.Be applicable to that the counter-current chromatograph with multiple model prepares catechin, for example: with a small amount of preparation of analytic type high-speed counter-current chromatograph and the relatively large preparation that partly prepares high-speed counter-current chromatograph.
The invention will be further described below in conjunction with embodiment and accompanying drawing.
Fig. 1 a is that ethyl acetate, alcohol and water (25: 1: 25) system prepares the adverse current chromatogram figure that separates EGCG from the Tea Polyphenols crude product
Fig. 1 b is with normal hexane-ethyl acetate-water (1: 4: 5) system, separates the adverse current chromatogram figure of preparation GCG and ECG from the residue that separates EGCG
Fig. 2 separates EGCG, the adverse current chromatogram figure of GCG and ECG with ethyl acetate, alcohol and water system gradient elution
Fig. 3 is the adverse current chromatogram that separates EGCG, GCG and ECG with normal hexane-ethyl acetate-water (1: 10: 10) system
Fig. 4 is the adverse current chromatogram figure that separates EGCG with ethyl acetate-aqueous systems
Example 1. adopts and partly prepares high-speed counter-current chromatograph, chooses the ethyl acetate, alcohol and water system and separates preparation
EGCG (see Fig. 1 a), change then n-hexane-ethyl acetate-aqueous systems separate preparation GCG and
ECG (seeing Fig. 1 b).
At first above-mentioned dicyandiamide solution is formulated in the separatory funnel, shakes up the back standing demix with 25: 1: 25 volume ratios.Ready to balance after a period of time separates upper and lower phase, gets fixedly phase of conduct mutually, and is following mutually as the phase that flows.
Taking by weighing 1 gram Tea Polyphenols study, to be dissolved in 30ml stand-by in flowing mutually.
The GS10A2 type that adopts Beijing Institute for New Technologies to develop partly prepares high-speed counter-current chromatograph, and is furnished with pump, 10-30ml sampling valve, UV-detector and recorder.The column capacity of its multi-lay winding polyfluortetraethylene pipe is about 260ml.
Be full of whole pillar mutually with fixing earlier; Adjust engine speed 800rpm, will flow with the flow velocity of 2.0ml/min pumps in the post mutually; After treating that whole system is set up dynamic equilibrium, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition.After freeze drying obtains the about 280mg of the EGCG cotton-shaped solid of white, its HPLC purity can reach about 98%, and its rate of recovery can reach for 93% (being about 300mg according to EGCG content in the HPLC peak area percentage calculation study).
Then with 1: 4: 5 volume ratio preparation n-hexane-ethyl acetate-aqueous systems, more than be fixing phase mutually, be mobile phase down mutually.With above-mentioned separate raffinate in the EGCG rear pillar after concentrating as sample feeding.Ditto operate, can obtain the cotton-shaped solid of 140mg GCG and 130mg ECG white at last respectively, its HPLC purity all can reach about 98%.
Example 2. adopts the analytic type counter-current chromatograph, chooses the ethyl acetate, alcohol and water system and separates EGCG, and is right
The back adopts the gradient elution mode to separate GCG and ECG (see figure 2).
At first with respectively with 25: 1: 25,10: 1: 10,5: 1: 5 volume ratio was prepared three parts of above-mentioned dicyandiamide solutions in separatory funnel, shook up the back standing demix.Ready to balance separated upper and lower phase respectively after a period of time, got fixedly phase of conduct mutually, and is following mutually as the phase that flows.
Taking by weighing Tea Polyphenols study 16mg, to be dissolved in 1ml stand-by in flowing mutually.
The GS20 type analysis type high-speed counter-current chromatograph that adopts Beijing Institute for New Technologies to develop, and be furnished with pump, sampling valve, UV-detector and recorder.The column capacity of its multi-lay winding polyfluortetraethylene pipe is about 35ml.
Be full of whole pillar with going up mutually of 25: 1: 25 systems mutually as fixing earlier; Adjust engine speed 1800rpm, mutually pump into post in as mobile the following phase of this system with the flow velocity of 1.0ml/min; After treating that whole system is set up dynamic equilibrium, by the sampling valve sample introduction; Change the following phase wash-out of 10: 1: 10 systems when being eluted to p1 point shown in Figure 2, change the following phase wash-out of 5: 1: 5 systems during to the p2 point; Simultaneously according to the detector uv atlas, the receiving target composition.
Example 3. adopts the analytic type counter-current chromatograph, and choose n-hexane-ethyl acetate-aqueous systems and separate EGCG,
GCG and ECG (see figure 3).
Dispose above-mentioned dicyandiamide solution with 1: 10: 10 volume ratio, carry out the operation same, just midway with example 2
Changing solvent system not.Sample size also is 16mg.
Example 4. adopts the analytic type counter-current chromatographs, chooses ethyl acetate-aqueous systems and separates EGCG and (see figure
4)。
Dispose above-mentioned system with 1: 1 volume ratio, carry out lock out operation with example 3.Sample size is 17mg.
Claims (10)
1, a kind of highspeed counter-current chromatographic separation process for preparing of high purity catechin, it comprises: preparation constitutes the fixedly dicyandiamide solution of phase, the phase that flows; Make and be full of fixedly phase in the counter-current chromatograph pillar; Its main frame is rotated, and will flow pumps in the post mutually again; By the sampling valve sample introduction; According to detector spectrogram receiving target composition, it is characterized in that: described dicyandiamide solution is by fatty ester, fatty alcohol or aliphatic ketone, and water is formed.
2, the highspeed counter-current chromatographic separation process for preparing of high purity catechin according to claim 1, it is characterized in that: described fatty ester is an ethyl acetate, described fatty alcohol is an ethanol, in the ethyl acetate, alcohol and water system, their consumption volume ratio is (1-10): (0-1): 10, below be fixing phase mutually, be mobile phase down mutually.
3, the highspeed counter-current chromatographic separation process for preparing of high purity catechin according to claim 2, it is characterized in that: described dicyandiamide solution, after isolating EGCG, when separating GCG and GCG, increase the following volume ratio of middle ethanol/water mutually gradually, promptly adopt the gradient elution mode; Or solvent burden ratio in the change system, promptly improve middle mutually down ethanol/water volume ratio; Or use normal hexane-ethyl acetate-aqueous systems instead, back two kinds are called the substep separate mode.
4, the adverse current chromatogram method for separating and preparing of high purity catechin according to claim 3, it is characterized in that: the volume ratio of described separation EGCG dicyandiamide solution ethyl acetate, alcohol and water is 20: 1: 20,25: 1: 25,50: 1: 50, separating GCG and ECG system is normal hexane-ethyl acetate-water, and their volume ratio is 1: 4: 5 or 1: 3: 4.
5, a kind of highspeed counter-current chromatographic separation process for preparing of high purity catechin, it comprises: preparation constitutes the fixedly dicyandiamide solution of phase, the phase that flows; Make and be full of fixedly phase in the counter-current chromatograph pillar; Making its main frame rotate will flow then pumps in the post mutually again; By the sampling valve sample introduction; According to detector spectrogram receiving target composition, it is characterized in that: described dicyandiamide solution is made up of alkane, fatty ester, water.
6, the adverse current chromatogram method for separating and preparing of high purity catechin according to claim 5, it is characterized in that: described alkane is that normal hexane, described fatty ester are ethyl acetate, in normal hexane-ethyl acetate-aqueous systems, their consumption volume ratio is (0-1): 10: (10-100), more than mutually for fixing phase, be mobile phase mutually down.
7, the adverse current chromatogram method for separating and preparing of high purity catechin according to claim 6, it is characterized in that: described system, after separation EGCG goes out, when separating GCG and ECG, the component that wash-out is not gone out is as new sample, carry out secondary separation after concentrating, its dicyandiamide solution is constant, but the volume ratio of normal hexane/ethyl acetate improves.
8, according to the highspeed counter-current chromatographic separation process for preparing of the high purity catechin of claim 7, it is characterized in that: when separating GCG, ECG, the volume ratio of normal hexane/ethyl acetate is 1: 4 or 1: 3.
9, the adverse current chromatogram method for separating and preparing of high purity catechin according to claim 1 or 5 is characterized in that: describedly separating and collecting in the target composition process and adopt N
2Gas shiled.
10, the adverse current chromatogram method for separating and preparing of high purity catechin according to claim 1 or 5, it is characterized in that: the described product of collecting will carry out freeze drying immediately, places the closed container low-temperature dark to preserve then.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1333249C (en) * | 2004-12-07 | 2007-08-22 | 清华大学 | Counterflow chromatograph analyzing and separating preparation with micro-emulsion as solvent |
| CN100582102C (en) * | 2007-12-07 | 2010-01-20 | 西南大学 | Process for preparing methylation catechin by tea |
| CN101830882A (en) * | 2010-05-13 | 2010-09-15 | 遵义陆圣康源科技开发有限责任公司 | Method for simultaneously preparing seven catechin monomers from tea leaves |
| CN102070594B (en) * | 2009-11-24 | 2013-08-21 | 上海中药制药技术有限公司 | Separation preparation method for high-purity agarotetrol and 4'-methoxy agarotetrol |
| CN105601606A (en) * | 2016-03-08 | 2016-05-25 | 浙江省计量科学研究院 | Method for preparing high-purity gallnut catechin gallate (GCG) |
| CN113933405A (en) * | 2021-09-02 | 2022-01-14 | 大海粮油工业（防城港）有限公司 | Method for detecting content of tea polyphenol palmitate based on liquid chromatograph |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427501C (en) * | 2005-08-25 | 2008-10-22 | 浙江大学 | Method for separating and preparing ursolic acid and its derivative from persimmon leaf using counter current chromatography |
-
1999
- 1999-06-14 CN CNB991090314A patent/CN1136024C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1333249C (en) * | 2004-12-07 | 2007-08-22 | 清华大学 | Counterflow chromatograph analyzing and separating preparation with micro-emulsion as solvent |
| CN100582102C (en) * | 2007-12-07 | 2010-01-20 | 西南大学 | Process for preparing methylation catechin by tea |
| CN102070594B (en) * | 2009-11-24 | 2013-08-21 | 上海中药制药技术有限公司 | Separation preparation method for high-purity agarotetrol and 4'-methoxy agarotetrol |
| CN101830882A (en) * | 2010-05-13 | 2010-09-15 | 遵义陆圣康源科技开发有限责任公司 | Method for simultaneously preparing seven catechin monomers from tea leaves |
| CN105601606A (en) * | 2016-03-08 | 2016-05-25 | 浙江省计量科学研究院 | Method for preparing high-purity gallnut catechin gallate (GCG) |
| CN105601606B (en) * | 2016-03-08 | 2017-11-21 | 浙江省计量科学研究院 | A kind of method for preparing high-purity nutgall catechin gallic acid ester GCG |
| CN113933405A (en) * | 2021-09-02 | 2022-01-14 | 大海粮油工业（防城港）有限公司 | Method for detecting content of tea polyphenol palmitate based on liquid chromatograph |
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