CN102311419A - Refining and purification method of high content EGCG - Google Patents
Refining and purification method of high content EGCG Download PDFInfo
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- CN102311419A CN102311419A CN201110266483A CN201110266483A CN102311419A CN 102311419 A CN102311419 A CN 102311419A CN 201110266483 A CN201110266483 A CN 201110266483A CN 201110266483 A CN201110266483 A CN 201110266483A CN 102311419 A CN102311419 A CN 102311419A
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Abstract
The invention provides a refining and purification method of a high content EGCG. A raw material of low content EGCG tea polyphenol is treated with solving, column loading, washing, concentration, refining and drying. The EGCG prepared by the method of the invention is white powder with a content not lower than 98% and a yield not lower than 80%. The technology of the invention employs a mature silicagel column chromatography technology, which is easily operated, energy saving, with a large throughput and suitable for industrialized production.
Description
Technical field
The present invention relates to the process for refining and purifying of a kind of high-content EGCG, belong to the natural product chemistry technical field.
Background technology
EGCG, i.e. NVP-XAA 723, structural formula is seen figure-1, is from green tea, to extract refining and get.The staple of green tea tea-polyphenol is a catechin.Wherein comprise l-Epicatechol (EC), NVP-XAA 723 (EGCG), L-Epicatechin gallate (ECG) etc. again, and EGCG accounts for 50% of catechin total amount.Research shows that EGCG has the function of stronger oxidation-resistance and preventing cancer and cardiovascular disorder, and can improve the susceptibility of cancer cells to chemotherapy, reduces the toxicity of chemotherapy to heart simultaneously.Show that according to documents and materials EGCG is in the treatment cancer, there is stronger pharmacological action DNA plerosis, aspect such as antitumor; Function that can the enhancing immunity system; Suppress the growth of liver fat and SUV, suppress growth of tumor, dysentery injury, streptococcus aureus are also had extremely strong restraining effect.EGCG is mainly used in products such as foodstuff additive, healthcare products at present.
Figure-1
Because its chemical structure of polyphenols that from green tea, extracts is all extremely similar with character, all has strong polarity, general separation method all is difficult to it is separated.
The method of from tea-polyphenol, directly extracting epi-nutgall catechin gallic acid ester monomer at present mainly contains column chromatography, high-speed counter-current partition chromatography, ionic reaction method, acetylation method etc.The column chromatography of existing patent report such as CN1465572, CN1470510A remove most of impurity through loaded down with trivial details pre-treating process earlier, go up VISOSE SephadexLH-20 post again and purify; CN101386614A, CN101723927A etc. carry out chromatography to reach the purification purpose through repeatedly using different sorbent materials.Patent CN1367171A is through behind the counter-current extraction repeatedly; Carry out ionic reaction through adding divalence (trivalent) metal-salt behind the adjustment pH value; Obtain throw out and dissolve through washing, acid, extraction, depickling, concentrate, series of steps such as recrystallization finally obtains EGCG content and reaches 90%.Patent CN1733753 purifies through reach refining with the saturated deionized water dissolving tea-polyphenol sample of rare gas element, membrane filtration, upper prop etc.Patent CN10182319A crosses silicagel column after utilizing the phenolic hydroxyl group acetylize on the tea-polyphenol, and deacetylated again, acetylization reaction need carry out on ice bath, and reaction is accomplished also needs a large amount of frozen water, and long reaction time, and this method only is applicable to prepared in laboratory.Because reaction conditions is special, complex operation, shortcomings such as length consuming time can't be promoted the use of on big production.Patent CN1277068A has introduced a kind of high-speed counter-current branch color scheme of high purity catechin, and the amount of countercurrent chromatography flash liberation sample is little at present, and separation cycle is long, is difficult to be applied to industrial production.Patent CN1724530A discloses the method that a kind of continuous medium pressure column chromatography prepares high purity EGCG, and this method only is fit to EGCG content is purified greater than 50% tea-polyphenol, and yield reaches about 70%.
In sum, though above method of purification can reach the high-load requirement of EGCG, it remains in many problems.For addressing these problems, the present invention has developed a kind of simple to operate, and is energy-conservation, the method for purification that yield is high.
Summary of the invention
The present invention will provide a kind of process for purification that from tea-polyphenol, extracts high-content EGCG.Tea-polyphenol with low levels is a raw material, through dissolving, upper prop, wash-out, concentrate, refining, dry and get.The prepared EGCG of this method is a white powder, and HPLC detects, content >=98%.
Of the present invention is raw material with the low levels tea-polyphenol, is meant that tea-polyphenol EGCG content is at 40～50% green tea extract.
The process for purification that from tea-polyphenol, extracts high-content EGCG of the present invention comprises following main operational steps:
A. by the tea-polyphenol of 1:3～1:8 (m/m) and the weight ratio of silica gel tea-polyphenol is carried out silica gel column chromatography.
B. ETHYLE ACETATE-sherwood oil-acetate the mixed solvent in 4:6:1～5:5:1 ratio carries out wash-out, collects each elutriant respectively.
C. confirm the EGCG elutriant collected in the b step with thin-layer chromatography, and its high purity elutriant merged concentrate, EGCG medicinal extract.
D. in this solid, add 60～90% ethanol in 1:6～10 (m/v) ratio, adding was stirred 2～3 hours in 50 ℃～70 ℃ water-baths, placed the crystallization of spending the night, and filtered, and collected crystal, and 80 ℃ of drying under reduced pressure get high-content EGCG.
Wherein step a, the described column chromatography condition of b are: the silica gel specification is 60～100 orders.
The process for purification of high-content EGCG provided by the present invention, products obtained therefrom content >=98%, yield >=80%.
Embodiment:
Following examples are used to further specify the present invention, but not represent be unique channel of the present invention to said embodiment, also do not mean that the present invention is done any restriction.
Embodiment one: adopt dry column-packing to be loaded on internal diameter 5cm 60～100 order chromatographic silica gel 120g, in the height 80cm glass chromatography column.Get tea-polyphenol (containing EGCG40.8%) 20g, add ETHYLE ACETATE-sherwood oil mixed solvent 100ml dissolving, add in the silicagel column and carry out chromatography.Use ETHYLE ACETATE-sherwood oil-acetate (5:5:1) mixed solvent to carry out wash-out then, collect each wash-out section liquid respectively.Confirm behind the EGCG wash-out section liquid its merging to be concentrated with thin-layer chromatography, get EGCG medicinal extract.This solid is placed the Erlenmeyer flask of 250ml, in this solid, add 70% ethanol 160ml, the crystallization of spending the night is placed in 60 ℃ of stirred in water bath dissolvings 2 hours, filters, and collects crystal.80 ℃ of drying under reduced pressure get 6.7g, and detecting its content through HPLC is 98.23%.
Embodiment two: adopt dry column-packing to be loaded on internal diameter 5cm 60～100 order chromatographic silica gel 140g, in the height 80cm glass chromatography column.Get tea-polyphenol (containing EGCG40.8%) 20g, add ETHYLE ACETATE-sherwood oil mixed solvent 100ml dissolving, pour into and carry out chromatography in the silicagel column.Use ETHYLE ACETATE-sherwood oil-acetate (5:5:1) mixed solvent to carry out wash-out then, collect each wash-out section liquid respectively.Confirm behind the EGCG wash-out section liquid its merging to be concentrated with thin-layer chromatography, get EGCG medicinal extract.This solid is placed the Erlenmeyer flask of 250ml, in this solid, add 85% ethanol 160ml, the crystallization of spending the night is placed in 60 ℃ of stirred in water bath dissolvings 2 hours, filters, and collects crystal.80 ℃ of drying under reduced pressure get 6.7g, and detecting its content through HPLC is 98.53%.
Embodiment three: adopt dry column-packing to be loaded in the chromatography column 60～100 order chromatographic silica gel 600g.Get tea-polyphenol (containing EGCG40.8%) 100g, add ETHYLE ACETATE-sherwood oil mixed solvent 500ml dissolving, pour into and carry out chromatography in the silicagel column.Use ETHYLE ACETATE-sherwood oil-acetate (4:6:1) mixed solvent to carry out wash-out then, collect each wash-out section liquid respectively.Confirm behind the EGCG wash-out section liquid its merging to be concentrated with thin-layer chromatography, get EGCG medicinal extract.This solid is placed the Erlenmeyer flask of 2000ml, in this solid, add 85% ethanol 800ml, the crystallization of spending the night is placed in 70 ℃ of stirred in water bath dissolvings 2 hours, filters, and collects crystal.80 ℃ of drying under reduced pressure get 33.8g, and detecting its content through HPLC is 98.74%.
Embodiment four: adopt dry column-packing to be loaded in the chromatography column 60～100 order chromatographic silica gel 120kg.Get tea-polyphenol (containing EGCG40.8%) 20kg, add ETHYLE ACETATE-sherwood oil mixed solvent 1000L stirring and made dissolving in 2 hours, add in the silicagel column and adsorb.Use ETHYLE ACETATE-sherwood oil-acetate (5:5:1) mixed solvent to carry out wash-out then; Detect elutriant purity with thin-layer chromatography simultaneously; Be collected into low-purity elutriant and high purity elutriant, wash-out back segment in wash-out leading portion jar, EGCG section jar and the wash-out back segment jar respectively, reclaim solvent respectively.Wherein EGCG section medicinal extract is used for that next step is refining.This solid is placed treatment tank, add 80% ethanol 160L, 60 ℃ of stirring and dissolving 3 hours are placed the crystallization of spending the night, and filter, and collect crystal.80 ℃ of drying under reduced pressure get 6.71kg, and detecting its content through HPLC is 98.31%.
Embodiment five: adopt dry column-packing to be loaded in the chromatography column 60～100 order chromatographic silica gel 300kg.Get tea-polyphenol (containing EGCG40.8%) 60kg, add ETHYLE ACETATE-sherwood oil mixed solvent 3000L stirring and made dissolving in 2 hours, add in the silicagel column and adsorb.Use ETHYLE ACETATE-sherwood oil-acetate (5:5:1) mixed solvent to carry out wash-out then; Detect elutriant purity with thin-layer chromatography simultaneously; Be collected into low-purity elutriant, high purity elutriant and wash-out back segment in wash-out leading portion jar, EGCG section jar and the wash-out back segment jar respectively, reclaim solvent respectively.Wherein EGCG section medicinal extract is used for that next step is refining.This solid is placed treatment tank, add 85% ethanol 420L, 60 ℃ of stirring and dissolving 2 hours are placed the crystallization of spending the night, and filter, and collect crystal.80 ℃ of drying under reduced pressure get 20.1kg, and detecting its content through HPLC is 98.90%.
Claims (7)
1. the process for refining and purifying of a high-content EGCG is that the tea-polyphenol with low levels EGCG carries out silica gel column chromatography, carries out wash-out through ETHYLE ACETATE-sherwood oil-acetate mixed solvent again, collects each section elutriant respectively; Confirm the purity of collected EGCG elutriant with thin-layer chromatography, and its highly purified elutriant merged concentrate, EGCG medicinal extract, in this solid, add ethanolic soln; 50 ℃～70 ℃ stirred in water bath were dissolved 2～3 hours, placed crystallization, filtered; Collect crystal, 80 ℃ of drying under reduced pressure get high-content EGCG; This method has easy and simple to handle, and is energy-conservation, the characteristics of yield high (>=80%); Product detects through HPLC, and EGCG content >=98% is prone to realize producing in enormous quantities.
2. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, its characteristics are: it is that tea-polyphenol with low levels (40%～50%) is a raw material, through upper prop, wash-out, concentrate, crystallization, dry and get.
3. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, the solvent that its characteristics are to dissolve tea-polyphenol is the mixed solvent of ETHYLE ACETATE-sherwood oil (1:1).
4. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, the solvent load that its characteristics are to dissolve tea-polyphenol is 5 times of sample size.
5. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, its characteristics are to adopt 60～100 order chromatographic silica gels, and the silica gel consumption is 3～8 times of sample size, adopt dry column-packing.
6. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, its characteristics are that the blending ratio of eluent ETHYLE ACETATE-sherwood oil-acetate is 4:6:1～5:5:1.
7. according to the process for refining and purifying of claim 1 described a kind of high-content EGCG, its characteristics are that with 60～90% ethanolic solns its consumption is 8 times of medicinal extract.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643260A (en) * | 2012-04-25 | 2012-08-22 | 中国农业科学院茶叶研究所 | Method for separating epigallocatechin gallate by continuous column chromatography |
| CN102875691A (en) * | 2012-10-19 | 2013-01-16 | 黄冈师范学院 | Method for synchronously preparing active substances from tea dregs |
| CN103550412A (en) * | 2013-11-19 | 2014-02-05 | 管天球 | Method for extracting tea polyphenol through using mixed solvent |
| CN104130232A (en) * | 2014-08-12 | 2014-11-05 | 四川天予植物药业有限公司 | EGCG (epigallocatechin gallate) purification method, EGCG obtained by same and medicinal composition prepared from EGCG |
| CN104356105A (en) * | 2014-10-22 | 2015-02-18 | 北京康育博尔生物科技有限公司 | Preparation method for high-content EGCG |
| WO2019105216A1 (en) * | 2017-11-30 | 2019-06-06 | 江苏天晟药业股份有限公司 | Epigallocatechin gallate crystal form i and method for preparing same |
| US11420954B2 (en) | 2017-12-05 | 2022-08-23 | Crystalmorphix Technologies Pvt. Ltd | Process for the separation of gallated epicatechins (EGCG and ECG) from green tea extract or green tea dust |
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| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
| CN1733753A (en) * | 2005-08-12 | 2006-02-15 | 上海诺德生物实业有限公司 | Epigallocatechin gallate monomer purification method |
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2011
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| US6210679B1 (en) * | 1999-01-07 | 2001-04-03 | Hauser, Inc. | Method for isolation of caffeine-free catechins from green tea |
| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643260A (en) * | 2012-04-25 | 2012-08-22 | 中国农业科学院茶叶研究所 | Method for separating epigallocatechin gallate by continuous column chromatography |
| CN102875691A (en) * | 2012-10-19 | 2013-01-16 | 黄冈师范学院 | Method for synchronously preparing active substances from tea dregs |
| CN103550412A (en) * | 2013-11-19 | 2014-02-05 | 管天球 | Method for extracting tea polyphenol through using mixed solvent |
| CN104130232A (en) * | 2014-08-12 | 2014-11-05 | 四川天予植物药业有限公司 | EGCG (epigallocatechin gallate) purification method, EGCG obtained by same and medicinal composition prepared from EGCG |
| CN104130232B (en) * | 2014-08-12 | 2015-11-18 | 四川天予植物药业有限公司 | The method of purification of a kind of EGCG and the EGCG of acquisition and pharmaceutical composition |
| CN104356105A (en) * | 2014-10-22 | 2015-02-18 | 北京康育博尔生物科技有限公司 | Preparation method for high-content EGCG |
| WO2019105216A1 (en) * | 2017-11-30 | 2019-06-06 | 江苏天晟药业股份有限公司 | Epigallocatechin gallate crystal form i and method for preparing same |
| US11420954B2 (en) | 2017-12-05 | 2022-08-23 | Crystalmorphix Technologies Pvt. Ltd | Process for the separation of gallated epicatechins (EGCG and ECG) from green tea extract or green tea dust |
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Application publication date: 20120111 |