CN103910705A - Method for rapidly extracting, separating and purifying epigallocatechin gallate(EGCG) from leftover of green tea - Google Patents
Method for rapidly extracting, separating and purifying epigallocatechin gallate(EGCG) from leftover of green tea Download PDFInfo
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- CN103910705A CN103910705A CN201410103960.0A CN201410103960A CN103910705A CN 103910705 A CN103910705 A CN 103910705A CN 201410103960 A CN201410103960 A CN 201410103960A CN 103910705 A CN103910705 A CN 103910705A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention relates to a method for rapidly extracting, separating and purifying epigallocatechin gallate from leftover of green tea, which comprises the following steps: 1)extracting the leftover of the green tea by ethanol through a conventional method; 2)performing pressure reduction on an extract below the temperature of 80 DEG C and recovering ethanol until no alcohol taste; 3)absorbing an alcohol extract by polyamide resin and separating; 4)collecting epigallocatechin gallate fraction, decolouring by active carbon, performing pressure reduction at low temperature and concentrating; and 5)recrystallizing by ethanol to obtain the high purity epigallocatechin gallate. According to the invention, the method is simple to operate, only one resin is employed to reach one-step completion of adsorption, separation and purification, the used solvent has the advantages of no toxicity, safety and reliability, and the product has the advantages of high purity and low cost, and is suitable for industrial production.
Description
Technical field
The present invention relates to medical applications field, particularly relate to a kind of from the tankage of green tea the method for rapid extraction separation and purification NVP-XAA 723.
Background technology
Polyphenols in tealeaves accounts for the 18%-36% of dry weight of tea leaves, comprises take catechin as main flavanol compound, flavones and flavonols, anthocyanidin and leucoanthocyanidin and phenolic acid and depside.The main body composition that wherein catechin compounds is tea-polyphenol, accounts for the 12%-24% of dry weight of tea leaves, accounts for the 70%-80% of tea-polyphenol total amount.Modern medicine study discovery, tea-polyphenol is a kind of novel natural antioxidants, has antitumor, anti-ageing, anti-sudden change, suppresses the physiological actions such as tumor growth, Weight-lossing antihypertensive and preventing cardiovascular disease.And these physiological actions are mainly to show by catechin wherein, effect that wherein NVP-XAA 723 in ester catechin (EGCG) shows in many aspects is obviously better than other catechin compositions.
The molecular formula of EGCG is C
22h
18o
11,molecular weight 458.4, contains multiple phenolic hydroxyl groups in the molecule of EGCG, active centre, on two rings, therefore has the ability of very strong anti-oxidant and removing free radical.EGCG sterling is white powder, and bitter and puckery flavor, nontoxic, very easily water-soluble, can be dissolved in methyl alcohol, ethanol, acetonitrile and ethyl acetate, is insoluble to chloroform, and fusing point is 218 ℃.Easily oxidized in air, become micro mist look, also easily oxidized along with the rising of temperature.
Along with going deep into that people are familiar with tea component nourishing function, the extraction of tea-polyphenol separates the research obtaining extensively and profoundly, and the catechin monomers research that especially prepared by the separation of ester catechin EGCG also more and more receives people's concern.At present, the process patent of extraction separation EGCG is more.Wherein preparation method's patent of high purity EGCG mainly contains:
CN1465572A epi-nutgall catechin gallic acid ester monomer purifying method
CN1470510A epi-nutgall catechin gallic acid ester monomer purifying process
CN102702163A prepares the method for high purity catechin from the tankage of tealeaves
The separation purification method of CN1603319A catechin monomers
The method of CN101386614A preparing epigallocatechin-3-gallate by resin adsorption method
CN1733753A epi-nutgall catechin gallic acid ester monomer purifying method
Mono-kind of CN101381359A extracts the method for high-purity epigallocatechin-3-gallate from green tea
CN101723927A method for batch production, separation and purification of catechin monomers EGCG
The preparation method of the EGCG of a CN101074224A high-content
Eight above-mentioned patents, sample is all to adopt conventional method to extract, comprise solvent-extraction process, precipitation by metallic ion method, resin adsorption method, supercritical fluid extraction, ultrasonic-leaching method etc., due in tea-polyphenol except EGCG, also contain other materials of certain content, the EGCG purity therefore extracting is not high.To the purge process of single component EGCG, mostly first to adopt resin absorption technology to carry out removal of impurities, comprising macroporous adsorptive resins, anion-cation exchange resin post etc., conventionally adopt the resin column of a post or two different in kinds to connect, main effect is that sample is adsorbed, and take water as elutriant, carries out desorption again after removing water-soluble impurity with rare alcohol.Only according to above-mentioned means, cannot reach the purity requirement of EGCG, and the crucial purification step of above-mentioned 8 patents is that last a few step has all adopted chromatographic separation technology means, comprise means such as using supercritical fluid chromatography, sephadex chromatography, mesolow silica gel column chromatography chromatogram, C18 liquid chromatography technology of preparing, finally, by the method for recrystallization, make EGCG reach very high purity.
The weak point of 8 above-mentioned patents is operating process complexity, to requirement for experiment condition harshness, owing to adopting high-end laboratory apparatus and expensive chemical reagent, makes preparation cost very high and operate wayward.And repeatedly adopted post adsorption separation process, and lose greatlyr, make product yield lower, and introduced during the course a large amount of organic solvents, operation steps only limits to laboratory, and product can not be realized industrialization.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of tankage that can make full use of green tea carrys out the method for rapid extraction separation and purification NVP-XAA 723, make the tankage of green tea obtain recycling as waste, also solved EGCG in purge process simultaneously, step complexity, length consuming time, solvent for use toxicity is large, and product yield is low, can not realize the problems such as industrialization.
Of the present invention from the tankage of green tea the method for rapid extraction separation and purification NVP-XAA 723, comprise following steps:
(1) tankage of green tea extract with the ultrasonic concussion of 60% ethanol, and extraction time is 30 minutes, extracts twice, united extraction liquid;
(2) extracting solution reclaims ethanol to without alcohol taste, is concentrated into relative density and is 1.20 to 1.22 clear cream;
(3) by the clear cream polyamide resin column fractionation by adsorption obtaining in (2);
(4) thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 flow point, with after activated carbon decolorizing, filters, and filtrate, in 60 ℃ of-80 ℃ of low-temperature reduced-pressures, is led to nitrogen protection, and being concentrated into relative density is 1.15-1.18, lyophilize;
(5) with ethyl alcohol recrystallization, obtain highly purified NVP-XAA 723.
2, method according to claim 1, it is characterized in that: the tankage of described step (1) green tea, comprise the thick old bar of some except a large amount of fresh leaves in the process of picking tea-leaves, prune branch or the tea dust producing in the grade course of processing or in storage overstocked some old tea, tea grounds.
3, method according to claim 1, is characterized in that the tankage of described step (1) green tea and the ratio of 60% ethanol are 1:8-1:20(g/ml).
4, method according to claim 1, is characterized in that described step (2) extracting solution reclaims ethanol to without alcohol taste, refers to 80 ℃ of following reclaim under reduced pressure.
5, method according to claim 1, is characterized in that the granularity of described step (3) polyamide resin is 60-100 order, and consumption is: resin: clear cream=10-20:1(g/g), the blade diameter length ratio of resin column is 1:20.
6, method according to claim 1, is characterized in that the elutriant of using in described step (3) fractionation by adsorption comprises 95% ethanol and 36% acetic acid, wherein 95% ethanol: 36% acetic acid is 5:1-1:5(v/v).
7, method according to claim 1, the 2%-3% (g/100ml) that the consumption that it is characterized in that described step (4) gac is liquor capacity, the amount of adding gac in every 100ml solution is 2g-3g.
The invention has the beneficial effects as follows: the present invention does not need the preliminary purification of macroporous resin, by controlling chromatographic separation condition, only adopt a kind of separation system, can directly carry out the separation of polymeric amide chromatography, therefore, follow the tracks of detection by thin-layer chromatography, can collect EGCG sterling flow point, make like this separation, purifying one step complete, make easy to operation; And in whole process, only used etoh solvent, acetic acid and the gac of food grade, guarantee product safety, nontoxic.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of EGCG reference substance;
Fig. 2 is the sample high-efficient liquid phase chromatogram that the method for rapid extraction separation and purification NVP-XAA 723 obtains from the tankage of green tea of the present invention.
embodiment
embodiment 1
Green tea tankage 1 kg, adds the ultrasonic concussion of 8L60% ethanol and extracts, and extracts twice, each each 30min, united extraction liquid; The following decompression recycling ethanols of 80 ℃ of extracting solutions are to without alcohol taste, are concentrated into relative density and are 1.20 to 1.22 clear cream; Cooling; (resin demand is heavy 10 times of cream to the absorption of upper polyamide resin column; the blade diameter length ratio of post is 1:20); take 95% ethanol: 36% acetic acid (volume ratio is as 5:1) wash-out, thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 (EGCG) flow point; after activated carbon decolorizing with 2% (g/100ml); filter, it is 1.15-1.18 that filtrate low temperature under 60 ℃ of-80 ℃ of logical nitrogen protections is evaporated to relative density, lyophilize.With ethyl alcohol recrystallization, obtain purity and be 98.3% NVP-XAA 723 (EGCG), yield is 10%.
embodiment 2
Green tea tankage 1 kg, adds the ultrasonic concussion of 12L60% ethanol and extracts, and extracts twice, each each 30min, united extraction liquid.The following decompression recycling ethanols of 80 ℃ of extracting solutions are to without alcohol taste, are concentrated into relative density and are 1.20 to 1.22 clear cream.Cooling; (resin demand is heavy 15 times of cream to the absorption of upper polyamide resin column; the blade diameter length ratio of post is 1:20); take 95% ethanol: 36% acetic acid (volume ratio is as 2:1) wash-out, thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 (EGCG) flow point; after charcoal absorption with 2.5% (g/100ml); filter, it is 1.15-1.18 that filtrate low temperature under 60 ℃ of-80 ℃ of logical nitrogen protections is evaporated to relative density, lyophilize.With ethyl alcohol recrystallization, obtain purity and be 98.6% NVP-XAA 723 (EGCG), yield is 12%.
embodiment 3
Green tea tankage 1 kg, adds the ultrasonic concussion of 16L60% ethanol and extracts, and extracts twice, each each 30min, united extraction liquid.The following decompression recycling ethanols of 80 ℃ of extracting solutions are to without alcohol taste, are concentrated into relative density and are 1.20 to 1.22 clear cream.Cooling; (resin demand is heavy 20 times of cream to the absorption of upper polyamide resin column; the blade diameter length ratio of post is 1:20); take 95% ethanol: 36% acetic acid (volume ratio is as 1:1) wash-out, thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 (EGCG) flow point; after charcoal absorption with 3% (g/100ml); filter, it is 1.15-1.18 that filtrate low temperature under 60 ℃ of-80 ℃ of logical nitrogen protections is evaporated to relative density, lyophilize.With ethyl alcohol recrystallization, obtain purity and be 98.9% NVP-XAA 723 (EGCG), yield is 16%.
embodiment 4
Green tea tankage 1 kg, adds the ultrasonic concussion of 20L60% ethanol and extracts, and extracts twice, each each 30min, united extraction liquid.The following decompression recycling ethanols of 80 ℃ of extracting solutions are to without alcohol taste, are concentrated into relative density and are 1.20 to 1.22 clear cream.Cooling; (resin demand is heavy 20 times of cream to the absorption of upper polyamide resin column; the blade diameter length ratio of post is 1:20); take 95% ethanol: 36% acetic acid (volume ratio is as 1:2) wash-out, thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 (EGCG) flow point; after charcoal absorption with 3% (g/100ml); filter, it is 1.15-1.18 that filtrate low temperature under 60 ℃ of-80 ℃ of logical nitrogen protections is evaporated to relative density, lyophilize.With ethyl alcohol recrystallization, obtain purity and be 99.0% NVP-XAA 723 (EGCG), yield is 15%.
The tankage of the green tea in above-described embodiment, comprise some thick old bars of removing in the process of picking tea-leaves outside a large amount of fresh leaves, prune some old tea overstocked in branch or the tea dust producing and storage, tea grounds in the grade courses of processing.
The beneficial effect of above-described embodiment is: the present invention does not need the preliminary purification of macroporous resin, because EGCG is polyphenolic compound, by controlling chromatographic separation condition, adopt a kind of separation system, can directly carry out the separation of polymeric amide chromatography, therefore, follow the tracks of detection by thin-layer chromatography, can collect EGCG sterling flow point, rather than mixture, make like this separation, purifying one step complete, make easy to operation; And in whole process, only used etoh solvent, acetic acid and the gac of food grade, guarantee product safety, nontoxic.
Above-described embodiment is only explanation technical conceive of the present invention and feature; its object is to allow person skilled in the art can understand content of the present invention and be implemented; can not limit the scope of the invention with this; all equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (7)
1. the method for rapid extraction separation and purification NVP-XAA 723 from the tankage of green tea, comprises following steps:
(1) tankage of green tea extract with the ultrasonic concussion of 60% ethanol, and extraction time is 30 minutes, extracts twice, united extraction liquid;
(2) extracting solution reclaims ethanol to without alcohol taste, is concentrated into relative density and is 1.20 to 1.22 clear cream;
(3) by the clear cream polyamide resin column fractionation by adsorption obtaining in (2);
(4) thin-layer chromatography is followed the tracks of and is detected, and collects NVP-XAA 723 flow point, with after activated carbon decolorizing, filters, and filtrate, in 60 ℃ of-80 ℃ of low-temperature reduced-pressures, is led to nitrogen protection, and being concentrated into relative density is 1.15-1.18, lyophilize;
(5) with ethyl alcohol recrystallization, obtain highly purified NVP-XAA 723.
2. method according to claim 1, it is characterized in that: the tankage of described step (1) green tea, comprise some thick old bars of removing in the process of picking tea-leaves outside a large amount of fresh leaves, prune branch or the tea dust producing in the grade course of processing and in storage overstocked some old tea, tea grounds.
3. method according to claim 1, is characterized in that the tankage of described step (1) green tea and the ratio of 60% ethanol are 1:8-1:20(g/ml).
4. method according to claim 1, is characterized in that described step (2) extracting solution reclaims ethanol to without alcohol taste, refers to 80 ℃ of following reclaim under reduced pressure.
5. method according to claim 1, is characterized in that the granularity of described step (3) polyamide resin is 60-100 order, and consumption is: resin: clear cream=10-20:1(g/g), the blade diameter length ratio of resin column is 1:20.
6. method according to claim 1, is characterized in that the elutriant of using in described step (3) fractionation by adsorption comprises 95% ethanol and 36% acetic acid, wherein 95% ethanol: 36% acetic acid is 5:1-1:5(v/v).
7. method according to claim 1, the 2%-3% (g/100ml) that the consumption that it is characterized in that described step (4) gac is liquor capacity, the amount of adding gac in every 100ml solution is 2g-3g.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104171157A (en) * | 2014-09-10 | 2014-12-03 | 嘉兴市得百科新材料科技有限公司 | Green tea extract as well as preparation method and application thereof |
| CN104435369A (en) * | 2014-11-28 | 2015-03-25 | 桂林市和胤祥新型材料有限公司 | Preparation method of tea polyphenol |
| CN104926680A (en) * | 2015-05-08 | 2015-09-23 | 李会杰 | Method for extracting bio-active substances from low-grade tea and tea-manufacturing leftovers |
| 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 |
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| CN1286252A (en) * | 1999-08-16 | 2001-03-07 | 弗·哈夫曼-拉罗切有限公司 | Process for preparing epigallocatechin gallate |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104171157A (en) * | 2014-09-10 | 2014-12-03 | 嘉兴市得百科新材料科技有限公司 | Green tea extract as well as preparation method and application thereof |
| CN104171157B (en) * | 2014-09-10 | 2016-06-15 | 河源市绿环生物科技有限公司 | A kind of green tea extract and preparation method thereof, application |
| CN104435369A (en) * | 2014-11-28 | 2015-03-25 | 桂林市和胤祥新型材料有限公司 | Preparation method of tea polyphenol |
| CN104926680A (en) * | 2015-05-08 | 2015-09-23 | 李会杰 | Method for extracting bio-active substances from low-grade tea and tea-manufacturing leftovers |
| CN104926680B (en) * | 2015-05-08 | 2016-08-24 | 李会杰 | The method extracting bioactive substance from low-grade tea and tea making leftover bits and pieces |
| 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 |
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| CN103910705B (en) | 2015-10-28 |
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