CN103613575A - Method for purifying high-content epigallocatechin gallate (EGCG) - Google Patents
Method for purifying high-content epigallocatechin gallate (EGCG) Download PDFInfo
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- CN103613575A CN103613575A CN201310626320.3A CN201310626320A CN103613575A CN 103613575 A CN103613575 A CN 103613575A CN 201310626320 A CN201310626320 A CN 201310626320A CN 103613575 A CN103613575 A CN 103613575A
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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 discloses a method for purifying high-content epigallocatechin gallate (EGCG). The method comprises the following step: crystallizing a liquid or extractum containing a target component, wherein the temperature in crystallization is controlled at 4-8 DEG C in the crystallization process; the overall crystallization process is carried out under an agitation condition; the agitation speed is 100-300rpm (revolutions per minute). Crystal is slowly and evenly separated out in a manner of carrying out agitating crystallization at the rotating speed of 100-300rpm at a specific temperature under the condition of not adding seed crystal. The obtained crystal particles are thick and even; separation of crystal and removal of impurities are more easily achieved; the obtained crystal is higher in EGCG content and high in recovery rate.
Description
Technical field
The present invention relates to natural product chemistry technical field, be specifically related to the method for purification of a kind of high-content EGCG.
Background technology
EGCG, NVP-XAA 723, is a kind of composition extracting from green tea, it is the main activity of green tea and water-soluble components, accounts for 9%～13% of green tea gross weight; EGCG is the chief component composition of Green Tea Polyphenols, is also the main component of green tea catechins class, and it accounts for 10～15% of green tea catechins class total amount.Because there is special stereochemical structure; EGCG is the most effective anti-oxidant polyphenol in green tea; there is anti-oxidant, anticancer, anti-mutation isoreactivity; anti-oxidant activity is at least ascorbic more than 100 times; 25 times of vitamin-E; can Cell protection and DNA undermined, this infringement is believed relevant with other major diseases with cancer, heart disease, these effects of EGCG ascribe them to the removing of oxyradical (anti-oxidant) ability.
The existing extracting and purifying method to EGCG mainly contains solvent-extraction process, ion precipitation method, supercritical extraction, high-speed counter-current layer and membrane separation technique attached gel post method for separating and preparing.In the existing method of purification that contains crystallisation step, when the liquid that contains target component or medicinal extract are carried out to crystallization, crystallization is all carried out under standing condition, but the mode of standing crystallization is not easy crystallize out.Employing adds a small amount of high-content EGCG crystal as the method for crystal seed in standing crystallisation process, although can reach the object of bearing more quickly crystal, but, after adding crystal seed, the easy embedding impurity of crystal of separating out, this can reduce the content of EGCG in crystal to a certain extent, particularly when the elutriant of low levels EGCG tea-polyphenol gained after macroporous resin column purification, or the medicinal extract being obtained after concentrated by this gained elutriant is while directly carrying out crystallization, in gained crystal, the content of EGCG is lower, conventionally in 90% left and right, do not reach 95% content requirement; On the other hand, adopt and in standing crystallisation process, to add crystal that the crystallization mode of crystal seed obtains thinner, easily occur latch up phenomenon when isolation of crystalline, the washing of crystal is inconvenience also.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for purification of a kind of high-content EGCG.The method is by changing crystallization condition, do not needing to add the crystal that can obtain high EGCG content under the condition of crystal seed, and the rate of recovery of crystal is high.
The method of purification of high-content EGCG of the present invention, comprise the step that the liquid that contains target component or medicinal extract are carried out to crystallization, unlike the prior art: in crystallisation step, temperature during crystallization is controlled at 4～8 ℃, whole crystallisation process carries out under agitation condition, and stir speed (S.S.) is 100～300rpm.By adopting the rotating speed of 100～300rpm to carry out the mode of stirred crystallization under specified temp, do not needing to add under the condition of crystal seed, crystal is slowly evenly separated out, the thick and uniform particles of crystal, more easily realize the separation of crystal and the removal of impurity, and the crystal EGCG content obtaining is high, and the rate of recovery of crystal is also high.
In technique scheme, the temperature when crystallization is controlled at 4～6 ℃, and when stir speed (S.S.) is controlled at 150～250rpm, in the crystal that can make to obtain, content and the yield of EGCG are higher.
In technique scheme, the time of whole crystallisation process is 10～30h, and when whole crystallization time is within the scope of 10～24h, along with the prolongation of time, the crystal of separating out increases obviously, and when crystallization time surpasses after 24h, increasing of crystallize out is not obvious.
In technique scheme, the described liquid that contains target component or medicinal extract can be by existing routine to the liquid that contains EGCG target component or the medicinal extract that obtain in the extracting and purifying method of EGCG, but when the liquid that contains target component or medicinal extract refer to the elutriant by low levels EGCG tea-polyphenol gained after macroporous resin column purification, or during the medicinal extract being obtained after concentrated by this gained elutriant, adopt the effect of the method for the invention the most remarkable, can make the crystal EGCG content obtaining reach more than 95% (HPLC method); Standing crystallization mode identical with other condition and employing interpolation crystal seed is compared, and the high raising of the rate of recovery of crystal is more than 15%.Here the macroporous resin of indication refers in prior art that, for separating of the conventional macroporous adsorbent resin of EGCG, the model that is generally macroporous adsorbent resin can be D101, D102, D103, HP-20, HPD-600, AB-8, HP20SS, SP20SS or S-8 etc.On the liquid that contains target component or medicinal extract after macroporous resin column chromatography, solvent for wash-out is same as the prior art, conventionally using 20～30% (v/v) ethanol as eluent, in elution process, with thin-layer chromatography, follow the tracks of and detect to collect the elutriant containing target component.Here the low levels EGCG tea-polyphenol of indication typically refers to the tea-polyphenol that EGCG content is 5～40wt%, can directly buy from the market, also can adopt existing ordinary method to prepare voluntarily.When take tea-polyphenol that EGCG content is 10～20wt%, adopt the effect of the method for the invention more remarkable comparatively speaking during as raw material.
In order to obtain higher yield, technique scheme preferably also comprises re-crystallization step, and in re-crystallization step, the temperature of recrystallization is controlled at 4～8 ℃, and whole recrystallization process carries out under agitation condition, and stir speed (S.S.) is 100～300rpm.Here same as the prior art for the solvent of recrystallization, be generally the ethanol of 20～95% (v/v).In re-crystallization step, the temperature of recrystallization is preferably controlled at 4～6 ℃, and stir speed (S.S.) is preferably 150～250rpm.
The concrete steps of the method for the invention comprise:
1) take low levels EGCG tea-polyphenol is raw material, is dissolved in water;
2) macroporous resin column on gained lysate, first washes resin column with water colourless to effluent liquid, then uses 20～30% (v/v) ethanol elution, and thin-layer chromatography is followed the tracks of and detected, and collects alcohol eluen;
3) alcohol eluen of collection is concentrated into medicinal extract (40～60% solid substances), medicinal extract is sent in crystallizer (also the alcohol eluen of collection directly can be sent in crystallizer carry out crystallization), in crystallization control tank, temperature is 4～8 ℃, open and stir, whole crystallisation process is carried out under agitation condition, and control stir speed (S.S.) is 100～300rpm;
4) by crystallisate suction filtration, crystal washing, dry, obtain high-content EGCG.
When the method for the invention also comprises re-crystallization step, as long as the high-content EGCG of above-mentioned acquisition is dissolved with recrystallization solvent, send into again and in crystallizer, carry out recrystallization, in recrystallization process, in crystallization control tank, temperature is 4～8 ℃, whole recrystallization process is carried out under agitation condition, and control stir speed (S.S.) is 100～300rpm.
Compared with prior art, feature of the present invention is:
1, by adopting the rotating speed of 100～300rpm to carry out the mode of stirred crystallization under specified temp, do not needing to add under the condition of crystal seed, crystal is slowly evenly separated out, the thick and uniform particles of gained crystal grain, more easily realize the separation of crystal and the removal of impurity, and the crystal EGCG content obtaining is high, and the rate of recovery of crystal is high.
2, when the liquid that contains target component or medicinal extract are defined as, be the elutriant by low levels EGCG tea-polyphenol gained after macroporous resin column purification, or during the medicinal extract being obtained after concentrated by this gained elutriant, adopt the effect of the method for the invention the most remarkable, can make the crystal EGCG obtaining reach more than 95% (HPLC method); Standing crystallization mode identical with other condition and employing interpolation crystal seed is compared, and the high raising of the rate of recovery of crystal is more than 15%.When carrying out the step of recrystallization after crystallization, the rate of recovery of crystal standing crystallization mode identical with other condition and employing interpolation crystal seed is compared and can be improved more than 20% again.
Embodiment
With specific embodiment, the invention will be further described below, but the present invention is not limited to these embodiment.
Embodiment 1
1) get the tea-polyphenol that EGCG content is 10wt% (Guilin Laiyin Biotechnology Co., Ltd.'s production) 200g and be placed in 400ml pure water ultrasonic dissolution 30min;
2) D101 macroporous resin column on gained lysate, first washes resin column with water colourless to effluent liquid, then uses 20% (v/v) ethanol elution, and thin-layer chromatography is followed the tracks of and detected, and collects alcohol eluen;
3) alcohol eluen of collection is evaporated to medicinal extract (50% solid substance), medicinal extract is sent in crystallizer, use refrigeration cycle water for cooling, in crystallization control tank, temperature is 4 ℃, open to stir and carry out stirred crystallization 24h, whole crystallisation process is carried out under agitation condition, and control stir speed (S.S.) is 100rpm;
4) by crystallisate suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 10.68g.HPLC detects EGCG content 95.33%.
Comparative example 1
1) get the tea-polyphenol that EGCG content is 10wt% (Guilin Laiyin Biotechnology Co., Ltd.'s production) 200g and be placed in 400ml pure water ultrasonic dissolution 30min;
2) D101 macroporous resin column on gained lysate, first washes resin column with water colourless to effluent liquid, then uses 20% (v/v) ethanol elution, and thin-layer chromatography is followed the tracks of and detected, and collects alcohol eluen;
3) alcohol eluen of collection is evaporated to medicinal extract (50% solid substance), medicinal extract is sent in crystallizer, to the high-content EGCG (98.5%) that adds 0.2g in crystallizer, promote crystallization, use refrigeration cycle water for cooling, in crystallization control tank, temperature is 4 ℃, places 24h;
4) by crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 9.20g.HPLC detects EGCG content 90.83%.
Comparative example 2
Repeat comparative example 1, different, whole crystallisation process carries out at normal temperatures.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 8.12g.HPLC detects EGCG content 86.31%.
Comparative example 3
Repeat embodiment 1, different, the speed stirring in crystallisation process is 350rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 8.62g.HPLC detects EGCG content 91.35%.
Comparative example 4
Repeat embodiment 1, different, whole crystallisation process carries out at 2 ℃.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 11.25g.HPLC detects EGCG content 92.69%.
Embodiment 2
Repeat embodiment 1, different, the speed stirring in crystallisation process is 200rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 11.38g.HPLC detects EGCG content 96.53%.
Embodiment 3
Repeat embodiment 1, different, the speed stirring in crystallisation process is 300rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 10.24g.HPLC detects EGCG content 95.87%.
Embodiment 4
Repeat embodiment 1, different, the tea-polyphenol that the EGCG content of take is 20wt% (Wuxi Taiyo Green Power Co., Ltd.'s production) 100g is raw material, and the temperature in crystallisation process in crystallizer is 8 ℃, the time of stirred crystallization is 12h, and the speed of stirring is 200rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 10.21g.HPLC detects EGCG content 96.11%.
Embodiment 5
Repeat embodiment 1, different, the tea-polyphenol 67g that the EGCG content of take is 30wt% (Guilin Xingda Xingda Pharmaceutical Factory's production), the temperature in crystallisation process in crystallizer is 6 ℃, and the time of stirred crystallization is 20h, and the speed of stirring is 250rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 10.54g.HPLC detects EGCG content 95.98%.
Embodiment 6
Repeat embodiment 1, different, the tea-polyphenol 50g that the EGCG content of take is 40wt% (production of San Fu bio tech ltd, Changsha), the temperature in crystallisation process in crystallizer is 6 ℃, and the time of stirred crystallization is 30h, and the speed of stirring is 150rpm.
By crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 10.66g.HPLC detects EGCG content 95.96%.
Embodiment 7
1), 2), 3) step is with embodiment 1;
4) by crystal suction filtration, water rinses, collecting crystal sends in crystallizer again, 20% (v/v) ethanol heating for dissolving that adds 5 times of crystal weight, then use refrigeration cycle water for cooling, in crystallization control tank, temperature is 4 ℃, opens to stir and carries out stirred crystallization 24h, whole crystallisation process is carried out under agitation condition, and control stir speed (S.S.) is 100rpm;
5) by crystal suction filtration, water rinses, and lyophilize, obtains white powder crystallisate 8.95g.HPLC detects EGCG content 97.80%.
From the various embodiments described above comparative example, only have when temperature is controlled under the condition that 4～8 ℃, stir speed (S.S.) are 100～300rpm and carry out stirred crystallization, could obtain the crystal of high-content and high yield.
Claims (9)
1. the method for purification of a high-content EGCG, comprise the step that the liquid that contains target component or medicinal extract are carried out to crystallization, it is characterized in that: in crystallisation step, temperature during crystallization is controlled at 4～8 ℃, whole crystallisation process carries out under agitation condition, and stir speed (S.S.) is 100～300rpm.
2. the method for purification of high-content EGCG according to claim 1, is characterized in that: described stir speed (S.S.) is 150～250rpm.
3. the method for purification of high-content EGCG according to claim 1, is characterized in that: temperature during described crystallization is controlled at 4～6 ℃.
4. the method for purification of high-content EGCG according to claim 1, is characterized in that: the time of whole crystallisation process is 10～30h.
5. the method for purification of high-content EGCG according to claim 1, it is characterized in that: the described liquid that contains target component or medicinal extract refer to: by the elutriant of low levels EGCG tea-polyphenol gained after macroporous resin column purification, or the medicinal extract being obtained after concentrated by this gained elutriant.
6. the method for purification of high-content EGCG according to claim 5, is characterized in that: described low levels EGCG tea-polyphenol refers to that EGCG content is the tea-polyphenol of 5～40wt%.
7. according to the method for purification of the high-content EGCG described in any one in claim 1～6, it is characterized in that: also comprise re-crystallization step, in re-crystallization step, the temperature of recrystallization is controlled at 4～8 ℃, whole recrystallization process carries out under agitation condition, and stir speed (S.S.) is 100～300rpm.
8. the method for purification of high-content EGCG according to claim 7, is characterized in that: described stir speed (S.S.) is 150～250rpm.
9. the method for purification of high-content EGCG according to claim 7, is characterized in that: the temperature of described recrystallization is controlled at 4～6 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109053668A (en) * | 2018-07-25 | 2018-12-21 | 云南茶农生物产业有限责任公司 | A method of ester catechin is prepared with tea polyphenols |
| CN109180630A (en) * | 2018-08-06 | 2019-01-11 | 华茗国际健康产业（香港）有限公司 | A kind of crystallite catechin and preparation method thereof |
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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 |
| CN101074224A (en) * | 2007-04-13 | 2007-11-21 | 桂林莱茵生物科技股份有限公司 | Production of high-content EGCG |
| US20080015248A1 (en) * | 2003-01-24 | 2008-01-17 | Dou Q P | Polyphenol proteasome inhibitors, synthesis, and methods of use |
| KR20120024236A (en) * | 2010-09-06 | 2012-03-14 | (주)모아캠 | Method for separation and purification of egcg from camellia sinensis leaf by ultra high pressure recrystallization |
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2013
- 2013-11-29 CN CN201310626320.3A patent/CN103613575B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1286252A (en) * | 1999-08-16 | 2001-03-07 | 弗·哈夫曼-拉罗切有限公司 | Process for preparing epigallocatechin gallate |
| US20080015248A1 (en) * | 2003-01-24 | 2008-01-17 | Dou Q P | Polyphenol proteasome inhibitors, synthesis, and methods of use |
| CN101074224A (en) * | 2007-04-13 | 2007-11-21 | 桂林莱茵生物科技股份有限公司 | Production of high-content EGCG |
| KR20120024236A (en) * | 2010-09-06 | 2012-03-14 | (주)모아캠 | Method for separation and purification of egcg from camellia sinensis leaf by ultra high pressure recrystallization |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109053668A (en) * | 2018-07-25 | 2018-12-21 | 云南茶农生物产业有限责任公司 | A method of ester catechin is prepared with tea polyphenols |
| CN109180630A (en) * | 2018-08-06 | 2019-01-11 | 华茗国际健康产业（香港）有限公司 | A kind of crystallite catechin and preparation method thereof |
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Effective date of registration: 20201120 Address after: No.40, Lane 2777, Jinxiu East Road, pilot Free Trade Zone, Pudong New Area, Shanghai Patentee after: Shanghai Biyan Biotechnology Co., Ltd Address before: 541100, Guilin County, Xicheng County, the Guangxi Zhuang Autonomous Region, Lingui Patentee before: GUILIN LAYN NATURAL INGREDIENTS Corp. |
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