CN103613575B - The method of purification of a kind of high-load EGCG - Google Patents
The method of purification of a kind of high-load EGCG Download PDFInfo
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- CN103613575B CN103613575B CN201310626320.3A CN201310626320A CN103613575B CN 103613575 B CN103613575 B CN 103613575B CN 201310626320 A CN201310626320 A CN 201310626320A CN 103613575 B CN103613575 B CN 103613575B
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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
Abstract
The invention discloses the method for purification of a kind of high-load EGCG, comprise the step that the liquid that contains target component or medicinal extract are carried out to crystallization, wherein: in crystallisation step, temperature when crystallization is controlled at 4～8 DEG C, whole crystallization process carries out under stirring condition, and stir speed (S.S.) is 100～300rpm. The present invention 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.
Description
Technical field
The present invention relates to natural product chemistry technical field, be specifically related to carrying of a kind of high-load EGCGPure method.
Background technology
EGCG, i.e. Epigallo-catechin gallate (EGCG), 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 green teaThe chief component composition of Tea Polyphenols, is also the main component of green tea catechins class, and it accounts for green tea catechins10～15% of class total amount. Because have special stereochemical structure, EGCG is the most effective in green teaAnti-oxidant polyphenol, there is anti-oxidant, anticancer, anti-sudden change isoreactivity, antioxidation activity be at least dimensionMore than 100 times of raw plain C, is 25 times of vitamin E, can Cell protection and DNA undermined,This infringement is believed relevant with other major diseases with cancer, heart disease, and these effects of EGCG are returnedTie the removing to oxygen radical (anti-oxidant) ability in them.
The existing method for extraction and purification to EGCG mainly contains solvent extraction method, ion precipitation method, superCritical extraction, high-speed counter-current layer and membrane separation technique attached gel post method for separating and preparing. ExistingIn the method for purification that contains crystallisation step, the liquid that contains target component or medicinal extract are being carried out to crystallizationTime, crystallization is all carried out under the condition of leaving standstill, but the mode of standing crystallization is not easy crystallize out. AdoptIn standing crystallization process, add the method for a small amount of high-load EGCG crystal as crystal seed, although canReach the object of bearing more quickly crystal, still, adding after crystal seed, the crystal of separating out easily wrapsBury impurity, this can reduce the content of EGCG in crystal to a certain extent, particularly as low content EGCGThe eluent of Tea Polyphenols gained after macroporous resin column purifying, or concentrated by this gained eluent warpAfter the medicinal extract that obtains while directly carrying out crystallization, in gained crystal, the content of EGCG is lower, conventionally 90%Left and right, does not reach 95% content requirement; On the other hand, adopt in standing crystallization process and add crystalline substanceThe crystal that the crystallization mode of planting obtains is thinner, easily occurs clogging, crystal in the time of isolation of crystallineWashing also inconvenience.
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-load EGCG. The partyMethod, by changing crystallization condition, can obtain high EGCG content under the condition that does not need to add crystal seedCrystal, and the rate of recovery of crystal is high.
The method of purification of high-load EGCG of the present invention, comprises and will contain the liquid of target componentOr medicinal extract carries out the step of crystallization, unlike the prior art: in crystallisation step, when crystallizationTemperature is controlled at 4～8 DEG C, and whole crystallization process carries out under stirring 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, crystalThe rate of recovery also high.
In technique scheme, the temperature in the time of crystallization is controlled at 4～6 DEG C, and stir speed (S.S.) is controlled atWhen 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 crystallization process is 10～30h, when whole crystallization timeWithin the scope of 10～24h time, along with the prolongation of time, the crystal of separating out increases obviously, in the time of crystallizationBetween exceed 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 existingConventional to the liquid that contains EGCG target component obtaining in the method for extraction and purification of EGCG or soakCream, but set through macropore when the liquid that contain target component or medicinal extract refer to by low content EGCG Tea PolyphenolsThe eluent of gained after fat post purifying, or the medicinal extract being obtained after concentrated by this gained eluentTime, adopt the effect of the method for the invention the most remarkable, can make the crystal EGCG content obtaining reachMore than 95% (HPLC method); Standing crystallization mode identical with other condition and employing interpolation crystal seed is compared,The rate of recovery of crystal improves more than 15%. Here the macroreticular resin of indication refers in prior art for dividingFrom the conventional macroporous absorbent resin of EGCG, the model that is generally macroporous absorbent resin can be D101,D102, D103, HP-20, HPD-600, AB-8, HP20SS, SP20SS or S-8 etc. When containingHave on the liquid of target component or medicinal extract after macroporous resin column chromatography, for solvent and the existing skill of wash-outArt is identical, conventionally, follows in elution process with thin-layer chromatography as eluant, eluent using 20～30% (v/v) ethanolTrack detects to collect the eluent containing target component. Here the low content EGCG Tea Polyphenols of indication is commonRefer to that EGCG content is the Tea Polyphenols of 5～40wt%, can directly buy from the market, also can adoptExisting conventional method is prepared voluntarily. When the Tea Polyphenols taking EGCG content as 10～20wt% is adopted during as raw materialMore remarkable comparatively speaking by the effect of the method for the invention.
In order to obtain higher yield, technique scheme preferably also comprises re-crystallization step, is heavily tyingIn brilliant step, the temperature of recrystallization is controlled at 4～8 DEG C, and whole recrystallization process enters under stirring conditionOK, stir speed (S.S.) is 100～300rpm. It is here same as the prior art for the solvent being recrystallized,Be generally the ethanol of 20～95% (v/v). In re-crystallization step, the temperature of recrystallization is preferably controlled at 4～6 DEG C, stir speed (S.S.) is preferably 150～250rpm.
The concrete steps of the method for the invention comprise:
1) taking low content EGCG Tea Polyphenols as raw material, be dissolved in water;
2) macroporous resin column on gained lysate, first washes resin column with water colourless to efflux, then uses20～30% (v/v) ethanol elution, thin-layer chromatography is followed the tracks of and is detected, and collects alcohol eluen;
3) alcohol eluen of collection is concentrated into medicinal extract (40～60% solid contents), medicinal extract is sent into knotIn brilliant tank (also the alcohol eluen of collection directly can be sent in crystallizing tank carry out crystallization), crystallization controlIn tank, temperature is 4～8 DEG C, opens and stirs, and whole crystallization process is carried out under stirring condition, controlsStir speed (S.S.) is 100～300rpm;
4) by crystal suction filtration, crystal washing, dry, obtain high-load EGCG.
In the time that the method for the invention also comprises re-crystallization step, as long as by the high-load of above-mentioned acquisitionEGCG dissolves with recrystallization solvent, then sends in crystallizing tank and be recrystallized, at recrystallization processIn, in crystallization control tank, temperature is 4～8 DEG C, and whole recrystallization process is carried out under stirring condition,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 slowly evenly separated out, gained crystal grain thick andUniform particles, more easily realizes the separation of crystal and the removal of impurity, and the crystal EGCG obtaining containsMeasure highly, the rate of recovery of crystal is high.
2, be through large by low content EGCG Tea Polyphenols when the liquid that contains target component or medicinal extract are defined asThe eluent of gained after the resin column purifying of hole, or soaking of being obtained after concentrated by this gained eluentWhen cream, adopt the effect of the method for the invention the most remarkable, can make the crystal EGCG obtaining reach 95%(HPLC method) above; Standing crystallization mode identical with other condition and employing interpolation crystal seed is compared, crystalline substanceThe high raising more than 15% of the rate of recovery of body. When after crystallization, be recrystallized again step time, crystalThe rate of recovery identical with other condition and adopt and add the standing crystallization mode of crystal seed and compare and can improve 20%Above.
Detailed description of the invention
With specific embodiment, the invention will be further described below, but the present invention is not limited to theseEmbodiment.
Embodiment 1
1) (Guilin Laiyin Biotechnology Co., Ltd. is raw to get the Tea Polyphenols that EGCG content is 10wt%Producing) 200g is placed in 400ml pure water ultrasonic dissolution 30min;
2) D101 macroporous resin column on gained lysate, first washes resin column with water colourless to efflux,Use 20% (v/v) ethanol elution, thin-layer chromatography is followed the tracks of and is detected, and collects alcohol eluen again;
3) alcohol eluen of collection is evaporated to medicinal extract (50% solid content), medicinal extract is sent into knotIn brilliant tank, use cool cycles water for cooling, in crystallization control tank, temperature is 4 DEG C, opens and stirsMix crystallization 24h, whole crystallization process is carried out under stirring condition, control stir speed (S.S.) is 100rpm;
4) by crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal10.68g. HPLC detects EGCG content 95.33%.
Comparative example 1
1) (Guilin Laiyin Biotechnology Co., Ltd. is raw to get the Tea Polyphenols that EGCG content is 10wt%Producing) 200g is placed in 400ml pure water ultrasonic dissolution 30min;
2) D101 macroporous resin column on gained lysate, first washes resin column with water colourless to efflux,Use 20% (v/v) ethanol elution, thin-layer chromatography is followed the tracks of and is detected, and collects alcohol eluen again;
3) alcohol eluen of collection is evaporated to medicinal extract (50% solid content), medicinal extract is sent into knotIn brilliant tank, to the high-load EGCG (98.5%) that adds 0.2g in crystallizing tank, promote crystallization, with coolingCirculating water cooling, in crystallization control tank, temperature is 4 DEG C, places 24h;
4) by crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 9.20g.HPLC detects EGCG content 90.83%.
Comparative example 2
Repeat comparative example 1, different, whole crystallization process carries out at normal temperatures.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 8.12g.HPLC detects EGCG content 86.31%.
Comparative example 3
Repeat embodiment 1, different, the speed stirring in crystallization process is 350rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 8.62g.HPLC detects EGCG content 91.35%.
Comparative example 4
Repeat embodiment 1, different, whole crystallization process carries out at 2 DEG C.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 11.25g.HPLC detects EGCG content 92.69%.
Embodiment 2
Repeat embodiment 1, different, the speed stirring in crystallization process is 200rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 11.38g.HPLC detects EGCG content 96.53%.
Embodiment 3
Repeat embodiment 1, different, the speed stirring in crystallization process is 300rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 10.24g.HPLC detects EGCG content 95.87%.
Embodiment 4
Repeat embodiment 1, different, Tea Polyphenols (the Wuxi sun taking EGCG content as 20wt%Lv Bao Science and Technology Ltd. produces) 100g is raw material, the temperature in crystallization process in crystallizing tank is 8 DEG C,The time of stirred crystallization is 12h, and the speed of stirring is 200rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 10.21g.HPLC detects EGCG content 96.11%.
Embodiment 5
Repeat embodiment 1, different, (Guilin is emerging for the Tea Polyphenols 67g taking EGCG content as 30wt%Reaching pharmaceutical factory produces), the temperature in crystallization process in crystallizing tank is 6 DEG C, the time of stirred crystallization is20h, the speed of stirring is 250rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 10.54g.HPLC detects EGCG content 95.98%.
Embodiment 6
Repeat embodiment 1, different, the Tea Polyphenols 50g (Changsha three taking EGCG content as 40wt%Good fortune bio tech ltd produces), the temperature in crystallization process in crystallizing tank is 6 DEG C, stirred crystallizationTime be 30h, the speed of stirring is 150rpm.
By crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 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, and collects crystal and sends in crystallizing tank again, adds crystal weight20% (v/v) ethanol heating for dissolving of 5 times, then uses cool cycles water for cooling, temperature in crystallization control tankDegree is 4 DEG C, opens to stir and carries out stirred crystallization 24h, and whole crystallization process is entered under stirring conditionOK, controlling stir speed (S.S.) is 100rpm;
5) by crystal suction filtration, water rinses, and freeze drying, obtains white powder crystal 8.95g.HPLC detects EGCG content 97.80%.
From the various embodiments described above comparative example, only have when temperature is controlled at 4～8 DEG C, stir speed (S.S.) to beUnder the condition of 100～300rpm, carry out stirred crystallization, could obtain the crystal of high-load and high yield.
Claims (7)
1. a method of purification of high-load EGCG, comprises the liquid that contains target component or medicinal extractThe step of carrying out crystallization, is characterized in that: in crystallisation step, temperature when crystallization is controlled at 4～8DEG C, whole crystallization process carries out under stirring condition, and stir speed (S.S.) is 100～300rpm;
The described liquid that contains target component or medicinal extract refer to: large by low content EGCG Tea Polyphenols warpThe eluent of gained after the resin column purifying of hole, or soaking of being obtained after concentrated by this gained eluentCream; Described low content EGCG Tea Polyphenols refers to that EGCG content is the Tea Polyphenols of 5～40wt%;
Described high-load EGCG refers to that EGCG content is more than 95%.
2. the method for purification of high-load EGCG according to claim 1, is characterized in that: instituteStating stir speed (S.S.) is 150～250rpm.
3. the method for purification of high-load EGCG according to claim 1, is characterized in that: instituteTemperature while stating crystallization is controlled at 4～6 DEG C.
4. the method for purification of high-load EGCG according to claim 1, is characterized in that: wholeThe time of individual crystallization process is 10～30h.
5. according to the method for purification of the high-load EGCG described in any one in claim 1～4, itsBe characterised in that: also comprise re-crystallization step, in re-crystallization step, the temperature of recrystallization is controlled at 4～8 DEG C, whole recrystallization process carries out under stirring condition, and stir speed (S.S.) is 100～300rpm.
6. the method for purification of high-load EGCG according to claim 5, is characterized in that: instituteStating stir speed (S.S.) is 150～250rpm.
7. the method for purification of high-load EGCG according to claim 5, is characterized in that: instituteThe temperature of stating recrystallization is controlled at 4～6 DEG C.
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| CN109053668B (en) * | 2018-07-25 | 2022-07-26 | 满山歌茶业(西双版纳)有限公司 | Method for preparing ester catechin from tea polyphenol |
| 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 |
Citations (3)
| 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 |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101123102B1 (en) * | 2010-09-06 | 2012-03-16 | (주)모아캠 | Method for separation and purification of EGCG from Camellia sinensis leaf by ultra high pressure recrystallization |
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Patent Citations (3)
| 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 |
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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. |