CN102276570B - Method for purifying epigallo catechin gallate (EGCG) - Google Patents
Method for purifying epigallo catechin gallate (EGCG) Download PDFInfo
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- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 title claims abstract description 86
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 36
- 229940030275 epigallocatechin gallate Drugs 0.000 title abstract 7
- 239000007788 liquid Substances 0.000 claims abstract description 53
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000012535 impurity Substances 0.000 claims abstract description 26
- 238000007670 refining Methods 0.000 claims abstract description 25
- 238000000926 separation method Methods 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 9
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 229940092665 tea leaf extract Drugs 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 238000001179 sorption measurement Methods 0.000 claims description 30
- 238000010828 elution Methods 0.000 claims description 26
- 238000000605 extraction Methods 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 21
- 238000004088 simulation Methods 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000011097 chromatography purification Methods 0.000 claims description 4
- 238000004807 desolvation Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 abstract description 9
- 150000008442 polyphenolic compounds Chemical class 0.000 abstract description 8
- 235000013824 polyphenols Nutrition 0.000 abstract description 8
- 241001122767 Theaceae Species 0.000 abstract 3
- 239000011259 mixed solution Substances 0.000 abstract 1
- 238000004064 recycling Methods 0.000 abstract 1
- 239000002904 solvent Substances 0.000 abstract 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 238000002156 mixing Methods 0.000 description 8
- 238000002390 rotary evaporation Methods 0.000 description 6
- 244000269722 Thea sinensis Species 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 4
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 4
- 235000005487 catechin Nutrition 0.000 description 4
- 229950001002 cianidanol Drugs 0.000 description 4
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 4
- 235000013616 tea Nutrition 0.000 description 4
- 238000005374 membrane filtration Methods 0.000 description 3
- 238000004237 preparative chromatography Methods 0.000 description 3
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 description 2
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 description 2
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 2
- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N anhydrous gallic acid Natural products OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- -1 catechin gallic acid ester Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 150000002206 flavan-3-ols Chemical class 0.000 description 2
- 229940074391 gallic acid Drugs 0.000 description 2
- 235000004515 gallic acid Nutrition 0.000 description 2
- 229930014124 (-)-epigallocatechin gallate Natural products 0.000 description 1
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 240000003152 Rhus chinensis Species 0.000 description 1
- 235000014220 Rhus chinensis Nutrition 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002019 anti-mutation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 150000001765 catechin Chemical class 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
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- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for separating and purifying epigallo catechin gallate (EGCG) by using a simulated moving bed (SMB) chromatography. The method is characterized in that: a tea leaf extract, which is tea polyphenol, is adopted as a raw material; EGCG is separated and purified by using an SMB chromatography system. According to the method, fundamental zones of the SMB are set as an eluting zone, a refining zone and an absorbing zone. The eluting zone is independent. Octadecylsilane chemically bonded silica ODS is adopted as a stationary phase, a mixed solution of alcohol and water is adopted as a mobile phase, and SMB separation are carried out two times upon a tea polyphenol raw material liquid, wherein one time is for removing impurities which are hard to elute, and the other time is for removing impurities which are easy to elute. The product is processed through a solvent removing process, such that the EGCG product is obtained. With the method, EGCG can be stably, continuously, and automatically purified from tea polyphenol in large scale, and with high efficiency. The recycling rate of the product is above 94%, and the purity of the product is above 96%. According to the invention, the stationary phase and the mobile phase can be repeatedly utilized, such that the cost is reduced. The invention belongs to green and environment-friendly separation engineering.
Description
Technical field
The present invention relates to the separating and purifying method of natural phant active principle, especially a kind of method of utilizing three-section simulated moving bed chromatography purification NVP-XAA 723 (EGCG).
Background technology
NVP-XAA 723 (is called for short EGCG, hereinafter referred to as EGCG) be a kind of oxidation-resistance and the very strong active substance of removing free radical function, as the new drug of a kind of antitumor, anti-mutation, the purposes such as hypotensive, it extracts with application and is subject to domestic and international extensive concern.EGCG is that the Polyphenols mixture that tealeaves extracts is the main component of tea-polyphenol, in tea-polyphenol, also has other flavanols compounds very close with character with the EGCG chemical structure, as: l-Epicatechol (EC); L-Epicatechin gallate (ECG); Epigallocatechin (EGC); Nutgall catechin gallic acid ester (GCG); DL-catechin (DL-C), and the very easily oxidation of these flavanols compounds, poly-(contracting) closes, and makes separation and purification EGCG monomer difficulty.
At present, the major technique that obtains high purity EGCG monomer (being higher than 95%) is the preparative chromatography partition method, as: the separation method of catechin compounds in CN1319597(tea-polyphenol), the separation purification method of CN1603319(catechin monomers) and CN1465572(epi-nutgall catechin gallic acid ester monomer purifying method) chromatography of employing dextrane gel SephadexLH-20 post; CN101381359(method of from green tea, extracting high-purity epigallocatechin-3-gallate) chromatography of employing flicker silicagel column, CN101723927A(method for batch production, separation and purification of catechin monomers EGCG) chromatography of employing KR100-5C18 post, although these methods can be used for the industrial production high purity EGCG, but all be that batch process is the purifying technique at intermittence, can not automatically produce continuously, and the utilization ratio of sorbent material is low, the eluent consumption is large, the treatment capacity of raw material is less, consuming time, production efficiency is low.
Simulated moving bed chromatography is the powerful separation means with advantage of of purification compound.Simulated moving bed chromatography has been introduced special operating mechanism on the basis of preparative chromatography, make chromatographic separation process from intermittently becoming continuously, can carry out high efficiency automatization and separate with mass-producing.There is no at present the report that adopts simulated moving bed chromatography technology separating-purifying EGCG.
Summary of the invention
The invention provides the method for a kind of simulation moving-bed (being called for short SMB, hereinafter referred to as SMB) chromatographic purification NVP-XAA 723 (being called for short EGCG, hereinafter referred to as EGCG), EGCG continuously, automatically, efficiently can purify.
The method of a kind of EGCG that purifies is characterized in that take the tea leaf extract tea-polyphenol as raw material, adopts SMB chromatographic purification EGCG, the theing contents are as follows of the method:
(1) SMB equipment
SMB equipment consists of chromatographic column, elutriant transferpump, feeding liquid transferpump, internally piloted valve, micro-chip, automatic control system, many logical, pipelines, joint, container for storing liquid, computer;
The chromatographic column specification is column length 10～50cm, and column length is 8～20 with the diameter ratio;
Stationary phase uses octadecylsilane chemically bonded silica ODS, 10～60 μ m;
Internally piloted valve is comprised of magnetic valve and non-return valve.
(2) SMB working conditions
SMB operational mode: the chromatographic column number sum N of SMB system is set, 3≤N≤8, the base region band that SMB is set is elution band, refining band and adsorption zone, each band is formed by 1～4 identical Coupled columns, and a is elution band chromatographic column number, b is refining band chromatographic column number, c is adsorption zone chromatographic column number, a+b+c=N, and operational mode is expressed as a-b-c;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the homogeneous phase solution that alcohol mixes with water, the volumn concentration C of alcohol
DBe 10～40%; The moving phase P of elution band is the homogeneous phase solution that alcohol mixes with water, the volumn concentration C of alcohol
PBe C
D～100%;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 1～20cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 1～15cm/min, sample introduction liquid F flow velocity U
FBe 0.1～6cm/min, raffinate R flow velocity U
R=U
D+ U
FU
D≤ U
p≤ 3U
DU
F＜2U
D
Simulation moving-bed service temperature: room temperature.
(3) separating step
Use as (1) as described in SMB equipment as (2) described condition under, carry out twice SMB separation: once be to remove difficult wash-out impurity from extraction liquid E, switching time t
S: 5～30min; Another time removed easy wash-out impurity from raffinate R, switching time t
S: 3～25min;
Tea-polyphenol is 0～100% aqueous solution dissolving with pure content, and configuration concentration is the homogeneous phase stock liquid of 10～100mg/mL, as the sample introduction liquid F that the first time, SMB separated; For the first time SMB separates the raffinate R that obtains or the sample introduction liquid F through after concentrated or directly separating as SMB for the second time;
The EGCG solution of removing impurity after desolvation is processed, is obtained the EGCG product.
(4) detection method
Adopt Shimadzu LC-10AT chromatogram pump, SPD-10A UV-detector, Agilent Extend chromatographic column, 4.6 * 150mm, the ODS filler, particle diameter 5 μ m, moving phase is acetonitrile and water, volume ratio is 15: 85, Glacial acetic acid volumn concentration 0.1%, flow velocity 1.0mL/min detects wavelength 278nm.Demarcated the content of EGCG in the product by the EGCG standard substance.
The method of described a kind of EGCG that purifies, the alcohol in the moving phase are methyl alcohol, ethanol.
The method of described a kind of EGCG that purifies, stationary phase ODS, 20～30 μ m; The moving phase D of refining band and adsorption zone adopts methanol aqueous solution, wherein methyl alcohol volumn concentration C
DBe 25～35%.
The method of described a kind of EGCG that purifies, stationary phase ODS, 20～30 μ m; The moving phase D of refining band and adsorption zone adopts aqueous ethanolic solution, wherein ethanol volumn concentration C
DBe 10～20%.
The method of described a kind of EGCG that purifies, the process of being separated the EGCG solution desolvation obtain by SMB is: perhaps lyophilize or recrystallization or vacuum-drying or pressure reducing film distillation.
Method with simulated moving bed chromatography separating-purifying EGCG provided by the invention is compared with existing preparative chromatography isolation technique; its significant advantage is can mass-producing, EGCG steadily and surely, continuously, automatically and efficiently purifies from tea-polyphenol; product recovery rate surpasses 94%; purity surpasses 96%; stationary phase and mobile being on good terms are recycled; reduce cost, belong to the environmental protection separation engineering.
Description of drawings
Fig. 1 is twice simulation moving-bed sepn process schematic diagram;
Fig. 2 is the HPLC spectrogram of tea polyphenol raw materials liquid;
Fig. 3 is the SMB separating resulting first time, the HPLC spectrogram of difficult wash-out impurity;
Fig. 4 is the SMB separating resulting first time, removes the HPLC spectrogram of the EGCG of difficult wash-out impurity;
Fig. 5 is the SMB separating resulting second time, easily the HPLC spectrogram of wash-out impurity;
Fig. 6 is the SMB separating resulting second time, the HPLC spectrogram of the EGCG of imurity-removal.
Embodiment
(1) SMB equipment
8 root chromatogram columns, moving phase D transferpump flow 0-10mL/min, pressure 0-10Mpa; Stock liquid F pumping capacity 0-30mL/h, pressure 0-8Mpa; Moving phase P transferpump flow 0-10mL/min, pressure 0-10Mpa, 48 internally piloted valves, a cover single chip computer automatic control system, 5 container for storing liquids (each needs a container for storing liquid moving phase D, stock liquid F, moving phase P, extraction liquid E, raffinate R), many logical, pipelines, joint, computer; Chromatographic column is specification 10cm * 1cm; Stationary phase uses octadecylsilane chemically bonded silica ODS, 20～30 μ m.
(2) SMB working conditions
The SMB working conditions is as follows for the first time:
The SMB operational mode: the chromatographic column number sum N=4 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 1 root chromatogram column, adsorption zone 2 root chromatogram columns, operational mode is 1-1-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of methyl alcohol and water, the volumn concentration C of methyl alcohol
DBe 30%; The moving phase P of elution band is the mixing solutions of methyl alcohol and water, the volumn concentration C of alcohol
PBe 80%;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 3.2cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 1.9cm/min, sample introduction liquid F flow velocity U
FBe 0.13cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 15min.
Simulation moving-bed service temperature: 15-20 ℃.
The SMB working conditions is as follows for the second time:
The SMB operational mode: the chromatographic column number sum N=4 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 1 root chromatogram column, adsorption zone 2 root chromatogram columns, operational mode is 1-1-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of methyl alcohol and water, the volumn concentration C of methyl alcohol
DBe 30%; The moving phase P of elution band is the mixing solutions of methyl alcohol and water, the volumn concentration C of alcohol
PBe 60%;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 3.8cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 1.9cm/min, sample introduction liquid F flow velocity U
FBe 0.13cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 10min.
Simulation moving-bed service temperature: 15-20 ℃.
(3) separating step
Use as (1) as described in SMB equipment as (2) described condition under, carry out twice SMB separation, as shown in Figure 1.For the first time SMB separation is to remove difficult wash-out impurity from extraction liquid E, get 5 gram tea polyphenol raw materials (EGCG purity 55.5%), be the dissolving of 30% the aqueous solution with the methyl alcohol volumn concentration, 0.45 μ m membrane filtration, configuration concentration is the solution of 5g/100mL, as the sample introduction liquid F that the first time, SMB separated, EGCG is 22.3mg/mL, its HPLC spectrogram as shown in Figure 2, with moving phase P difficult wash-out impurity is removed in extraction liquid E, its HPLC spectrogram is obtained to contain the raffinate R of easy wash-out impurity and EGCG as shown in Figure 3 by moving phase D, its HPLC spectrogram as shown in Figure 4, the purity of EGCG is 92.19%, and yield 99.7% is concentrated with this raffinate rotary evaporation, EGCG is 18mg/mL, as the sample introduction liquid F of for the second time SMB separation.For the second time SMB separation is to remove easy wash-out impurity, with moving phase D easy wash-out impurity is removed in raffinate R, and its HPLC spectrogram as shown in Figure 5, obtained the extraction liquid E of EGCG by moving phase P, its HPLC spectrogram as shown in Figure 6, the purity of EGCG is 97.8%, yield 99.8%.The extraction liquid of EGCG is behind rotary evaporation, and lyophilize obtains the EGCG product.
(4) detection method
Adopt Shimadzu LC-10AT chromatogram pump, SPD-10A UV-detector, Agilent Extend chromatographic column, 4.6 * 150mm, the ODS filler, particle diameter 5 μ m, moving phase is acetonitrile and water, volume ratio is 15: 85, Glacial acetic acid volumn concentration 0.1%, flow velocity 1.0mL/min detects wavelength 278nm.Demarcated the content of EGCG in the product by the EGCG standard substance.
(1) SMB equipment: with embodiment 1.
(2) SMB working conditions
The SMB working conditions is as follows for the first time:
The SMB operational mode: the chromatographic column number sum N=5 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 2 root chromatogram columns, adsorption zone 2 root chromatogram columns, operational mode is 1-2-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the volumn concentration C of ethanol
DBe 15%; The moving phase P of elution band is ethanol;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 3.8cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 2.9cm/min, sample introduction liquid F flow velocity U
FBe 0.25cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 14min.
Simulation moving-bed service temperature: 10-15 ℃.
The SMB working conditions is as follows for the second time:
The SMB operational mode: the chromatographic column number sum N=5 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 2 root chromatogram columns, adsorption zone 2 root chromatogram columns, operational mode is 1-2-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the volumn concentration C of ethanol
DBe 15%; The moving phase P of elution band is ethanol;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 3.8cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 1.3cm/min, sample introduction liquid F flow velocity U
FBe 1.9cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 4min.
Simulation moving-bed service temperature: 10-15 ℃.
(3) separating step
Use as (1) as described in SMB equipment as (2) described condition under, carry out twice SMB separation.For the first time SMB separation is to remove difficult wash-out impurity from extraction liquid E, get 5 gram tea polyphenol raw materials (EGCG purity 65.8%), be the dissolving of 15% the aqueous solution with the ethanol volumn concentration, 0.45 μ m membrane filtration, configuration concentration is the solution of 5g/100mL, SMB separates sample introduction liquid F as the first time, EGCG is 28.9mg/mL, with moving phase P difficult wash-out impurity is removed in extraction liquid E, obtained to contain the raffinate R of easy wash-out impurity and EGCG by moving phase D, the purity of EGCG is 92.43%, yield 99.2%, this raffinate rotary evaporation is concentrated, and EGCG is 8.3mg/mL, as the sample introduction liquid F of for the second time SMB separation.For the second time SMB separation is to remove easy wash-out impurity, with moving phase D easy wash-out impurity is removed in raffinate R, is obtained the extraction liquid E of EGCG by moving phase P, and the purity of EGCG is 96.5%, yield 99.4%.The extraction liquid of EGCG is behind rotary evaporation, and lyophilize obtains the EGCG product.
(4) detection method is with embodiment 1
Embodiment 3
(1) SMB equipment: with embodiment 1.
(2) SMB working conditions
The SMB working conditions is as follows for the first time:
The SMB operational mode: the chromatographic column number sum N=6 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 3 root chromatogram columns, adsorption zone 2 root chromatogram columns, operational mode is 1-3-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the volumn concentration C of ethanol
DBe 15%; The moving phase P of elution band is ethanol;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 5.1cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 2.9cm/min, sample introduction liquid F flow velocity U
FBe 0.25cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 6min.
Simulation moving-bed service temperature: 10-15 ℃.
The SMB working conditions is as follows for the second time:
The SMB operational mode: the chromatographic column number sum N=7 of SMB system is set, SMB elution band 1 root chromatogram column is set, make with extra care and be with 3 root chromatogram columns, adsorption zone 3 root chromatogram columns, operational mode is 1-3-3;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the volumn concentration C of ethanol
DBe 15%; The moving phase P of elution band is ethanol;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 5.1cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 2.0cm/min, sample introduction liquid F flow velocity U
FBe 1.1cm/min, raffinate R flow velocity U
R=U
D+ U
F
Be switching time: 5min.
Simulation moving-bed service temperature: 10-15 ℃.
(3) separating step
Use as (1) as described in SMB equipment as (2) described condition under, carry out twice SMB separation.For the first time SMB separation is to remove difficult wash-out impurity from extraction liquid E, get 5 gram tea polyphenol raw materials (EGCG purity 65.8%), be the dissolving of 15% the aqueous solution with the ethanol volumn concentration, 0.45 μ m membrane filtration, configuration concentration is the solution of 5g/100mL, SMB separates sample introduction liquid F as the first time, EGCG is 28.9mg/mL, with moving phase P difficult wash-out impurity is removed in extraction liquid E, obtained to contain the raffinate R of easy wash-out impurity and EGCG by moving phase D, the purity of EGCG is 94.4%, yield 97.5%, this raffinate rotary evaporation is concentrated, and EGCG is 11.3mg/mL, as the sample introduction liquid F of for the second time SMB separation.For the second time SMB separation is to remove easy wash-out impurity, with moving phase D easy wash-out impurity is removed in raffinate R, is obtained the extraction liquid E of EGCG by moving phase P, and the purity of EGCG is 98.9%, yield 97.4%.The extraction liquid of EGCG is behind rotary evaporation, and lyophilize obtains the EGCG product.
(4) detection method is with embodiment 1
Claims (5)
1. the NVP-XAA 723 of purifying is called for short the method for EGCG, it is characterized in that take the tea leaf extract tea-polyphenol as raw material, adopts simulation moving-bed abbreviation SMB chromatographic purification EGCG, the theing contents are as follows of the method:
(1) SMB equipment
SMB equipment consists of chromatographic column, elutriant transferpump, feeding liquid transferpump, internally piloted valve, micro-chip, automatic control system, many logical, pipelines, joint, container for storing liquid, computer;
The chromatographic column specification is column length 10～50cm, and column length is 8～20 with the diameter ratio;
Stationary phase uses octadecylsilane chemically bonded silica ODS, 10～60 μ m;
Internally piloted valve is comprised of magnetic valve and non-return valve;
(2) SMB working conditions
SMB operational mode: the chromatographic column number sum N of SMB system is set, 3≤N≤8, the base region band that SMB is set is elution band, refining band and adsorption zone, each band is formed by 1～4 identical Coupled columns, and a is elution band chromatographic column number, b is refining band chromatographic column number, c is adsorption zone chromatographic column number, a+b+c=N, and operational mode is expressed as a-b-c;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the homogeneous phase solution that alcohol mixes with water, the volumn concentration C of alcohol
DBe 10～40%; The moving phase P of elution band is the homogeneous phase solution that alcohol mixes with water, the volumn concentration C of alcohol
PBe C
D～100%;
Flow rate of mobile phase: in elution band, elutriant P flow velocity U
pBe 1～20cm/min, extraction liquid E flow velocity U
E=U
pIn refining band and adsorption zone, elutriant D flow velocity U
DBe 1～15cm/min, sample introduction liquid F flow velocity U
FBe 0.1～6cm/min, raffinate R flow velocity U
R=U
D+ U
FU
D≤ U
p≤ 3U
DU
F＜2U
D
Simulation moving-bed service temperature: room temperature;
(3) separating step
Use as (1) as described in SMB equipment as (2) described condition under, carry out twice SMB separation: once be to remove difficult wash-out impurity from extraction liquid E, switching time t
S: 5～30min; Another time removed easy wash-out impurity from raffinate R, switching time t
S: 3～25min;
Tea-polyphenol is 0～100% aqueous solution dissolving with pure volumn concentration, and configuration concentration is the homogeneous phase stock liquid of 10～100mg/mL, as the sample introduction liquid F that the first time, SMB separated; For the first time SMB separates the raffinate R that obtains or the sample introduction liquid F through after concentrated or directly separating as SMB for the second time;
The EGCG solution of removing impurity after desolvation is processed, is obtained the EGCG product;
(4) detection method
Adopt Shimadzu LC-10AT chromatogram pump, the SPD-10A UV-detector, Agilent Extend chromatographic column, 4.6 * 150mm, the ODS filler, particle diameter 5 μ m, moving phase is acetonitrile and water, volume ratio is 15: 85, Glacial acetic acid volumn concentration 0.1%, flow velocity 1.0mL/min detects wavelength 278nm, is demarcated the content of EGCG in the product by the EGCG standard substance.
2. a kind of NVP-XAA 723 of purifying according to claim 1 is called for short the method for EGCG, and it is characterized in that: the alcohol in the moving phase is methyl alcohol, ethanol.
According to claim 1 or a kind of NVP-XAA 723 of purifying claimed in claim 2 be called for short the method for EGCG, it is characterized in that: stationary phase ODS, 20～30 μ m; The moving phase D of refining band and adsorption zone adopts methanol aqueous solution, wherein methyl alcohol volumn concentration C
DBe 25～35%.
According to claim 1 or a kind of NVP-XAA 723 of purifying claimed in claim 2 be called for short the method for EGCG, it is characterized in that: stationary phase ODS, 20～30 μ m; The moving phase D of refining band and adsorption zone adopts aqueous ethanolic solution, wherein ethanol volumn concentration C
DBe 10～20%.
5. a kind of NVP-XAA 723 of purifying according to claim 1 is called for short the method for EGCG, and it is characterized in that: the process of being separated the EGCG solution desolvation that obtains by SMB is: lyophilize or recrystallization or vacuum-drying or pressure reducing film distillation.
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| 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 |
| CN106986851B (en) * | 2017-05-04 | 2019-03-08 | 广西轻工业科学技术研究院有限公司 | A kind of preparation method of high purity EGCG |
| KR20190048502A (en) * | 2017-10-31 | 2019-05-09 | (주)아모레퍼시픽 | Composition for improving circulatory diseases comprising tea extraction which has modified amount of ingredients |
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 |
| CN1546485A (en) * | 2003-12-12 | 2004-11-17 | 贵州家诚药业有限责任公司 | Method for separating tea polyphenol mixture |
| CN101732890A (en) * | 2009-12-08 | 2010-06-16 | 辽宁科技大学 | Three-section simulated moving bed chromatography device |
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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 |
| CN1546485A (en) * | 2003-12-12 | 2004-11-17 | 贵州家诚药业有限责任公司 | Method for separating tea polyphenol mixture |
| CN101732890A (en) * | 2009-12-08 | 2010-06-16 | 辽宁科技大学 | Three-section simulated moving bed chromatography device |
Non-Patent Citations (2)
| Title |
|---|
| 传统SMB、Varicol和Partial-discard工艺分离纯化ECG和EGCG的比较研究;黄永东等;《茶叶科学》;20110615;第31卷(第3期);第201-210页 * |
| 黄永东等.传统SMB、Varicol和Partial-discard工艺分离纯化ECG和EGCG的比较研究.《茶叶科学》.2011,第31卷(第3期),第201-210页. |
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