CN102276570B - Method for purifying epigallo catechin gallate (EGCG) - Google Patents

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

A method for purifying epigallocatechin gallate (EGCG)

technical field

The invention relates to a method for separating and purifying effective components of natural plants, in particular to a method for purifying epigallocatechin gallate (EGCG) by using three-band simulated moving bed chromatography.

Background technique

Epigallocatechin gallate (abbreviated as EGCG, hereinafter referred to as EGCG) is an active substance with strong antioxidant and free radical scavenging functions. It has been used as a new drug for anti-tumor, anti-mutation, and blood pressure reduction. Its extraction and application have attracted widespread attention at home and abroad. EGCG is the polyphenol mixture extracted from tea leaves, that is, the main component of tea polyphenols. In tea polyphenols, there are also other flavanol compounds that are very similar in chemical structure and properties to EGCG, such as: epicatechin (EC) ; epicatechin gallate (ECG); epigallocatechin gallate (EGC); gallocatechin gallate (GCG); DL-catechin (DL-C), and these flavanols It is very easy to oxidize and poly(condensate), which makes it difficult to separate and purify EGCG monomer.

At present, the main technology to obtain high-purity EGCG monomer (higher than 95%) is to prepare chromatographic separation method, such as: CN1319597 (a separation method of catechin compounds in tea polyphenols), CN1603319 (catechin monomer separation and purification method) and CN1465572 (purification method of epigallocatechin gallate monomer) using dextran gel SephadexLH-20 column chromatography; CN101381359 (a high-purity epigallocatechin extracted from green tea The method of plain gallate) adopts the chromatography of flashing silica gel column, and CN101723927A (a kind of catechin monomer EGCG industrial production separation and purification method) adopts the chromatography of KR100-5C18 column, although these methods can be used in industry The production of high-purity EGCG is a batch process, that is, a batch purification process, which cannot be produced continuously and automatically. Moreover, the utilization rate of the adsorbent is low, the consumption of the eluent is large, the processing amount of the raw material is small, time-consuming, and the production efficiency is low.

Simulated moving bed chromatography is a powerful and advantageous separation tool for purifying compounds. On the basis of preparative chromatography, simulated moving bed chromatography introduces a special operating mechanism, which makes the chromatographic separation process change from batch to continuous, enabling efficient automation and large-scale separation. At present, there is no report on the separation and purification of EGCG by simulated moving bed chromatography.

Contents of the invention

The present invention provides a method for chromatographically separating and purifying epigallocatechin gallate (EGCG, hereinafter referred to as EGCG) in a simulated moving bed (abbreviated as SMB, hereinafter referred to as SMB), which can continuously, automatically and efficiently purify EGCG.

A method for purifying EGCG, characterized in that using tea extract tea polyphenols as raw material, adopting SMB chromatography to separate and purify EGCG, the content of the method is as follows:

(1) SMB device

SMB equipment is composed of chromatographic column, eluent delivery pump, feed solution delivery pump, automatic control valve, single-chip microcomputer, automatic control system, multi-pass, pipeline, joint, liquid storage tank, computer;

The column specification is 10-50cm long, and the ratio of column length to diameter is 8-20;

The stationary phase uses octadecylsilane bonded silica gel ODS, 10-60 μm;

The automatic control valve is composed of a solenoid valve and a check valve.

(2) SMB working conditions

SMB operation mode: set the total number of columns in the SMB system N, 3≤N≤8, set the basic zone of SMB as the elution zone, refining zone and adsorption zone, each zone consists of 1 to 4 identical columns connected in series a is the number of chromatographic columns in the eluting band, b is the number of chromatographic columns in the refining band, c is the number of chromatographic columns in the adsorption band, a+b+c=N, and the operating mode is expressed as a-b-c;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a homogeneous solution mixed with alcohol and water, and the volume percentage content _CD of alcohol is 10-40%; the mobile phase P of the eluting zone is a mixture of alcohol and water Homogeneous solution, the volume percentage of alcohol C _P is C _D ~ 100%;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 1-20 cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 1～15cm/min, the flow rate U _F of the sample liquid F is 0.1～6cm/min, the flow rate U R of the raffinate _R = U _D + U _F ; U _D ≤ U _p ≤ 3 U _D ; U _F < 2 U _D ;

Simulated moving bed operating temperature: room temperature.

(3) Separation step

Use the SMB equipment as described in (1) and under the conditions as described in (2), perform two SMB separations: one is to remove difficult-to-elute impurities from the extract E, switching time t _S : 5-30min; The raffinate R removes easily eluted impurities, switching time t _S : 3-25min;

Tea polyphenols are dissolved in an aqueous solution with an alcohol content of 0-100%, and a homogeneous raw material solution with a concentration of 10-100mg/mL is prepared as the sample solution F for the first SMB separation; the raffinate obtained from the first SMB separation Solution R is either concentrated or directly used as the injection solution F for the second SMB separation;

EGCG products are obtained after the EGCG solution from which impurities have been removed is treated with solvent removal.

(4) Detection method

Shimadzu LC-10AT chromatographic pump, SPD-10A ultraviolet detector, Agilent Extend chromatographic column, 4.6 × 150mm, ODS filler, particle size 5μm, mobile phase is acetonitrile and water, the volume ratio is 15:85, and the volume of glacial acetic acid is 100%. Content of 0.1%, flow rate 1.0mL/min, detection wavelength 278nm. The EGCG content in the product is calibrated by the EGCG standard.

A kind of described method of purifying EGCG, the alcohol in mobile phase is methyl alcohol, ethanol.

A method for purifying EGCG, the stationary phase ODS, 20-30 μm; the mobile phase D of the refining zone and the adsorption zone adopts methanol aqueous solution, wherein the methanol volume percentage _CD is 25-35%.

A method for purifying EGCG, the stationary phase ODS, 20-30 μm; the mobile phase D of the refining zone and the adsorption zone adopts ethanol aqueous solution, wherein the ethanol volume percentage _CD is 10-20%.

In the method for purifying EGCG, the process of removing the solvent from the EGCG solution obtained by SMB separation is: or freeze-drying, or recrystallization, or vacuum drying, or vacuum membrane distillation.

Compared with the existing preparation chromatographic separation technology, the method of using simulated moving bed chromatographic separation and purification of EGCG provided by the present invention has significant advantages in that it can purify EGCG from tea polyphenols in a large-scale, robust, continuous, automatic and efficient manner, and the product recovery rate is More than 94%, the purity is more than 96%, the stationary phase and the mobile phase can be reused, and the cost is reduced, which belongs to the green separation project.

Description of drawings

Fig. 1 is the schematic diagram of two simulated moving bed separation processes;

Fig. 2 is the HPLC spectrogram of tea polyphenol raw material liquid;

Figure 3 is the first SMB separation result, the HPLC spectrum of difficult-to-elute impurities;

Figure 4 is the first SMB separation result, the HPLC spectrogram of EGCG that removes difficult-to-elute impurities;

Figure 5 is the second SMB separation result, the HPLC spectrogram of the easily eluted impurities;

Fig. 6 is the second SMB separation result, the HPLC spectrogram of the EGCG that removes impurity.

Detailed ways

Example 1

(1) SMB device

8 chromatographic columns, mobile phase D delivery pump flow rate 0-10mL/min, pressure 0-10Mpa; material liquid F pump flow rate 0-30mL/h, pressure 0-8Mpa; mobile phase P delivery pump flow rate 0-10mL/min, Pressure 0-10Mpa, 48 automatic control valves, a set of single-chip automatic control system, 5 liquid storage tanks (one liquid storage tank is required for mobile phase D, raw material F, mobile phase P, extract E, and raffinate R) , multi-pass, pipelines, connectors, computer; the chromatographic column is 10cm×1cm; the stationary phase uses octadecylsilane bonded silica gel ODS, 20-30μm.

(2) SMB working conditions

The first SMB working conditions are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=4, set the SMB elution zone to 1 chromatographic column, the refining zone to 1 chromatographic column, the adsorption zone to 2 chromatographic columns, and the operating mode is 1-1-2;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of methanol and water, and the volume percentage content _CD of methanol is 30%; the mobile phase P of the eluting zone is a mixed solution of methanol and water, and the alcohol Volume percentage C _P is 80%;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 3.2 cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 1.9 cm/min, the flow rate U _F of the sample solution F is 0.13 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

The switching time is: 15min.

Simulated moving bed operating temperature: 15-20°C.

The working conditions of the second SMB are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=4, set the SMB elution zone to 1 chromatographic column, the refining zone to 1 chromatographic column, the adsorption zone to 2 chromatographic columns, and the operating mode is 1-1-2;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of methanol and water, and the volume percentage content _CD of methanol is 30%; the mobile phase P of the eluting zone is a mixed solution of methanol and water, and the alcohol Volume percentage C _P is 60%;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 3.8cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 1.9 cm/min, the flow rate U _F of the sample solution F is 0.13 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

Switching time: 10min.

Simulated moving bed operating temperature: 15-20°C.

(3) Separation step

Using the SMB equipment described in (1) under the conditions described in (2), perform two SMB separations, as shown in Figure 1. The first SMB separation is to remove difficult-to-elute impurities from the extract E. Take 5 grams of tea polyphenol raw material (EGCG purity 55.5%), dissolve it in an aqueous solution with a volume percentage of methanol of 30%, filter it through a 0.45 μm filter membrane, and configure A solution with a concentration of 5g/100mL is used as the injection solution F for the first SMB separation, and the EGCG is 22.3mg/mL. Remove in, its HPLC spectrogram as shown in Figure 3, obtain the raffinate R containing easily eluted impurity and EGCG by mobile phase D, its HPLC spectrogram as shown in Figure 4, the purity of EGCG is 92.19%, the yield 99.7%, the raffinate was concentrated by rotary evaporation, and the EGCG was 18mg/mL, which was used as the injection solution F for the second SMB separation. The second SMB separation is to remove the easily eluted impurities, and the easily eluted impurities are removed in the raffinate R with the mobile phase D, and its HPLC spectrum is shown in Figure 5. The extract E of EGCG is obtained from the mobile phase P, Its HPLC spectrogram is shown in Figure 6, and the purity of EGCG is 97.8%, and the yield is 99.8%. After the extract of EGCG is evaporated by rotary evaporation, the EGCG product is obtained by freeze-drying.

(4) Detection method

Shimadzu LC-10AT chromatographic pump, SPD-10A ultraviolet detector, Agilent Extend chromatographic column, 4.6 × 150mm, ODS filler, particle size 5μm, mobile phase is acetonitrile and water, the volume ratio is 15:85, and the volume of glacial acetic acid is 100%. Content of 0.1%, flow rate 1.0mL/min, detection wavelength 278nm. The EGCG content in the product is calibrated by the EGCG standard.

Example 2

(1) SMB device: same as embodiment 1.

(2) SMB working conditions

The first SMB working conditions are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=5, set the SMB elution zone to 1 chromatographic column, the refining zone to 2 chromatographic columns, the adsorption zone to 2 chromatographic columns, and the operating mode is 1-2-2;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of ethanol and water, and the volume percentage content _CD of ethanol is 15%; the mobile phase P of the eluting zone is ethanol;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 3.8cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 2.9 cm/min, the flow rate U _F of the sample solution F is 0.25 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

The switching time is: 14min.

Simulated moving bed operating temperature: 10-15°C.

The working conditions of the second SMB are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=5, set the SMB elution zone to 1 chromatographic column, the refining zone to 2 chromatographic columns, the adsorption zone to 2 chromatographic columns, and the operating mode is 1-2-2;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of ethanol and water, and the volume percentage content _CD of ethanol is 15%; the mobile phase P of the eluting zone is ethanol;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 3.8cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 1.3 cm/min, the flow rate U _F of the sample solution F is 1.9 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

Switching time: 4min.

Simulated moving bed operating temperature: 10-15°C.

(3) Separation step

Using the SMB equipment as described in (1) under the conditions as described in (2), perform two SMB separations. The first SMB separation is to remove difficult-to-elute impurities from the extract E. Take 5 grams of tea polyphenol raw material (EGCG purity 65.8%), dissolve it in an aqueous solution with an ethanol volume percentage of 15%, filter it through a 0.45 μm filter membrane, and configure A solution with a concentration of 5g/100mL is used as the first SMB separation injection solution F, and the EGCG is 28.9mg/mL. The difficult-to-elute impurities are removed in the extract E with mobile phase P, and the easy-to-wash content is obtained from mobile phase D. The raffinate R from impurity removal and EGCG, the purity of EGCG is 92.43%, and the yield is 99.2%. The raffinate is concentrated by rotary evaporation, and the EGCG is 8.3mg/mL, which is used as the injection liquid F for the second SMB separation . The second SMB separation is to remove the easily eluted impurities. The mobile phase D is used to remove the easily eluted impurities in the raffinate R, and the extract E of EGCG is obtained from the mobile phase P. The purity of EGCG is 96.5%, and the yield is 99.4%. %. After the extract of EGCG is evaporated by rotary evaporation, the EGCG product is obtained by freeze-drying.

(4) Detection method is the same as in Example 1

Example 3

(1) SMB device: same as embodiment 1.

(2) SMB working conditions

The first SMB working conditions are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=6, set the SMB elution zone to 1 chromatographic column, the refining zone to 3 chromatographic columns, the adsorption zone to 2 chromatographic columns, and the operating mode is 1-3-2;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of ethanol and water, and the volume percentage content _CD of ethanol is 15%; the mobile phase P of the eluting zone is ethanol;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 5.1cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 2.9 cm/min, the flow rate U _F of the sample solution F is 0.25 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

The switching time is: 6min.

Simulated moving bed operating temperature: 10-15°C.

The working conditions of the second SMB are as follows:

SMB operation mode: set the total number of columns of the SMB system to N=7, set the SMB elution zone to 1 chromatographic column, the refining zone to 3 chromatographic columns, the adsorption zone to 3 chromatographic columns, and the operating mode is 1-3-3;

Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a mixed solution of ethanol and water, and the volume percentage content _CD of ethanol is 15%; the mobile phase P of the eluting zone is ethanol;

Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 5.1 cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 2.0 cm/min, the flow rate U _F of the sample solution F is 1.1 cm/min, and the flow rate U _R of the raffinate R = U _D + U _F .

Switching time: 5min.

Simulated moving bed operating temperature: 10-15°C.

(3) Separation step

Using the SMB equipment as described in (1) under the conditions as described in (2), perform two SMB separations. The first SMB separation is to remove difficult-to-elute impurities from the extract E. Take 5 grams of tea polyphenol raw material (EGCG purity 65.8%), dissolve it in an aqueous solution with an ethanol volume percentage of 15%, filter it through a 0.45 μm filter membrane, and configure A solution with a concentration of 5g/100mL is used as the first SMB separation injection solution F, and the EGCG is 28.9mg/mL. The difficult-to-elute impurities are removed in the extract E with mobile phase P, and the easy-to-wash content is obtained from mobile phase D. The raffinate R from impurity removal and EGCG, the purity of EGCG is 94.4%, and the yield is 97.5%. The raffinate is concentrated by rotary evaporation, and the EGCG is 11.3 mg/mL, which is used as the injection liquid F for the second SMB separation . The second SMB separation is to remove the easily eluted impurities. The mobile phase D is used to remove the easily eluted impurities in the raffinate R, and the extract E of EGCG is obtained from the mobile phase P. The purity of EGCG is 98.9%, and the yield is 97.4%. %. After the extract of EGCG is evaporated by rotary evaporation, the EGCG product is obtained by freeze-drying.

(4) Detection method is the same as in Example 1

Claims (5)

1. a method for purifying epigallocatechin gallate is called for short EGCG, is characterized in that with tealeaves extract polyphenols as raw material, adopts simulated moving bed to be called for short SMB chromatographic separation and purification EGCG, the content of this method is as follows: (1) SMB device SMB equipment is composed of chromatographic column, eluent delivery pump, feed solution delivery pump, automatic control valve, single-chip microcomputer, automatic control system, multi-pass, pipeline, joint, liquid storage tank, computer; The column specification is 10-50cm long, and the ratio of column length to diameter is 8-20; The stationary phase uses octadecylsilane bonded silica gel ODS, 10-60 μm; The automatic control valve is composed of a solenoid valve and a check valve; (2) SMB working conditions SMB operation mode: set the total number of columns in the SMB system N, 3≤N≤8, set the basic zone of SMB as the elution zone, refining zone and adsorption zone, each zone consists of 1 to 4 identical columns connected in series a is the number of chromatographic columns in the elution band, b is the number of chromatographic columns in the refining band, c is the number of chromatographic columns in the adsorption band, a+b+c =N, and the operation mode is expressed as a-b-c; Mobile phase composition: the mobile phase D of the refining zone and the adsorption zone is a homogeneous solution mixed with alcohol and water, and the volume percentage content _CD of alcohol is 10-40%; the mobile phase P of the eluting zone is a mixture of alcohol and water Homogeneous solution, the volume percentage of alcohol C _P is C _D ~ 100%; Mobile phase flow rate: in the elution zone, the flow rate U _p of the eluent P is 1-20 cm/min, and the flow rate U _E = U _p of the extract E; in the refining zone and the adsorption zone, the flow rate U D of the eluent _D is 1～15cm/min, the flow rate U _F of the sample liquid F is 0.1～6cm/min, the flow rate U R of the raffinate _R = U _D + U _F ; U _D ≤ U _p ≤ 3 U _D ; U _F < 2 U _D ; Simulated moving bed operating temperature: room temperature; (3) Separation step Use the SMB equipment as described in (1) and under the conditions as described in (2), perform two SMB separations: one is to remove difficult-to-elute impurities from the extract E, switching time t _S : 5-30min; The raffinate R removes easily eluted impurities, switching time t _S : 3-25min; Tea polyphenols are dissolved in an aqueous solution with an alcohol volume percentage of 0-100%, and a homogeneous raw material solution with a concentration of 10-100mg/mL is prepared as the sample solution F for the first SMB separation; the first SMB separation is obtained The raffinate R is either concentrated or directly used as the sample liquid F for the second SMB separation; After the EGCG solution that removes impurities is treated by removing the solvent, the EGCG product is obtained; (4) Detection method Shimadzu LC-10AT chromatographic pump, SPD-10A ultraviolet detector, Agilent Extend chromatographic column, 4.6 × 150mm, ODS filler, particle size 5μm, mobile phase is acetonitrile and water, the volume ratio is 15:85, and the volume of glacial acetic acid is 100%. Content of 0.1%, flow rate 1.0mL/min, detection wavelength 278nm, the content of EGCG in the product is calibrated by EGCG standard. 2. a kind of method for purifying epigallocatechin gallate according to claim 1 is called for short EGCG, it is characterized in that: the alcohol in mobile phase is methyl alcohol, ethanol. 3. A method for purifying epigallocatechin gallate for short EGCG according to claim 1 or claim 2, characterized in that: stationary phase ODS, 20-30 μm; mobile phase D of refining zone and adsorption zone Methanol aqueous solution is used, wherein the methanol volume percentage _CD is 25-35%. 4. A method for purifying epigallocatechin gallate for short EGCG according to claim 1 or claim 2, characterized in that: stationary phase ODS, 20-30 μm; mobile phase D of refining zone and adsorption zone An aqueous ethanol solution is used, wherein the ethanol volume percentage _CD is 10-20%. 5. a kind of method for purifying epigallocatechin gallate according to claim 1 is called for short EGCG, it is characterized in that: the process that the EGCG solution that obtains by SMB separation desolventizes is: freeze-drying or recrystallization, Or vacuum drying, or vacuum membrane distillation.

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