CN106632207A

CN106632207A - Preparation method of high-purity GCG (gallocatechin gallate)

Info

Publication number: CN106632207A
Application number: CN201611198080.1A
Authority: CN
Inventors: 季浩; 张宇; 阚建伟; 胡亚京; 闫成亮; 孔繁博; 徐娟
Original assignee: Jiangsu Tiansheng Pharmaceutical Co Ltd
Current assignee: Jiangsu Tiansheng Pharmaceutical Co Ltd
Priority date: 2016-12-22
Filing date: 2016-12-22
Publication date: 2017-05-10
Anticipated expiration: 2036-12-22
Also published as: CN106632207B

Abstract

The invention discloses a preparation method of a high-purity GCG. The method comprises the following steps (1) taking EGCG (epigallocatechin gallate) as the raw material, adding in solvent to obtain reaction liquid A; (2) concentrating the reaction liquid A into an aqueous solution, and adding in ethyl acetate for extraction to obtain a solution B; (3) gradually adding the solution B into a chromatographic column filled with macroporous absorbent resin for gradient elution, and concentrating elutant with target components until no alcohol flavor exists to obtain a concentrated solution C; (4) adding n-butyl alcohol inside the concentrated solution C for extraction, combining an n-butyl alcohol solution, performing concentration until extracts are formed, dissolving the extracts into water until forming clear liquor, and performing cooling for crystallization, filtering and drying to obtain high-purity GCG powder. According to the preparation method the high-purity GCG, the EGCG serves as the raw materials and is subjected to reactive transformation at high temperature, and through solvent extraction and macroporous absorbent resin column chromatographic technology, the high-purity GCG can be obtained.

Description

A kind of preparation method of high-purity GCG

Technical field

The present invention relates to field of medicine and chemical technology, and in particular to a kind of preparation method of high-purity GCG.

Background technology

Epigallo-catechin gallate (EGCG) (EGCG) is one of major catechin in tealeaves, used as natural anti-oxidation Agent and free radical scavenger, it is various with obvious anti-aging, anti-mutation, cancer-resisting, prevention of cardiovascular disease, radiation proof etc. BA, is widely used in food processing, medicines and health protection and filed of daily-use chemical industry.However, due to its superpower oxidation and Architectural characteristic, EGCG is also highly prone to the impact of environmental factor, such as easily aoxidizes in atmosphere, is extremely easy in decomposition in alkali, in high temperature Condition stability inferior difference, etc..

Nutgall catechin gallic acid ester (GCG) is a kind of isomer of EGCG, and it is deposited among natural green tea It is considerably less measuring, it is difficult to directly the very high GCG of purity be directly obtained from green tea, and then limit the progress of its application study. It can be appreciated that the research for GCG at present is still seldom, overwhelming majority research is concentrated on based on its isomers EGCG.

Find in EGCG research process, processed by some specific conditions, EGCG can be effectively converted into GCG, then by a series of purification techniques, the high GCG samples of purity are finally given, and tentatively find the shape existed with GCG configurations Under formula, its good stability is more beneficial for the exploitation and application of the kind.

The content of the invention

Goal of the invention：For the problem that prior art is present, the present invention provides a kind of preparation method of high-purity GCG, leads to Crossing the preparation method can obtain high-purity GCG, and technique is simple and direct, easy to operate, be fully able to be satisfied with industrialization production requirements.

Technical scheme：To achieve these goals, a kind of preparation method of high-purity GCG as described in the present invention, including Following steps：

(1) with EGCG as raw material, after adding solvent dissolving, thermostatic mixer is transferred to, control temperature is in 60-100 DEG C Reaction obtains reactant liquor A for 4-6 days, lets cool standby；

(2) by reactant liquor A in 60-100 DEG C of concentration to the aqueous solution, let cool, add ethyl acetate extracted, merge Aqueous, continue to concentrate the ethyl acetate for removing residual at 60-100 DEG C, obtain solution B, let cool standby；

(3) solution B is taken, is added gradually in the chromatographic column equipped with macroporous absorbent resin, successively with water and different volumes point Several ethanol carries out gradient elution, and 2-10 column volume of each gradient elution simultaneously receives respectively each several part eluent, and selection contains The eluent of target component, is concentrated into without alcohol taste, obtains concentrate C, standby；

(4) concentrate C is taken, adds n-butanol to be extracted, merge butanol solution, at 60-100 DEG C medicinal extract is concentrated into, Add water to be dissolved at 60-100 DEG C clearly, let cool to crystallization completely, filtering drying is simultaneously crushed, and obtains final product highly purified GCG powder.

Preferably, HPLC purity >=50% of step (1) EGCG.

Preferably, at least one of step (1) solvent in purified water, methyl alcohol, ethanol or the acetic acid, raw material with The w/v of solvent is 1：(5-20)g/ml.

Add ethyl acetate to carry out 3-5 extraction preferably, step (2) is described, every time the ethyl acetate of addition with it is anti- Liquid A volume ratios are answered to be 0.2-2.

Preferably, step (2) solution B is (5-20) with raw material volume weight ratio:1ml/g.

Preferably, step (3) resin model be HPD-100, HPD-200, HPD-400, HPD-826 at least It is a kind of.

Preferably, the ethanol of step (3) the different volumes fraction is 5-20% ethanol, 30-40% ethanol and 50- 90% ethanol.

N-butanol is added to carry out 3-5 extraction, the n-butanol for adding every time and concentrate C preferably, step (4) is described Volume ratio is 0.2-2.

Step (4) water is (0.5-10) with the envelope-bulk to weight ratio of medicinal extract：1ml/g.

Beneficial effect：Compared with prior art, the invention has the advantages that：

1st, the present invention is processed EGCG raw materials by thermostatic mixer, and simple and safe operation, reaction temperature is moderate, no Only avoid causes EGCG Partial digestions to be other non-targeted compositions because temperature is too high, and also assures that filling for configuration conversion Divide property.

2nd, the present invention takes full advantage of the catechin such as EGCG, EC, ECG composition and GCG and dissolves in ethyl acetate and water Property difference, by ethyl acetate abstraction technique, adjust extractant ratio, and control targe constituent concentration, can be by major part Catechin impurity component efficiently separate ethyl acetate layer, and by GCG retain in aqueous, so as to reach efficiently separate it is pure The purpose of change.

3rd, the present invention also applies macroreticular resin technology and target component is purified, and not remove only most colors Element, also by different gradients, target component purity is further improved.

4th, method involved in the present invention can be amplified in equivalent manner, and technique is simple and direct, easy to operate, be fully able to It is satisfied with industrialization production requirements.

Specific embodiment

With reference to embodiments the invention will be further described.

Embodiment 1

(1) it is raw material to weigh the EGCG powder 50g that HPLC purity is 55%, after adding 10% methyl alcohol 1000ml dissolvings, is turned In moving on to 80 DEG C of thermostatic mixer water-baths, coreaction 5 days obtains reactant liquor A, lets cool standby；

(2) by reactant liquor A in 70 DEG C of concentrations to without the alcohol taste aqueous solution, add ethyl acetate to carry out 3 extractions after letting cool, The ethyl acetate for adding every time is 0.5 with reactant liquor A volume ratios, merges aqueous, continues to concentrate the acetic acid for removing residual at 70 DEG C Ethyl ester, it is 500ml to mend purified water and make final solution cumulative volume, obtains solution B, is let cool standby；

(3) solution B is taken, is added gradually to that (resin model is HPD- in the chromatographic column equipped with 1000g macroporous absorbent resins 100 press quality 1 with HPD-400:1 mixing), successively gradient elution is carried out with water, 20% ethanol, 40% ethanol and 60% ethanol, 4 column volumes of each gradient elution simultaneously receive respectively each several part eluent, choose the eluent containing target component, are concentrated into nothing Alcohol taste, obtains concentrate C, standby；

(4) concentrate C is taken, adds n-butanol to carry out 3 extractions, the n-butanol for adding every time is with concentrate C volume ratios 0.2, merge butanol solution, medicinal extract is concentrated at 80 DEG C, adding water, it is clear to be dissolved at 80 DEG C, (the envelope-bulk to weight ratio of water and medicinal extract For 2ml/g), let cool to crystallization completely, filtering drying is simultaneously crushed, and obtains final product white powder 6.1g, and Jing differentiates, is defined as GCG, Jing HPLC detects that its purity is 92.3%.

Embodiment 2

(1) it is raw material to weigh the EGCG powder 50g that HPLC purity is 60%, after adding 10% ethanol 250ml dissolvings, transfer To in 60 DEG C of thermostatic mixer water-baths, coreaction 4 days obtains reactant liquor A, lets cool standby；

(2) by reactant liquor A in 60 DEG C of concentrations to without the alcohol taste aqueous solution, add ethyl acetate to carry out 5 extractions after letting cool Take, the ethyl acetate for adding every time is 0.2 with reactant liquor A volume ratios, merges aqueous, continue to concentrate the second for removing residual at 60 DEG C Acetoacetic ester, it is 250ml to mend purified water and make final solution cumulative volume, obtains solution B, is let cool standby；

(3) solution B is taken, is added gradually to that (resin model is HPD- in the chromatographic column equipped with 1000g macroporous absorbent resins 200), with water, 5% ethanol, 30% ethanol and 50% ethanol gradient elution is carried out successively, 2 column volumes of each gradient elution are simultaneously Each several part eluent is received respectively, the eluent containing target component is chosen, is concentrated into without alcohol taste, obtain concentrate C, it is standby；

(4) concentrate C is taken, adds n-butanol to carry out 5 extractions, the n-butanol for adding every time is with concentrate C volume ratios 0.5, merge butanol solution, medicinal extract is concentrated at 60 DEG C, adding water, it is clear to be dissolved at 60 DEG C, (the envelope-bulk to weight ratio of water and medicinal extract For 10ml/g), let cool to crystallization completely, filtering drying is simultaneously crushed, and obtains final product white powder 5.4g, and Jing differentiates, is defined as GCG, Jing HPLC detects that its purity is 94.5%.

Embodiment 3

(1) it is raw material to weigh the EGCG powder 50g that HPLC purity is 58%, after adding 10% acetic acid 500ml dissolvings, transfer To in 100 DEG C of thermostatic mixer water-baths, coreaction 6 days obtains reactant liquor A, lets cool standby；

(2) by reactant liquor A in 100 DEG C of concentrations to the aqueous solution, after letting cool add ethyl acetate carry out 4 extractions, every time The ethyl acetate of addition is 2 with reactant liquor A volume ratios, merges aqueous, continues to concentrate the ethyl acetate for removing residual at 100 DEG C, It is 1000ml to mend purified water and make final solution cumulative volume, obtains solution B, is let cool standby；

(3) solution B is taken, is added gradually to that (resin model is HPD- in the chromatographic column equipped with 1000g macroporous absorbent resins 826), with water, 20% ethanol, 40% ethanol and 90% ethanol gradient elution is carried out successively, 2 column volumes of each gradient elution are simultaneously Each several part eluent is received respectively, the eluent containing target component is chosen, is concentrated into without alcohol taste, obtain concentrate C, it is standby；

(4) concentrate C is taken, adds n-butanol to carry out 4 extractions, the n-butanol for adding every time is with concentrate C volume ratios 2, merge butanol solution, medicinal extract is concentrated at 100 DEG C, adding water, it is clear to be dissolved at 100 DEG C, (the envelope-bulk to weight ratio of water and medicinal extract For 0.5ml/g), let cool to crystallization completely, filtering drying is simultaneously crushed, and obtains final product white powder 7.2g, and Jing differentiates, is defined as GCG, Jing HPLC detects that its purity is 90.3%.

Claims

1. a kind of preparation method of high-purity GCG, it is characterised in that comprise the steps：

(1) with EGCG as raw material, after adding solvent dissolving, thermostatic mixer is transferred to, control temperature is reacted in 60-100 DEG C Reactant liquor A is obtained within 4-6 days, is let cool standby；

(2) by reactant liquor A in 60-100 DEG C of concentration to the aqueous solution, let cool, add ethyl acetate extracted, merge water Liquid, continues to concentrate the ethyl acetate for removing residual at 60-100 DEG C, obtains solution B, lets cool standby；

(3) solution B is taken, is added gradually in the chromatographic column equipped with macroporous absorbent resin, successively with water and different volumes fraction Ethanol carries out gradient elution, and 2-10 column volume of each gradient elution simultaneously receives respectively each several part eluent, and selection contains target The eluent of composition, is concentrated into without alcohol taste, obtains concentrate C, standby；

(4) concentrate C is taken, adds n-butanol to be extracted, merge butanol solution, medicinal extract is concentrated at 60-100 DEG C, added Water is dissolved to clearly at 60-100 DEG C, lets cool to crystallization completely, and filtering drying is simultaneously crushed, and obtains final product highly purified GCG powder.

2. preparation method according to claim 1, it is characterised in that HPLC purity >=50% of step (1) EGCG.

3. preparation method according to claim 1, it is characterised in that step (1) solvent selected from purified water, methyl alcohol, At least one in ethanol or acetic acid, raw material is 1 with the w/v of solvent：(5-20)g/ml.

4. preparation method according to claim 1, it is characterised in that step (2) is described to add ethyl acetate to carry out 3-5 time Extraction, the ethyl acetate for adding every time is 0.2-2 with reactant liquor A volume ratios.

5. preparation method according to claim 1, it is characterised in that step (2) solution B and raw material volume weight ratio For (5-20):1ml/g.

6. preparation method according to claim 1, it is characterised in that step (3) resin model be HPD-100, At least one in HPD-200, HPD-400, HPD-826.

7. preparation method according to claim 1, it is characterised in that the ethanol of step (3) the different volumes fraction is 5-20% ethanol, 30-40% ethanol and 50-90% ethanol.

8. preparation method according to claim 1, it is characterised in that step (4) is described to be added n-butanol to carry out 3-5 time to extract Take, the n-butanol for adding every time is 0.2-2 with concentrate C volume ratios.

9. preparation method according to claim 1, it is characterised in that the envelope-bulk to weight ratio of step (4) water and medicinal extract For (0.5-10)：1ml/g.