CN101723927A - Method for batch production, separation and purification of catechin monomers EGCG - Google Patents

Abstract

The invention discloses a method for batch production, separation and purification of catechin monomers EGCG, which comprises the following steps of: taking KR100-5C18 as a column packing and taking 25 percent and 75 percent ethanol-water solutions as eluents respectively to perform column chromatography; collecting catechin monomer EGCG target solutions by time or volume; and recycling, concentrating, crystallizing and freeze-drying the catechin monomer EGCG target solutions to obtain the catechin monomer EGCG finished products. The method overcomes difficulty in the conversion from achievements in laboratories to the bath production, and has the advantages of great output, simplicity, convenience, low cost, high purity, greatly shortened operation time, no pollution in the whole production process, 99.9 percent of monomer extraction rate and 99.9 percent of production purity.

Description

A kind of method for batch production, separation and purification of catechin monomers EGCG

Technical field

The present invention relates to a kind of improvement of catechin monomers extraction and separation technology technology, particularly a kind of method for batch production, separation and purification of catechin monomers EGCG.

Background technology

Catechin is a class natural active matter important in the tealeaves, belongs to flavanols compounds, nineteen twenty-nine by the Japanese first extraction separation come out.It is made up of about eight kinds monomer, be D, L-catechin (D, L-C), L-l-Epicatechol (L-EC), L-epigallocatechin (L-EGC), D, L-l-Epigallocatechol (D, L-GC), L-L-Epicatechin gallate (L-ECG), L-catechin acid esters (L-CG), L-NVP-XAA 723 (L-EGCG), L-nutgall catechin gallic acid ester (L-GCG).Owing among CG, ECG, GCG and the EGCG esterification of catechin and gallic acid has taken place, so be called ester catechin; And C, EC, GC, EGC that esterification does not take place are called non-ester catechin, and the structure of each catechin is comparatively similar, and some is an isomers, and the key distinction of structure is that the quantity of hydroxyl replacement is different with the position.The content of various units is also different because of the tea leaf quality difference, in general two class ester catechin-epigallocatechingallate (EGCG) wherein and-epicatechin gallate (ECG) content is bigger, pharmacologically active is the strongest.The host compound catechin that studies show that tea-polyphenol is multiple unique effects such as ideal natural antioxidants, anti-ageing and antibacterial, AIDS virus resisting.In recent years, world developed country actively pushes forward the exploitation of catechin application product, as natural antioxidants and free-radical scavengers, has been widely used in fields such as food-processing, medicines and health protection and daily-use chemical industry with it.

The method of separating catechin generally was divided into for two steps at present: at first extract the tea-polyphenol raw product (TP) that contains catechin from tea dust; Next purifies and separates catechin composition from crude extract.If will obtain the various monomer components in the catechin, then need further pass through column chromatography, half preparative high-performance liquid chromatographic method (HPLC) purifying or Other Instruments method purifying.Because the chemical structure and the character of each component of catechin are very close, adopt general method to be difficult to separate, according to data analysis, separate the method for preparing catechin monomers at present and mainly contain following several:

(1) column chromatography for separation method

This method be with the tea-polyphenol crude product after certain dissolution with solvents, upper prop separates, and use the eluent wash-out, the collection catechin.The key of column chromatography is the selection of column packing and eluent.The column packing of report has the three major types type both at home and abroad: adsorptive type post, ion exchange column, gel column.This separation method is loaded down with trivial details, time-consuming, product purity is not high, also have dissolvent residual.

(2) high performance liquid phase (HPLC) preparative chromatography

Preparation HPLC is mainly used in the monomer component that separation and purification goes out catechin.Technical process is that the TP crude product goes out the catechin mixture through dextrane gel Sephadex LH-20 column separating purification, generally make eluent with the aqueous acetone solution of different gradients, separate and obtain ester catechin and two groups of flow points of non-ester catechin, again through the separation and purification of HPLC preparative column, the employing methanol is an eluent, is further purified to obtain the catechin monomers component, and gained catechin product purity is higher, but have the cost height, the cycle is grown shortcomings such as the later stage solvent is difficult to eliminate fully.

(3) high-speed countercurrent chromatography (HSCCC)

High-speed countercurrent chromatography is the new developing technology eighties, the gravity field that produces that is characterized in running up makes the high reservation of realization in separator column of fixedly being on good terms, thereby improved separating power greatly, this instrument partition method owned by France, after each solvent mixes in separating funnel in the solvent systems, the upper strata is a stationary phase, pours into chromatography column; Lower floor is a moving phase, the TP dissolving crude product is become the solution of 5%-10%.When moving phase was flowed through chromatographic column, each component of catechin obtained separating.This separation method carries out between two-phase, has overcome to use the sample irreversible adsorption that solid adsorption material causes or the shortcoming of degraded, and sample can be reclaimed to greatest extent.But this method cost height, preparation amount is little, is difficult at present popularize.

Summary of the invention

The objective of the invention is provides the method for batch production, separation and purification of catechin monomers EGCG that a kind of separation and purification operation steps is simple, separation efficiency is high, preparation amount is big by the improvement to existing catechin monomers extraction and separation technology technology.

The object of the present invention is achieved like this: a kind of method for batch production, separation and purification of catechin monomers EGCG may further comprise the steps

A, single-column extracting and separating: with rough tea-polyphenol solution and eluent upper prop, the high 15m of post, diameter 55cm, make eluent with 75% ethanol, control tea-polyphenol solution flow rate 700-900L/min, ethanol flow velocity 1500-1700L/min extracted time 1.5-2 hour, reclaim the elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG with reduced vacuum, again chromatography is treated in its storage;

B, low pressure column chromatography purifying: with KR100-5C18 is column packing, the high 2.8m of post, with 25% aqueous ethanolic solution is eluent, elution flow rate 450L/min, carry out column chromatography to reclaiming the concentrated back elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG to be purified, collect catechin monomers EGCG target liquid by time or volume, to catechin monomers EGCG target liquid reclaim concentrated, crystallization, lyophilize gets catechin monomers EGCG finished product.

The present invention adopts the KR100-5C18 column chromatography, respectively with 25%, 75% ethanol as eluent, carry out the experiment of pre-separation and purifying, main six catechins (EGCG/GCG/ECG/DL-C/EG/GCG/EGC) in the rough tea-polyphenol can be realized the separation of continuous high-efficient.This method is easy and simple to handle, and output is big, with low cost, solvent is nontoxic, and monomer extraction yield and product purity are all higher, and can realize the preparation that the disposable feather weight of EGCG is above, and economic benefit is very considerable.

The present invention has the useful technique effect of three aspects:

(1) separation is for the first time adopted 25% ethanol to make eluent and has been substituted other aqueous solution eluents such as acetone commonly used at present, has the following advantages:

A, the main ester catechin EGCG of disposable separation (containing GCG) and ECG, separating effect and separation efficiency are all higher, and non-ester catechin, caffeine can be separated simultaneously with tea pigment, solved and reported and obtain tea-polyphenol behind the glycan gel Sephadex LH-20 chromatography of crawling, generally the catechin in the raw material tea-polyphenol can only be divided into ester type and non-ester catechin two classes, and EGCG and ECG, EGC can't isolating problems, can reduce the subsequent purification formality greatly;

B, good separating effect, colour band is even, is difficult for hangover and produces bubble;

C, convenient solvent reclaiming owing to collect the water-ethanol liquid that liquid is catechin monomers, adopt vacuum decompression to concentrate and reclaim, reclaim very quick and convenient, shortened the connection procedure with subsequent purification, and other solvent aqueous solution eluting liquids are collected liquid and are difficult for fully concentrating owing to contain water constituent.

(2) EGCG column chromatography purification method adopts column chromatography technology, that has simplified present report greatly partly prepares technologies such as HPLC purifying, the separation that only needs to finish monomer EGCG through conventional column chromatography system is purified, chromatographic purity 99%, and the disposable purifying preparation reduction of EGCG production cost; Whole separation, purge process adopt ethanol and 25% aqueous ethanolic solution, and compatibility and continuity are strong, and very easily reclaim, and non-toxic inexpensive has avoided partly preparing the safety problem that the HPLC purification solvent is brought.

Embodiment

A kind of method for batch production, separation and purification of catechin monomers EGCG may further comprise the steps

A, single-column extracting and separating: with rough tea-polyphenol solution and eluent upper prop, the high 15m of post, diameter 55cm, make eluent with 75% ethanol, control tea-polyphenol solution flow rate 700-900L/min, ethanol flow velocity 1500-1700L/min, eluent consumption 4000L, 1.5-2 hour extraction time, reclaim the elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG with reduced vacuum, again chromatography is treated in its storage;

B, low pressure column chromatography purifying: with KR100-5C18 is column packing, the high 2.8m of post, with 25% aqueous ethanolic solution is eluent, elution flow rate 450L/min, wash-out consumption 800L, concentrate the back elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG to be purified and carry out column chromatography reclaiming, collect catechin monomers EGCG target liquid by time or volume, to catechin monomers EGCG target liquid reclaim concentrated, crystallization, lyophilize gets catechin monomers EGCG finished product.

Embodiment 1:

(1) at first drops into 1200kg tealeaves,, soak three times with the deionized water immersion tealeaves of heating

(2) suction filtration tea

(3) respectively twice suction filtration mixed solution concentrated

(4) go up chromatography column

1. the pre-treatment of filler: take by weighing KR100-5C18 300kg, with eluent 25% ethanol 400kg abundant swelling (also can with 80 ℃ of warm water soaking 1.5 hours).

2. swelling is good filler is slowly poured chromatography column into, makes its uniform settling, when waiting to be deposited to 2.75m, closes feeding port, with transferpump 25% ethanol of 5-8 times of column volume is imported drip washing in the post, with the balance pillar.

3. upper prop: about 200L extraction polyphenol medicinal extract solution, in transferpump input post, after treating that sample infiltrates in the post fully, connect collector again, import 25% ethanol with the low-pressure delivery pump and begin to collect, first section is EGC, is followed successively by DL-C, CAF, EGCG, EC, GCG, ECG, elution flow rate is 450L/min, collects 800L altogether.

4. adopt high performance liquid chromatograph, respectively composition and the content of collecting liquid is carried out qualitative analysis.

(5) elutriant of merging EGCG (containing ECG) catechin monomers concentrates 40 minutes (solid content is about 45) at 100L vacuum concentration unit.

1. adorn post: the filler that swelling is good is slowly poured in the post, makes its uniform settling, when waiting to be deposited to 2.75m, closes and fills out post mouth valve, carries 5-8 50% ethanol drip washing doubly with transferpump, with the balance pillar.

2. upper prop: will concentrate the back catechin monomers of EGCG (ECG) solid content about 50 and collect liquid, in transferpump input post, after treating that sample infiltrates in the post fully, adopt senior liquid phase chromatography, composition, the content of collecting liquid is carried out qualitative analysis, merge the elutriant that contains the EGCG catechin monomers, at 50L concentrating under reduced pressure unit, be concentrated into contain 45% solid content after, be further purified crystallization, lyophilize gets finished product 10.065kg, and purity is 99.9%.

Embodiment 2:

The pre-separation method of catechin monomers L-L-Epicatechin gallate EGCG and L-L-Epicatechin gallate ECG: KR100-5C18 is a column packing, with 25% ethanol is eluent, processing condition are: the high 2.8m of post, elution flow rate 450L/min, disposable applied sample amount (TP) can reach 400L, eluent consumption 800L, the effective separation that can realize catechin in the rough tea polyphenol raw materials (EGCG and ECG/GCG).Technique effect: EGCG (containing EGC) monomer eluting rate is 98.45%, and purity can reach 98.66%, and ECG monomer eluting rate is 98.64%, and purity reaches 98.62%, extraction yield 88.57%.

Catechin monomers L-L-Epicatechin gallate EGCG purification process: separate the EGCG (ECG) that obtains, vacuum decompression is concentrated into 40L, through the KR100-5C18 column chromatographic isolation and purification, concrete processing condition are: eluent is 50% ethanol, the high 2.8m of post again, elution flow rate 450L/min, eluent consumption 800L, eluting rate can reach more than 98%, and purity reaches more than 99.9%, extraction yield 88.57%, and non-pigment is residual.

Claims (1)

1. a method for batch production, separation and purification of catechin monomers EGCG is characterized in that: may further comprise the steps

A, single-column extracting and separating: with rough tea-polyphenol solution and eluent upper prop, the high 15m of post, diameter 55cm, make eluent with 75% ethanol, control tea-polyphenol solution flow rate 700-900L/min, ethanol flow velocity 1500-1700L/min extracted time 1.5-2 hour, reclaim the elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG with reduced vacuum, again chromatography is treated in its storage;

B, low pressure column chromatography purifying: with KR100-5C18 is column packing, the high 2.8m of post, with 25% aqueous ethanolic solution is eluent, elution flow rate 450L/min, carry out column chromatography to reclaiming the concentrated back elutriant that contains L-L-Epicatechin gallate catechin monomers EGCG to be purified, collect catechin monomers EGCG target liquid by time or volume, to catechin monomers EGCG target liquid reclaim concentrated, crystallization, lyophilize gets catechin monomers EGCG finished product.