CN1603319A - Separation purification method of catechin monomer - Google Patents

Abstract

This invention relates to separation and purification method of catechin monomer EGCG and ECG. The features are that dextrane gel Sephadex LH-20 is column filling and eluant is absolute ethyl alcohol. Then column chromatography is made by dextrane gel Sephadex LH-20 and 40% ethanol aqueous solution be eluant, that is chromatography column non- gradient expendable separation is adopted. Comparing to original method, equipments are facilitated greatly and the method is simple, cost is low. The solvent is nontoxic and separation period is short, and monomer extraction rate and product purity are both high.

Description

The separation purification method of catechin monomers

Technical field

The present invention relates to important catechin monomers L-NVP-XAA 723 EGCG of two classes in the tealeaves and the separation purification method of L-L-Epicatechin gallate ECG.

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.Various monomeric content are also different because of the tea leaf quality difference, in general two classes ester catechin wherein-epigallocatechin gallate (EGCG) and-epicatechin gallate (ECG) content is bigger, pharmacologically active is the strongest, also is the emphasis of research.The host compound catechin that studies show that tea-polyphenol is the ideal natural antioxidants, be again the extremely valuable natural drug raw material of a class, it has multiple unique effects such as antitumor, hypotensive, reducing blood-fat, anti-oxidant, anti-ageing and antibacterial, AIDS virus resisting.In recent years, world developed country actively pushes forward the exploitation of catechin application product, with it as natural antioxidants and free-radical scavengers, be widely used in fields such as food-processing, medicines and health protection and daily-use chemical industry, research monomeric extracting and purifying method of tea catechin and production technique have important practical significance.Over past ten years, both at home and abroad, particularly China and Japan are to exploring new catechin extraction and separation process growing interest.Except that traditional method, some new extraction and separation technologies have been developed again, as supercritical extraction, high speed adverse current chromatogram partition method or the like.

The method of separating catechin generally was divided into for two steps at present: at first extract the tea-polyphenol crude 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, and solvent is poisonous.

(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.Gained catechin product purity is higher, but has the cost height, and the cycle is grown shortcomings such as the later stage solvent is difficult to eliminate fully.

(3) high-speed countercurrent chromatography (HSCCC)

The HSCCC method is the new developing technology eighties, and 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 has improved separating power greatly.This instrument partition method owned by France, after each solvent mixed in separating funnel in the solvent systems, the upper strata was 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.

Problem in the catechin monomers separating and purifying technology

In recent years, the catechin extraction and separation technology has obtained constantly improving and development, shows that the product yield improves constantly on the one hand; Be product diversification on the other hand, be that each technology can both be to the catechin separation of purifying to some extent, and then obtaining the catechin series product that purity differs, product colour also has different models such as yellowish brown, green, yellow-white simultaneously, to satisfy the needs in market.The catechin monomers separation purification method that we will report at present is summarized in table 1.

Table 1 catechin monomers separation purification method relatively

Separation method advantage shortcoming

The column chromatography cost is low, and the relatively large operation steps of preparation amount is many, and the cycle is long, and extraction yield is low, condition

Wayward, product purity is low

High performance liquid preparative chromatography method purity height, preparation amount simple to operate is little, the cost height, preparative column easily pollutes

The expensive height of high-speed countercurrent chromatography separating power, preparation amount is little, is difficult at present popularize

Wherein, high performance liquid chromatography (HPLC) preparation method is that report is maximum, also is the most frequently used method, and mainly there is following several respects problem in this technology:

(1) separating step is loaded down with trivial details, and separation efficiency is not high

Use sephadex chromatography, the aqueous acetone solution gradient elution can roughly be separated ester catechin and non-ester catechin, and the EGCG and the ECG monomer that account for main content do not separate, and need advance half preparative chromatography instrument and be further purified.Separate for also having only by preparation HPLC with the present report of isomers GCG of EGCG.Used eluent also exists elution time longer in the sephadex chromatography method in addition, shortcomings such as the easy oxidation of product.

(2) separation costs height

What catechin monomers separation and purification was at present generally adopted is the preparation HPLC method, be that the TP crude product is after dextrane gel Sephadex LH-20 column separating purification goes out the catechin mixture, through concentrate drying, obtain the catechin monomers component through the separation and purification of HPLC preparative column again.Because the preparation HPLC chromatographic column costs an arm and a leg, and very easily pollute and stop up, the life-span is short, and production cost is higher, and disposable applied sample amount very little (about each about 1mL of sample size), and the purifying cycle is long.

(3) noxious solvent is residual

At present both at home and abroad all over adopt partly to prepare HPLC method later stage purification solvent comparatively complicated, mostly be ternary mixed solvent, and contain noxious solvents such as methyl alcohol, tetrahydrofuran (THF), be difficult to reclaim and eliminate, restricted the Application Areas of product.

To sum up, be still waiting further raising for catechin monomers extraction and separation technology efficient integrated, promptly on the catechin separation purifying technique, make the process high efficiency as far as possible, obtain highly purified product with the simplest process, simplify the separation and purification operation steps, to obtain maximum economic benefits.

Summary of the invention

The present invention is intended to by the improvement to existing catechin monomers extraction and separation technology technology, provide a kind of separation and purification operation steps is simple, separation efficiency is high, preparation amount is big catechin monomers L-NVP-XAA 723 EGCG and L-L-Epicatechin gallate ECG separation purification method.

The technical solution that realizes the foregoing invention purpose is as follows:

The separation purification method of catechin monomers comprises and extract the rough tea-polyphenol that contains catechin from tea dust, secondly purifies and separates catechin composition from crude extract; It is characterized in that: described from crude extract the purifies and separates catechin composition, be meant purifies and separates catechin L-NVP-XAA 723 and L-L-Epicatechin gallate component, concrete processing method comprises: column chromatography pre-separation, column chromatography purification and separation and purification;

A, described column chromatography pre-separation operation are: being column packing with dextrane gel Sephadex LH-20, the high 50cm of its post, get rough tea-polyphenol 5-6g, dehydrated alcohol is an eluent, elution flow rate 0.75ml/min, eluent consumption 1500mL, 30-38 hour column chromatography time, collect liquid and merge the elutriant that contains L-L-Epicatechin gallate ECG catechin monomers in the back after testing, concentrated postlyophilization gets finished product; Merge the elutriant that contains L-NVP-XAA 723 EGCG (containing L-nutgall catechin gallic acid ester GCG) catechin monomers, rotary evaporation is concentrated into 15-20ml in 45 ℃ of water-baths, and is to be purified;

B, described column chromatography purification operation are: again with dextrane gel Sephadex LH-20 post, the high 50cm of post, with 40% aqueous ethanolic solution is eluent, elution flow rate 0.60ml/min, wash-out consumption 1800mL, the concentrated back elutriant that contains L-NVP-XAA 723 EGCG (containing GCG) catechin monomers to be purified is carried out column chromatography, collect liquid and merge L-NVP-XAA 723 EGCG monomer component in the back after testing, the dry finished product that gets of concentrated frozen.

The present invention adopts dextrane gel Sephadex LH-20 column chromatography, respectively with dehydrated alcohol and 40% ethanol as eluent, carry out pre-separation and purification experiment, the main ester catechin in the rough tea-polyphenol (EGCG, GCG, ECG) can be realized the separation of continuous high-efficient.This method is easy and simple to handle, and is with low cost, and solvent is nontoxic, and monomer extraction yield and product purity are all higher, and can realize the preparation that the disposable gram magnitude of EGCG is above, and economic benefit is very considerable.

(1) pre-separation method and the technique effect of catechin monomers L-NVP-XAA 723 EGCG and L-L-Epicatechin gallate ECG: dextrane gel Sephadex LH-20 is a column packing, with the dehydrated alcohol be: the high 50cm of post for the eluent processing condition, elution flow rate 0.75ml/min can realize effective pre-separation of main ester catechin (EGCG and ECG, GCG) in the rough tea polyphenol raw materials.EGCG (containing GCG) monomer eluting rate is 98.45%, and purity can reach 88.66%; ECG monomer eluting rate is 97.64%, and purity reaches 97.62%, extraction yield 85.67%; Disposable applied sample amount (TP) can reach 6g, eluent consumption 1500ml.

(2) catechin monomers L-NVP-XAA 723 EGCG purification process and technique effect: separate EGCG (GCG) cut that obtains and be concentrated into 20ml through appropriateness, again through Sephadex LH-20 column chromatographic isolation and purification, concrete processing condition are: eluent is 40% ethanol, the high 50cm of post, elution flow rate 0.60ml/min, eluent consumption 1500ml, can prepare the high purity EGCG monomer, eluting rate can reach more than 98%, purity reaches more than 99.9%, extraction yield 85.31%, and non-pigment is residual.

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

(1) preliminary separation stage adopts dehydrated alcohol to make eluent and has substituted aqueous acetone solution eluent 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 and tea pigment can separate simultaneously to be removed, solved and reported and obtain tea-polyphenol behind dextrane gel Sephadex LH-20 chromatography, generally the catechin in the raw material tea-polyphenol can only be divided into ester type and non-ester catechin two classes, and EGCG with can't isolating problem with ECG, can reduce the subsequent purification formality greatly;

B, agent non-toxic inexpensive, elution time are short, have reduced production cost, and a wash-out cycle only needs more than 30 hour, and aqueous acetone solution is made eluent and roughly needed 77 hours;

C, take off effectively, colour band is even, is difficult for hangover and produces bubble;

D, convenient solvent reclaiming owing to collect the anhydrous alcohol solution that liquid is catechin monomers, adopt Rotary Evaporators, reclaim very quick and convenient, shortened the connection procedure with subsequent purification, and the aqueous acetone solution eluting liquid is collected liquid and is 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 the HPLC purifying process, the separation that only needs to finish monomer EGCG through conventional column chromatography system is purified, extraction yield (24.56%) and chromatographic purity (99%) all are close to or higher than present report both domestic and external, and purifying preparation amount of EGCG can reach more than 1 gram, considerably beyond the preparation amount of milligram level of the HPLC that partly purchases, this has reduced production cost to a great extent, has increased productive rate;

(3) whole separation, purge process adopt dehydrated alcohol and 40% 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.

Description of drawings

Fig. 1 is a process route chart of the present invention.

Embodiment

Below in conjunction with accompanying drawing, the present invention is described in detail by embodiment.

Embodiment:

Referring to Fig. 1, as follows according to the operational path operation,

(1) at first from tea dust, extracts the tea-polyphenol crude product (TP) that contains catechin;

(2) catechin monomers L-NVP-XAA 723 EGCG and L-L-Epicatechin gallate ECG pre-separation:

The pre-treatment of a, gel column: take by weighing 70g dextrane gel Sephadex LH-20 xerogel, with the abundant swelling of eluent dehydrated alcohol 300ml (2 hours swellings of available boiling water bath heating shorten swelling time);

B, dress post: the gel that swelling is good is slowly poured chromatography column into, makes its uniform settling.Wait to be deposited to behind the desired height about 50cm, continue dehydrated alcohol drip washing with 3-5 times of column volume with the balance pillar;

C, last sample: take by weighing 5.8140 gram raw material tea-polyphenol and be dissolved in fully dissolving in the 20ml dehydrated alcohol, after the ultrasonic exhaust, slowly it is added in the post with dropper under (25 ℃) under the normal temperature, treating that sample infiltrates fully connects automatic Fraction Collector behind the gel and begins to collect, elution flow rate 0.75ml/min, the 20min/ pipe, the 12ml/ pipe is collected 1500ml altogether;

D, employing high performance liquid chromatography detect composition and the content of collecting liquid;

E, merging contain the elutriant of ECG catechin monomers, and rotary evaporation is concentrated into 15ml-20ml in 45 ℃ of water-baths, and frozen drying gets finished product 0.3975g;

F, merging contain the elutriant of EGCG (containing GCG) catechin monomers, and rotary evaporation is concentrated into 20ml in 45 ℃ of water-baths.

(3) catechin monomers L-NVP-XAA 723 EGCG purifying

The pre-treatment of a, gel column: take by weighing 30g dextrane gel Sephadex LH-20 xerogel, with the abundant swelling of eluent 40% aqueous ethanolic solution 150ml (2 hours swellings of available boiling water bath heating shorten swelling time);

B, dress post: the gel that swelling is good is slowly poured chromatography column into, makes its uniform settling.Wait to be deposited to behind the desired height about 50cm, continue 40% ethanol drip washing with 3-5 times of column volume with the balance pillar;

C, last sample: take by weighing and concentrate back EGCG (containing GCG) catechin monomers collection liquid 20ml, after the ultrasonic exhaust, slowly it is added in the post with dropper under (25 ℃) under the normal temperature, treating that sample infiltrates fully connects automatic Fraction Collector behind the gel and begins to collect, elution flow rate 0.60ml/min, the 20min/ pipe, the 12ml/ pipe is collected 1800ml altogether;

D, employing high performance liquid chromatography detect composition and the content of collecting liquid, merge the elutriant that contains the EGCG catechin monomers, and rotary evaporation is concentrated into 15ml-20ml in 45 ℃ of water-baths, and frozen drying gets finished product 1.4280g.

Claims (1)

1, the separation purification method of catechin monomers comprises and extract the rough tea-polyphenol that contains catechin from tea dust, secondly purifies and separates catechin composition from crude extract; It is characterized in that: described from crude extract the purifies and separates catechin composition, be meant purifies and separates catechin L-NVP-XAA 723 and L-L-Epicatechin gallate component, concrete processing method comprises: column chromatography pre-separation, column chromatography purification and separation and purification;

A, described column chromatography pre-separation operation are: being column packing with dextrane gel Sephadex LH-20, the high 50cm of its post, get rough tea-polyphenol 5-6g, dehydrated alcohol is an eluent, elution flow rate 0.75ml/min, eluent consumption 1500mL, 30-38 hour column chromatography time, collect liquid and merge the elutriant that contains L-L-Epicatechin gallate ECG catechin monomers in the back after testing, concentrated postlyophilization gets finished product; Merge the elutriant that contains L-NVP-XAA 723 EGCG (containing L-nutgall catechin gallic acid ester GCG) catechin monomers, rotary evaporation is concentrated into 15-20ml in 45 ℃ of water-baths, and is to be purified;

B, described column chromatography purification operation are: again with dextrane gel Sephadex LH-20 post, the high 50cm of post, with 40% aqueous ethanolic solution is eluent, elution flow rate 0.60ml/min, wash-out consumption 1800mL, the concentrated back elutriant that contains L-NVP-XAA 723 EGCG (containing GCG) catechin monomers to be purified is carried out column chromatography, collect liquid and merge L-NVP-XAA 723 EGCG monomer component in the back after testing, the dry finished product that gets of concentrated frozen.

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