CN101391990A - A method for extracting and purifying EGCG from tea fresh or dry leaves - Google Patents

Abstract

The present invention relates to a method for extracting and purifying functional components of Chinese herbal medicines, and the purpose is to provide a simple, safe, economical and efficient way of extracting and purifying monomeric compound (-) epigallocatechin-3-O-gallate from tea leaves (EGCG) method, the technical scheme adopted is: take tea dry leaf as raw material, adopt Soxhlet extractor extraction technology, recrystallization technology, silica gel normal phase partition chromatography technology and low-temperature recrystallization technology to combine, obtain purity average The (-) epigallocatechin-3-O-gallate reaches more than 99%, and its extraction rate is more than 1%. The raw materials used in the invention are fresh or dry tea leaves that are mostly discarded in agricultural production, which neither destroys the ecological balance of plants nor affects agricultural production, and can obtain high-purity tea functional components. Moreover, the method is simple and easy to implement after extraction, preliminary impurity removal and repurification, and the loss of the target compound is small. It is suitable for industrial applications and is of great significance to industrial production.

Description

A kind of method of extracting purifying EGCG from tealeaves the fresh or cured leaf

Technical field

The present invention relates to a kind of method of separation and purification functional ingredient from plant, specifically is to extract purifying (-) epigallocatechin-3-O-gallic acid ester from the fresh or dry leave of tealeaves.

Background technology

The Ji Yuan of tealeaves is the leaf of Theaceae Camellia tea plant [Camellia sinensis (L.) O.Ktze].Tealeaves contains the number of chemical composition, and type relates to tea-polyphenol class, alkaloids, tea amino acids, fats, enzyme, ucleotides, carbohydrate, mineral element, volatile matter (" China's book on Chinese herbal medicine ", 1999).Tea-polyphenol accounts for 25% of dry weight of tea leaves, mainly is made up of catechin, flavones (flavonol), cyanidin(e) and phenolic acid (depside) four big compounds.Wherein catechin is the main body component of tea-polyphenol, accounts for about 80% (tealeaves biological chemistry (second edition), 1998) of tea-polyphenol total amount.Tea-polyphenol has stronger antioxygenation (Zhao Baolu, 2002) pharmacological effect (Liu Xueming and widely, 1998), NVP-XAA 723 (EGCG wherein particularly, structure is seen Fig. 1) (Chen Zongmao occupies an important position at anti-oxidant, anti-mutation, aspect such as antitumor with its distinctive molecular structure, 2003), but because polyphenol mainly is made up of water miscible condensed tannin in the tealeaves, their unstable chemcial properties, difference often only is the difference of carbonoid between the molecular structure, mask work very difficult (Shi Guofang, 2001).

At present, in the process of separating tea multi-phenol monomer-(-) epigallocatechin-3-O-gallic acid ester, use the dextrane gel column chromatography, often need just can obtain monomer (Li Bingzheng, 1992 in conjunction with additive method such as recrystallization method, counter-current distribution method, high-pressure liquid phase preparation method etc.; Zhou Hongzhi etc., 2000).Because when recycle-water, need comparatively high temps, not oxidized for making tea-polyphenol, need usually to adopt freeze-drying to reclaim, so it is uneconomical to prepare a large amount of monomers with this method, and the cycle is long, production efficiency is low.Also Chang Yinxu is in conjunction with additive method to adopt high-pressure liquid phase method, and the cost height reclaims difficulty, is not suitable for suitability for industrialized production.High-speed countercurrent chromatography can separate catechin monomers well, but can only obtain a small amount of catechin monomers at present, if carry out suitability for industrialized production, also will further improve isolation technique, and its facility investment also very big (army of wearing, 1999; Du Qizhen etc., 1997).In sum, in the process of utilizing plant resources separation and purification (-) epigallocatechin-3-O-gallic acid ester, main influence factor is that the separation efficiency of ubiquity (-) epigallocatechin-3-O-gallic acid ester in the sepn process is low, removal process is very long, makes cost raise.Therefore, be necessary to seek the method that a kind of handy and safe economical and efficient ground extracts purifying (-) epigallocatechin-3-O-gallic acid ester.The present invention is intended to set up a kind of method that combines by the various modern separating and purifying technology, separation and purification (-) epigallocatechin-3-O-gallic acid ester that realize simply fast, loss is few.

Summary of the invention

The object of the present invention is to provide that a kind of fresh or dry leave is a raw material with tealeaves, by adopting apparatus,Soxhlet's abstraction technique, recrystallization technology, silica gel positive partition chromatography technology and low temperature recrystallization technology to combine, thereby obtain the processing method of high purity (-) epigallocatechin-3-O-gallic acid ester in a large number, fast.The objective of the invention is to reach by following scheme:

With the tea-drying leaf is raw material, adopts apparatus,Soxhlet's abstraction technique, recrystallization technology, silica gel positive partition chromatography technology and low temperature recrystallization technology to combine, and obtains purity and all reaches (-) epigallocatechin-3-O-gallic acid ester more than 99%.

Above-mentioned (-) epigallocatechin-3-O-gallic acid ester extracting and purifying method is characterized in that, gets a certain amount of dry tealeaves, and the used solvent of apparatus,Soxhlet's extraction impurity elimination is a chloroform, and volume is 1 times of solid masses, continuous extraction 48h.The used solvent of apparatus,Soxhlet's extraction is an ethyl acetate, and volume is 1 times of solid masses, continuous extraction 48h.

The ethyl acetate that concentrates apparatus,Soxhlet's reflux extraction arrives half of original volume, adds 3-5 and doubly measures chloroform, has (-) epigallocatechin-3-O-gallic acid ester crude product to separate out.

Used silica gel is a 100-150 order macroporous silica gel, upward moisture ether silica gel is partition column for the employing of silica gel positive partition chromatography step in this method, keep liquid level, with the molten moisture ether of the crude product of apparatus,Soxhlet's reflux extraction (-) epigallocatechin-3-O-gallic acid ester, the 1/10-1/20 of applied sample amount silica gel quality, be 6～8BV per hour, every part of 1/4～1/2BV with flow velocity.Detect the elutriant composition with high-pressure liquid phase, merge the same section fraction, HPLC method analysis condition: chromatographic column, ODS post (4.0 * 150mm, 5 μ m); Detect wavelength 280nm; 40 ℃ of column ovens; Moving phase, second is fine: ethyl acetate: 0.05 phosphoric acid (12: 2: 86); Flow velocity, 1mL/min.Fraction spends the night through a small amount of anhydrous sodium sulfate drying again, reclaims below 45 ℃, gets (-) epigallocatechin-3-O-gallic acid ester crude product.

The used solvent of recrystallization also comprises methylene dichloride, chloroform, sherwood oil except that ether, the low temperature recrystallization temperature is-10 ℃～4 ℃.Extraction yield is 1.0%, and purity is 99.0%.

Embodiment 2

Take by weighing fresh tea leaf in its 10kg (dry weight is 2kg), dry back with 1 times of volume chloroform continuous extraction 48h in apparatus,Soxhlet's, use 200mL ethyl acetate backflow 48h again, filter ethyl acetate solution, spend the night, concentrate half that ethyl acetate arrives original volume with anhydrous sodium sulfate drying, adding 3-5 doubly measures dry chloroform adularescent precipitation and separates out, use filter paper filtering, with a small amount of dry chloroform flushing, the dry EGCG crude product (about 150g) that gets.

100g (-) epigallocatechin-3-O-gallic acid ester crude product is dissolved in the moisture ether of 2000mL, the moisture ether silica gel of last 2000g partition column (internal diameter 20cm, high 150cm), and flow velocity is 10L per hour, fraction of 500mL, 2.5L fraction before merging.Fraction spends the night through a small amount of anhydrous sodium sulfate drying, reclaims to such an extent that sample is dissolved in moisture ether again below 45 ℃, the moisture ether silica gel of last 2000g partition column (internal diameter 20cm, high 150cm), and flow velocity is 10L per hour, fraction of 500mL, 2.5L fraction before merging.Fraction spends the night through a small amount of anhydrous sodium sulfate drying again, reclaims below 45 ℃, gets some bottles of white powder materials, about altogether 10g.

Each fraction detects with high-pressure liquid phase, merges same section.HPLC method analysis condition: get its 1mg and be dissolved in 1mL distilled water, cross millipore filtration (0.45 μ m), ultrasonic dissolution, sample size 20 μ L; Day island proper Tianjin chromatographic column, ODS post (4.0 * 150mm, 5 μ m); Detect wavelength 280nm; 40 ℃ of column ovens; Moving phase, second is fine: ethyl acetate: 0.05 phosphoric acid (12: 2: 86); Flow velocity, 1mL/min.The white powder material 4 ℃ of crystallizations, obtains (-) epigallocatechin-3-O-gallate monomer and separates out in ether.Extraction yield is 0.985%, and purity is 98.5%.

Used standard substance are available from Sigma company in this test.

Quantitative detecting analysis adopts surface condition down as a result:

Instrument	Tianjin, island LC-6A liquid chromatograph
		Chromatographic column	Day island proper Tianjin chromatographic column, ODS post (4.0 * 150mm, 5 μ m)
Column temperature	40℃
		Moving phase	Second is fine: ethyl acetate: 0.05 phosphoric acid (12: 2: 86)
Flow velocity	1mL/min
		Sampling volume	20μL
Detector	Tianjin, island SPD-6AV UV-VIS detector

Description of drawings

Fig. 1 is (-) epigallocatechin-3-O-gallic acid ester molecular formula figure.

Claims (6)

1. A method for extracting and purifying (-) epigallocatechin-3-O-gallate (EGCG) from tealeaves, its main feature is: tealeaves adopt Soxhlet extractor extraction technology, recrystallization technology, The silica gel normal phase partition chromatography technique and the low-temperature recrystallization technique are combined to obtain (-) epigallocatechin-3-O-gallic acid with a purity greater than 99%. 2. The tea according to claim 1, characterized in that: the tea mainly comes from fresh or dried leaves of various tea plants. 3. according to the Soxhlet extractor extraction technology described in claim 1, it is characterized in that: the solvent used for Soxhlet extractor extraction and impurity removal is chloroform, and the volume is 1 times of solid mass, continuous extraction 48h. Soxhlet extractor chloroform reflux extraction can efficiently remove fat-soluble impurities such as chlorophyll while saving solvent. The solvent used in the Soxhlet extractor extraction was ethyl acetate, the volume was 1 times the mass of the solid, and the extraction was continuously refluxed for 48 hours. When the Soxhlet extractor is used for ethyl acetate reflux extraction, tea polyphenols such as (-) epigallocatechin-3-O-gallate can be efficiently extracted while saving solvent. 4. according to the described recrystallization technology of claim 1, it is characterized in that: the ethyl acetate of concentrated Soxhlet extractor reflux extraction is to half of original volume, adds 3-5 times of amount chloroform, has (-) epigallocatechin The crude product of prime-3-O-gallate was precipitated. 5. According to the silica gel normal phase partition chromatography technology described in claim 1, it is characterized in that: the silica gel used is 100-150 mesh macroporous silica gel, and in the method, the silica gel normal phase partition chromatography step adopts upper water-containing ether silica gel normal phase partition column, keep the liquid level, and dissolve the crude product of (-) epigallocatechin-3-O-gallate in reflux extraction of Soxhlet extractor in diethyl ether with water, and the loading amount is 1/10-1/20 of the mass of silica gel, The flow rate is 6-8BV per hour, 1/4-1/2BV per serving. Use high-pressure liquid phase to detect eluent components, combine the same fractions, and analyze the conditions of HPLC method: chromatographic column, ODS column (4.0×150mm, 5μm); detection wavelength 280nm; column thermostat 40°C; mobile phase, acetonitrile: Ethyl acetate: 0.05 phosphoric acid (12:2:86); flow rate, 1 mL/min. The fraction was then dried overnight with a small amount of anhydrous sodium sulfate, and recovered below 45°C to obtain the crude product of (-) epigallocatechin-3-O-gallate. 6. according to the described low-temperature crystallization and recrystallization technology of claim 1, it is characterized in that: the used solvent of recrystallization also comprises dichloromethane, tetrachloromethane, chloroform, sherwood oil except diethyl ether, and low-temperature crystallization temperature is -10℃～4℃.

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