CN103910705B - The method of rapid extraction separation and purification NVP-XAA 723 from the tankage of green tea - Google Patents

Abstract

The invention relates to a method for quickly extracting, separating and purifying epigallocatechin gallate from green tea leftovers, which comprises the following steps: (1) extracting the green tea leftovers with ethanol in a conventional method; (2) extracting liquid at 80°C Recover ethanol under reduced pressure until there is no alcohol smell; (3) The alcohol extract is separated by adsorption with polyamide resin; (4) Collect epigallocatechin gallate (EGCG) fractions, decolorize with activated carbon, and concentrate under reduced pressure at low temperature; (5) Recrystallize with ethanol to obtain high-purity epigallocatechin gallate (EGCG); the invention is simple and easy, and only one resin can be used to achieve adsorption, separation and purification in one step, and the solvent used Non-toxic, safe, reliable, high product purity, low cost, suitable for large-scale industrial production.

Description

Method for rapidly extracting, separating and purifying epigallocatechin gallate from green tea leftovers

technical field

The invention relates to the field of medical applications, in particular to a method for rapidly extracting, separating and purifying epigallocatechin gallate from green tea leftovers.

Background technique

Polyphenols in tea account for 18%-36% of the dry weight of tea, including catechin-based flavanols, flavonoids and flavonols, anthocyanins and anthocyanins, and phenolic acids and depsipolic acids . Among them, catechins are the main components of tea polyphenols, accounting for 12%-24% of the dry weight of tea leaves and 70%-80% of the total amount of tea polyphenols. Modern medical research has found that tea polyphenols are a new type of natural antioxidant, which has physiological effects such as anti-tumor, anti-aging, anti-mutation, inhibition of tumor growth, weight loss and blood pressure prevention, and prevention of cardiovascular diseases. These physiological effects are mainly manifested by the catechins in it, among which the epigallocatechin gallate (EGCG) in the ester type catechins has significantly stronger effects than other catechins in many aspects. components.

The molecular formula of EGCG is C ₂₂ H ₁₈ O _{11, and} the molecular weight is 458.4. The molecule of EGCG contains multiple phenolic hydroxyl groups, and the active center is on two rings, so it has a strong ability to resist oxidation and scavenge free radicals. The pure product of EGCG is a white powder, bitter in taste, non-toxic, very soluble in water, soluble in methanol, ethanol, acetonitrile and ethyl acetate, insoluble in chloroform, with a melting point of 218°C. It is easily oxidized in the air and turns into a slightly pink color, and it is also easily oxidized as the temperature rises.

With the deepening of people's understanding of the health functions of tea ingredients, the extraction and separation of tea polyphenols has been extensively and deeply studied, and the research on the separation and preparation of catechin monomers, especially the ester catechin EGCG, has also attracted more and more attention. . At present, there are many patents on methods for extracting and separating EGCG. Among them, the patents for the preparation method of high-purity EGCG mainly include:

CN1465572A Method for purifying epigallocatechin gallate monomer

CN1470510A Purification process of epigallocatechin gallate monomer

CN102702163A Method for preparing high-purity monomer catechin from leftovers of tea leaves

CN1603319A Method for separation and purification of catechin monomer

CN101386614A Method for preparing epigallocatechin gallate by resin adsorption method

CN1733753A A method for purifying epigallocatechin gallate monomer

CN101381359A A method for extracting high-purity epigallocatechin gallate from green tea

CN101723927A Separation and purification method for factory production of catechin monomer EGCG

CN101074224A A kind of preparation method of high content EGCG

In the above-mentioned eight patents, the samples are all extracted by conventional methods, including solvent extraction, metal ion precipitation, resin adsorption, supercritical fluid extraction, ultrasonic extraction, etc., because tea polyphenols except EGCG , also contains a certain amount of other substances, so the purity of the extracted EGCG is not high. For the purification process of single-component EGCG, most of them first use resin adsorption technology to remove impurities, including macroporous adsorption resin columns, anion and cation exchange resin columns, etc., usually using one column or two resin columns with different properties to connect in series Complete, the main function is to adsorb the sample, use water as the eluent to remove water-soluble impurities, and then desorb with dilute alcohol. The above-mentioned means alone cannot meet the purity requirements of EGCG, and the key purification steps of the above-mentioned 8 patents are that chromatographic separation techniques are used in the last few steps, including the use of supercritical fluid chromatography, dextran gel chromatography, medium Low-pressure silica gel chromatography, C18 liquid chromatography preparation technology and other means, and finally through the method of recrystallization, make EGCG reach a high purity.

The disadvantages of the above 8 patents are that the operation process is complicated and the experimental conditions are harsh. Due to the use of high-end experimental instruments and expensive chemical reagents, the preparation cost is high and the operation is not easy to control. Moreover, the column adsorption separation process has been adopted many times, resulting in large losses, resulting in low product yields, and a large amount of organic solvents have been introduced in the process. The operation steps are limited to laboratories, and the products cannot be industrialized.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a method that can make full use of the leftovers of green tea to quickly extract, separate and purify epigallocatechin gallate, so that the leftovers of green tea can be recycled as waste, and at the same time solve the problem of In the purification process of EGCG, the steps are complicated, time-consuming, the solvent used is highly toxic, the product yield is low, and the industrialization cannot be realized.

The method for rapidly extracting, separating and purifying epigallocatechin gallate from the leftovers of green tea described in the present invention comprises the following steps:

(1) The leftovers of green tea were extracted by ultrasonic vibration with 60% ethanol, the extraction time was 30 minutes, extracted twice, and the extracts were combined;

(2) Recover ethanol from the extract until it has no alcohol smell, and concentrate it to a clear paste with a relative density of 1.20 to 1.22;

(3) Adsorption and separation of the clear paste obtained in (2) with a polyamide resin column;

(4) Thin-layer chromatography tracking detection, collecting epigallocatechin gallate fraction, decolorized with activated carbon, filtered, and the filtrate was decompressed at 60°C-80°C, protected by nitrogen, and concentrated to a relative density of 1.15- 1.18, freeze-dried;

(5) Recrystallize with ethanol to obtain high-purity epigallocatechin gallate;

The eluent used in the step (3) adsorption separation contains 95% ethanol and 36% acetic acid, wherein the volume ratio of 95% ethanol: 36% acetic acid is 5:1-1:5.

Preferably, the leftovers of the step (1) green tea include some thick and old slivers, trimmed branches or tea dust produced during the grade processing process or accumulated in the storage process except for a large number of fresh leaves during the tea picking process. Some old tea, tea dregs.

Preferably, the ratio of the leftovers of green tea in the step (1) to 60% ethanol is 1:8 g/ml-1:20 g/ml.

Preferably, the step (2) recovering the ethanol from the extract until it has no alcohol smell refers to recovery under reduced pressure below 80°C.

Preferably, the particle size of the polyamide resin in the step (3) is 60-100 mesh, and the dosage is: Resin: clear paste = 10-20:1 (g/g), and the diameter-to-height ratio of the resin column is 1:20 .

Preferably, the amount of activated carbon used in the step (4) is 2%-3% of the volume of the solution, that is, the amount of activated carbon added per 100ml of the solution is 2g-3g.

The beneficial effect of the present invention is: the present invention does not need the preliminary purification of macroporous resin, by controlling the chromatographic separation conditions, only one kind of separation system is used, polyamide chromatographic separation can be directly carried out, therefore, through thin-layer chromatographic tracking detection, The pure EGCG fraction can be collected, so that the separation and purification can be completed in one step, making the operation simple and easy; and only food-grade solvent ethanol, acetic acid and activated carbon are used in the whole process to ensure product safety and non-toxicity.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of EGCG reference substance;

Fig. 2 is a high performance liquid chromatogram of a sample obtained by the method for rapidly extracting, separating and purifying epigallocatechin gallate from green tea leftovers according to the present invention.

Detailed ways

Example 1

Green tea leftovers 1 kg, add 8L of 60% ethanol to extract by ultrasonic vibration, extract twice, each time for 30 minutes, and combine the extracts; the extracts are decompressed below 80°C to recover ethanol until there is no alcohol smell, and concentrate to a relative density of 1.20 to 1.22 Clear the cream; cool it, and adsorb it on a polyamide resin column (the amount of resin used is 10 times the weight of the cream, and the diameter-to-height ratio of the column is 1:20), elute with 95% ethanol: 36% acetic acid (volume ratio is 5:1) , thin-layer chromatography tracking detection, collect epigallocatechin gallate (EGCG) fraction, decolorize with 2% (g/100ml) activated carbon, filter, and filter the filtrate at 60°C-80°C under the protection of nitrogen. Concentrate under pressure to a relative density of 1.15-1.18, freeze-dry. Recrystallized with ethanol to obtain epigallocatechin gallate (EGCG) with a purity of 98.3% and a yield of 10%.

Example 2

Add 1 kg of green tea waste, add 12L of 60% ethanol to extract by ultrasonic vibration, extract twice, each time for 30min, and combine the extracts. The extract is recovered under reduced pressure below 80°C until there is no alcohol smell, and concentrated to a clear paste with a relative density of 1.20 to 1.22. Cool, adsorb on a polyamide resin column (the amount of resin used is 15 times the paste weight, and the diameter-to-height ratio of the column is 1:20), elute with 95% ethanol: 36% acetic acid (volume ratio: 2:1), thin layer Chromatography tracking detection, collecting epigallocatechin gallate (EGCG) fractions, adsorbing with 2.5% (g/100ml) activated carbon, filtering, and concentrating the filtrate under reduced pressure at 60°C-80°C under nitrogen protection at low temperature to The relative density is 1.15-1.18, freeze-dried. Recrystallized with ethanol to obtain epigallocatechin gallate (EGCG) with a purity of 98.6% and a yield of 12%.

Example 3

Green tea leftovers 1 kg, add 16L of 60% ethanol to extract by ultrasonic vibration, extract twice, each time 30min, and combine the extracts. The extract is recovered under reduced pressure below 80°C until there is no alcohol smell, and concentrated to a clear paste with a relative density of 1.20 to 1.22. Cool, adsorb on a polyamide resin column (the amount of resin used is 20 times the paste weight, and the ratio of diameter to height of the column is 1:20), elute with 95% ethanol: 36% acetic acid (volume ratio is 1:1), thin layer Chromatography tracking detection, collecting epigallocatechin gallate (EGCG) fractions, adsorbing with 3% (g/100ml) activated carbon, filtering, and concentrating the filtrate under reduced pressure at 60°C-80°C under nitrogen protection at low temperature to The relative density is 1.15-1.18, freeze-dried. Recrystallized with ethanol to obtain epigallocatechin gallate (EGCG) with a purity of 98.9% and a yield of 16%.

Example 4

Green tea leftovers 1 kg, add 20L of 60% ethanol to extract by ultrasonic vibration, extract twice, each time 30min, and combine the extracts. The extract is recovered under reduced pressure below 80°C until there is no alcohol smell, and concentrated to a clear paste with a relative density of 1.20 to 1.22. Cool, adsorb on a polyamide resin column (the amount of resin used is 20 times the paste weight, and the ratio of diameter to height of the column is 1:20), elute with 95% ethanol: 36% acetic acid (volume ratio is 1:2), thin layer Chromatography tracking detection, collecting epigallocatechin gallate (EGCG) fractions, adsorbing with 3% (g/100ml) activated carbon, filtering, and concentrating the filtrate under reduced pressure at 60°C-80°C under nitrogen protection at low temperature to The relative density is 1.15-1.18, freeze-dried. Recrystallized with ethanol to obtain epigallocatechin gallate (EGCG) with a purity of 99.0% and a yield of 15%.

The leftovers of the green tea in the above examples include some thick and old stalks except a large number of fresh leaves removed during the tea picking process, pruned branches or tea powder produced in the grade processing process and some old tea accumulated in the storage process, tea dregs.

The beneficial effect of above-mentioned embodiment is: the present invention does not need the primary purification of macroporous resin, because EGCG is a polyphenol compound, by controlling chromatographic separation condition, adopts a kind of separation system, can directly carry out polyamide chromatographic separation, therefore , through thin-layer chromatography tracking detection, EGCG pure product fractions can be collected instead of mixtures, so that the separation and purification can be completed in one step, making the operation simple and easy; and the whole process only uses food-grade solvents such as ethanol, acetic acid and Activated carbon ensures product safety and non-toxicity.

The above-mentioned embodiments are only to illustrate the technical conception and characteristics of the present invention, and its purpose is to allow those familiar with this technology to understand the content of the present invention and implement it, and cannot limit the protection scope of the present invention with this. Substantial equivalent changes or modifications shall fall within the protection scope of the present invention.

Claims (6)

1. the method for rapid extraction separation and purification NVP-XAA 723 from the tankage of green tea, comprises following steps:

(1) tankage of green tea extract with 60% EtOH Sonicate concussion, and extraction time is 30 minutes, extracts twice, united extraction liquid;

(2) extracting solution reclaims ethanol to without alcohol taste, is concentrated into the clear cream that relative density is 1.20 to 1.22;

(3) the clear cream polyamide resin column fractionation by adsorption will obtained in (2);

(4) thin-layer chromatography tracing detection, collects NVP-XAA 723 flow point, after activated carbon decolorizing, filters, and filtrate is in 60 DEG C of-80 DEG C of low-temperature reduced-pressures, and lead to nitrogen protection, being concentrated into relative density is 1.15-1.18, lyophilize;

(5) with ethyl alcohol recrystallization, highly purified NVP-XAA 723 is obtained;

Elutriant in described step (3) fractionation by adsorption comprises 95% ethanol and 36% acetic acid, wherein 95% ethanol: the volume ratio of 36% acetic acid is 5:1-1:5.

2. method according to claim 1, it is characterized in that: the tankage of described step (1) green tea, comprise in the process of picking tea-leaves some thick old bars, pruning branch or the tea dusts produced in the grade course of processing of removing outside a large amount of fresh leaf and some the old tea overstock in storage, tea grounds.

3. method according to claim 1, is characterized in that: the described tankage of step (1) green tea and the ratio of 60% ethanol are 1:8 g/ml-1:20 g/ml.

4. method according to claim 1, is characterized in that: described step (2) extracting solution reclaims ethanol to without alcohol taste, refers to reclaim under reduced pressure below 80 DEG C.

5. method according to claim 1, is characterized in that: the granularity of described step (3) polyamide resin is 60-100 order, and consumption is: resin: clear cream=10 g:1g-20 g:1g, the blade diameter length ratio of resin column is 1:20.

6. method according to claim 1, is characterized in that: the consumption of described step (4) gac is the 2%-3% of liquor capacity, and the amount of namely adding gac in every 100ml solution is 2g-3g.