KR100418605B1 - Process for the production of high purified (-)Epigallocatechin Gallate from Green tea extract - Google Patents

Abstract

The present invention relates to a method for high-volume separation of epigallocatechin gallate [(-) Epigallocatechin Gallate, EGCg] having excellent antioxidant efficacy from green tea extract. More specifically, the present invention selectively separates EGCg and ECg from the green tea catechin mixture by adding caffeine to the green tea extract, and precipitates the precipitate formed with EGCg-caffeine and ECg-caffeine in an aqueous solution containing an organic acid such as ascorbic acid. It is effective to provide a method for separating high purity EGCg with excellent stability and productivity by column chromatography using an eluent to which it is dissolved efficiently and added with an organic acid such as ascorbic acid.

Description

Process for the production of high purified (-) Epigallocatechin Gallate from Green tea extract}

The present invention relates to a method for separating high purity epigallocatechin gallate [(-) Epigallocatechin gallate, EGCg] derived from green tea extract. More specifically, by adding caffeine to the green tea extract, only EGCg and Epicatechin gallate (ECg) are selectively precipitated and separated from the green tea catechin mixture, which is loaded into a column chromatography filled with a synthetic adsorbent filler and then ascorbic. The present invention relates to a method for separating high-purity EGCg that elutes the stability and productivity of EGCg by eluting it in an eluent containing an organic acid such as ascorbic acid.

Compared to other plants, green tea contains a large amount of polyphenols among the constituents that are characteristically visible. Unlike other plants, green tea is known to have various physiological activities such as anticancer effect, antioxidant effect and antibacterial effect. Among the polyphenols, catechin is best known as an ingredient that shows the efficacy of green tea. Green tea catechins that exist in nature are largely composed of eight components, including epicatechin [(-) EC], epigallocatechin [(-) EGC], epicatechin gallate [(-) ECg] and epigallocatechin gal Rate [(-) EGCg] and the like are referred to as common tea catechins. Among them, EGCg is known to have the highest composition ratio among tea catechins, and is also excellent in various functional effects. In general, 10-15% of the total dry weight of tea leaves is the content of tea catechin, EGCg accounts for 50-60% of the tea leaves, but it is known that there is a difference in content depending on the cultivation climate, soil or species of green tea.

Recently, the health effects of green tea on the human body have been attracting attention from around the world, and research on bioefficacy has been actively conducted in countries around the world. Among the green tea catechins, EGCg has various effects from antioxidant activity to lower cholesterol, triglyceride elevation inhibition, anti-tumor, anti-cancer and skin beauty, obesity inhibition, heavy metal removal, mutagenicity inhibition, hypotension, central nervous system activation, cardiovascular disease The effect has been reported in vitro and in vivo clinical trials. Accordingly, a manufacturing technique for efficiently extracting and purifying high purity EGCg from green tea catechin and enabling mass production has been proposed.

A method for producing high purity EGCg in green tea known to date, in Europe EP1077211 (name: EGCg production process), a column chromatography filled with a porous polar filler and a process for separating high purity EGCg using an organic solvent such as alcohol as an eluent. Presented. The method using the in-process XAD-7 filler proposed in the patent used water / isopropanol (9: 1 v / v) as an eluent to isolate more than 90% of EGCg and recover 73.5% of the total EGCg content. . In addition, there is a feature using a thermostat to maintain the operating temperature of the column filled with the filling at 60 ℃. However, the patented method of separating high-purity EGCg using XAD-7 fill and thermostat was time consuming and costly in the purification process to powder the EGCg concentration in the main eluent to 0.064%. There is a problem that the overall manufacturing efficiency is somewhat lower. In addition, there is a problem of additional cost for equipping the apparatus by using a thermostat to keep the column temperature constant.

In the US US4613672 (name of the invention: tea catechin production process) EGCg by HPLC (high-speed liquid chromatography) method using acetone / tetrahydrofurene / chloroform as eluent and C ₁₈ as a reversed phase column As a result, more than 90% high purity EGCg was isolated and 72% of the total EGCg content was recovered. However, the method has many kinds of organic solvents used in the eluent and is very toxic, and there is a fear that such toxic organic solvents remain in the final product. In addition, since the HPLC method using a column filled with C ₁₈ fillers, which is not a manufacturing method for commercial use, separates EGCg of high purity, there is a problem in that re-evaluation of fillers has to be made to supply cheaper high purity EGCg.

US No. US6210679 (title of the invention: from non-caffeine green tea - catechin separation method) is a step of separating at least 95% decaffeinated -EGCg, 4 and step continuous process, polyamide (polyamide) CC6 or C ₁₈ filling each process A packed column is used to sequentially increase the purity of EGCg. However, in order to increase the purity of EGCg sequentially through a four-step continuous process, it is cumbersome to use two kinds of fillers, and the manufacturing process is rather complicated because it requires a four-step continuous process rather than a high-purity EGCg in one step. There is a problem.

In Korean Patent Laid-Open No. 1999-007629 (name of the invention: Extraction and Purification of Anticancer Components from Green Tea), the process of extracting water from green tea using water, the process of distributing by layer separation using chloroform and ethyl acetate, and quasi A catechin extraction process comprising a process for purifying EGCg using preparative liquid chromatography and a process for separating EGCg using reversed phase liquid chromatography are disclosed. In the present invention, more than 90% of EGCg was separated from green tea by HPLC using a column (250 × 4.60 mm) filled with a 15 μm Lichrospher 100RP-18. However, the method is also a result of experimental research, not commercial use, there is a problem that re-evaluation of the filling must be made in order to supply a large amount of cheaper high-purity EGCg for commercial use.

With all the above methods, the development of a high-purity EGCg separation method is required to supplement the problems that may be of concern when the high-purity EGCg is separated from the green tea extract and to increase the safety and commercial productivity for the human body.

Accordingly, the present inventors have studied to obtain a stable EGCg by increasing the productivity of the process by reducing the time and cost required to separate the green tea catechin EGCg, the active ingredient of green tea from the green tea extract, the first selective precipitation method using caffeine To selectively precipitate EGCg and ECg, and to increase the solubility of the precipitate, in order to increase the solubility of the precipitate, the organic acid was added and heated for a certain period of time, loaded in one column filled with a synthetic adsorbent including HP-20, and then the organic acid was added. By eluting in an alcoholic eluent with high purity EGCg of more than 90% from the green tea extract was completed the present invention.

Accordingly, an object of the present invention is to add caffeine to the green tea extract to selectively bind and precipitate only EGCg and ECg in the green tea catechin component, and then remove the artificially added caffeine using a halogenated organic solvent, and also solvent-solvent. The present invention provides a method of separating 90% or more of EGCg with high purity by adding organic acid to facilitate extraction, increase the solubility of the precipitate and increase the stability of the EGCg.

Hereinafter, the present invention will be described in more detail.

1 is a chromatogram of green tea extract containing more than 85% polyphenols.

Figure 2 is the change in precipitation rate of tea catechin with or without caffeine treatment (based on green tea extract 1g).

Figure 3 is the change of tea catechin in the supernatant according to the temperature change when mixing the green tea extract and caffeine.

4 is a chromatogram of the supernatant after caffeine treatment.

5 is a chromatogram of precipitate after addition of caffeine.

Figure 6 is a high purity EGCg chromatogram of more than 90% when eluted with 15% aqueous ethanol solution (0.01% ascorbic acid addition).

7 is a high purity ECg chromatogram eluted with 15% aqueous ethanol solution (0.01% ascorbic acid added).

8 is a high purity EGCg separation process of the present invention.

In order to achieve the above object, the process of the present invention (1) selectively precipitates only EGCg and ECg in the green tea catechin using caffeine to compensate for the disadvantages of time and cost to selectively separate the EGCg from the green tea catechin The first selective precipitation method was used, and (2) an organic acid was used to facilitate solvent-solvent extraction, increase the solubility of the precipitate and increase the stability of EGCg in the step of removing artificially added caffeine using a halogenated organic solvent. It was heated after the addition, and (3) there is a possibility that chemical denaturation occurs during elution of the caffeine-free precipitate aqueous solution in column chromatography, characterized in that it was used by adding an organic acid to the eluent to suppress it.

The present invention consists of the following steps.

1) preparing an aqueous solution by dissolving green tea extract (zhejiang Chemicals I./E. Corp., China) containing more than 85% polyphenol and caffeine, respectively.

2) mixing the green tea extract and an aqueous solution of caffeine at room temperature ~ 80 ℃ temperature range.

3) the green tea extract-caffeine aqueous solution mixed with the step of standing in the place where the light is blocked for 2 to 24 hours at 0 ~ 10 ℃ temperature to form a precipitate.

4) separating the formed precipitate with the supernatant within 24 hours to prepare a precipitate aqueous solution.

5) adding an acid to the precipitate aqueous solution and heating to dissolve the precipitate aqueous solution.

6) separating the caffeine contained in the aqueous solution of the precipitate using a halogenated organic solvent capable of selectively separating only caffeine.

7) loading the aqueous solution of the precipitate in which caffeine is removed in a column chromatography filled with a synthetic adsorbent, and then eluting with an organic acid-added water alcohol eluent.

8) Separating more than 90% of high purity EGCg while increasing the concentration of alcohol by 15% from the DW to which the organic acid is added.

9) After separating the EGCg to increase the alcohol concentration to 25% to separate the high purity ECg.

By preparing a green tea extract and a caffeine solution, respectively, by mixing, only the green tea polyphenols EGCg, ECg containing a galloyl group by the physicochemical properties of caffeine, hydrogenation and hydrophobic effect is selectively precipitated together with caffeine.

Solvent-solvent extraction is used to remove caffeine from caffeine-EGCg and caffeine-ECg precipitates. When the precipitate is dissolved in an aqueous solution, an aqueous solution containing an organic acid such as ascorbic acid, malic acid or boric acid is added, followed by heating to dissolve, thereby easily dissolving, thereby reducing time and cost. The caffeine is then easily removed by solvent-solvent extraction using a halogenated organic solvent such as chloroform.

In order to purely separate EGCg and ECg contained in the aqueous solution, column chromatography filled with a synthetic adsorbent such as Sephadex HP-20 is used. The eluent used at this time is a mixture of alcohol solvent and water, starting from 100% DW (distilled water) to separate high-purity EGCg from the eluent with increasing alcohol concentration. 99% or more of the obtained high purity EGCg aqueous solution using combiHT XDB-C18 column and THF eluent or 99% using Sephadex LH-20 column and 100% ethanol, 100% acetonitrile or ethanol-acetonitrile mixed organic solvent eluent The above EGCg can be separated experimentally.

In the conventional method, by treating the acid after separating EGCg, it was not possible to suppress oxidative denaturation of EGCg, which may be easily oxidized by water during purification and elution in column chromatography. In the present invention, an organic acid such as ascorbic acid was added to the eluent to compensate for these disadvantages, thereby increasing stability and recovery rate. At this time, even if the HP-20 adsorbent used in the column chromatography contains an organic acid in the eluent, there is no problem in resolution.

90% or more of high purity EGCg and ECg aqueous solution prepared in the present invention can be used for various purposes by dry powdering.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 Evaluation of Precipitation Rate According to Weight Ratio of Each Component in Mixing Green Tea Extract and Caffeine

Green tea extract (zhejiang Chemicals I./E. Corp., China) containing more than 85% polyphenol was used as a raw material. The component ratio of the green tea polyphenol containing caffeine was confirmed by HPLC using UV absorbance and the results are shown in FIG. 1. In addition, catechins and general components of the green tea extract containing more than 85% polyphenols are summarized in Table 1.

compound content (%) Caffeine 16.93 EGC 1.73 EC 5.61 EGCg 40.85 ECg 22.60 Ash <0.5% Arsenic (As, AS ₂ O ₃ ) <2PPM heavy Metals (ppm / max) <10 ppm Agricultural chemical residue <1000 CFU / gm Total bacteria <100 CFU / gm Yeast and mold negative E.coli negative Salmonela negative

The green tea extract was treated with caffeine to prepare a precipitate. As a result of measuring the change rate of green tea catechin with or without caffeine treatment, it can be seen that the content of catechin is high in the caffeine treated group (FIG. 2). Thus, by adding artificially varying the weight of caffeine to the above 85% polyphenol-containing green tea extract was evaluated the precipitation rate of tea catechins such as EGCg and ECg according to the change in the concentration of caffeine in the extract solution.

As a result of evaluating the precipitation rate after adding more than 85% of the polyphenol-containing green tea extract extracted at various concentrations of 1/6 to 7 times (w / w) based on caffeine 1, the weight ratio of caffeine: green tea extract is 1 At ≥ 1, EGCg reduction in the supernatant was more than 98%. In addition, when the green tea extract containing more than 85% of polyphenols was added in an amount of 0.02 to 1 times (w / w) based on the weight of caffeine, the green tea extract: caffeine weight ratio was 1: 0.2 (w / w) or more. The precipitation rate was the highest. Therefore, more than 85% of the polyphenol-containing green tea extract: caffeine showed the most efficient precipitation ratio by weight ratio of 5: 1.

When precipitated by mixing caffeine with more than 85% polyphenol-containing green tea extract, more than 85% polyphenol green tea extract has a specific gravity of 0.40 g / ml and low caffeine solubility at 0.2 g / 10 ml. Therefore, when the two materials are mixed together to prepare an aqueous solution, there is a possibility that precipitation occurs in a state in which the two materials are not completely dissolved, and thus the precipitation rate may be decreased. Then, green tea extract and caffeine were dissolved in different aqueous solutions, and then the two aqueous solutions were mixed.

Example 2 Evaluation of Precipitation Rate According to Settling Time in Mixing Green Tea Extract and Caffeine

Precipitation rates of EGCg and ECg over 85% of polyphenol-containing green tea extract and caffeine mixture were evaluated.

Green tea extract prepared in Example 1: caffeine mass ratio of 5: 1 to make an aqueous solution with a high precipitation rate, respectively, and mix the two aqueous solution, and then left in the place where the light is blocked 0 ~ 10 ℃, 2 to 24 hours after catechin Changes in precipitation rates of EGCg and ECg in water were measured.

As a result, the precipitation rate was 85.7% for 6 hours and 84.8% for 24 hours, and there was no significant difference in precipitation rate over time. Therefore, the green tea extract and caffeine were dissolved, and then mixed with each other, and allowed to stand for 6 hours to separate the precipitate and the supernatant.

Example 3 Evaluation of Precipitation Rate According to Operation Temperature in Mixing Green Tea Extract and Caffeine

Precipitation rates of EGCg and ECg according to the operating temperature of the polyphenol-containing green tea extract and caffeine mixture of more than 85% in the range of room temperature to 80 ℃ were evaluated.

The green tea extract and the caffeine mass ratio were each made into 5: 1 aqueous solution, the two aqueous solutions were mixed, and then bathed for 10 minutes at room temperature to 80 ° C., and then left to stand at 0 to 10 ° C. for 6 hours in a place where light was blocked. The results of measuring the change of tea catechin in the supernatant according to the temperature at the time of mixing the green tea extract and caffeine are shown in FIG. 3.

As a result, the difference of precipitation rate of green tea extract and caffeine did not appear significantly with temperature, and the content of green tea catechin did not change due to temperature increase, so it was precipitated at room temperature.

In addition, after mixing the green tea extract and caffeine at room temperature, the mixture should be left at a temperature of 0 ~ 10 ℃ in a place where it is not exposed to light, which is likely to cause chemical denaturation due to photooxidation reaction when green tea catechin is exposed to light, This is because the precipitate formed by the reaction of green tea extract and caffeine is in the form of a gel, and when it is left at 0-10 ° C., the properties of the precipitate are hardened, so that the precipitate and the supernatant can be more easily separated.

As a result of evaluating the total recovery rate of the supernatant and sediment when the green tea extract and caffeine were mixed under various optimum conditions, the recovery rate of EGCg and ECg was close to 100%, indicating no chemical change. %, 95.19%, lower than EGCg and ECg (Table 2). The analysis results are shown in FIGS. 4 and 5.

ingredient Total recovery (%) EGC 89.25 EC 95.19 EGCg 100.94 ECg 100.80 [Note] Conditions: immersion time 6 hours, operating temperature room temperature

Example 4 Evaluation of Solubility of Precipitates

Solvent-solvent extraction should be easy in order to efficiently remove caffeine contained in the precipitate with a halogenated organic solvent such as chloroform. The precipitate has a disadvantage in that it is insoluble in water in the form of a gel. You have to. Thus, the organic acid was added to evaluate the solubility of the precipitate.

Solubility in the case of adding an organic acid such as ascorbic acid was dissolved in the precipitate aqueous solution prepared under the above optimum conditions by adding an organic acid such as ascorbic acid at a mass ratio of 0.1 to 3 times based on the mass of the precipitate 1. Much increased. Therefore, in the case of adding the organic acid, it is possible to save the time required to dissolve the precipitate, and has the advantage of suppressing chemical changes that may occur when removing caffeine using a halogenated organic solvent such as chloroform. In particular, ascorbic acid had much higher solubility of precipitates than acids such as citric acid, malic acid, boric acid, and so on, and was completely dissolved when more than twice the weight of the precipitate. Table 3 shows the evaluation results of the solubility of the precipitate according to the type of organic acid and the mass ratio of precipitate and acid.

0 (mass ratio) 0.5 One 2 3 Ascorbic acid + +++ ++++ +++++ +++++ Citric acid + ++ +++ ++++ ++++ Malic acid + ++ +++ ++++ ++++ Boric acid + + ++ ++ +++ Note: Based on the mass ratio of sediment 1, normal temperature, maximum value: +++++

When only an organic acid such as ascorbic acid is added to dissolve, there is a disadvantage in that the added cost of ascorbic acid is high. Therefore, a parallel experiment was conducted by heating it. First, ascorbic acid was added to the precipitate and heated with shaking in a water bath. As a result, when the temperature is increased, the precipitate is dissolved even if less acid is added than the acid added for dissolving at room temperature. Especially, ascorbic acid is dissolved at least as much as 0.5 times of the mass of the precipitate and is heated to 50 ° C. This saves time and money compared to using organic acids such as corbinic acid.

In addition, caffeine separated by a halogenated organic solvent by a solvent-solvent extraction method has the advantage that it can be reused by vacuum concentration.

Example 5 Separation of High Purity EGCg in Different Eluent Volume Percentage Columns

An inner diameter of 10 cm, a length of 110 cm and a volume of 8.65 L were filled with 4.5 kg of synthetic filler HP-20 filler having a particle size of 0.32 mm or less. The packing was washed thoroughly with water and methanol, filled in a column, and stabilized with an aqueous solution added with 0.01% ascorbic acid as an initial eluent. Thereafter, the EGCg and ECg solution without caffeine was loaded into this column. 0.01% ascorbic acid was added as eluent to increase the stability of EGCg and ECg. When ascorbic acid is not added to the eluate, the final product containing more than 90% of high purity EGCg becomes reddish brown. Therefore, there is an advantage that can be used for various purposes such as cosmetics.

The first eluate eluted in chromatography is 100% DW with ascorbic acid added to remove caffeine that is not completely removed in halogenated organic solvents such as chloroform and to remove residual EGC. When 200 g of the precipitate was adsorbed on the column, the eluent was used as an aqueous solution containing 0.01% ascorbic acid, about 25 L, and flowed at a flow rate of 100 ml / min.

After removing caffeine and EGC, the remaining EC was removed using a 5% aqueous ethanol solution (0.01% ascorbic acid added). At this time, the eluent was flowed about 20 L at a flow rate of 20 ml / min.

After removing the remaining caffeine, EGC, EC with 5% aqueous ethanol solution (0.01% ascorbic acid), eluting with 15% ethanol (0.01% ascorbic acid) aqueous solution can separate more than 90% EGCg. At this time, 15% ethanol (0.01% ascorbic acid) aqueous solution was flowed at 20ml / min and was used about 18L. Over 70% of the total EGCg present in the initial precipitate was recovered, and the EGCg concentration in the eluate containing more than 90% high purity EGCg was 0.17%. The high purity EGCg chromatogram of 90% or more obtained as a result of eluting with 15% aqueous ethanol solution (0.01% ascorbic acid added) is shown in FIG. 6. In addition, high-purity EGCg was separated by column chromatography, followed by elution of 25% ethanol aqueous solution (0.01% ascorbic acid added) to high-purity ECg.

Eluate containing more than 90% high-purity EGCg was flowed using a column filled with combiHT XDB-C18 (150x21.2 mm) and Prep HPLC at a wavelength of 280 nm and 22% tetrahydrofurene (THF) at a flow rate of 10 ml / min. Primary 99% or more pure EGCg is isolated, or 90% or more EGCg is separated using 100% ethanol or 100% acetonitrile or ethanol-acetonitrile mixed organic solvent as a eluent in a column chromatography filled with Sephadex LH-20. It was.

The process described above is shown in FIG. 8.

Comparative Example 1

33.5 L (26 kg) of amberlite XAD-7 fill with a particle size of 0.3-1.2 mm or less was subjected to column chromatography of 15 cm inside diameter, 200 cm length and 35.4 L volume. This fill was thoroughly washed with water and water / isopropanol (9: 1 volume ratio), filled to the column and stabilized with the initial eluent of water / isopropanol (9: 1 volume ratio). Filled column chromatography was maintained at 60 ° C. using a thermostat. 0.4 kg of green tea extract containing 152.5 g pure EGCg was dissolved in 1.8 kg of water / isopropanol (9: 1 volume ratio) and eluted on the column. The flow rate was 50 kg / h flowed eluent. 144kg of the initial eluate was collected and 174kg of high purity EGCg was collected. At this time, the concentration of EGCg dissolved in the main eluate was 0.064%.

Comparative Example 2

33.5 L (26 kg) of amberlite XAD-7 fill with a particle size of 0.3-1.2 mm or less was subjected to column chromatography with an inner diameter of 15 cm, a length of 200 cm and a volume of 35.4 L. This fill was thoroughly washed with water and water / isopropanol (9: 1 volume ratio), filled to the column and stabilized with the initial eluent of water / isopropanol (9: 1 volume ratio). Filled column chromatography was maintained at 60 ° C. with a thermostat. 0.4 kg of green tea extract containing 140.5 g pure EGCg was dissolved in 1.8 kg of water / isopropanol (9: 1 volume ratio) and eluted on the column. The flow rate was 50 kg / h flowed eluent. 200kg of the initial eluate was collected and 117kg containing high purity EGCg was collected over 90%. At this time, the concentration of EGCg dissolved in the main eluate was 0.062%.

Comparative Example 3

450 ml of amberlite XAD-7 fill with a particle size of 0.3-1.2 mm or less was subjected to column chromatography having an inner diameter of 2.4 cm and a length of 100 cm. This fill was thoroughly washed with water and water / ethanol (9: 1 by volume), filled to the column and stabilized with water / ethanol (9: 1 by volume), the initial eluent. 20 g of green tea extract containing more than 95% polyphenol was dissolved in 20 ml. 14 g of solution containing 2.91 g pure EGCg eluted on the column. The flow rate was 16.9 mL / min flowed eluent [water / ethanol (9: 1 volume ratio)]. Filled column chromatography was maintained at 60 ° C. 2.48 L of the initial eluate was changed to 23.6 ml / min after flowing. 4.95L of the main eluate was collected, and the concentration of EGCg in the main eluate was 0.470g / L and the purity of EGCg was 86.1%.

Comparative Example 4

250 g of a polyamide 11 filler having a particle size of 5 to 40 microns was filled in column chromatography having an inner diameter of 5 cm and a length of 36 cm, and then maintained at 40 ° C. 3 g of green tea extract containing 21.11 g pure EGCg was dissolved in 153 ml of ethyl acetate. Eluents were ethyl acetate / ethanol gradient [500 ml ethyl acetate, 1000 ml ethyl acetate / ethanol (8.5: 1.5 by volume), 1000 ml ethyl acetate / ethanol (7: 3 by volume), 2000 ml ethyl acetate / ethanol (1: 1 by volume) )]. 550 ml of the main eluate was collected. At this time, the concentration of EGCg in the main eluate was 0.186% and the recovery of EGCg was 76%.

As described in the above Examples and Comparative Examples, the process of the present invention by adding caffeine to the green tea extract to selectively precipitate EGCg and ECg, by adding an organic acid to dissolve the precipitate by heating, using water or alcohol aqueous solution as eluent It is an excellent effect of commercially mass-producing high purity EGCg of more than 90% stabilized to near colorless color by column chromatography, which can be used not only as a natural antioxidant but also for skin beauty, and thus is a very useful invention in the cosmetic manufacturing industry. .

Claims (6)

In the method for separating EGCg from green tea extract, (1) dissolving green tea extract and caffeine in a mass ratio of 1: 1 to 0.01 (w: w), respectively, to prepare an aqueous solution; (2) After mixing the green tea extract and the aqueous solution of caffeine prepared in (1) and then bathed in a range of room temperature ~ 80 ℃ and left in a place not exposed to light at 0 ~ 10 ℃, 2 hours EGCg and green tea catechin component Selectively binding ECg and caffeine to precipitate and separate EGC and EC present in the supernatant; (3) After separating the precipitate from the supernatant, the precipitate was prepared in 5-15% (w / v) aqueous solution, and the organic acid was added to the precipitate aqueous solution in a mass ratio of 1: 0.1-3 (w: w), and then 20-20 Heating to 80 ° C. to dissolve the precipitate aqueous solution; (4) selectively removing caffeine from the aqueous solution of precipitate using an organic solvent; And (5) loading the precipitate aqueous solution without caffeine in a column filled with a synthetic adsorbent, and eluting a 0-20% alcohol aqueous solution eluent containing 0.0001-1% organic acid stepwise to separate a high purity EGCg aqueous solution. Characterized by high purity EGCg separation method. In the method of claim 1, (6) further comprising the step of separating the high-purity ECg by loading the separated high-purity EGCg aqueous solution into a column filled with a synthetic adsorbent and eluting an aqueous solution of 20% or more with 0.0001-1% organic acid added thereto using an eluent. High purity ECg separation method. The high purity EGCg separation method according to claim 1 or 2, wherein the organic acid of step (3), step (5) or step (6) is ascorbic acid. The high purity EGCg separation method according to claim 1 or 2, wherein the organic solvent of step (4) for eluting caffeine is a halogenated organic solvent. The high purity EGCg separation method according to claim 1 or 2, wherein the alcoholic solution eluate of step (5) is an aqueous ethanol solution. The high purity EGCg separation method according to claim 1 or 2, wherein the synthetic adsorbent of step (5) or step (6) is Sephadex LH-20 or HP-20.