KR100690928B1 - Method for separation and purification of epigallocatechin gallate - Google Patents

Abstract

The present invention relates to a method for high-volume separation and purification of epigallocatechin gallate (EGCG), which is known to be the most active substance from green tea extract.

Green Tea Extract, Epigallocatechin Gallate, DM 1020 Column, C-18 ODS Column

Description

Method for separation and purification of epigallocatechin gallate

In Figure 1 I is catechin, II is gallocatechin, III is epicatechin, IV is epicatechin gallate, V is epigallocatechin, and VI is epigallocatechin gallate.

2 shows the structure of DM 1020, where n is 5 on average.

3 shows an HPLC chart of purified epigallocatechin gallate.

The present invention relates to a method for high-volume separation and purification of epigallocatechin gallate (EGCG), which is known to be the most active substance from green tea extract.

Green tea, which has been drinking for about 5000 years, is recognized to improve blood circulation, remove toxic substances in the body, and increase resistance to various diseases. In recent years, many important researches have been conducted to scientifically prove the efficacy of green tea, and the research on the efficacy of the green tea has been continuously conducted.

In vitro and in vivo experiments reported that catechin is the most effective ingredient of green tea, and epigallocatechin gallate has the highest activity among the catechin components (A review of the latest research findings on the health promotion properties of tea, Christiane J. Dufresne, Edward R. Farnworth, J. Nutr. Bichem, 2001. 12, 404--421)

Six types of green tea catechins are known, which are catechin, gallocatechin (GC), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (epigallocatechin). : EGC), and epigallocatechin gallate, the structure of the compound is shown in FIG. 1.

Although the content of catechin in green tea generally varies depending on the cultivation environment and harvesting time of the tea growing area, the content and composition ratio of catechin are different, but on average, the catechin content is 10 ~ 15 of the total dry weight of tea leaves. %, And epigallocatechin gallate, which is considered to have the strongest activity, accounts for 50 to 60% of catechin components (chemical composition of Jiri wild green tea, Shin Mi-kyung, Korean Tea Culture Association, March-April 2000) , 30-33).

Various results have been reported for these green tea catechins, including antioxidants, anticancer, antibacterial, cholesterol lowering, cardiovascular disease suppression, obesity suppression, skin care, heavy metal removal and tooth decay and periodontitis. Among the catechins, epigallocatechin gallate Is considered to be the most active substance.

Epigallocatechin gallate has been reported to exhibit about 20 times more antioxidant activity than vitamin C, and has also been reported to be an excellent substance for cancer prevention. In particular, necrosis does not occur in normal cells, but it has been reported to cause apoptosis in cancer cells. Due to this selectivity of epigallocatechin gallate, many experiments have been conducted in humans for cancer prevention and treatment.

In addition, in order to obtain the efficacy of green tea, which appears only in long-term and high doses, in short-term doses, separation of the most active epigallocatechin gallate is required, and therefore, high purity of epigallocatechin gallate, which is economical and productive, is high. Separation and purification technology development is required.

Until now, a method of preparing high purity epigallocatechin gallate has been proposed using a porous polar filler, separation using HPLC (high-performance liquid chromatography), separation using caffeine, and separation using polyamide filler. .

Korean Patent Publication No. 2001-0050080 discloses macroporous polar resins, such as polyacrylates and polys such as AMBERLITE® XAD-7, available from Rohm and Haas. A method of separating and preparing epigallocatechin gallate by treating green tea extract on methacrylate and polyamide resin was presented. The process uses a thermostat that maintains the temperature of the column filled with filler in the process at 60 ° C. 0.4 kg containing 152.5 g of epigallocatechin gallate in 26 kg of Amberlite XAD-7 resin. After the green tea extract was loaded, about 318 kg of water / isopropanol (volume ratio 9: 1) was eluted to obtain epigallocatechin gallate. In the process of obtaining epigallocatechin gallate in the above method, a large amount of water / isopropanol (volume ratio 9: 1) is used, which leads to relatively low manufacturing efficiency, such as enlarging the enrichment equipment, and additional cost of maintaining the column temperature. There is a problem.

US Patent Publication No. US04613672 proposed an HPLC separation method using a C-18 reversed phase column using acetone / tetrahydrofuran / chloroform as eluent. Epigallocatechin gallate separation by HPLC with a column (250 × 4.60 mm) using -18 filler was presented.

These methods primarily cause problems such as safety in food manufacturing using solvents such as tetrahydrofuran. Also, the use of HPLC is a laboratory use, which requires a reassessment of the manufacturing cost and productivity for mass production. have.

U.S. Patent Publication No. US06210679 is a method of separating more than 95% of caffeine-epigallocatechin gallate, which is a four-step process, using a column filled with polyamide or C-18 filler for each process, and the manufacturing process is rather complicated. there is a problem.

In Korean Patent Publication No. 2003-0046122, caffeine is added to green tea extract to selectively separate epigallocatechin gallate and epicatechin gallate, and then caffeine is removed using an organic halogen solvent such as chloroform, and Sephadex HP. A method of obtaining epigallocatechin gallate with a purity of 90% or more by performing column chromatography using a synthetic adsorbent such as -20 has been proposed. In the above method, a large amount of caffeine is artificially added first, and an organic halogen solvent is used to remove the added caffeine again, which is a problem in evaluating safety in the manufacture of food and the like.

Therefore, there is a need for a method of isolating epigallocatechin gallate that is safe for the human body and economical and productive.

The present invention has been made in order to solve the conventional problems as described above, an object of the present invention is to provide a method for mass separation and purification of epigallocatechin gallate known as the most active substance from green tea extracts economically and high purity To provide.

One aspect of the invention is a method for the separation and purification of epigallocatechin gallate from green tea extract comprising the following steps:

 1) A green tea extract prepared by heating and extracting green tea leaves with water or ethanol into powder form, using a column using a filler having a size of 100 μm as a filling medium as a filler in which dichloromethyl silane is bonded to silica gel, and using 0.1 to 5.0 v Separation and purification of epigallocatechin gallate using an aqueous solution of / v% acetic acid or an aqueous solution of 1.0-20.0v / v% ethanol as eluent,

2) extracting the fraction obtained by concentrating the eluate containing epigallocatechin gallate in the eluate separated and eluted in step 1) with ethyl acetate, and

3) Recrystallizing the concentrate obtained by re-concentrating the ethyl acetate solution separated in step 2) with 0.1 ~ 5.0v / v% acetic acid aqueous solution.

In addition, the method for separating and purifying epigallocatechin gallate of the present invention further comprises a C-18 ODS column using a filler having a size of 100 μm as a filling medium between the steps 1) and 2). It may include the step of using.

Otherwise, the method of the present invention, C, using a filler having a size of 100 μm as a filling medium as a filler in which dichloromethyl silane is bonded to the silica gel, and a filler having a size of 100 μm as a filler as a filler medium Epigallocatechin gallate can be separated and purified further by connecting an -18 ODS column in series.

On the other hand, the eluent is preferably 0.5 ~ 1.0v / v% aqueous acetic acid solution or 5.0 ~ 10.0v / v% ethanol aqueous solution.

In addition, a column in which a filler having a size of 100 μm as a filling medium as a filler in which dichloromethyl silane is bonded to the silica gel is preferably, for example, a DM 1020 column (100 μm).

For example, by the following steps, a high amount of at least 90% high purity epigallocatechin gallate can be obtained in a short time:

1) A green tea extract containing 52% epigallocatechin gallate was dissolved in water or an aqueous solution of 0.1-2.0v / v% acetic acid, and the solution was a filler in which dichloromethyl silane was bonded to silica gel and its size was 100 μm. Phosphorus filler DM 1020 (Shiseido Fine Chemicals, Japan) was loaded onto a column using a filling medium (hereinafter referred to as a DM 1020 column), and 0.1-2.0v / v% acetic acid aqueous solution or 1.0-10.0v. eluting with an aqueous solution of / v% ethanol to separate the non-polar substances contained in the green tea extract,

2) The epigallocatechin gallate and epicatechin gallate fractionated in the DM 1020 column of step 1) are loaded on a C-18 ODS column, and 0.1 to 2.0 v / v% aqueous acetic acid solution or 1.0 to 10.0 v / v%. Eluting with an ethanol aqueous solution to separate epigallocatechin gallate and epicatechin gallate,

3) extracting the epigallocatechin gallate fractionated in step 2) with ethyl acetate, and concentrating and recrystallizing the concentrate obtained by dissolving in 0.1-2.0 v / v% acetic acid aqueous solution.

The production of green tea extract used as a raw material is generalized and can be easily obtained as a commodity (Huzhou Chengma Biological and chemical co. Ltd., China). The composition of this raw material is shown in Table 1.

Item standard result Catechin (HPLC) At least 80% 82.16% Epigallocatechin gallate (HPLC) 50% minimum 52.21% Gallocatechin 7.98% Epicatechin 0.17% Epicatechin gallate 15.71% Epigallocatechin 2.20% (+) (-) Catechin (DL-C) 1.89%

The catechin content of the extract was 82%, and epigallocatechin gallate and epicatechin gallate content by HPLC were 52% and 16%, respectively. As can be seen from the catechin component ratio of the extract, in the purification and separation of epigallocatechin gallate, epicatechin gallate has a similar structure to epigallocatechin gallate and contains the second largest amount after epigallocatechin gallate. Separation from is important. In addition, the green tea extract used as a raw material contains many substances which are more non-polar than epigallocatechin gallate, including caffeine, which was not removed during manufacture, and they are easily formed by general filtration or centrifugation because they form fine particles. It is insoluble and inseparable.

The inventors have found that DM 1020 medium is an inverse phase filler which is effective in removing non-polar green tea components and separating epigallocatechin gallate and epicatechin gallate. DM 1020 is a medium in which dichlorodimethyl silane is bonded to silica gel, and the structure thereof is shown in FIG. 2.

In addition, DM 1020 filler is 1/5 cheaper than C-18 filler, so it can be applied to the separation of epigallocatechin gallate by using DM 1020 column alone or connecting DM 1020 column and C-18 ODS column in series. This can compensate for the disadvantages of C-18 ODS columns in terms of economics and productivity.

In the weight of the filler and green tea extract, when using the DM 1020 column alone, for example, a filler of 15 to 20 times the weight of the green tea extract to be separated can be used, and the DM 1020 column and the C-18 ODS column are serialized. When used in conjunction with, for example, DM 1020 filler may be used in the same amount relative to the weight of the green tea extract, C-18 filler may be used 4 to 5 times the weight of the green tea extract. Regeneration of the column can be reused, for example after eluting 70% methanol, 50% ethanol aqueous solution or 50% isopropanol.

In the present invention, it is possible to maintain the stability of epigallocatechin gallate by adding a small amount of acetic acid in order to prevent the decomposition of epigallocatechin gallate in water easily.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples illustrate the present invention in detail, and the scope of the present invention is not limited by the following examples.

Experimental Materials and Methods

DM 1020 (100 μm) was purchased from Shiseido Fine Chemicals, Japan, and C-18 ODS (100 μm) was purchased from Timely.co., LTD., Japan. Green tea extracts were obtained from Huzhou Chengma Biological, China. and chemical co. ltd.).

Epigallocatechin gallate was identified during epigallocatechin gallate separation by TLC and HPLC.

TLC analysis conditions

TLC plate: Silica gel 60 F254 (Merck, USA)

Developing solvent: hexane / ethyl acetate (v / v = 1 / 3.5)

HPLC analysis conditions

HPLC solvent: 0.4% phosphoric acid / 27% aqueous methanol solution

Speed: 1.0 ml / min

Detector: UV (284 nm)

Column: 0.45 μm × 15 cm, C18 column (capcelpak: Shiseido Fine Chemicals, Japan)

Example 1

50 g of green tea extract containing 26 g of epigallocatechin gallate was dissolved in 50 ml of water and then loaded onto a DM 1020 column filled with 1 kg of DM 1020. The aqueous solution of 1.0v / v% acetic acid was eluted at 2.5 L at a rate of 100 mL / min, and the eluate was concentrated to 50 mL.

The concentrate was extracted with ethyl acetate (100 mL × 2), and the concentrate obtained by removing the ethyl acetate under reduced pressure was dissolved in 70 mL of 1.0v / v% acetic acid aqueous solution and recrystallized twice. 15.3 g of white epigallocatechin gallate was obtained. HPLC analysis showed that epigallocatechin gallate was 94% and moisture was 3.5%.

Example 2

1 kg of green tea extract containing 520 g of epigallocatechin gallate was dissolved in 3 l of 1.0v / v% acetic acid aqueous solution, loaded on a DM 1020 column (100 μm, 3 kg), and then at a flow rate of 100 ml / min. A 1.0 v / v% acetic acid aqueous solution was poured, and 10 L was eluted and concentrated to obtain a concentrate of 50 brix.

The concentrate was loaded on a C-18 ODS column (100 mesh, 5 kg), and then 40 L was eluted with 1.0 v / v% acetic acid solution at a flow rate of 800 ml / min. The eluate was concentrated to 1 L, extracted with ethyl acetate (2 L × 3), the ethyl acetate fractions were concentrated and recrystallized with 1.0v / v% acetic acid aqueous solution, 250 g of primary, 87.5 g of secondary Obtained (337.5 g, 65%). HPLC analysis showed that epigallocatechin gallate was 93% and moisture 3.3%. The column was eluted with 10 L of 50% aqueous ethanol solution and then reused.

Example 3

It carried out in the same manner as in Example 2. 1 kg of green tea extract containing 520 g of epigallocatechin gallate was dissolved in 3 l of an aqueous 0.5v / v% acetic acid solution and loaded on a DM 1020 column, followed by 5v / v% aqueous ethanol solution at a flow rate of 100 ml / min. Eluate and concentrate to 50 brix.

The concentrate was loaded on a C-18 ODS column and eluted with 1.0 l / v% acetic acid solution at a flow rate of 800 ml / min, followed by 20 l with 5 v / v% ethanol aqueous solution. The eluate was concentrated to 1 L, extracted with ethyl acetate, concentrated, and recrystallized with 1.0% acetic acid aqueous solution to obtain 389 g of a white solid. HPLC analysis showed that Epigallocatechin gallate was 92% and moisture was 4.4%.

Example 4

Further, a C-18 ODS column was connected in series to the DM 1020 column, 1 kg of green tea extract containing 520 g of epigallocatechin gallate was dissolved in 2.0 L of 1.0v / v% acetic acid solution, and then connected in series. Loaded on the column. After the loading was completed, 1.0 v / v% acetic acid aqueous solution was eluted at 800 ml / min. After dissolving 10 L, the DM 1020 column was separated from the C-18 ODS column, and 40 C was further eluted by flowing the C-18 ODS column at 800 mL / min with an aqueous 1.0v / v% acetic acid solution.

The combined eluates were concentrated to 2 L, extracted with ethyl acetate, and recrystallized with 1.0 v / v% acetic acid aqueous solution to obtain 357 g of a white solid. HPLC analysis showed that epigallocatechin gallate was 92% and moisture 4.5%.

Comparative Example 1

33.5 L (26 kg) of Amberlite XAD-7 resin having a particle size of 0.3 to 1.2 mm was charged into a column having a diameter of 150 mm and a length of 2 m. The column was equipped with a heating jacket and the packed column was kept at 60 ° C. 0.4 kg of green tea extract (152.5 g as epigallocatechin gallate) was dissolved in 1.8 kg of a mixture of water / isopropanol (volume 9: 1) and introduced to the top of the column. Epigallocatechin gallate was eluted by pumping a mixture of water / isopropanol (volume ratio 9: 1) at a pressure of 0.5 bar and a flow rate of 50 kg / h at a temperature of 60 ° C.

After an initial elution of 144 kg, 174 kg of eluate containing 112 g of epigallocatechin gallate was recovered. The epigallocatechin gallate concentration in the main eluate was 0.064%. The yield of isolated epigallocatechin gallate was 73.5%. The remaining catechins for resin regeneration were eluted with 78.3 kg of a mixture of water / isopropanol (volume ratio 4: 6).

Comparative Example 2

250 g of polyamide 11 filler having a particle size of 4 to 5 microns was suspended in 300 ml of ethyl acetate and transferred into a column 5 cm in diameter and 36 cm in length. The column was set up with a heating jacket and kept at 40 ° C. 3 g green tea extract (1.11 g as epigallocatechin gallate) was dissolved in 153 mg of ethyl acetate and introduced at the top of the column. 500 ml of ethyl acetate under pressure of 0.3 bar, 1000 ml of ethyl acetate / ethanol (volume ratio 8.5 / 1.5) 1000 ml of ethyl acetate / ethanol (volume ratio 7: 3), 2000 ml of ethyl acetate / ethanol (volume ratio 1: 1) Eluting) concentrated 550 ml of the main fraction to give 1.12 g of a solid containing 0.87 g of epigallocatechin gallate. Epigallocatechin gallate concentration in the main eluate was 0.186%. The yield of isolated epigallocatechin gallate was 76%.

Comparative Example 3

450 ml of Amberlite XAD-7 filler having a particle size of 0.3-1.2 mm or less was charged into a column having an internal diameter of 2.4 cm and a length of 100 cm, washed with water / ethanol (volume ratio 9: 1), and the initial eluent of water / Stabilized with ethanol (volume 9: 1). 20 g of green tea extract (2.91 g as epigallocatechin gallate) was kept at 60 ° C. and the eluent was flowed. After flowing 2.48 L of the initial eluent, 4.95 L of the eluent was obtained. Epigallocatechin gallate had a concentration of 0.47 g / l and epigallocatechin gallate had a purity of 86.1%.

When epigallocatechin gallate is separated by a conventional method, problems such as the attachment of a heating device to the facility and the enlarging of the concentration facility due to the use of a large amount of solvents are a problem. When separating and refining the rate, epigallocatechin gallate having a high purity of 90% or more can be economically and practically separated in bulk, and can be used as a raw material for cosmetics, food, and medicine, and the present invention is a very useful invention. .

According to the present invention, high purity epigallocatechin gallate can be separated from green tea extract economically and with high productivity.

In addition, the process of the present invention is loaded with green tea extract on a column using a filler (DM 1020) in which dichloromethylsilane is bonded to silica gel, and using an acetic acid aqueous solution as an eluent, high purity epigallocatechin gallate of 90% or more It is a process for producing large-scale separated production, and it is a method for preparing separated tablets of epigallocatechin gallate which has a safety which can be used for food production such as acetic acid, ethanol, ethyl acetate and the like.

Claims (4)

Separation and purification of epigallocatechin gallate from green tea extract comprising the following steps: 1) A green tea extract made by heating and extracting green tea leaves with water or ethanol into powder form, using a column using a filler having a size of 100 μm as a filling medium as a filler in which dichloromethyl silane is bonded to silica gel, and using 0.1 to 5.0 v Separation and purification of epigallocatechin gallate using an aqueous solution of / v% acetic acid or 1.0 to 20.0 v / v% ethanol as eluent, 2) extracting the fraction obtained by concentrating the eluate containing epigallocatechin gallate in the eluate separated and eluted in step 1) with ethyl acetate, and 3) Recrystallizing the concentrate obtained by re-concentrating the ethyl acetate solution separated in step 2) with 0.1 ~ 5.0v / v% acetic acid aqueous solution. The method of claim 1, further comprising the step of using a C-18 ODS column using a filler having a size of 100㎛ as a filler medium between the steps 1) and 2), the epigallocatechin gallate How to separate and purify. The method for separating and purifying epigallocatechin gallate according to claim 1, wherein an additional C-18 ODS column using a filler having a size of 100 µm as a filler is connected in series to the column. delete

Images