KR100859579B1 - Method for Extracting Catechin from Green Tea - Google Patents

Abstract

The present invention discloses a method for separating catechins from green tea. Specifically, (a) immersing green tea in a neutral solvent of 80 ° C. or higher, (b) maintaining the extraction time for at least 60 minutes, and (c) separating the catechin from the green tea extract obtained after step (b). Disclosed is a method for separating catechin from green tea.

Catechin, Green Tea

Description

Method for Extracting Catechin from Green Tea

1 is a graph showing the total catechin content of green tea extract by pH when the green tea is extracted with hot water.

Figure 2 is a graph showing the total catechin content of the green tea extract according to temperature when the green tea is extracted with hot water.

Figure 3 is a hot water extract picture of green tea by temperature (A is a green tea extract at 50 ℃ extraction temperature, B is a green tea extract picture at 80 ℃ extraction temperature and C is a green tea extract picture at 100 ℃ extraction temperature ).

FIG. 4 is a graph of HPLC analysis results of green tea extract obtained at an extraction temperature of 100 ° C. (parts indicated by circles represent impurities).

Figure 5 is a graph showing the total catechin content of the green tea extract over time when the green tea is extracted with hot water.

6 is a graph showing the catechin content of each solvent when the green tea is extracted with hot water and then the green tea extract is dispensed with a solvent.

The present invention relates to a method for separating catechin from green tea.

Green tea, which has been drinking for thousands of years, contains many functional substances such as caffeine, catechin, theanine, and amino acids. , Functional ingredients and physiological activity of tea, Journal of the Korean Magnetics Society, 1 (1), 175-191 (1995)).

Recent studies show that catechin is the most important ingredient in green tea's physiological activity (A review of the latest research findings on the healthpromotion properties of tea, Christiane J. Dufresne, Edward R. Farnworth , J. Nutr.Bichem, Dec. 2001, 404-421)

There are six known catechin components of green tea, including catechin, gallocatechin (GC), epicatechin (EC), epicatechin galate (ECG), epigallocatechin (EGC), And epigallocatechingalate (EGCG).

Although the content of catechin in green tea generally varies according to the cultivation environment and harvesting time of the tea growing region, and the harvesting time, the catechin content is, on average, 10 to 15 of the total dry weight of tea leaves. %, And epigallocatechin gallate, which is said to be the most active, accounts for 50 to 60% of catechin components (chemical composition of wild green tea of Jiri, Shin Mi-kyung, Korean Tea Culture Association, March-April 2000, 30-33).

Therefore, the technology for separating functional substances from green tea to date can be roughly classified into a technique for separating catechin from green tea and a technique for separating epigallocatechin gallate known to have the highest physiological activity among the catechins.

Korean Unexamined Patent Publication No. 2000-0046079 entitled "Production Method of Green Tea Extract with High Polyphenol Content" and Korean New Patent Publication No. 2001- titled "Novel Extraction Method of Catechins and Drinks Using the Same". 0035410 and US Pat. No. 4248789, titled Process for producing catechins, relates to a technique for separating total catechins from green tea, and the invention is titled "Isolation of Epigallocatechin gallate." Republic of Korea Patent Publication No. 2006-0058356 "Method of manufacturing epigallocatechin gallate" Republic of Korea Patent Publication No. 2001-0050080, Republic of Korea Patent of the invention "high purity EGCG separation method derived from green tea extract" No. 418605 and the like describe a method for separating epigallocatechin gallate, which is known to have the highest physiological activity among catechins.

The present invention discloses a technique for separating catechins from green tea.

It is an object of the present invention to provide a method for separating catechins which can separate catechin from green tea in higher yield.

Other objects, specific embodiments, and the like of the present invention will be presented below.

In order to achieve the above object, the present inventor first tested whether the content of catechin is changed according to pH, temperature, extraction time and dispensing solvent in catechin separation from green tea.

Specifically, the inventors adjusted the pH of the extraction solvent to 4.0, 5.5, 7.0 and 10.0 as can be seen in the following examples, the extraction temperature is 50 ℃, 80 ℃ and 100 ℃, and the extraction time is 30 minutes, When separation yields of catechin from green tea were observed at 60, 90, 120, 150, 180, 240 and 300 minutes, the pH was 7.0, the extraction temperature was 80 ° C, and the extraction time was 60 minutes. As described above, it was confirmed that the highest catechin separation efficiency was obtained with the least impurity (However, the extraction temperature showed maximum catechin separation efficiency at 100 ° C (1.4% higher than the extraction temperature at 80 ° C). When the extraction temperature was set at 100 ° C, it was found that a lot of impurities were mixed in the green tea extract, which was inappropriate for the optimum temperature).

In addition, the inventors obtained the green tea extract according to the above optimum conditions, and when it was mixed with the same volume of chloroform, stirred and fixed, and distributed, and the green tea extract was mixed with the same volume of chloroform, stirred and fixed, and then distributed. The extraction solvent layer (i.e. water layer) was again mixed with the same volume of chloroform, stirred and placed, and distributed, and finally the extraction solvent layer (i.e. water layer) obtained by dividing twice with chloroform was mixed with the same volume of ethyl acetate. As a result of analyzing the content of catechin at the time of dispensing, it was confirmed that the catechin separation efficiency was maximized (about 80%) while impurities such as caffeine were removed.

The present invention is based on the experimental results as described above, in one aspect, the present invention provides a method for separating catechin from green tea.

The method for separating catechin from green tea of the present invention comprises (a) immersing green tea in a neutral solvent of 80 ° C. or higher, (b) maintaining the extraction time for at least 60 minutes, and (c) green tea obtained after step (b). It characterized in that it comprises a step of separating the catechin from the extract.

The catechin extraction method from the green tea of the present invention has the above-described constitution so that the catechin can be separated with high yield while containing little impurities.

In the present specification, "green tea" is meant to include commercially available powdered green tea, tea bag green tea, leaf green tea, harvested and dried green tea leaves.

In addition, in the present specification, "neutral solvent" means an extraction solvent having a pH of 7.0, including, for example, water and distilled water, as long as pH 7.0, alcohol (preferably lower alcohol having 5 or less carbon atoms), ethyl acetate, acetone, It is meant to include a solvent whose pH is adjusted to 7.0 as a solvent in which two or more of chloroform and methylene chloride are mixed.

In the present specification, "catechin" refers to catechin (catechin), gallocatechin (GC), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin, which are known to date as catechin components of green tea. (epigallocatechin: EGC), and epigallocatechingalate (EGCG).

On the other hand, in the separation method of catechin from the green tea of the present invention, the step (a) is as described above and as confirmed in the following examples, the extraction yield increases as the temperature of the extraction solvent from 80 ℃ or higher to higher temperature However, since impurities increase, it is preferable to set the temperature of the extraction solvent to 80 ° C.

In addition, in the method for separating catechin from green tea of the present invention, the step (b) has a large change in extraction yield up to 300 minutes when the extraction time is 60 minutes or more as described above and as confirmed in the following examples. It can be seen that (from 60 minutes to 300 minutes total catechin content is 3.0% to 3.3%; see Fig. 5), considering the thermal efficiency, 60 to 90 minutes is appropriate, most preferably extraction This is the case with 60 minutes.

Also in the method for separating catechin from green tea of the present invention, the step of separating the catechin from the green tea extract of step (c) can be separated using any method known in the art, for example, silica gel, cepha as a filler. It can be performed using column chromatography packed with dex, RP-18, polyamide, toyopearl, XAD and the like.

In a preferred embodiment, the method of separating catechin from green tea of the present invention preferably further comprises the step of properly dispensing with a dispensing solvent after step (b) to increase the separation yield of catechin, specifically, dispensing solvent. The step of distributing the mixture was carried out by filtering the green tea extract obtained after step (b) with a filter paper to remove the extraction residue, and then mixing the filtrate with an equal volume of chloroform and stirring and distributing the mixture into a chloroform layer and an extraction solvent layer. The partitioned extraction solvent layer was again mixed with the same volume of chloroform, stirred and fixed, and then secondly partitioned into the chloroform layer and the extraction solvent layer, and then the extracted solvent layer was mixed with the same volume of ethyl acetate and stirred and fixed. It comprises the step of obtaining an ethyl acetate layer containing the catechin component.

When the method for separating catechin from green tea of the present invention includes dispensing with the dispensing solvent, the catechin can be separated at a higher yield, wherein the separation of catechin is an ethyl acetate layer containing the catechin component. And can be accomplished using methods known in the art such as column chromatography as described above.

In another aspect, the present invention relates to a method for producing green tea extract having a high catechin content.

The method for producing a green tea extract having a high catechin content of the present invention is obtained by (a) immersing green tea in a neutral solvent of 80 ° C. or higher, (b) maintaining the extraction time for at least 60 minutes, (c) (b) It characterized in that it comprises a step of separating the catechin from the green tea extract.

In the method for producing green tea extract having a high catechin content of the present invention, the meaning of "green tea", "neutral solvent" and "catechin", and the preferred embodiment, the above-described method for separating catechin from green tea of the present invention The statement is valid as it is.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

EXAMPLES Establishment of Optimum Catechin Extraction from Green Tea

Example 1 Establishment of Optimum pH for Catechin Extraction from Green Tea

100 ml of pure water is added to 3.0 g of tea bag green tea sold in Jeju Dawon, Seogwipo-si, Jeju Island, and a small amount of HCl or NaOH is added to adjust the pH of the extraction solvent to pH 4.0, pH 5.5, pH 7.0 and pH 10.0. It was. Extract the extraction temperature for 1 hour at 80 ℃ to obtain a green tea extract while stirring in a stirrer, using a filter paper (Fifter papers whatman, 90mm Dia, 2.5 μm, Whatman international Ltd.) to remove the extraction residue, filtered under reduced pressure and concentrated Green tea extract was obtained.

The total catechin content of the green tea extract was measured according to the catechin quantitative analysis described in the food additive revolution. Specifically, 3 ml of phosphate buffer solution (pH 7.5) and 1 ml of iron stannate solution were added to 1 ml of the diluted green tea extract diluted with 1/10 by adding water to the green tea extract, which was used as a test solution. Water was used as a reference solution at a wavelength of 540 nm. Absorbance was measured. In this case, prepare 5 ml, 10, 15, 20, and 25 mg of ethyl acetate (standard foam) per 100 ml of water separately as standard solution, and use 5 ml of this standard solution in the same manner as the test solution. After absorbance was measured at a wavelength of 540 nm, a standard curve was obtained. Total catechin content in 1 ml of the test solution was determined from the absorbance and standard curve of the test solution.

Total catechin content (%) = C X 1.5 X 100 / sample taken (mg) X (100-W) X 100

The total catechin content of green tea by pH is shown graphically in <Figure 1>. As shown in FIG. 1, 8.0% of catechins were obtained at pH 4.0, and 8.4%, 8.7%, and 6.3% of catechins at pH 5.5, 7.0, and 10.0, respectively.

The above results show that pH 7.0 is the optimal pH for catechin extraction from green tea.

Example 2 Establishment of Optimal Temperature for Catechin Extraction from Green Tea

Put 100ml of pure water into 3.0g of tea bag green tea sold in Jeju Dawon (Seogwi-dong, Seogwipo-si, Jeju-do), extract time was 1 hour, and the extraction temperature was set at 50 ℃, 80 ℃ and 100 ℃, The extract was removed using filter paper (Fifter papers whatman, 90mm Dia, 2.5 μm, Whatman international Ltd.), filtered under reduced pressure, and concentrated to obtain green tea extract.

The catechin content of the green tea extract thus obtained was measured in the same manner as in <Example 1>.

The results are shown graphically in Figure 2, while the catechin content at the extraction temperature of 50 ℃ is very low as about 5%, while the catechin content of 15.8% and 17.4% at the extraction temperature of 80 ℃ and 100 ℃, respectively, extraction temperature 50 It was higher than ℃. By the way, the green tea extract at the extraction temperature of 100 ℃ is foamed as shown in Figure 3 (A is the extract at the extraction temperature 50 ℃, B is the extract at the extraction temperature 80 ℃ and C is the extract at the extraction temperature 100 ℃) This occurred a lot and opaque and analyzed by high performance liquid chromatography (HPLC), as shown in Figure 4 it was confirmed that a lot of impurities (parts shown as circles) are mixed. On the other hand, in the case of the extraction temperature 80 ℃ as shown in Figure 3 it can be seen that the extract is transparent and almost no impurities.

In conclusion, the optimum temperature for catechin extraction was determined to be 80 ° C.

Example 3 Establishment of Optimal Time for Catechin Extraction from Green Tea

The optimal time of catechin extraction was confirmed by applying the optimum pH and optimum temperature identified above. First, 100ml of pure water is added to 3.0g of tea bag green tea sold in Jeju Dawon (Seogwi-dong, Seogwipo-si, Jeju-do), and the extraction temperature is 80 ℃ at pH 7.0 and the extraction time is 0, 30, 60, 90, 120, 150, 180 , 240, 300 minutes to obtain a green tea extract while stirring in a stirrer and filter paper (Fifter papers whatman, 90mm Dia, 2.5 μm, Whatman international Ltd.) to remove the extraction residue, filtered under reduced pressure and concentrated to obtain a green tea extract.

The catechin content of the green tea extract thus obtained was measured in the same manner as in <Example 1>.

5 shows that the catechin content of green tea extract rapidly increases to 60 minutes, but after 60 minutes, it slowly increases or hardly increases (the catechin content is about 60 minutes). 3.0% to 3.3%). Based on these results, the optimal extraction time was determined to be 60 ~ 90 minutes in consideration of extraction efficiency and thermal efficiency.

Example 4 Establishment of Optimal Distribution Method for Catechin Extraction from Green Tea

Green tea extract was obtained by applying the optimum pH, optimum temperature, and optimal time identified above, and the optimal distribution method of catechin extraction was confirmed.

First, 100 ml of pure water is added to 3.0 g of tea bag green tea sold in Jeju Dawon (Seogwi-dong, Seogwipo-si, Jeju Island), and the extraction temperature is 80 ° C and the extraction time is 60 minutes at pH 7.0. Extraction residue was removed using (Fifter papers whatman, 90 mm Dia, 2.5 μm, Whatman international Ltd.).

The filtered green tea extract was mixed with the same volume of chloroform and stirred for 30 minutes in a stirrer to separate layers (primary chloroform distribution). Here, the separated water layer was mixed with the same volume of chloroform again to perform layer separation (secondary chloroform distribution), and the same volume of ethyl acetate was again mixed with the obtained water layer to perform layer separation. The reason why two distributions have been attempted with chloroform is to remove impurities such as caffeine.

After each distribution above, the catechin content of each layer was measured. The catechin content was measured in the same manner as in <Example 1>.

As shown in FIG. 6, the catechin content of the filtered green tea extract was 31% (confirmed), 33% in the primary and secondary chloroform layers, and 80% in the ethyl acetate layer. .

This implies that the green tea extract is twice distributed with chloroform and then distributed with ethyl acetate.

As described above, according to the present invention, a method for separating catechin from green tea can be provided.

The method for separating catechin from green tea of the present invention can separate catechins having various physiological activities in high yield, and using column chromatography known to those skilled in the art with catechins separated according to the present invention or HPLC, etc. By using it can be further separated into each catechin component.

Claims (6)

(a) immersing green tea in water or distilled water at 80 ° C., (b) extracting for 60 minutes, (c) filtering the obtained green tea extract with filter paper to remove the extraction residue, and then filtering the filtrate to Mixing with chloroform and stirring to fix the chloroform layer and extracting solvent layer, and (d) dividing the extracted extracting solvent layer with the same volume of chloroform and stirring and fixing to make the chloroform layer and the extraction solvent layer secondary. (E) mixing the extractant layer with an equal volume of ethyl acetate, stirring and standing to obtain an ethyl acetate layer containing the catechin component, and (f) separating the catechin from the ethyl acetate layer. Method for separating catechin from green tea comprising a. delete delete The method of claim 1, Separating the catechin from the green tea extract of step (f) is a column chromatography filled with a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, toyopearl and XAD as a filler Isolation method of catechin, characterized in that carried out using. delete delete

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