JPH04182479A - Production of tea catechins - Google Patents

Abstract

PURPOSE:To obtain high-purity tea catechins at a low cost by extracting a water soluble ingredient from tea leaf, passing the ingredient through a chromatographic column in which a synthetic absorbent is packed and then eluting the adsorbed ingredient and dissolving the eluted ingredient in other solvent and distilling the organic solvent. CONSTITUTION:For example, tea leaf is subjected to extraction treatment using a hot water heated to >=80 deg.C to provide a water soluble ingredient. Then the ingredient is passed through a chromatographic column in which an absorbent composed mainly of styrene, divinyllvenzene, etc., or gel filtrating agent composed mainly of dextran derivative, etc., is packed to make the ingredient adsorbed into the chromatographic column. Then the adsorbed ingredient is eluted by 40-100% aqueous solution consisting of one kind of methanol, ethanol or acetone or mixture thereof and the eluted liquid is dissolved in one kind of ethyl acetate, methylisobutly ketone or diethyl ether or mixture thereof and the organic solvent is distilled to provide the objective tea catechins (consisting of epicatechin, epigallocatechin and epicatechingallate).

Description

[Detailed Description of the Invention] [Industrial Field of Application] The present invention relates to a method for producing tea catechins, and in particular to a method for producing tea catechins using tea leaves as a raw material, which can produce highly purified tea catechins at low cost. This relates to a manufacturing method.

[Prior Art] Catechins, which are abundantly contained in tea leaves, are polyphenol compounds and are a type of flavanol. The main catechins contained in tea leaves are epicatechin, epigallocatechin,
and epicatechin gallate, which is a gallic acid ester thereof, and) 4 of epigallocatechin gallate.
There are seeds. It is known that the tea catechins of these plants have antioxidant, antibacterial, and other powerful physiologically active effects.

In order to produce these tea catechins with high purity, it is necessary to remove the coexisting caffeine, brown pigment, amino acids, sugars, steroids,
Separation of impurities such as catechin oxide is essential.

Conventionally, a method using a liquid-liquid extraction method is known as a technique for recovering tea catechins with high purity from tea leaves. In this method, tea catechins extracted from tea leaves using warm water or hot water are transferred and dissolved in another organic solvent.

For example, auspicious days (Tea Industry Research Report No. 13, p. 4, 195)
(April 9) was used as an extraction operation for a polyphenol mixture.
This article introduces a method of extracting green tea with water, washing with chloroform, and then extracting with ethyl acetate.

Further, in Japanese Patent Publication No. 1-44234, a natural antioxidant is produced by a similar method.

Furthermore, in JP-A No. 64-9922, a tea extract is washed with hexane and chloroform, and then the desired product is extracted with ethyl acetate.

These methods are based on the fact that tea catechins are wood-loving and
Unlike W and free amino acids, tea catechins are being selectively extracted and recovered using both of these solvents, taking advantage of their strong affinity with hydrophobic organic solvents. It requires various organic solvents, and it takes a long time to obtain high-purity tea catechins.
There is a problem with production costs.

On the other hand, in a reference example disclosed in JP-A No. 63-135484, liquid-liquid extraction was performed on tea extract using water and soybean oil, and only fat-soluble components were dissolved in the soybean oil and removed. The aqueous phase is collected to obtain the desired tea catechins. This method has the problem that the purity of tea catechins is naturally low because these recovered components contain large amounts of sugars and amino acids together with tea catechins. We have developed an adsorption separation technology that recovers high-purity tea catechins by selectively adsorbing catechins and eluting them using an organic solvent (Japanese Patent Application Laid-open No. 1-175978, Japanese Patent Application No. 1-135463).
. According to these methods, tea extract is injected into a chromatographic column packed with a synthetic adsorbent, and impurities are first removed by sequential elution with varying concentrations of water and organic solvent, and the remaining tea catechins are highly concentrated. It is something that can be recovered in concentrated amounts.

However, subsequent research revealed that brown catechins produced by selective adsorption using fillers contain brown substances that cause coloration. It was estimated that it was an oxidized polymer of Furthermore, these brown substances have no antioxidant power or other physiologically active effects, and it has become necessary to remove these brown substances in order to further improve the purity of tea catechins.

[Problems to be Solved by the Invention] The present invention has been made in view of the above-mentioned problems. By removing the oxidized polymers from tea catechins using the above-mentioned adsorption separation method, high-purity products can be obtained. To provide a method for producing tea catechins that can produce tea catechins with high antioxidant capacity at low cost! ! The subject is

cam] That is, the first invention includes a first extraction step of extracting water-soluble components from tea leaves, and a chromatography column filled with a synthetic adsorbent or a gel filtration agent. Fill and
The adsorption process of adsorbing components onto this chromatography column and these adsorbed components.

An elution step in which elution is performed with an approximately 40% to 100% aqueous solution of one of methanol, ethanol, or acetone, or a mixture thereof; This is a method for producing tea catechins, which comprises a second extraction step of distilling off organic components after dissolving the tea catechins in the mixture.

Furthermore, the extraction step of dissolving into an organic solvent and the adsorption step of passing through a chromatography column can be reversed.

That is, the second invention consists of a first extraction step of extracting water-soluble components from tea leaves, and after dissolving this extract in one of ethyl acetate, methyl isobutyl ketone, diethyl ether, or a mixture thereof, organic A second extraction step in which the components are distilled off, an adsorption step in which the extracted components are packed into a chromatography column packed with a synthetic adsorbent or gel filtration agent, and the components are adsorbed on the chromatography column; Approximately 40% consisting of one of methanol, ethanol or acetone, or a mixture thereof
This is a method for producing tea catechins, which is characterized by having an elution step in which each of the tea catechins is eluted with a 100% to 100% aqueous solution.

The tea catechins described above are preferably a mixture whose main components are epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate.

In addition, the above synthetic adsorbents include styrene/divinylbenzene,
Alternatively, an adsorbent based on methacrylic acid ester is preferable.

Further, the gel filtration agent is preferably an adsorbent based on a dextran derivative or a hydrophilic vinyl polymer.

The present invention will be explained in more detail below.

FIG. 1 particularly shows the schematic steps of the method for producing tea catechins according to the first invention.

The present inventors have developed a selective method using a conventional filler in order to remove colored substances such as the brown substances mentioned above and further improve the purity of tea catechins compared to conventional methods to obtain a white recovered product. We considered a method that combines adsorption and liquid-liquid extraction.

That is, the tea that is the raw material of the present invention includes fresh leaves, unfermented tea,
Various forms of tea, such as semi-fermented tea and sencha, are used, and water-soluble components are obtained by bolting for about 5 to 10 minutes using boiling water of 80° C. or higher as a first extraction step. In addition, when using a water-containing organic solvent such as methanol, the extraction operation is generally performed at a low temperature of about 60° C. for a longer time.

After concentrating these water-soluble components or tea extract,
The mixture is injected into a chromatography column filled with an adsorbent, and the various components are adsorbed onto the adsorbent (adsorption step).

As the adsorbent, a synthetic adsorbent and a gel filtration agent can be employed. Synthetic adsorbents include common synthetic adsorption resins such as HP-IMG and HP-2MG (
Mitsubishi Kasei), XAD-7 and XAD-8 (organo)
There are methacrylic ester-based adsorbents such as methacrylic acid ester adsorbents, and styrene divinylbenzene-based adsorbents such as S-761 (Duolite). Gel filtration agents include resins suitable for gel filtration, such as adsorbents based on dextran derivatives or hydrophilic vinyl polymers, such as Sephadex LH-20 (Pharmacia) and Toyopearl HW-40 (Tosoh).

Note that sugars, metals, proteins, etc. are separated from the chromatography column as an aqueous phase by the above-described injection.

Next, the components remaining in the chromatographic column are treated with about 40% to 70% of a hydrophilic organic solvent such as methanol, ethanol, or acetone, or a mixture thereof.
Elute using an eluent with a concentration of 80% (elution step).

The above-mentioned hydrophilic organic solvents are suitable as solvents for elution, but
Concentrations of 40% to 80% are effective for eluting tea catechins. If it is less than 40%, elution will be delayed, and if it is less than 80%
% or more, the effect of increasing concentration cannot be obtained, and in either case it is uneconomical. Around 60% is desirable.

The solvent from which the tea catechins were eluted is once concentrated to remove the organic solvent, and then subjected to the next step (extraction step 12). Note that if the organic solvent is not removed, the extraction efficiency in the first step decreases, and the amount of solvent used in the next step increases, which is uneconomical.

The extracted components are then dissolved in an organic solvent from the liquid from which the solvent has been removed (second extraction step).

As for the organic solvent, it is desirable that the amount of solubility in water is small, that the boiling point is not so high due to the necessity of removing the solvent, and that the brown substance is not dissolved at all although the tea catechins are dissolved to some extent. Suitable solvents that satisfy these requirements include ethyl acetate, methyl isobutyl ketone, and diethyl ether, with ethyl acetate being particularly preferred.

On the other hand, ethers other than diethyl ether, such as isopropyl ether, benzene, chloroform, petroleum ether, and hexane, have low solubility for tea catechins, and alcohols such as n-butanol are unsuitable because they also dissolve brown substances. be.

In this second extraction step, the oxidized polymer of tea catechins is dissolved in the aqueous phase, and the tea catechins are transferred and dissolved in the organic solvent, thereby separating the two.

Generally, the organic solvent in which the tea catechins are dissolved is distilled off under reduced pressure, and then the tea catechins are pulverized by freeze-drying or spray drying, and this is processed into various product forms.

As mentioned above, by adopting a two-stage processing method that sequentially performs selective adsorption using a filler and liquid-liquid extraction,
It has become possible to produce highly pure tea catechins.

[Function] In the method for producing tea catechins according to the present invention, tea catechins are adsorbed and separated by treating the tea extract with column chromatography, and liquid-liquid extraction is carried out in the next step. It becomes possible to separate brown substances that were previously impossible to separate, and high-purity tea catechins can be produced at low cost without requiring a large amount of organic solvent.

[Example] Next, the present invention will be explained in detail.

(Example 1) Extract Ikg of Sencha with 10 liters of hot water for 10 minutes,
After squeezing, vacuum concentration and centrifugal separation were performed to produce 2 liters of tea extract.

The entire amount of this tea extract was injected into a chromatography column packed with approximately 700 ml of a synthetic adsorbent based on methacrylic acid ester (Diaion HP-2MG manufactured by Mitsubishi Kasei), and then eluted with 1.7 liters of 70% ethanol. .

This liquid is analyzed by high performance liquid chromatography.

The analysis conditions for high performance liquid chromatography are as follows. That is, Chromato column: Capsule pack Cl8 (Shiseido) 4.6 mm diameter x 250 mm length Eluent for elution: Eluent A: Methanol/water/phosphoric acid (22%/78% 10.1%) Eluent B: Methanol 100% Gradient: 0 to 15 minutes B solution 0% 15 to 35 minutes B solution O→20% 35 to 50 minutes B solution 20→100% (Initial elution with eluent A, and between sample injection v115 and 35 minutes, eluent 0 to 20%, and 20% during 35 to 55 minutes.
Flow rate: 1 ml/min Sample volume: 5 microliters Detector 2'M external absorption spectrophotometer Wavelength: 0-22 minutes UV 280 nm 22-50 minutes UV 350 nm .

The analysis results from the above high performance liquid chromatography are
As shown in the figure. As shown, tea catechins (1, 2, 4,
5) with a purity of 68%. The eluate containing this extract was concentrated under reduced pressure to remove ethanol and
Collected milliliters.

This liquid was treated three times with the same kind of ethyl acetate, and the ethyl acetate layers were combined, concentrated under reduced pressure, and then lyophilized according to a conventional method to obtain 80 g of a white solid.

When this powder was analyzed by high performance liquid chromatography,
As shown in FIG. 3, the peak derived from brown substances disappeared, and the purity of the tea catechins was about 85%.

(Application example) A small amount of each of the tea catechins after column fractionation obtained by the method of Example 1 and the tea catechins after it's ethyl purification is dissolved in ethanol and added to linoleic acid. The antioxidant capacity was investigated. The antioxidant capacity was determined by peroxide value (POV).

That is, 10 ml of each of the additive-free and 1100 pp added samples were placed in a 100 ml beaker, left in a constant temperature room at 40°C, and the peroxide value (pov) was measured by iodostarch reaction according to the usual method. .

FIG. 4 shows this antioxidant capacity curve. As shown in the figure, it can be seen that the antioxidant effect of the product is stronger than that of the column-fractionated product.

(Comparative Example 1) For comparison with the present invention, extraction was performed using a conventionally known method.

After washing 1.5 liters of the same tea extract as in Example 1 with the same amount of chloroform, it was treated with 1.5 liters of ethyl acetate three times (4.5 liters in total) to transfer the extract components.

After combining the ethyl acetate layers and distilling off the solvent under reduced pressure,
104 g of tea catechins were produced by freeze-drying. The purity of tea catechins was 75%. The method of the present invention
Both the amount of solvent used and the purity were excellent.

(Example 2) It was carried out in the same manner as in Example 1. However, methyl isobutyl ketone and diethyl ether were used as organic solvents for purification instead of ethyl acetate.

As a result, the amount of tea catechins was 75g and 6g, respectively.
5 g, and the purity was 83% and 79%, respectively.

(Example 3) It was carried out in the same manner as in Example 1. However, as a column packing material, LH-20 (Sephadex), HW-40
(Toyopearl) and S-761 (Duolite) were used.

The results are shown in the table of Figure jI5 together with the results of Example 1. With the exception of S-761, there was not much difference in performance depending on the resin under these conditions.

(Example 4) Two liters of tea extract was produced in the same manner as in Example 1, and this tea extract was extracted three times with the same amount of ethyl acetate.

Approximately 6 liters of the ethyl acetate layer was combined and concentrated under reduced pressure to remove ethyl acetate, yielding 120 g of solid material. Add water to dissolve it to make 500 ml, and inject the entire amount into a chromatography column packed with about 175 ml of the same synthetic adsorbent (HP-2MG) as in Example 1.
Elution was performed with 450 milliliters of ethanol.

This liquid was freeze-dried in the same manner as in Example 1 to form a white solid.
Obtained 0g.

Therefore, if the manufacturing procedure is changed, that is, the water-soluble components extracted from tea leaves are first dissolved in an organic solvent, and after removing this organic solvent, they are dissolved again in water and passed through a chromatography column. Although a large amount of organic solvent is used for dissolution, the amount of adsorbent is small and the amount of adsorbent can be increased.

Note that the amount and purity of the obtained product are the same as the results obtained in Example 1.

[Effects of the Invention] As described above, according to the present invention, tea catechins having high antioxidant ability and other useful physiologically active functions can be produced as never before through the two-step process of the chromatography column adsorption separation process and the liquid-liquid extraction process. It has become possible to produce it with high purity and at low cost.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an outline of the steps of the method for purifying tea catechins according to the present invention, and the jIZ diagram is an explanatory diagram showing a chromatogram by adsorption separation. Figure 13 is an explanatory diagram of a chromatogram obtained by liquid-liquid extraction after adsorption separation. Figure 4 is a graph showing the antioxidant ability of tea catechins produced by the method of the present invention against linoleic acid. Figure 5 is a graph showing tea catechins. It is a table showing recovery amount and purity. Patent applicant: Food Industry High Separation System Technology Research Association

Claims (5)

[Claims] (1) A first extraction step in which water-soluble components are extracted from tea leaves, and an adsorption step in which the water-soluble components are packed into a chromatography column filled with a synthetic adsorbent or gel filtration agent, and the components are adsorbed on this chromatography column. and an elution step in which these adsorbed components are eluted with an approximately 40% to 100% aqueous solution consisting of one of methanol, ethanol or acetone, or a mixture thereof, and such an eluate is eluted with ethyl acetate, methyl isobutyl ketone or diethyl. 1. A method for producing tea catechins, which comprises dissolving them in one type of ether or a mixture thereof, and then a second extraction step of distilling off organic components. (2) A first extraction step in which water-soluble components are extracted from tea leaves, and after dissolving this extract in one of ethyl acetate, methyl isobutyl ketone, diethyl ether, or a mixture thereof, the organic components are distilled off. a second extraction step in which the extracted components are packed into a chromatography column packed with a synthetic adsorbent or gel filtration agent, and an adsorption step in which the components are adsorbed on the chromatography column; 1. A method for producing tea catechins, comprising an elution step of eluting with an approximately 40% to 100% aqueous solution of one type of acetone or a mixture thereof. (3) Claim (1) or (2) characterized in that the tea catechins are a mixture whose main components are epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. The method for producing tea catechins described above. (4) The method for producing tea catechins according to claim 1 or 2, wherein the synthetic adsorbent is an adsorbent based on styrene/divinylbenzene or methacrylic acid ester. (5) The method for producing tea catechins according to claim 1 or 2, wherein the gel filtration agent is an adsorbent based on a dextran derivative or a hydrophilic vinyl polymer.