JPH0770105A - Production of tea catechins - Google Patents

Abstract

PURPOSE:To provide production method capable of inexpensively manufacturing high-purity tea catechins by separating and purifying ester type catechins, free type catechins and individual tea catechins. CONSTITUTION:This production method of tea catechins has a first separating process in which an extracted solution of tea is poured into a first chromatography column to give a recovered solution from which admixtures are removed by the first chromatographic column and a second separating process in which the recovered solution is poured into a second chromatography column and tea catechins are separated and purified by the second chromatographic column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing tea catechins, and more particularly to a method for producing tea catechins for producing highly pure tea catechins from tea leaves.

[0002]

2. Description of the Related Art Tea catechins, which are contained in a large amount in tea leaves, are a kind of polyphenol compound, and include (-) epicatechin (EC) and (-) epigallocatechin (EG).
C), and their gallic acid esters (-)
The four main types are epicatechin gallate (ECg) and (−) epigallocatechin gallate (EGCg), and the dry tea leaves contain 10 to 15 wt%.

These tea catechins have an antioxidant effect,
It is known to have a chemical / physiologically active action such as an antibacterial action, a deodorizing action, and a blood cholesterol suppressing action.
It is also reported that these effects are stronger in the ester type catechins than in the free type catechins, and particularly (-) epigallocatechin gallate.

In order to produce such tea catechins with high purity, it is indispensable to separate impurities that coexist in the tea leaves with the tea catechins. The contaminants include caffeine (Caf), sugars, amino acids, organic acids and catechin oxidation polymers.

Conventionally, as a technique for recovering tea catechins from tea leaves, a method using liquid-liquid extraction using an organic solvent or chromatographic separation has been known.

For example, Japanese Patent Publication No. 1-44234 discloses that
It is described that a natural antioxidant can be industrially produced by removing caffeine with chloroform and extracting tea catechins with ethyl acetate.

Further, in Japanese Patent Application Laid-Open No. 2-311474, tea catechins are produced by using a chromatographic separation to selectively adsorb tea catechins to a filler and eluting them with a hydrophilic organic solvent.

Further, JP-A-3-14572 describes that it is possible to produce tea catechins by using gel beads by a multi-stage batch process capable of high load conditions.

However, the products obtained by these conventional production methods are a mixture of tea catechins, that is,
(−) Epicatechin, (−) epigallocatechin, (−)
It is obtained as a mixture of tea catechins mainly consisting of epicatechin gallate and (−) epigallocatechin gallate.

In the liquid-liquid extraction method, since the partition coefficient between tea catechins and other contaminants is about 1 in a normal solvent, multistage extraction is required, which leads to a decrease in the recovery rate and the tea catechin. Only mixtures of classes can be recovered.

On the other hand, in chromatographic separation, tea catechins are highly efficiently recovered as a mixture by low pressure chromatographic separation using a resin having a relatively large particle size as a filler.
Also in the above batch process, tea catechins are recovered as a mixture.

[0012] In addition, the separation of ester catechins and free catechins is possible by using medium pressure preparative chromatographic separation, and the individual tea catechins can be separated by using high pressure preparative chromatographic separation. It is possible to get

However, in the conventional medium-high pressure preparative chromatographic column device, there is a problem that the chromatographic column pressure loss increases, making it difficult to pass the liquid, and the load amount is smaller than in the low-pressure chromatographic separation. It was For this reason, there is a problem that the manufacturing cost of tea catechins becomes very high, and the apparatus itself is expensive due to the pressure resistance specification.

[0014]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and in the production of tea catechins on a commercial scale, in the prior art, tea catechins can be recovered only as a mixture. To provide a method for producing tea catechins that can be produced at a high purity and at low cost by improving what was not present, separating and purifying ester catechins, free catechins, and individual tea catechins. Is an issue.

Specifically, the problems of increasing column pressure loss and difficulty of liquid passage in medium- and high-pressure preparative chromatographic separation are solved, and high-load treatment is enabled to enhance productivity, thereby improving tea catechins. An object of the present invention is to provide a method for producing tea catechins that can be produced with high purity and at low cost.

[0016]

Means for Solving the Problems That is, the present invention focuses on removing contaminants in advance by using a chromatographic column in multiple stages, and an extraction step for obtaining a tea extract by extracting a water-soluble component from tea leaves. And a first separation step of injecting this tea extract into a first chromatographic column to obtain a recovered liquid from which contaminants have been removed by the first chromatographic column, and the recovered liquid to a second chromatographic column. A second separation step of injecting and separating and purifying tea catechins by the second chromatographic column, and a method for producing tea catechins.

The tea extract is injected into the first chromatographic column and the first chromatographic column is washed with water to remove the contaminants and adsorb to the first chromatographic column. At the same time, a component that is not washed with water is recovered as a solvent by using an aqueous solution of one of a methanol, ethanol or acetone solvent or a mixture thereof as a solvent to obtain a recovered liquid.

Further, after removing the solvent of the recovered liquid, the recovered liquid is injected into the second chromatographic column and eluted with an aqueous solution of one of a methanol, ethanol or acetone solvent or a mixture thereof. ,
Tea catechins can be purified and separated.

Further, the above tea catechins are (-)
It may be epicatechin, (−) epigallocatechin, (−) epicatechin gallate, or (−) epigallocatechin gallate.

The packing material of the first chromatographic column may be hydrophilic polymer particles of styrene / divinylbenzene type, methacrylic acid ester type, or hydrophilic vinyl type.

The packing material for the second chromatographic column may be silica gel-based or hydrophilic polymer particles.

The present invention will be specifically described below. The present invention uses a tea extract obtained by extracting the water-soluble component of tea leaves with warm water as a raw material, removes impurities by the first chromatographic column shown in FIG. 1, and removes tea catechins by the second chromatographic column. A method for producing tea catechins, which comprises separating and purifying.

In the present invention, a particularly important point is that impurities contained in the tea extract are removed in advance by the first chromatographic column.

The present inventors have so far found a method for producing tea catechins at low cost on a commercial scale, though they are a mixture, by chromatographic separation (Japanese Patent Laid-Open No. 2-3114).
74, JP-A-4-182479). According to such a method, since a polymer having a large particle size is used as a packing material, there are no problems such as an increase in column pressure loss and packing material packing.

On the other hand, in parallel with this, a separation experiment was started using a medium- and high-pressure preparative chromatographic column in order to recover individual tea catechins. There was a problem that the pressure loss of the column increased and, in the worst case, it was not possible to pass the liquid.

Initially, it was thought that such a problem would occur because a filler having a small particle size was used. However, after that, when the viscosity of the chromatographic fraction was measured in the experiment of the low-pressure chromatographic column, the viscosity of the fraction containing contaminants was surprisingly high, but the viscosity of the fraction containing tea catechins was surprisingly small. I got the result.

In general, the pressure loss of the column is Kozeny.
Since it is proportional to viscosity as can be estimated from the -Carman equation, it is possible to suppress an increase in pressure loss by supplying a raw material from which impurities have been removed in advance to a medium-high pressure chromatographic column. As a result of further studies, the present invention has been completed.

The present invention will be described in detail below. As shown in FIG. 1, first, the water-soluble component of tea leaves is extracted with warm water to obtain a tea extract.

The tea extract is injected into the first chromatographic column, and then the water is used to wash the chromatographic column to remove impurities. The adsorbed component is recovered with an aqueous solution containing one of methanol, ethanol or acetone solvent or a mixture thereof.

Next, the solvent is removed by concentrating the recovered liquid obtained by the first chromatographic column under reduced pressure.

The concentrated liquid is passed through a second chromatographic column and eluted with an aqueous solution of one of a methanol, ethanol or acetone solvent or a mixture thereof to purify tea catechins.

The present invention will be described in more detail below. As raw material, raw tea or unfermented dried leaves are usually extracted with hot water having a temperature of 80 ° C. or higher, and then filtered,
Use the tea extract that has been centrifuged and concentrated.

As the packing material for the first chromatographic column,
A hydrophilic polymer such as styrene / divinylbenzene-based, methacrylic acid ester-based, or hydrophilic vinyl-based polymer having a particle diameter of about 200 to 1200 μm can be applied.

For example, Diaion HP20, HP2
1, HP40, Sepa beads SP800, SP850, S
P900, SP206, SP207, HP1MG, HP
2MG (above, Mitsubishi Kasei), Duolite S861,
S862, S876 (above, made by Rohm & Haas), Levatit OC1062 (made by Bayer), Amber Light X
AD7, XAD8 (above, manufactured by Organo), and Toyopearl HW40EC (manufactured by Tosoh Corporation).

By passing the tea extract through the first chromatographic column packed with these packings, tea catechins are selectively adsorbed on the packings, while non-adsorbing contaminants such as sugars and amino acids. Is discharged from the column outlet.

Next, by washing the column with water,
The impurities remaining in the voids in the column are discharged. The amount of water used for washing at this time may be 0.5 to 4 times the column volume, although it depends on the amount of load on the column.

Next, the tea catechins adsorbed on the column are recovered by eluting with a hydrophilic organic solvent, for example, one of methanol, ethanol or acetone solvent, or a 50 to 90 vol% aqueous solution of a mixture thereof.

The components of the recovered liquid thus obtained are a mixture of tea catechins, caffeine, and a catechin oxidation polymer, although the component ratio varies depending on the type of filler and the separation conditions.

Next, the recovered liquid of the first chromatographic column is concentrated under reduced pressure to remove the solvent.

Then, the concentrated liquid is passed through the second chromatographic column.

As the packing material for the second chromatographic column,
Silica gel type or hydrophilic polymer with particle size of 5 to 1
Those having a diameter of about 00 μm can be applied.

For example, ODS, Capcell Pa
k C-18 (above, Shiseido product), Toyopearl HW40
C, Toyopearl HW40F (above, made by Tosoh), Sun
Pearl ODP, S-ODS, I-ODS, GS,
VP, S-SIL, I-SIL (all manufactured by Asahi Kasei Corporation), Sephadex LH-20 (all manufactured by Pharmacia), Amber Chrome CG71 (manufactured by Toso Haas), and the like.

Then, one of methanol, ethanol or acetone solvent or a mixture of these is used.
Elution is performed using a 0 to 100 vol% aqueous solution to sequentially obtain the fractions containing the target tea catechins.

[0044]

In the method for producing tea catechins according to the present invention, the tea extract as a raw material is passed through the first chromatographic column to remove impurities first, and then the second chromatographic column. The tea catechins are obtained by passing the solution through.

Therefore, it is possible to remove impurities having a relatively large molecular weight, so that the increase in column pressure loss and the difficulty of passing the liquid can be eliminated especially when the chromatographic separation is carried out at medium and high pressure or when a large amount of liquid is passed. In addition, the treatment with a high load can be performed, and the target tea catechins can be separated and purified with high purity and at low cost in the second chromatographic column.

[0046]

EXAMPLES Next, examples of the present invention will be described. [Example 1] 1 Kg of green tea was extracted with 15 liters of warm water at a temperature of 95 ° C for 10 minutes, the extraction residue was removed, and then the mixture was filtered through a 200-mesh filter to remove 5 minute fixed components in the filtrate.
It was removed by centrifugation at 000 rpm and finally concentrated under reduced pressure to obtain 1.5 Kg of a 15 Brix% tea extract. The viscosity of this extract was 53 cP at a temperature of 25 ° C.

1 liter of the tea extract was injected into a chromatographic column filled with 1 liter of the packing material HP1MG, and the column effluent at this time was recovered. Then, the column was washed with 2 liters of water and the column effluent was collected.

Finally, 2.5 liters of 70 vol% aqueous ethanol solution was passed through to obtain a recovered liquid containing tea catechins. The viscosities of the respective fractions at this time were 8.7, 1.9, and 2.2 cP at a temperature of 25 ° C, respectively.

Next, the tea catechins recovery liquid is treated at a temperature of 45 ° C.
The mixture was concentrated under reduced pressure with to remove ethanol to obtain 750 ml of a concentrated liquid. The viscosity of this concentrated liquid was 1.8 cP.

Conventional high performance liquid chromatography (HP
As a result of quantifying (−) epicatechin, (−) epigallocatechin, (−) epicatechin gallate, and (−) epigallocatechin gallate by LC), 37.9 g was obtained in the concentrated liquid.
Containing these tea catechins, the recovery was 96.4%.

Thus, by removing the contaminants from the tea extract, a concentrated solution having a viscosity reduced to about 1/30 times could be obtained.

Example 2 Concentrated liquid 100 obtained in Example 1
Pass the milliliter through ODS R354 (column volume 687 ml) manufactured by YMC, and then add 25 vol.
% Liter aqueous solution 4.6 liter was passed, then the eluent was switched to 100% methanol and 1.0 liter was passed.

At this time, the liquid passing condition was good, and the chromatogram of the column outlet liquid measured with a UV detector (wavelength 280 to 350 nm) is shown in FIG.

[Example 3] Concentrated liquid 500 obtained in Example 1
Pass milliliters through Toyopearl HW40C (column volume 147 milliliters) manufactured by Tosoh Corporation, and then 30v
294 ml of an ol% acetone aqueous solution was passed, and then 294 ml of a 60 vol% acetone aqueous solution was passed.

The column outlet liquid at this time was sampled at regular intervals, developed with ferric chloride, and the absorbance at 540 nm was measured. The measurement result is shown in FIG.

Elution peaks A and B in FIG. 3 were confirmed to be ester catechin and free catechin by HPLC according to a conventional method.

Compared with the conditions when using the conventional tea extract, the injection amount of tea catechins can be increased 1.3 times and the flow rate can be increased 10 times, and the productivity can be improved by removing impurities. Improved.

Example 4 Concentrated liquid 200 obtained in Example 1
CapillPak C manufactured by Shiseido
18 (column volume 8.6 liters), then 3
45 liters of a 0 vol% methanol aqueous solution was passed, and then the eluent was switched to 100% methanol to pass 18 liters.

At this time, the liquid passing condition was good, the column inlet pressure was stable at 50 Kg / cm ² G or less, and the pressure loss did not increase. UV detector (wavelength 2)
The chromatogram measured at 80 to 350 nm) is shown in FIG.

As can be seen from FIG. 4, it was confirmed that the tea catechins can be individually separated, and that they are well separated from the coexisting caffeine and catechin oxidation polymer in the concentrate.

When the conventional tea extract was used, the column pressure loss increased and it was difficult to pass the liquid even if the injection amount of the tea catechins was set to about 1/3 of the above.

[0062]

Industrial Applicability As described above, according to the present invention, the problem of column pressure loss in medium and high pressure preparative chromatographic separation is solved by selectively removing highly viscous impurities contained in the tea extract. The free catechin and the ester catechin are separated, or (−) epicatechin,
By separating (-) epigallocatechin, (-) epicatechin gallate, and (-) epigallocatechin gallate individually, it has become possible to inexpensively produce purified tea catechins.

[0063]

[Brief description of drawings]

FIG. 1 is an explanatory view schematically showing steps of a method for producing tea catechins according to the present invention.

FIG. 2 is a UV chromatogram of the column outlet liquid according to Example 2.

FIG. 3 is a diagram in which the column outlet liquid according to Example 3 was sampled at regular intervals, developed with ferric chloride, and the absorbance at 540 nm was measured.

FIG. 4 is a UV chromatogram of the column outlet liquid of Example 4.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Fumihisa Ranabe, 21 Goddess, Sagara-cho, Hara-gun, Shizuoka Prefecture, Itoen Central Research Institute Co., Ltd. In the laboratory

Claims (6)

[Claims] 1. An extraction step of obtaining a tea extract by extracting a water-soluble component from tea leaves, and injecting the tea extract into a first chromatographic column to remove impurities by the first chromatographic column. The first separation step for obtaining the recovered liquid, and the injection of this recovered liquid into the second chromatographic column
A second separation step of separating and purifying tea catechins by the chromatographic column of 1., and a method for producing tea catechins, the method comprising: 2. The tea extract is injected into the first chromatographic column, and the contaminants are removed by washing the first chromatographic column with water to adsorb the tea on the first chromatographic column. 2. The production of tea catechins according to claim 1, further comprising recovering a component which is not washed with water, as a solvent, an aqueous solution consisting of one of methanol, ethanol or acetone solvent or a mixture thereof. Method. 3. After removing the solvent of the recovered liquid, the recovered liquid is injected into the second chromatographic column and eluted with an aqueous solution consisting of a solvent of methanol, ethanol or acetone, or a mixture thereof. 3. The tea catechins are purified and separated.
A method for producing the tea catechins described. 4. The tea catechins are (−) epicatechin, (−) epigallocatechin, (−) epicatechin gallate, and (−) epigallocatechin gallate, according to claim 1. A method for producing tea catechins. 5. The packing material for the first chromatographic column is made of styrene / divinylbenzene-based, methacrylic acid ester-based, or hydrophilic vinyl-based hydrophilic polymer particles. 1. The method for producing tea catechins according to 1. 6. The method for producing tea catechins according to claim 1, wherein the packing material of the second chromatographic column is silica gel-based or hydrophilic polymer particles.