JP3052174B2 - Method for producing tea catechins - Google Patents

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 high-purity tea catechins from tea leaves.

[0002]

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

[0003] These tea catechins have an antioxidant effect,
It is known that it has chemical and physiological activities such as an antibacterial effect, a deodorant effect, and a blood cholesterol suppressing effect.
Further, it is reported that these effects are stronger in ester-type catechins than in free-type catechins, and in particular, (-) epigallocatechin gallate is stronger.

[0004] In order to produce such tea catechins with high purity, it is essential to separate impurities coexisting with the tea catechins in tea leaves. The impurities include caffeine (Caf), saccharides, amino acids, organic acids, and catechin oxidized 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.

[0006] For example, Japanese Patent Publication No. 1-44234 discloses that
It is described that natural antioxidants can be industrially produced by removing caffeine with chloroform and extracting tea catechins with ethyl acetate.

[0007] In Japanese Patent Application Laid-Open No. 2-311474, tea catechins are produced by selectively adsorbing tea catechins to a filler using a chromatographic separation and eluting them with a hydrophilic organic solvent.

Further, Japanese Patent Application Laid-Open No. Hei 3-14572 discloses that tea catechins can be produced using gel beads by a multi-stage batch process capable of high load conditions.

[0009] However, the products obtained by these conventional production methods are a mixture of tea catechins, ie,
(-) Epicatechin, (-) epigallocatechin, (-)
It is obtained as a mixture of tea catechins mainly comprising epicatechin gallate and (−) epigallocatechin gallate.

In the liquid-liquid extraction method, a normal solvent has a partition coefficient of about 1 between tea catechins and other contaminants, so that multi-stage extraction is required and the recovery rate is reduced. Only mixtures of this kind can be recovered.

On the other hand, in the chromatographic separation, tea catechins are recovered as a mixture with high efficiency 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 collected as a mixture.

Further, the separation of ester-type catechins and free catechins is possible by using medium-pressure preparative chromatographic separation, and individual tea catechins are possible by using high-pressure preparative chromatographic separation. It is possible to get

However, the conventional medium- and high-pressure preparative chromatographic column apparatus has problems such as an increase in chromatographic column pressure loss, making it difficult to pass the liquid, and a smaller load compared to low-pressure chromatographic separation. Was. For this reason, there is a problem that the production cost of tea catechins becomes extremely high, and the device itself is expensive because of the pressure resistance specification.

[0014]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned problems. In producing commercial catechins, tea catechins can be recovered only as a mixture in the prior art. To provide a method for producing tea catechins which can be separated and purified into ester-type catechins, free-type catechins, and individual tea catechins, and which can be produced with high purity and at low cost. As an issue.

[0015] Specifically, it eliminates the problems of increased column pressure loss and difficulties in liquid flow in preparative chromatographic separation at medium and high pressures, and enables high-load treatment to increase productivity and improve tea catechins. It is an object of the present invention to provide a method for producing tea catechins which can produce catechins with high purity and at low cost.

[0016]

That is, the present invention focuses on removing impurities in advance by using a multistage chromatography column, and an extraction step of obtaining a tea extract by extracting water-soluble components from tea leaves. And a first separation step of injecting the tea extract into a first chromatographic column to obtain a recovered liquid from which contaminants have been removed by the first chromatographic column, and transferring the recovered liquid to a second chromatographic column. A second separation step of injecting and separating and purifying tea catechins using the second chromatographic column.

The tea extract is injected into the first chromatographic column, and the first chromatographic column is washed with water to remove the contaminants and to be adsorbed to the first chromatographic column. In addition, a recovered liquid can be obtained by recovering a component that cannot be washed with water as a solvent using an aqueous solution composed of one of methanol, ethanol, and acetone, or a mixture thereof.

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

Further, the above tea catechins can be obtained by adding (-)
Epicatechin, (-) epigallocatechin, (-) epicatechin gallate, and (-) epigallocatechin gallate can be used.

The filler for the first chromatographic column can be made of hydrophilic polymer particles of styrene / divinylbenzene type, methacrylate type, or hydrophilic vinyl type.

The filler for the second chromatographic column can be silica gel or hydrophilic polymer particles.

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

A particularly important point in the present invention is that impurities contained in the tea extract are removed in advance by the first chromatography column.

The present inventors have so far found a method for producing tea catechins at low cost on a commercial scale, though they are mixtures, by chromatographic separation (JP-A-2-3114).
No. 74, JP-A-4-182479). According to such a method, a polymer having a large particle diameter is used as a filler, and no problems such as an increase in column pressure loss and a problem of consolidation of the filler occur.

On the other hand, in parallel with this, a separation experiment was started even by using a medium-high pressure preparative chromatography column in order to recover individual tea catechins. However, when the load was increased as described above, There is a problem that the pressure loss of the column increases, and in the worst case, the liquid cannot be passed.

It was initially thought that such a problem would occur due to the use of a filler having a small particle size. However, after that, when the viscosity of the chromatographic fraction was measured in the low-pressure chromatographic column experiment, unexpectedly, the viscosity of the fraction containing contaminants was high, but the viscosity of the fraction containing tea catechins was small. The result was obtained.

Generally, the pressure loss of a column is determined by Kozeny.
As can be estimated from the Carman equation or the like, since the viscosity is proportional to the viscosity, 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 chromatography column. As a result, the present inventors have completed the present invention.

Hereinafter, the present invention will be described in detail. As shown in FIG. 1, a tea extract is first obtained by extracting a water-soluble component of tea leaves with warm water.

The tea extract is poured into the first chromatographic column, and then the chromatographic column is washed with water to remove impurities, and cannot be washed with water. The adsorbed components are recovered with an aqueous solution consisting of one of methanol, ethanol or acetone solvents, or a mixture thereof.

Next, the solvent is removed by concentrating the recovered solution obtained in the first chromatography column under reduced pressure.

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

Hereinafter, the present invention will be described more specifically. As raw materials, usually, raw tea or unfermented dried leaves are extracted using, for example, boiling water at a temperature of 80 ° C. or higher, and then filtered,
Centrifuge and use the concentrated tea extract.

As the packing material of the first chromatography column,
A hydrophilic polymer such as a styrene / divinylbenzene type, a methacrylic acid ester type, or a hydrophilic vinyl type having a particle size of about 200 to 1200 μm can be used.

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

By passing the tea extract through the first chromatographic column packed with these fillers, tea catechins can be selectively adsorbed on the filler, while non-adsorbable impurities such as saccharides and amino acids can be obtained. From the column outlet.

Next, by washing the column with water,
The contaminants remaining in the voids in the column are discharged. The amount of water used for washing at this time varies depending on the load on the column, but may be 0.5 to 4 times the column volume.

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

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

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

Next, the concentrate is passed through a second chromatography column.

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

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

Next, one of methanol, ethanol or acetone solvent or a mixture thereof is used.
Elution is carried out using a 0 to 100 vol% aqueous solution to obtain a fraction containing the desired tea catechins sequentially.

[0044]

In the method for producing tea catechins according to the present invention, a tea extract as a raw material is passed through a first chromatographic column to remove contaminants first and then to a second chromatographic column. Through which the tea catechins are obtained.

Therefore, it is possible to remove contaminants having a relatively large molecular weight, and it is possible to eliminate an increase in column pressure loss and difficulty in passing a liquid, especially when chromatographic separation is performed at a medium or high pressure or when a large amount of liquid is passed. At the same time, it is possible to carry out the treatment with a high load, and to separate and purify the target tea catechins with high purity and low cost in the second chromatographic column.

[0046]

Next, embodiments of the present invention will be described. [Example 1] 1 kg of green tea was extracted with 15 liters of hot water at a temperature of 95 ° C for 10 minutes, and after removing the extraction residue, the mixture was filtered with a 200-mesh filter.
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.

One liter of the above tea extract was injected into a chromatographic column filled with one liter of HP1MG, and the column effluent was collected. Next, the column was washed with 2 liters of water, and the column effluent was collected.

Finally, 2.5 liters of a 70 vol% ethanol aqueous solution was passed to obtain a recovered solution containing tea catechins. The viscosity of each fraction at this time was 8.7, 1.9, and 2.2 cP at a temperature of 25 ° C., respectively.

Next, the tea catechins recovered liquid was heated at a temperature of 45 ° C.
The mixture was concentrated under reduced pressure to remove ethanol, and 750 ml of a concentrated solution was obtained. The viscosity of this concentrate was 1.8 cP.

Conventional high performance liquid chromatography (HP
(-) Epicatechin, (-) epigallocatechin, (-) epicatechin gallate, and (-) epigallocatechin gallate were quantified by LC).
Of these tea catechins, and the recovery was 96.4%.

In this way, by removing impurities from the tea extract, it was possible to obtain a concentrate having a viscosity reduced to about 1/30 times.

"Example 2" The concentrate 100 obtained in Example 1 was used.
Milliliter was passed through YMC ODS R354 (column volume 687 ml), then 25 vol
4.6 liters of an aqueous methanol solution were passed through, and then 1.0 liters of the eluent was switched to 100% methanol.

At this time, the liquid passing state was good, and the chromatogram obtained by measuring the liquid at the outlet of the column with a UV detector (wavelength: 280 to 350 nm) is shown in FIG.

Example 3 The concentrated liquid 500 obtained in Example 1
Milliliter was passed through Tosoh Pearl HW40C (column volume: 147 ml) manufactured by Tosoh Corporation.
294 ml of an aqueous solution of acetone in 100% by volume was passed, and then 294 ml of an aqueous solution of 60% by volume acetone was passed in.

The liquid at the outlet of the column at this time was sampled at regular intervals, colored with ferric chloride, and the absorbance at 540 nm was measured. FIG. 3 shows the measurement results.

The elution peaks A and B in FIG. 3 were confirmed to be ester-type catechin and free-type catechin, respectively, by conventional HPLC.

Compared with the conditions when a conventional tea extract is used, the amount of tea catechins to be injected can be increased 1.3 times and the flow rate can be increased 10 times. Improved.

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

At this time, the liquid passing state was good, the column inlet pressure was stable at 50 kg / cm ² G or less, and no increase in pressure loss occurred. The liquid at the column outlet is measured with a UV detector (wavelength
The chromatogram measured at 80-350 nm is shown in FIG.

As can be seen from FIG. 4, it was confirmed that the tea catechins could be individually separated, and that the separation from the caffeine and catechin oxidized polymer coexisting in the concentrated solution was good.

When a conventional tea extract was used, the column pressure loss increased even if the amount of tea catechins to be injected was about 1/3 as described above, making it difficult to pass the tea.

[0062]

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 contaminants contained in tea extract. To separate free catechin and ester catechin, or (-) epicatechin,
(-) Epigallocatechin, (-) epicatechin gallate, and (-) epigallocatechin gallate can be separated individually, and thus, purified tea catechins can be produced at low cost.

[0063]

[Brief description of the 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 a column outlet liquid according to Example 2;

FIG. 3 is a view showing a column outlet liquid according to Example 3 collected at regular intervals, colored with ferric chloride, and measured for absorbance at 540 nm.

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

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Fumihisa Ranabe 21 Goddess of Sagara-cho, Haibara-gun, Shizuoka Pref. (56) References JP-A-1-299224 (JP, A) JP-A-2-311474 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 311 / 62

Claims (5)

(57) [Claims] 1. An extraction step of obtaining a tea extract by extracting a water-soluble component from tea leaves, and using the tea extract as a filler having a particle size of about 200 to 1200 μm.
Into the first chromatographic column, and use water to
By washing the first chromatographic column, the first chromatographic column is washed.
The first chromatka was removed using a mat column to remove impurities.
The components that are absorbed by the ram and are not
A type of ethanol, ethanol or acetone solvent
Or 50-90 vol% aqueous solution consisting of these mixtures
A first separation step of obtaining a recovering solution by recovering the liquid as a solvent, the particle size of the filler of this recovery liquid is about 5 to 100 [mu] m 2
A second separation step in which tea catechins are separated and purified by injecting the catechins into the chromatographic column, and a tea catechins are separated and purified by the second chromatographic column. 2. After removing the solvent from the recovered liquid, the recovered liquid is injected into the second chromatographic column, and eluted with an aqueous solution comprising one of methanol, ethanol or acetone solvents, or a mixture thereof. , claim 1, characterized in that the purified and separated tea catechins
A method for producing the tea catechins according to the above. 3. The tea catechin according to claim 1, wherein the tea catechins are (−) epicatechin, (−) epigallocatechin, (−) epicatechin gallate, and (−) epigallocatechin gallate. A method for producing tea catechins. 4. The filler for the first chromatographic column is made of hydrophilic polymer particles of styrene / divinylbenzene type, methacrylate ester type or hydrophilic vinyl type. 2. The method for producing the tea catechins according to 1. 5. The method for producing tea catechins according to claim 1, wherein the filler for the second chromatographic column is silica gel or hydrophilic polymer particles.