JP5295744B2 - Method for producing theacinensin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing theasinensin, capable of obtaining the theasinensin more stably and in a higher yield than those of conventional methods, while being simple. <P>SOLUTION: This theasinensin is produced by oxidizing epigallocatechin and/or epigallocatechin gallate in the presence of a non-homogeneous catalyst, and then reducing the obtained oxidation product, in a higher yield than those of the conventional methods. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

The present invention relates to a method for producing theacinensin stably and in a high yield as compared with a conventional method, though it is simple.

There are a wide variety of teas that are drunk around the world, but black tea and oolong tea are produced by fermenting fresh leaves of Camellia sinensis . In this fermentation process, epigallocatechin (EGC), epigallocatechin gallate (EGCg), epicatechin (EC) and epicatechin gallate (ECg) are produced by the action of oxidase (polyphenol oxidase and / or peroxidase) contained in the raw leaves. Representative catechins undergo oxidative polymerization to produce polymerized polyphenols such as theaflavins and theasinensins. The structures of the four catechins are shown in the following chemical formula.

(In the formula, R ₁ represents a hydrogen atom or a galloyl group, and R ₂ represents a hydrogen atom or a hydroxyl group.)

Theacinensin has a structure in which catechins are bonded to each other at the B-rings. The well-known theacinensins are the following five types: theacinensin A and its atropisomers theasinensin D and theasinensin B and theasinensin C and its atropisomers. One theasinensin E is mentioned. Their structures are shown in the chemical formula below.

(In the formula, R ₁ and R ₂ each independently represent a hydrogen atom or a galloyl group.)

In recent years, theacinensin has an apoptosis-inducing action (Patent Document 1), a lipase inhibitory action (Patent Document 2), an anti-inflammatory action (Patent Document 3), a blood glucose level increase inhibiting action (Patent Document 4), a matrix metalloprotease inhibitory action (Patent Document 4) It has been reported to have functions such as literature 5), and further research development is expected.

Theacinensin can be obtained by oxidative polymerization of catechin corresponding to the structure enzymatically or non-enzymatically. For example, theacinensin A is generated from two molecules of EGCg, theacinensin B is generated from EGCg and EGC, and theacinensin C is generated from two molecules of EGC.

Specifically, the following method is disclosed as a method for producing theacinensin. A method using potassium ferricyanide as an oxidizing agent (Patent Document 1, Non-Patent Document 1), a method of radical oxidation using 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Non-Patent Document 2), alkaline conditions There is a method (Non-Patent Document 3) in which air oxidation is performed below. Moreover, the method (nonpatent literature 4) oxidized using a pear fruit homogenate is also disclosed. It has been reported that when pear fruit homogenate is allowed to act on EGCg, dehydroteasinensin A, which is a precursor of theacinensin A, is produced, and dehydrotheacinensin A is separated and then reduced to yield theacinensin A. The chemical reaction formula is shown below.

(In the formula, R represents a galloyl group.)

JP-A-2005-75790 WO2006 / 004114 JP2007-119414A JP2007-231009 JP 2000-226329 A Fumio Hashimoto, Gen-Ichiro Nonaka, Ittsu Nishioka, Chem. Pharm. Bull. , 36, 1676-1684 (1988) Nankun Zhu, Mingfu Wang, Guo-Jien Wei, Jen-Kun Lin, Chung S. Yang, Chi-Tang Ho, Food Chem. 73, 345-349 (2001) Tsutomu Hatano, Miwako Kusuda, Mami Hori, Sumiko Shiota, Tomofusa Tsuchiya, Takashi Yoshida, Plant Med. , 69, 984-989 (2003) Takashi Tanaka, Sayaka Watarumi, Yosuke Matsuo, Midori Kamei, Isao Kouno, Tetrahedron, 59, 7939-7947 (2003)

Regarding the method for producing theacinensin, the method as described above is disclosed. However, these methods have at least the following drawbacks.

For example, in the method using potassium ferricyanide of Patent Document 1 and Non-Patent Document 1, since the potassium ferricyanide solution is dropped little by little, it is difficult to control the reaction unless the dripping amount and time are strictly controlled. In fact, Patent Document 1 obtains 392 mg of theacinensin A from 10 g of EGCg, and Non-patent Document 1 obtains 1.18 g of theacinensin A from 10 g of EGCg, but the yields of theacinensin A are 3.9% and 11.8 respectively. It is difficult to obtain theacinensin in a stable yield. Further, it is considered that theacinensin B is preferentially produced when the mixture of EGC and EGCg is oxidized. However, even if the mixture of EGC and EGCg is oxidized by this method, the production of theasinensin B does not take precedence over the production of theasinensin C. The yield of B is as low as 3.8%. In the method using DPPH of Non-Patent Document 2, the reaction time is as long as 2 days, and the yield of theacinensin is as low as 7% or less. The yield in the method of air oxidation under alkaline conditions of Non-Patent Document 3 is low and 4% or less. In the method of using non-patent document 4 pear fruit homogenate, it takes time to prepare the homogenate, and the enzyme activity varies depending on the type of fruit, the difference in harvest time and the preparation of the homogenate, and theasinensin is obtained in a stable yield. Is difficult. The yield from EGCg to theacinensin A is calculated to be about 10%.

As described above, the conventional methods for producing theacinensin cannot easily and stably yield theacinensin in a high yield. Accordingly, an object of the present invention is to provide a method for producing theacinensin that can overcome the above-mentioned drawbacks.

As a result of intensive studies to achieve the above object, the present inventor unexpectedly oxidizes EGC and / or EGCg in the presence of a heterogeneous catalyst, and then reduces the obtained oxidation product to reduce the oxidation product. The present inventors have found that the problems can be solved and have completed the present invention.

That is, the present invention according to claim 1 is a method for producing theasinensin comprising a step of oxidizing epigallocatechin and / or epigallocatechin gallate in the presence of a heterogeneous platinum group catalyst and then reducing the obtained oxidation product. A method is provided.

Claims the invention of claim 2 is to provide a Teashinenshin manufacturing method of claim 1 wherein heterogeneous platinum group catalyst is a palladium catalyst and / or platinum catalyst.

The present invention according to claim 3 provides the method for producing theasinensin according to claim 1 or 2, wherein the theasinensin is at least one selected from the group consisting of theasinensin A, theasinensin B, and theasinensin C.

The theasinensin in the present invention refers to a compound having a structure of the general formula (I) represented by theasinensin A, theasinensin B, theasinensin C, theasinensin D, and theainensin E.

(In the formula, R ₁ and R ₂ each independently represent a hydrogen atom or a galloyl group, and the configuration of the biphenyl moiety may be either R configuration or S configuration.)

In the present invention, epigallocatechin (EGC) and epigallocatechin gallate (EGCg) are compounds having the structure of general formula (II).

(In the formula, R represents a hydrogen atom or a galloyl group.)

The method of the present invention does not require a special apparatus and can easily obtain theacinensin with a high yield in a stable manner compared with the conventional method, while being simple using a general production facility.

The present invention is described in detail below.
Theasinensin obtained by the method of the present invention refers to a compound having the structure of the general formula (I) typified by theacinensin A, theasinensin B, theasinensin C, theasinensin D, and theasinensin E.

(In the formula, R ₁ and R ₂ each independently represent a hydrogen atom or a galloyl group, and the configuration of the biphenyl moiety may be either R configuration or S configuration.)

The raw material catechin used in the present invention is epigallocatechin (EGC) and / or epigallocatechin gallate (EGCg) having the structure of the general formula (II). EGC and / or EGCg used as a raw material is preferable because the amount of theacinensin obtained is higher as the purity is higher, and the amount of by-products due to the reaction is further reduced.

(In the formula, R represents a hydrogen atom or a galloyl group.)

The heterogeneous catalyst used in the present invention is an insoluble solid catalyst that can be easily removed from the reaction system, and among them, a heterogeneous platinum group catalyst is preferably used. Examples of the heterogeneous platinum group catalyst include a palladium catalyst, a platinum catalyst, a rhodium catalyst, an iridium catalyst, a ruthenium catalyst, and an osmium catalyst, and a palladium catalyst and a platinum catalyst are particularly preferable. These catalysts may be supported on a porous carrier that does not participate in the reaction such as activated carbon, alumina or zeolite. Although a dry product may be used, it is preferable to use a water-containing product moistened with water or the like in consideration of safety. Specific examples of the palladium catalyst include palladium / carbon, palladium hydroxide / carbon, palladium / alumina, palladium oxide, and palladium black. Examples of the platinum catalyst include platinum / carbon, platinum / alumina, platinum oxide, and platinum black. Can be illustrated.

The amount of the heterogeneous catalyst to be used may be determined appropriately according to other conditions (reaction pH, reaction temperature, reaction time, etc.) so that the production amount of theacinensin is increased. Is preferably 0.001 to 10 molar equivalents, more preferably 0.01 to 7.5 molar equivalents, and still more preferably 0.05 to 1.5 molar equivalents. If the amount of the catalyst is too small, the reaction rate decreases and the reaction takes a long time. If the amount is too large, the catalyst cost increases.

Examples of the reducing agent used in the present invention include tris (2-carboxyethyl) phosphine hydrochloride (TCEP · HCl), dithiothreitol, dithioerythritol, 2-mercaptoethanol, 2-mercaptoethanolamine hydrochloride, ascorbic acid, Examples include sodium alcorbate, cysteine hydrochloride, sodium sulfite and the like. TCEP · HCl and dithiothreitol having high reducing power are suitable, and TCEP · HCl having reducing power in a wide pH range is most suitable.

The amount of the reducing agent to be used is not particularly limited, but is preferably 0.1 to 10 molar equivalents, more preferably 0.2 to 5 molar equivalents, and still more preferably 0.4 to 2. mol. 5 molar equivalents. If the amount of the reducing agent is too small, the production amount of theacinensin decreases, and if it is too large, the cost of the reducing agent increases.

The solvent used in the method of the present invention is water or a solvent containing water as a main component, and it is preferable to use water alone or a buffer solution that does not contain an organic solvent because the production amount of theacinensin increases. When a mixed solvent of water and an organic solvent is used, it is preferable that water is present at least 50% or more. As an example of such an organic solvent, an organic solvent arbitrarily mixed with water such as methanol, ethanol, propanol, and acetonitrile can be used.

The amount of the solvent to be used is not particularly limited as long as the raw material catechin can be dissolved, but is 25 to 2800 parts by weight, preferably 50 to 1400 parts by weight, and more preferably 100 to 700 parts by weight with respect to 1 part by weight of catechin.

The pH of the solvent used in the present invention is not particularly limited, but the reaction is preferably performed at pH 4 or higher, more preferably pH 4-8, and still more preferably pH 5-7. When the pH is 4 or more, the amount of theacinensin produced increases. However, when the pH exceeds 7, the stability of theacinensin produced decreases.

The reaction temperature of the oxidation reaction or reduction reaction is not particularly limited as long as theacinensin can be obtained. For example, the reaction temperature is 0 to 90 ° C, preferably 10 to 60 ° C, more preferably 20 to 20 ° C. 40 ° C.

The reaction time of the oxidation reaction is not particularly limited, but the desired reaction time is 10 minutes to 8 hours, preferably 30 minutes to 6 hours. The reaction time for the reduction reaction is not particularly limited, but the desired reaction time is 1 minute to 16 hours, preferably 5 minutes to 3 hours, and more preferably 10 minutes to 1 hour.

If theacinensin produced by the method of the present invention is purified by column chromatography such as an adsorption resin, theacinensin with higher purity can be obtained.

As the adsorption resin, a synthetic adsorption resin having a high adsorption ability is preferable. Specifically, the solution after reaction is passed through a column packed with a synthetic adsorption resin to adsorb theasinensin to the resin, and the desired theacinensin is eluted from the resin using an organic solvent to obtain a purified product of theacinensin. Usually, this operation is carried out using a column filled with a synthetic adsorption resin, but it can also be carried out batchwise without using a column. As the synthetic adsorption resin usable at this time, resins such as styrene divinylbenzene, methacrylic, styrene, modified styrene, acrylic, amide, dextran, cellulose, and polyvinyl can be used. Commercially available products include, for example, styrene divinylbenzene-based Diaion HP-20, Diaion HP-21 (above, manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-2, Amberlite XAD-4 (above, Rohm and Manufactured by Haas Co., Ltd.), methacrylic Diaion HP-2MG (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-16 (manufactured by Rohm and Haas Co.) as styrene, and Sepabeads SP-207 (Mitsubishi) as modified styrene Chemical Co., Ltd.), acrylic Diaion WK-20 (Mitsubishi Chemical Co., Ltd.), amide XAD-11 (Rohm and Haas), dextran Sephadex LH-20 (Pharmacia) ), Cellulosic Indion DS-3 (Phoenix Chemicals), Polyvinyl Toyoper HW-40 (Tosoh Co., Ltd.), and the like. As other adsorbents, silica gels can be used. For example, commercially available products include silica gel 40, silica gel 60 (spherical) (manufactured by Kanto Chemical Co., Inc.), silica gel medium pressure fractionation (Yamazen Co., Ltd.) Chromatolex (manufactured by Fuji Silysia Chemical Co., Ltd.), octadecyl medium pressure fractionation (manufactured by Yamazen Co., Ltd.) and the like are examples of types in which alkyl groups such as octadecylsilane (ODS) are chemically bonded. It is possible, but it is not limited to this.

As a solvent used for elution, an organic solvent arbitrarily mixed with water such as methanol, ethanol, propanol, acetone, acetonitrile, etc. can be used in addition to water.

As a combination of the resin and the elution solvent, any of the above can be used in combination. This eluate can be dried or crystallized by a conventional method to obtain the desired theacinensin.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these. The yield of theacinensin was calculated by the following formula, yield (%) = {(number of moles of theacinensin produced or obtained) / (number of moles of raw material catechin × 0.5)} × 100. .

(Examination of reaction pH)
15 mL of 20 mM EGCg aqueous solution and 15 mL of McIlvine buffer were placed in a 50 mL conical beaker, 150 mg of 20% palladium hydroxide / carbon (about 50% water wet product, manufactured by Aldrich) was added, and the mixture was stirred at room temperature in an air atmosphere. The buffer was tested at pH 3, 4, 5, 6, 7, and 8. In tests of pH 3-6, 10, 20, 40, 60, 90, 120, 150, 180, 240, 300 and 360 minutes after the start of reaction, and in tests of pH 7 and 8, 10, 20, 40, 60, The reaction solution was sampled at 90 and 120 minutes. After diluting 500 μL of the sampled reaction solution 10 times with water, the catalyst was filtered off. To 500 μL of the filtrate, 250 μL of 3.5 mM TCEP · HCl aqueous solution was added and shaken vigorously, and 250 μL of 4 wt% acetic acid aqueous solution was added and further shaken. This solution was used as a sample for HPLC analysis, and the production amount of theacinensin A was measured by HPLC. The HPLC conditions were as follows, and a standard product of theacinensin A (manufactured by Mitsui Norin Co., Ltd.) was used for quantification.

Column: Mightysil RP18-GP (4.6 mm ID × 75 mm, particle size 3 μm, manufactured by Kanto Chemical Co., Inc.), column temperature: 40 ° C., mobile phase: (A) water / phosphoric acid / acetonitrile = 100 / 0.05 / 2.5, (B) water / phosphoric acid / acetonitrile / methanol = 100 / 0.05 / 2.5 / 50, (C) methanol, gradient program: 0 to 1.5 minutes at 100% A Until 1.5 to 12.5 minutes, A / B / C = 100/0/0 to 0/100/0 linear gradient elution, 12.5 to 25 minutes A / B / C = 0 Gradient elution linearly from 0/100/0 to 0/0/100, held at C 100% for 25-27 minutes, returned to A 100% at 27-27.5 minutes, A 100 up to 27.5-35 minutes % Equilibration, flow rate: 1 mL / min Detection wavelength: 275 nm, injection volume: 10 μL.

Table 1 shows the amount of theacinensin A produced and the yield of theacinensin A at each pH. The values in the table were the reaction time values when the production amount of theacinensin A was maximized. Above pH 4, the yield exceeded 20%.

(Catalyst comparison)
15 mL of 20 mM EGCg aqueous solution and 15 mL of McIlvine buffer (pH 5) were placed in a 50 mL conical beaker. After adding a catalyst to this solution, it stirred for 240 minutes at room temperature by air atmosphere. Three types of catalysts were used in the test, 20% palladium hydroxide / carbon (about 50% water wet product, manufactured by Aldrich) was 150 mg, 10% palladium / carbon (about 50% water wet product, manufactured by Aldrich) was 100 mg, 10 90 mg of% platinum / carbon (manufactured by Aldrich) was added. The reaction solution was sampled at 10, 30, 60, 90, 120, 150, 180 and 240 minutes after the start of the reaction, and the amount of theacinensin A produced was measured by the method described in Example 1.

Table 2 shows the production amount of theacinensin A and the yield of theacinensin A. The values in the table were the reaction time values when the production amount of theacinensin A was maximized. The highest yield was obtained when palladium hydroxide / carbon was used, but the yield of other catalysts was not as great as about 30%.

Theasinensin A, theasinensin B and theasinensin C were synthesized by the following method. Further, the reaction solution after the reduction reaction was analyzed under the HPLC conditions described in Example 1 to determine the production amount of each theacinensin. For the determination, a standard product of each theacinensin (manufactured by Mitsui Norin Co., Ltd.) was used.

(Production Example 1: Synthesis of theacinensin A)
413 mg (900 μmol) of EGCg was dissolved in 45 mL of water. To each of three 50 mL conical beakers, add 15 mL of EGCg aqueous solution and 15 mL of McIlvine buffer (pH 5), add 150 mg of 20% palladium hydroxide / carbon (about 50% water-wet product, manufactured by Aldrich), and then add 150 mg at room temperature in an air atmosphere. Stir for minutes. After the catalyst was filtered off, 287 mg (1000 μmol) of TCEP · HCl was added to the filtrate and stirred for 10 minutes (production amount 116 μmol, yield 25.7%). This reaction solution was extracted four times with 50 mL of ethyl acetate, and the ethyl acetate layer was dehydrated with sodium sulfate. After sodium sulfate was filtered off, the filtrate was concentrated to dryness on a rotary evaporator. Subsequently, 308 mg of the obtained ethyl acetate fraction was subjected to Toyopearl HW-40S column chromatography (2.2 cm ID × 28 cm, mobile phase: methanol, flow rate 5 mL / min). 137 mg of the resulting crude theasinensin A fraction was purified by preparative HPLC, and 90 mg (98.6 μmol, yield 21.9%) of theasinensin A with high purity (HPLC area percentage of 98% or more) was turned off-white powder. Got as.

(Production Example 2: Synthesis of theasinensin B)
92 mg (300 μmol) of EGC and 138 mg (300 μmol) of EGCg were dissolved in 30 mL of water. To each of two 50 mL conical beakers, add 15 mL of EGC and EGCg aqueous solution and 15 mL of McIlvine buffer (pH 5), add 150% of 20% palladium hydroxide / carbon (about 50% water-wet product, Aldrich), and then in an air atmosphere Stir at room temperature for 150 minutes. After the catalyst was filtered off, 172 mg (600 μmol) of TCEP · HCl was added to the filtrate and stirred for 10 minutes (production amount 54.6 μmol, yield 18.2%). The reaction solution was directly subjected to Diaion HP-20 column chromatography (2.0 cm ID × 13.5 cm), washed with 200 mL of water, and eluted with 200 mL of 40% methanol. 182 mg of 40% methanol fraction was subjected to Toyopearl HW-40S column chromatography (2.2 cm ID × 28 cm, mobile phase: methanol, flow rate 5 mL / min) to obtain 61 mg of crude theasinensin B fraction. The resulting crude theasinensin B fraction was purified by preparative HPLC to obtain 35 mg (45.2 μmol, 15.1% yield) of theasinensin B with high purity (HPLC area percentage of 98% or more) as an off-white powder. It was.

(Production Example 3: Synthesis of theasinensin C)
184 mg (600 μmol) of EGC was dissolved in 30 mL of water. To each of two 50 mL conical beakers, add 15 mL of EGC aqueous solution and 15 mL of McIlvine buffer (pH 5), add 150% of 20% palladium hydroxide / carbon (about 50% water wet product, manufactured by Aldrich), and then add 150 mg at room temperature in an air atmosphere Stir for minutes. After the catalyst was filtered off, 258 mg of TCEP · HCl (900 μmol was added to the filtrate and stirred for 10 minutes (production amount 142 μmol, yield 47.4%). The reaction solution was directly subjected to Diaion HP-20 column chromatography (2. 0 cm ID × 13.5 cm), washed with 200 mL of water, and eluted with 200 mL of methanol, and 173 mg of the obtained methanol fraction was purified by preparative HPLC to obtain a high purity (HPLC area percentage of 98% or more). 66 mg (108 μmol, yield 36.1%) of theasinensin C was obtained as an off-white powder.

The method of the present invention does not require any special apparatus, and can easily produce theasinensin with high yield in a stable and simple manner using general equipment.

Claims (3)

A method for producing theacinensin, comprising oxidizing epigallocatechin and / or epigallocatechin gallate in the presence of a heterogeneous platinum group catalyst and then reducing the resulting oxidation product. Method for producing Teashinenshin of claim 1 wherein heterogeneous platinum group catalyst is a palladium catalyst and / or platinum catalyst. The method for producing theasinensin according to claim 1 or 2, wherein theasinensin is at least one selected from the group consisting of theacinensin A, theasinensin B, and theasinensin C.