JP2003033196A - Method for producing ester of cinnamic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily, safely and efficiently producing esters of cinnamic acid or the like. SOLUTION: The esters of the cinnamic acid are produced by subjecting the cinnamic acids, and quinic acid or an ester of a saccharide to an ester- interchange reaction by using an enzyme in an aqueous solution of an alcohol. Cinnamic acid, hydroxycinnamic acid, caffeic acid, or ferulic acid is cited as the example of the cinnamic acids. A hexose, a pentose, or an oligosccharide or a polysaccharide containing them as constituting elements is cited as the example of the saccharide. The aqueous solution of methanol, ethanol, propanol, isopropanol, n-butanol, or phenylethyl alcohol is cited as the example of the aqueous solution of the alcohol. Tannase, chlorogenic acid esterase, or ferulic acid esterase is used as the enzyme. The esters of the cinnamic acid having excellent antiinflammatory and antitumor activities are easily produced by using the ester-exchange reaction.

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 cinnamic acid esters having excellent anti-inflammatory and anti-cancer effects.

[0002]

BACKGROUND OF THE INVENTION Cinnamic acid esters such as ethyl ester of caffeic acid generally exist widely in nature and are excellent substances having anti-inflammatory, anti-cancer, anti-oxidant, anti-bacterial and disease resistance effects on plants. It has been known. As a method for producing cinnamic acid esters, there are a method of extracting from a natural product and a method of chemically synthesizing. As a method of chemically synthesizing, a method of reacting cinnamic acid and alcohols in the presence of an acid catalyst (patent) Japanese Patent No. 3155325), a method of chlorinating cinnamic acids with thionyl chloride and the like and then reacting with alcohols (Japanese Patent Publication No. 10-501216).

However, the method of extracting from a natural product can produce only a small amount because the amount of cinnamic acid ester present in the natural product is very small. Also, in the case of chemically synthesizing, cinnamic acids such as caffeic acid, which is a raw material, are often difficult to obtain, and thus cannot be said to be suitable for production. further,
Since an acid catalyst and a chlorinating agent are used, it is necessary to remove them after the reaction, which is problematic in terms of product safety.

On the other hand, Japanese Patent Publication No. 10-501216 discloses a method for producing caffeic acid esters by reacting chlorogenic acid and the like with alcohols in the presence of a transesterification enzyme. However, in this method, an organic solvent such as hexane is used as a solvent for the transesterification reaction, and it is necessary to remove the organic solvent after the reaction, so there is still a problem in terms of product safety. In addition, many hydrophilic organic solvents serve as protein denaturants, which often reduce the charge of protein molecules, induce their aggregation and precipitation, and inactivate the enzyme.Therefore, transesterification enzymes such as chlorogenic acid are used. The reaction in the hydrophilic organic solvent or the aqueous solution thereof has not been studied so far.

[0005]

An object of the present invention is to provide a method for producing cinnamic acid esters simply, safely and efficiently.

[0006]

In order to achieve the above object, the inventors of the present invention investigated various transesterification reactions of cinnamic acids and quinic acid or saccharide esters and alcohols, which are easily available from natural products. As a result, they have found that the transesterification reaction by an enzyme such as tannase of cinnamic acid and quinic acid or saccharide ester in an aqueous solution of alcohol proceeds efficiently without deactivating the enzyme, and complete the present invention. Came to. That is, the present invention is a method for producing cinnamic acid esters, which comprises subjecting an ester of cinnamic acid and quinic acid or a saccharide to an enzymatic transesterification reaction in an aqueous alcohol solution.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below. The cinnamic acid and quinic acid or saccharide ester used as a raw material in the present invention is not particularly limited,
Those existing as a component in natural products such as plants are preferable, and examples thereof include chlorogenic acid, isochlorogenic acid, caffeoylquinic acids such as cinnaline, cinnamic acid, ferulic acid, o.
-Hydroxycinnamic acid, m-hydroxycinnamic acid, p-hydroxycinnamic acid, hesperitic acid, 3,4-dimethoxycinnamic acid, etc. and quinic acid or hexoses such as glucose, fructose, mannose, galactose, xylose, arabinose, ribose, etc. Examples thereof include pentose and esters with sugars such as oligosaccharides and polysaccharides having these components as constituents. From the viewpoint of easy availability, caffeoylquinic acids and ferulic acid arabinoxylan ester are preferable, and chlorogenic acid is most preferable. These can be obtained as commercial products or as extracts from natural products such as coffee.

When an extract is used, the crude extract can be used as it is as a raw material without isolating and purifying the cinnamic acids and quinic acid or saccharide ester contained in the extract.

The alcohol aqueous solution used in the present invention uses an alcohol corresponding to the intended cinnamic acid ester as an aqueous solution. For example, an aqueous solution of ethanol is used to produce the ethyl ester, and an aqueous solution of methanol is used to produce the methyl ester. As described above, in the present invention, alcohol can be arbitrarily selected to produce a desired cinnamic acid ester. The alcohol used is not particularly limited, but since it is used as an aqueous solution, a lower alcohol is preferable from the viewpoint of the solubility of alcohol in water. Especially methanol, ethanol,
Propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol are preferred, and ethanol is most preferred. It is also possible to use two or more alcohols as a mixture, and in this case the product is obtained as a mixture of two or more esters. When using an alcohol with low solubility in water, such as phenylethyl alcohol, in order to increase the water solubility of the alcohol, add a highly hydrophilic alcohol or a hydrophilic organic solvent to the extent that enzyme denaturation does not occur. It can also be used.

The alcohol content of the aqueous alcohol solution is not particularly limited. However, when the alcohol content is low, the yield of the desired cinnamic acid esters decreases, and when the alcohol content is high, the reaction rate decreases due to denaturation of the enzyme. . Therefore, the alcohol content is preferably 5 to 50% by weight, and 1
0 to 50% by weight is more preferable, and 20 to 40% by weight is most preferable.

The enzyme used in the transesterification reaction is not particularly limited as long as it can carry out the transesterification reaction of cinnamic acids, and examples of such an enzyme include tannase, chlorogenic acid esterase, and ferulic acid esterase. These enzymes, filamentous fungi such as Aspergillus, yeast, can be used those produced by microorganisms such as bacteria, Aspergillus genus, tannase produced by the genus Penicillium or Aspergillus genus, Penicillium genus, chlorogen produced by Botrytis genus Acid esterases are preferred, especially Aspe
tannase produced by rgillus oryze or Aspergillus j
The chlorogenic acid esterase produced by Aponics and Aspergillus niger is most preferable. As these enzymes, commercially available products can be used, and for example, tannase and chlorogenic acid esterase manufactured by Kikkoman Corporation can be used. In addition, by using ones obtained by immobilizing these enzymes by various immobilization methods, it becomes possible to further increase the reaction efficiency.

The conditions of the transesterification reaction are not particularly limited, but preferably 30 to 50 ° C. for 1 to 48 hours and 35 to 4
More preferably at 5 ° C. for 2 to 24 hours. Unreacted cinnamic acids and esters of quinic acid or saccharides and cinnamic acids such as caffeic acid that form a complex can be easily removed from the reaction solution by solvent extraction or silica gel chromatography.

[0013]

EXAMPLES The present invention will be specifically described below with reference to reference examples and examples, but the present invention is not limited to the description of the examples.

Reference Example Bioorganic & Medicinal Chemistry, Vol. 9, 199-209
According to the method described in (2001), caffeic acid and ethanol are reacted in the presence of p-toluenesulfonic acid,
Caffeic acid ethyl ester was synthesized. The obtained caffeic acid ethyl ester was used as a standard in the following examples.

Example 1 To 35.4 mg (100 μmol) of chlorogenic acid was added 10 ml of an aqueous solution of 35% by weight of ethanol, and 2 ml of an aqueous solution of 1% of chlorogenic acid esterase (manufactured by Kikkoman Corp.) was added, and the mixture was added at 40 ° C. for 4 hours. Incubated. After the incubation, the reaction solution was diluted to 100 ml with ethanol, and the reaction solution was analyzed by high performance liquid chromatography under the following measurement conditions. It was confirmed that the product was caffeic acid ethyl ester by comparing the retention time and the ultraviolet absorption spectrum of high performance liquid chromatography with that of the standard caffeic acid ethyl ester. The high-performance liquid chromatograms of the reaction liquid and the standard ethyl caffeate ester are shown in FIGS. 1 and 2, and the ultraviolet absorption spectra are shown in FIGS. 4 and 5, respectively.

High-performance liquid chromatographic analysis conditions Column: COSMOSIL 5C18-AR-II 4.6mm id X 250mm Eluent: Water: Methanol = 85:15 → 15:85 (20min; linear gradient) Flow rate: 1ml / min. Detection Instrument: UV 325nm Column temperature: 40 ℃

The results are shown in Table 1. The molar ratio of chlorogenic acid, caffeic acid, and caffeic acid ethyl ester in Table 1 is
It is a value calculated from the peak area of the chromatogram of FIG. 1 using a calibration curve prepared in advance using commercially available products of chlorogenic acid, caffeic acid, and a standard product of caffeic acid ethyl ester.

[0018]

[Table 1]

Example 2 To 35.4 mg (100 μmol) of chlorogenic acid was added 10 ml of a 25 wt% propanol aqueous solution, and 2 ml of a 1 wt% aqueous solution of chlorogenic acid esterase (manufactured by Kikkoman Corporation) was added.
Was added and incubated at 40 ° C. for 4 hours. After the incubation, a part of the reaction solution was sampled and diluted 10 times with ethanol, and high performance liquid chromatography analysis was performed under the same measurement conditions as in Example 1.
The chromatogram is shown in FIG. UV absorption spectrum of peak at retention time of 17.6 min (Fig. 6) and mass spectral data of LC / MS ((M-1) ⁺ m / z = 221)
From this, it was confirmed that this component was caffeic acid propyl ester. The reaction solution was concentrated under reduced pressure, the insoluble material was removed, and the product was separated by high performance liquid chromatography under the same conditions as above to obtain 6 mg of caffeic acid propyl ester.

Example 3 500 g of green coffee beans were finely pulverized, 5000 ml of a 30 wt% aqueous ethanol solution was added, and the mixture was heated under reflux for 2 hours.
After cooling, solid-liquid separation was performed with a centrifugal filter, and 5000 units of tannase (manufactured by Kikkoman Corp.)
The unit was the amount of enzyme that hydrolyzes the ester bond contained in tannic acid in the water at 30 ° C. for 1 μmol / min, and the mixture was stirred at 40 ° C. for 3 hours. Insoluble matter was removed from the reaction solution by centrifugation, ethanol was removed by concentration under reduced pressure, and then synthetic adsorbent (Diaion HP-20) 1
The reaction solution concentrated at SV = 1.0 was passed through a column filled with 000 ml to adsorb caffeic acid ethyl ester, and then the column was flushed with water to wash it, and then 0.5% aqueous sodium carbonate solution was flushed to remove acidic substances. Was eluted. Next, 95% by weight ethanol was passed through the column at SV = 0.5 to desorb caffeic acid ethyl ester. Ethanol was distilled off from the eluted solution under reduced pressure to obtain 4.2 g of purified caffeic acid ethyl ester. This purified caffeic acid ethyl ester was confirmed to have a purity of 90% or higher by a calibration curve prepared using a standard caffeic acid ethyl ester.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, cinnamic acid esters can be produced by subjecting an ester of cinnamic acid and quinic acid or a saccharide to an enzymatic transesterification reaction in an aqueous alcohol solution. As an aqueous alcohol solution, methanol, ethanol, propanol, isopropanol, n
-Butanol, isobutanol, sec-butanol,
By using an aqueous solution of tert-butanol, phenylethyl alcohol, etc., cinnamic acid esters having excellent actions such as anti-inflammatory and anti-cancer can be easily produced.

[Brief description of drawings]

FIG. 1 is a high performance liquid chromatogram of a reaction solution of an enzyme reaction in Example 1.

FIG. 2 is a high performance liquid chromatogram of authentic caffeic acid ethyl ester.

FIG. 3 is a high performance liquid chromatogram of a reaction solution of the enzyme reaction in Example 2.

FIG. 4 is an ultraviolet absorption spectrum of caffeic acid ethyl ester produced by the enzymatic reaction in Example 1.

FIG. 5 is an ultraviolet absorption spectrum of a standard caffeic acid ethyl ester.

6 is an ultraviolet absorption spectrum of caffeic acid propyl ester produced by the enzymatic reaction in Example 2. FIG.

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Claims (6)

[Claims] 1. A method for producing a cinnamic acid ester, which comprises subjecting an ester of cinnamic acid and quinic acid or a saccharide to an enzymatic transesterification reaction in an aqueous alcohol solution. 2. The cinnamic acids are cinnamic acid, hydroxycinnamic acid,
Caffeic acid, ferulic acid, hesperitic acid, 3,4-dimethoxycinnamic acid, The method for producing cinnamic acid esters according to claim 1, which is characterized in that. 3. The method for producing a cinnamic acid ester according to claim 1 or 2, wherein the saccharide is hexose, pentose, or an oligosaccharide or a polysaccharide having these as constituents. 4. The aqueous alcohol solution is methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-.
The method for producing a cinnamic acid ester according to any one of claims 1 to 3, which is an aqueous solution of at least one alcohol selected from the group consisting of butanol and phenylethyl alcohol. 5. The alcohol content of the alcohol aqueous solution is 5
5 to 50% by weight, The method for producing a cinnamic acid ester according to any one of claims 1 to 4, wherein 6. The method for producing a cinnamic acid ester according to claim 1, wherein the enzyme is tannase, chlorogenic acid esterase, or ferulic acid esterase.