JP2016108269A - Cyclooxygenase 2 inhibitor derived from tea - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel COX-2 inhibitor derived from tea, to provide tea drinks in which the COX-2 inhibitory activity is enhanced, and to provide a production method thereof.SOLUTION: The invention provides a cyclooxygenase 2 inhibitor containing hydroxyoctadecadienoic acid and/or hydroxyoctadecatrienoic acid as an active ingredient.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a tea-derived cyclooxygenase-2 inhibitor, a tea beverage with enhanced cyclooxygenase-2 inhibitory activity, and methods for producing them.

  A tea beverage obtained by hot water extraction of tea leaves is a favorite beverage excellent in various effects. For example, green tea drinks obtained from green tea have antibacterial and antiviral effects, blood pressure lowering effects, blood cholesterol lowering effects, antiallergic effects, cancer prevention effects, etc. It is expected that a preventive effect against hypertension, arteriosclerosis, allergy, cancer and the like can be obtained (see Non-Patent Document 1).

  It is considered that the effects of tea beverages are mainly derived from tea polyphenols contained in the beverages. Representative of tea polyphenols is catechin and its derivatives. The catechins originally contained in fresh tea leaves are (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epicatechin, (-)-gallocatechin Galate, (−)-catechin gallate, (±) -gallocatechin, (±) -catechin and the like are known. Theacinensins, theaflavins, and theafragalins produced by oxidative polymerization of the catechins in the process of processing fresh tea leaves (for example, the fermentation process for producing black tea, oolong tea, etc.) are also representative tea polyphenols. Patent Document 1).

  In the above-mentioned diseases for which a preventive effect is expected by ingesting green tea beverages, in recent years, the involvement of cyclooxygenase-2 (hereinafter sometimes abbreviated as COX-2) has received much attention. COX-2 is a cyclooxygenase (EC1.14.99.1) isozyme that produces prostaglandin G2 (Prostaglandin G2, hereinafter abbreviated as PGG2) and prostaglandin H2 (Prostaglandin H2, hereinafter abbreviated as PGH2) from arachidonic acid. One of them is characterized in that expression is induced by cytokines, growth factors, tumor promoters, endotoxins and the like. For example, celecoxib (trade name: Celecox) and rofecoxib (trade name: Biox), which are COX-2 activity inhibitors, are effective in inhibiting tumors (celecoxib) and adenoma recurrence (celecoxib, rofecoxib) in colorectal cancer clinical trials. Therefore, the application of COX-2 inhibitors to cancer prevention and treatment is expected.

  However, since celecoxib and rofecoxib were both reported to increase cardiovascular events, large-scale trials aimed at expanding indications were discontinued and rofecoxib was discontinued in the United States. Therefore, in order to obtain a COX-2 inhibitor with few side effects, a search for a new COX-2 inhibitory active ingredient has been carried out using natural products, preferably foods and beverages that are already commonly used.

Regarding tea beverages, (-)-epigallocatechin gallate (Non-patent document 2), (+)-catechin and (+)-gallocatechin (Non-patent document 3) have a COX-2 inhibitory activity so far. It was revealed. We also reported that prodelphinidin B-2,3'-O-gallate (epicatechin polymer) and theasinensin E isolated from tea leaves have the effect of suppressing PGE2 production in mouse macrophage-like cells after LPS stimulation (Patent Document 1). Patent Document 2 discloses a COX-2 inhibitor containing epitheaflavalin 3-O-gallate or purpurogallin produced from theaflavin or a derivative thereof as an active ingredient.
However, the tea catechins have no high COX-2 inhibitory activity, and it seems difficult to obtain a sufficient COX-2 inhibitory effect by ingesting a tea beverage produced by a normal method. Thus, Patent Document 3 discloses a method for enhancing the physiological activity by separating a composition that enhances the physiological activity of catechins from the hot water extract of green tea leaves and ingesting it with catechin. Inhibitory activity has not been studied.

  Under these circumstances, a new COX-2 inhibitor derived from tea and a method for enhancing the COX-2 inhibitory activity of tea beverages have been demanded.

JP 2007-330190 A Japanese Patent No. 5424424 JP 2002-220340 A JP 2007-330190 A

Kazuo Ina, Kanzo Sakata, Chemistry and Functions of Green Tea, Chinese Tea, and Black Tea, IKE Corporation Publishing, 2007 Hussain T., et al, Int. J. Cancer, 113: 660-669, 2005 Noreen Y., et al, Planta Med., 64: 520-524, 1998 Anan Toyomasa, Nakagawa Toshiyuki, Changes in Lipid Content and Fatty Acid Composition with Tea Season and Maturity, Tea Technology Research, 53, 82-87, 1977 Sato I., et al, J., Vet. Med. Sci., 69 (7): 709-712, 2007

  The present invention was made in view of the problems of the prior art, and provides a new COX-2 inhibitor derived from tea, a tea beverage with enhanced COX-2 inhibitory activity, and a method for producing them. With the goal.

  As a result of intensive studies by the inventor in order to achieve the above-mentioned object, COX-2 inhibitory activity has been greatly improved by extracting tea leaves from 40 ° C. to 100 ° C. using an aqueous solvent having a pH of 12.5 or higher. It was found that an enhanced tea beverage was obtained. Furthermore, it has been found that hydroxyoctadecadienoic acid and hydroxyoctadecatrienoic acid produced in the extraction step have excellent COX-2 inhibitory activity, and the present invention has been completed.

That is, the present invention includes the following.
[1] A cyclooxygenase-2 inhibitor containing hydroxyoctadecadienoic acid and / or hydroxyoctadecatrienoic acid as an active ingredient.
[2] The hydroxyoctadecadienoic acid is (10E, 12E) -9-hydroxy-10,12-octadecadienoic acid, (9E, 11E) -13-hydroxy-9,11-octadecadienoic acid, 10E, 12Z) -9-hydroxy-10,12-octadecadienoic acid, or (9Z, 11E) -13-hydroxy-9,11-octadecadienoic acid, or a mixture of one or more thereof [1 ]. The cyclooxygenase-2 inhibitor described in the above.
[3] The cyclooxygenase-2 inhibitor according to [1], wherein the hydroxyoctadecatrienoic acid is (10E, 12Z, 15Z) -9-hydroxy-10,12,15-octadecatrienoic acid.
[4] A food containing the cyclooxygenase-2 inhibitor according to any one of [1] to [3].
[5] A tea beverage containing the cyclooxygenase-2 inhibitor according to any one of [1] to [3].
[6] A quasi drug containing the cyclooxygenase-2 inhibitor according to any one of [1] to [3].
[7] A method for producing a cyclooxygenase-2 inhibitor from tea leaves, comprising the step of extracting tea leaves in an aqueous solvent having a pH of 12.5 or higher kept at 40-100 ° C.
[8] A method for producing a tea beverage with enhanced cyclooxygenase-2 inhibitory activity from tea leaves, comprising a step of extracting tea leaves in an aqueous solvent having a pH of 12.5 or higher kept at 40-100 ° C.
[9] The method according to [7] or [8] above, wherein the tea leaves are tea leaves heated and extracted with an aqueous solvent having a pH of 12.0 or less.

  The present invention provides a novel COX-2 inhibitor, a tea beverage having enhanced COX-2 inhibitory activity, and a method for easily producing them from tea leaves. Furthermore, foods and quasi drugs containing the COX-2 inhibitor are provided.

The HPLC chart developed by reverse phase chromatography is shown about the fraction 5 obtained by performing various chromatography with respect to the alkaline extract (Example 14) of the tea leaf for green tea (Test Example 5). The HPLC chart developed by reverse phase chromatography is shown about the fraction 6 obtained by performing various chromatography with respect to the alkaline extract (Example 14) of the tea leaf for green tea (Test Example 5).

  Hereinafter, preferred embodiments of the present invention will be described. In this document, when a numerical range is expressed using a hyphen, the numerical value before and after the hyphen is included. For example, the description “40-100 ° C.” means “40 ° C. or more and 100 ° C. or less”.

<Cyclooxygenase-2 (COX-2) inhibitor>
The COX-2 inhibitor in the present invention refers to a compound that can inhibit the enzyme activity of COX-2, ie, at least one activity of fatty acid cyclooxidase activity and hydroperoxidase activity. Although it is known that a compound capable of inhibiting the induction of transcription of COX-2 is included as a COX-2 inhibitor in a broad sense, the COX-2 inhibitor in this document means an inhibitor of the enzyme activity. The COX-2 inhibitory activity can be evaluated easily and with high sensitivity using a commercially available kit (for example, COX Inhibitor Screening Assay Kit (manufactured by Cayman Chemical)).

  The COX-2 inhibitor of the present invention comprises hydroxyoctadecadienoic acid and / or hydroxyoctadecatrienoic acid as an active ingredient. These hydroxylated unsaturated fatty acids are known to be generated by the oxidation of linoleic acid or α-linolenic acid in vivo, but have not been reported as components of tea leaves. It is also a fact that the present inventors have revealed for the first time that hydroxyoctadecadienoic acid and hydroxyoctadecatrienoic acid have remarkable COX-2 inhibitory activity. Below, these components are explained in full detail.

・ Hydroxyoctadecadienoic acid Hydroxyoctadecadienoic acid may have multiple stereoisomers, but when linoleic acid is oxidized by free radicals or enzymes in vivo, a hydroxyl group is added at the 9th or 13th position. Four types of isomers (compound 1: (10E, 12Z) -9-hydroxy-10,12-octadecadienoic acid), compound 2: (9Z, 11E) -13-hydroxy-9,11-octadecadiene Acid), compound 3: (10E, 12E) -9-hydroxy-10,12-octadecadienoic acid), compound 4: (9E, 11E) -13-hydroxy-9,11-octadecadienoic acid) . The chemical formula of each isomer is shown below (Chemical 1-4).

  These isomers are further divided into isomers (compounds 1 and 2) in which two double bonds are cis and trans, and isomers (compounds) in which both double bonds are cis, depending on the configuration of the double bond. Divided into 3 and 4). Compounds 1 and 2 are known to be tautomeric in acidic solution and exist in a molar ratio of approximately 1: 1. Similarly, compounds 3 and 4 are also present in an approximate 1: 1 molar ratio in acidic solution due to tautomerism.

Hydroxyoctadecadienoic acid contained as an active ingredient in the COX-2 inhibitor according to the present invention may be any of the above four types of stereoisomers, and may be a mixture of these stereoisomers, More preferably, compound 3 and / or compound 4 are included. The isomers in which both the double bonds are in the cis position (compounds 3 and 4) are more COX-2 than the isomer in which the double bond is in the cis and trans positions (compounds 1 and 2). This is because the inhibitory activity is much higher.
In addition, although all of the above compounds may have (R) and (S) optical isomers, hydroxyoctadecadienoic acid contained in the COX-2 inhibitor of the present invention contains (R) and ( S) Any body or a mixture of both may be used.

Hydroxyoctadecatrienoic acid that can be produced from α-linolenic acid includes (10E, 12Z, 15Z) -9-hydroxy-10,12,15-octadecatrienoic acid, (10Z, 12Z, 15Z ) -9-hydroxy-10,12,15-octadecatrienoic acid, (9Z, 12Z, 14E) -16-hydroxy-9,12,14-octadecatrienoic acid, (9Z, 12Z, 14Z) -16- Hydroxy-9,12,14-octadecatrienoic acid, (9Z, 11E, 15Z) -13-hydroxy-9,11,15-octadecatrienoic acid, (9Z, 11Z, 15Z) -13-hydroxy-9, There are 6 types of stereoisomers of 11,15-octadecatrienoic acid, and it is not necessary to distinguish these stereoisomers as the active ingredients of the COX-2 inhibitor of the present invention. Mixtures thereof can be used. Among these, it is particularly preferable to contain (10E, 12Z, 15Z) -9-hydroxy-10,12,15-octadecatrienoic acid represented by the following chemical formula (Formula 5).

Hydroxyoctadecatrienoic acid has (R) and (S) optical isomers, but hydroxyoctadecatrienoic acid contained in the COX-2 inhibitor of the present invention contains (R) and ( S) Any body or a mixture of both may be used.

<Method for producing tea beverage with enhanced COX-2 inhibitory activity>
According to the present invention, it is possible to easily produce a tea beverage with significantly enhanced COX-2 inhibitory activity by extracting tea leaves with an aqueous solvent having a pH of 12.5 or higher kept at 40-100 ° C. it can.

  Since catechins, which are the main components responsible for the effects of tea, are altered and decomposed in an alkaline solution, an alkaline solvent is usually not used in the production of tea beverages. As an exceptional production method, tea leaves are extracted with an alkaline aqueous solvent with a pH of 8.0-9.0 in order to degrade strictinin, which is the causative substance, in order to reduce secondary odors that occur during storage of tea beverages. A method is shown (Patent Document 4). However, this method also describes that the pH should be 8.9 or less in order to suppress excessive alteration of catechin (see [0023] and [0025] of Patent Document 4).

In the present invention, a general-purpose method for producing a tea beverage can be used except that an alkaline aqueous solvent having a pH of 12.5 or more is used as an extraction solvent. The requirements to be particularly noted are described in detail below.
In this document, the extraction method using an alkaline aqueous solvent having a pH of 12.5 or more according to the present invention may be referred to as “alkaline extraction method”, and in contrast to this, a pH around neutral (usually pH 4. The normal extraction method using the aqueous solvent of 5-8.0) is sometimes called “neutral extraction method”.

Tea leaves The tea leaves that can be used in the present invention may be those made from tea leaves (Cemellia sinensis), for example, green tea that is non-fermented tea and tea leaves for roasted tea (for example, tea leaves, tea leaves) Tea, etc.), tea leaves for oolong tea that are semi-fermented teas (for example, wushu narcissus, suisuisen, narcissus, etc.), tea leaves for tea that are fermented teas (for example, kimmon), and puer tea that is post-fermented tea Tea leaves (for example, Yunnan large-leaf type etc.) etc. can be used conveniently. Of these, tea leaves for non-fermented tea or semi-fermented tea are particularly preferred, and tea leaves for non-fermented tea are most preferred.
Dry tea leaves can be suitably used for extraction of tea beverages, and examples include rough tea and finished tea. As used herein, “dry tea leaves” means “tea leaves having a water content of 1-10% by weight”.

-Extraction solvent The solvent for extracting tea leaves is preferably an aqueous solvent having a pH of 12.5 or higher, more preferably a pH of 13.0 or higher, and most preferably an aqueous solvent having a pH of 13.5 or higher. From the standpoint of enhancing COX-2 inhibitory activity, it is not necessary to set an upper limit for the pH of the extraction solvent, but considering that it should be used as a beverage (afterwards neutralization), a pH of up to about 14.0 is appropriate. is there. The aqueous solvent having the pH should dissolve at least one compound selected from sodium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium hydroxide, calcium bicarbonate, and calcium carbonate in water. It can adjust suitably. Among these, sodium hydroxide, sodium hydrogen carbonate, potassium carbonate, or a mixture of sodium carbonate and potassium carbonate is preferable. Moreover, natural water, purified water (for example, ion-exchange water) etc. can be used as water used for the adjustment of the aqueous solvent.

Extraction conditions Heat the extraction solvent to 40 ° C to 100 ° C, preferably 50 ° C to 100 ° C, more preferably 60 ° C to 100 ° C, most preferably 70 ° C to 100 ° C, and add dried tea leaves And extract. In the extraction step, it is preferable to stir the mixture of the extraction solvent and tea leaves, and the extraction may be performed under pressure. The amount of the extraction solvent added to the tea leaves is not particularly limited, but an extraction solvent having a weight of about 5 to 50 times the weight of the dry tea leaves is appropriate. In the extraction solvent, the component having COX-2 inhibitory activity is rapidly extracted from the tea leaves, so it is not particularly necessary to set the extraction time, but it is preferably 1 minute or more, more preferably 3 minutes or more, and even more preferably 5 Minutes or more, most preferably 10 minutes or more. Since the increase in COX-2 inhibitory activity by extraction for 1 hour or more is very small, the upper limit of extraction time is preferably about 1 hour.

・ Neutralization treatment After the extraction step, the tea leaves are removed according to a conventional method (for example, filtration or centrifugation), and the pH of the resulting extract is adjusted to around neutral (preferably about PH5-7). A tea drink can be obtained. The pH can be adjusted suitably by adding, for example, ascorbic acid, hydrochloric acid, or an edible acid (eg, acetic acid, citric acid, etc.). The extract can be diluted as necessary, and sugars (including dextrin), (artificial) sweeteners, thickeners, emulsifiers, salts, amino acids, fragrances, colorants, acidulants, antioxidants A preservative or the like may be added. Furthermore, it is good also as a container-packed drink by heat-sterilizing.

・ COX-2 inhibitory activity of tea beverages By using the above alkaline extraction method, a tea beverage having COX-2 inhibitory activity increased by about 1.5 to 7.0 times compared to the tea beverage obtained by the neutral extraction method is obtained. Can do. In particular, when tea leaves for non-fermented tea or semi-fermented tea are used, it is also possible to obtain a tea beverage having an increase of about 4.5 to 7.0 times.
The COX-2 inhibitory activity of the tea beverage obtained by the method according to the present invention is mainly due to α-linolenic acid, linoleic acid, gallic acid, hydroxyoctadecadienoic acid, and hydroxyoctadecatrienoic acid, not catechins It is. In the extraction step using the alkaline extraction solvent of the present invention, the catechins are decomposed (partially becomes gallic acid), α-linolenic acid and linoleic acid are newly generated, and hydroxyoctadecadienoic acid is further formed from these fatty acids. It is thought that hydroxyoctadecatrienoic acid was produced. It is not known that hydroxyoctadecadienoic acid and hydroxyoctadecatrienoic acid are produced by alkali treatment of the fatty acid, and this is the mechanism that has been clarified for the first time by the present invention.

<Method for producing COX-2 inhibitor>
Hydroxyoctadecadienoic acid and hydroxyoctadecatrienoic acid can be easily produced by heat-treating tea leaves at 40-100 ° C. in an aqueous solution having a pH of 12.5 or higher. Therefore, the COX-2 inhibitor according to the present invention can be easily produced from tea leaves according to the above-mentioned “method for producing a tea beverage with enhanced COX-2 inhibitory activity”. Furthermore, unlike tea drinks that require a flavor, extraction may be performed under wider conditions when the purpose is to produce a COX-2 inhibitor. Below, the requirements which can be expanded from the manufacturing method of a tea drink are demonstrated.

・ Tea leaves As with the method for producing tea beverages, it is not necessary to specify the type of tea leaves to be used, tea leaves for unfermented tea or semi-fermented tea are particularly preferred, and tea leaves for unfermented tea are the most preferred. . However, for the purpose of producing a COX-2 inhibitor, not only dry tea leaves but also extraction residues (tea husks) produced by extraction of fresh tea and normal tea beverages (ie, neutral extraction method) are used. be able to. When using dry tea leaves or fresh tea, it is preferable that they are finely chopped. Moreover, it is preferable to use an extraction residue as a raw material from a viewpoint of effective utilization of unused resources.

-Extraction solvent The solvent used for extraction is an aqueous solvent having a pH of 12.5 or more, more preferably pH 13.0 or more, and most preferably pH 13.5 or more, as in the above-described method for producing a tea beverage. The upper limit of the pH of the extraction solvent is not particularly required, but a pH of about 15.0 is appropriate. In addition, the said extraction solvent can be suitably produced using the compound for pH adjustment illustrated with the manufacturing method of the said tea beverage, and water.

-Extraction conditions The same extraction temperature and extraction time as the tea beverage production method are suitable. Moreover, the point which extraction with stirring is preferable and the point which may extract by pressurizing are also the same. The amount of the extraction solvent added to tea leaves is the same for dry tea leaves (about 5-50 times the weight of dry tea leaves (weight / weight)), and about 10-100 times for fresh tea leaves. (Weight / weight), about 5-50 times (weight / weight) is preferable for the extraction residue.

-Purification of hydroxyoctadecadienoic acid and / or hydroxyoctadecatrienoic acid In the present invention, the tea leaves or tea leaves are removed from the extract obtained in the extraction step according to a conventional method (for example, filtration or centrifugation). It can be used as a tea leaf-derived COX-2 inhibitor. The extract may be subjected to the above-described neutralization treatment, drying treatment, concentration treatment, and the like, if necessary. Further, using a method well known to those skilled in the art, hydroxyoctadecadienoic acid and / or hydroxy Octadecatrienoic acid may be (crude) purified.
For the (crude) purification of hydroxyoctadecadienoic acid or hydroxyoctadecatrienoic acid from the extract, for example, the filtrate obtained by filtering the extract is fractionated using a high performance liquid chromatography method (HPLC method). Then, a fraction having COX-2 inhibitory activity is selected from the obtained fraction, and a known COX-2 inhibitory activity component (for example, catechins, gallic acid, linoleic acid, linolenic acid, etc. is selected from the fraction. ).

  Depending on the type of tea leaves, for example, when an extract is prepared from tea leaves for green tea using the above method and fractionated / purified by the HPLC method, about 0.1-0.4 mg of hydroxyoctadeca It is possible to obtain dienoic acid and about 0.1-0.3 mg of hydroxyoctadecatrienoic acid.

  COX-2 inhibitors according to the present invention include excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, magnesium aluminate metasilicate, etc.), disintegrants ( Calcium glycolate, etc.), lubricants (magnesium stearate, etc.), stabilizers, solubilizers (glutamic acid, aspartic acid, etc.), etc. may be mixed and formulated according to conventional methods.

  Examples of the preparation containing the COX-2 inhibitor according to the present invention include oral solid or semi-solid preparations (tablets, scattered powders, granules, gels, etc.) and oral liquid preparations (solutions, suspensions, emulsions, Syrups, elixirs, etc.) are preferred, and quasi-drugs such as troches, oral gels, dentifrices, mouthwashes, mouthwashes, mouth fresheners, etc. are exemplified. Daily use of these quasi drugs is expected to enhance the anti-inflammatory effect and improve the oral environment.

  Moreover, the foodstuff with which COX-2 inhibitory activity was enhanced by adding the COX-2 inhibitor which concerns on this invention is provided. The food suitable for this purpose is not particularly limited. For example, beverages such as soft drinks, carbonated drinks, nutrition drinks, fruit drinks, lactic acid drinks, candies, gums, chocolates, snacks, biscuits, jellies, jams, creams And confectionery such as baked confectionery, frozen confectionery such as ice cream, sorbet, shaved ice, and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited by these examples. In the Examples of the present application, the COX-2 activity inhibition rate of the test substance was measured according to the following method, and evaluated as the COX-2 inhibitory activity per unit weight of the test substance.

<Measurement of COX-2 activity inhibition rate>
COX-2 is an enzyme that produces PGG2 from arachidonic acid by cyclooxidase activity and produces PGH2 from PGG2 by hydroperoxidase activity. In the examples of the present application, the amount of PGH2 generated in the presence of COX-2, arachidonic acid, and a test substance (specifically, the amount of the reduction product) was measured using a COX Inhibitor Screening Assay Kit (Cayman Chemical). As a test substance, a tea leaf extract or a (crude) purified product thereof was freeze-dried and dissolved in dimethyl sulfoxide so as to be 500 microgram / ml. The difference between the amount of PGH2 generated when each test substance is added, with the amount of PGH2 generated when negative control (dimethylsulfoxide only) is added as 100% (= COX-2 activity is 100%) Was the COX-2 activity inhibition rate (%).

[Test Example 1: Examination of pH of extraction solvent]
The pH of aqueous solvents used in the process of extracting tea beverages and COX-2 inhibitors with enhanced COX-2 inhibitory activity from tea leaves was investigated.
Powdered commercially available green tea (finished tea) is suspended in 10 times the amount (weight / weight) of ion-exchanged water (pH 7.0) or aqueous sodium hydroxide (pH 10.0-pH 14.5). It became cloudy and extracted at 80 ° C. for 30 minutes. Thereafter, the mixture of the tea leaves and the extraction solvent was filtered, and the obtained filtrate was neutralized with hydrochloric acid to obtain an extract. Each extract was freeze-dried, and the COX-2 activity inhibition rate was measured according to the method described above for the obtained dry extract. The COX-2 activity inhibition rate obtained in each test example was expressed as a relative value to the COX-2 activity inhibition rate (Comparative Example 1) obtained when extracted with ion-exchanged water.

Table 1 shows that the extract obtained by extracting tea leaves with neutral ion-exchanged water has an activity of inhibiting COX-2 activity by 44.7% (Comparative Example 1). When the pH of the extraction solvent is increased to pH 10.0-pH 12.0, the COX-2 inhibitory activity of the resulting extract is lower than that when a neutral solvent is used (Comparative Example 2-4). Is thought to be due to alkaline decomposition of catechins. Surprisingly, however, when an extraction solvent having a pH of 13.0 was used, the inhibition rate of COX-2 activity of the obtained extract increased rapidly to 73.1% (Example 1), and as the pH further increased. The extract further increased (Examples 2 and 3), and when an extraction solvent having a pH of 14.5 was used, an extract capable of suppressing COX-2 activity by 98.8% was obtained (Example 4).
From these results, it was revealed that when tea leaves were extracted using an aqueous solvent having a pH of 12.5 or higher, components capable of inhibiting COX-2 activity could be extracted from tea leaves.

[Test Example 2: Examination of extraction temperature]
Next, the extraction temperature was examined.
The powdered commercial green tea is suspended in a 150 mM aqueous sodium hydroxide solution (pH 13.5) of 10 times the amount (weight / weight) of the tea leaves, and room temperature (25 ° C.), 40 ° C., 60 ° C., or Extraction was performed at 80 ° C. for 30 minutes. Thereafter, filtration was performed, and the obtained filtrate was neutralized with hydrochloric acid to obtain an extract. Each extract was lyophilized and the COX-2 activity inhibition rate was measured according to the method described above. The results are shown in Table 2.

From Table 2, it can be seen that the effect of using the alkaline extraction solvent cannot be obtained at room temperature, and the COX-2 activity inhibition rate of the extract is similar to that in the case of using the neutral solvent (Comparative Example 1). . On the other hand, it was shown that the COX-2 inhibitory activity of the resulting extract greatly increases when extraction is performed at 40 ° C. or higher.
Therefore, it was clarified that a temperature of 40 ° C. or higher is necessary to elute components capable of inhibiting COX-2 activity from tea leaves using an alkaline aqueous solvent having a pH of 12.5 or higher.

[Test Example 3: Examination of extraction time]
Subsequently, the extraction time was examined.
The powdered commercial green tea was suspended in 150 mM aqueous sodium hydroxide (pH 13.5) 10 times (weight / weight) of the tea leaf, and extracted at 80 ° C. for 5 minutes to 2 hours. Thereafter, filtration was performed, and the obtained filtrate was neutralized with hydrochloric acid to obtain an extract. Table 3 shows the results of freeze-drying each extract and measuring the COX-2 activity inhibition rate according to the method described above.

Table 3 shows that when extraction is performed in the alkaline aqueous solvent, most of the components that can inhibit COX-2 activity have already eluted from the tea leaves after 5 minutes.
Therefore, in a hot alkaline aqueous solvent at pH 12.5 or higher, components that can inhibit COX-2 activity are rapidly extracted from tea leaves. It became clear that components could be extracted.

[Test Example 4: Examination of types of tea leaves]
Next, the types of tea leaves were examined.
As representatives of non-fermented tea, semi-fermented tea, fermented tea and post-fermented tea, the following 3 using green tea, oolong tea, black tea, tea leaves for puer tea (all are used as finished tea, not powdered) The same extraction method was performed to obtain a dry extract.

<Extraction method>
(I) Neutral extraction To 5 g of tea leaves, 100 ml of ion-exchanged water (pH 7.0) kept at 80 ° C. was added, and extraction was performed at 80 ° C. for 30 minutes. Thereafter, filtration, concentration and freeze-drying were performed to obtain a dry extract.
(Ii) Alkaline extraction of neutral extraction residue To the residue (extraction residue) generated after filtration in (i) above, 100 ml of 150 mM aqueous sodium hydroxide solution (pH 13.5) kept at 80 ° C was added, and 30 ° C at 80 ° C was added. Extraction was performed for minutes. Thereafter, filtration, neutralization (using hydrochloric acid), concentration, and freeze-drying were performed to obtain a dry extract.
(Iii) Alkaline extraction To 3 g of tea leaves, 100 ml of a 150 mM aqueous sodium hydroxide solution (pH 13.5) kept at 80 ° C. was added, and extraction was performed at 80 ° C. for 30 minutes. Thereafter, filtration, concentration and freeze-drying were performed to obtain a dry extract.

  The weight of each dry extract obtained by the above method was measured, the weight of the dry extract obtained from 1 g of tea leaves was calculated, and the relative value to the value obtained by the method (i) ((in Table 4 a) Value) Moreover, the COX-2 activity inhibition rate per unit weight of each dry extract was measured according to the above-mentioned method, and the relative value (value (b) in Table 4) to the value obtained by method (i) was expressed. . Therefore, the product of the values (a) and (b) (= (a) × (b)) represents the relative value of the COX-2 inhibitory activity obtained from 1 g of tea leaves by each extraction method. These measured values and calculated values are shown in Table 4. Acetylsalicylic acid used as a positive control is a typical COX-2 inhibitor (product name: aspirin).

    From Table 4, in any tea leaf, the amount of extract obtained by neutral extraction is the smallest, and more extract than neutral extraction can be obtained by alkaline extraction of the extraction residue (1.5 Furthermore, it was revealed that when tea leaves were alkali extracted from the beginning, it was possible to extract the same or more components as the extract obtained by neutral extraction and alkaline extraction of the residue (Comparative Example 6 and implementation) Comparison of Examples 13 and 14, Comparative Example 7 and Examples 15 and 16, Comparative Example 8 and Examples 17 and 18, and Comparative Example 9 and Examples 19 and 20).

  Next, paying attention to the COX-2 inhibitory activity contained in each extract, in green tea and oolong tea, the alkaline extract and alkaline extract of the extraction residue per unit weight are more neutral than the neutral extract. It can be seen that COX-2 inhibitory activity is high (1.3 to 1.9 times) (Comparison between Comparative Example 6 and Examples 13 and 14, and Comparative Example 7 and Examples 15 and 16). In addition, with regard to Pu'er tea, the alkaline extract of the extraction residue was superior in COX-2 inhibitory activity per unit weight than the neutral extract (comparison between Comparative Example 9 and Example 19). .

  Furthermore, when comparing the relative value of COX-2 inhibitory activity obtained from 1 g of tea leaves (= (a) × (b) value), in any tea leaves, alkaline extraction was more effective than the value obtained by neutral extraction. The obtained value was 1.7 to 6.5 times higher, and it was shown that a large amount of COX-2 activity inhibitory substance can be extracted from tea leaves by performing alkaline extraction. Especially for green tea and oolong tea, the effect of alkaline extraction is very large, and only 2.7 times the COX-2 inhibitory activity is recovered (by neutral extraction). Furthermore, it was shown that COX-2 inhibitory activity 4.6 to 6.5 times (COX-2 inhibitory activity obtained by neutral extraction) can be obtained when alkaline extraction is performed from the beginning. Also, for black tea and puer tea, alkaline extraction of the neutral extraction residue is very beneficial, and COX-2 inhibitory activity obtained by neutral extraction is 0.6 times (black tea) and 1.7 times (puer tea) COX-2 It has been shown that further inhibitory activity can be recovered.

  From the above results, the components extracted from the tea leaves are greatly increased by using an alkaline aqueous solvent having a pH of 12.5 or higher for non-fermented tea, semi-fermented tea, fermented tea, and post-fermented tea. It was revealed that the total COX-2 inhibitory activity obtained was significantly increased (1.6 to 6.5 times that of neutral extraction) (2.4 to 3.6 times that of neutral extraction). Moreover, in any tea leaves, it was confirmed that a component having COX-2 inhibitory activity can be further extracted by alkaline extraction of the neutral extraction residue.

[Test Example 5: Identification of a component having COX-2 inhibitory activity]
As mentioned above, since catechins are almost completely decomposed under alkaline conditions at pH 12.5 or higher, the COX-2 inhibitory activity of the extract obtained by the alkaline extraction method is considered to be due to components other than catechins. It is done. Therefore, an attempt was made to identify the component responsible for the COX-2 inhibitory activity.

  Add 5 liters of 150 mM sodium hydroxide aqueous solution (pH 13.5) to 250 g of dried tea leaves (finished tea) for green tea, extract at 80 ° C for 30 minutes, and then neutralize the filtrate obtained by filtration (1N Using hydrochloric acid), concentration and freeze-drying were performed to obtain 125 g of a dry extract. 80 g of the dried extract was subjected to column chromatography using a synthetic adsorption resin (column is Diaion HP-20 (manufactured by Mitsubishi Kasei), elution solvent is water: methanol = 100: 0, 80:20, 50:50, 0: The resulting fraction was concentrated and dried, and the COX-2 activity inhibition rate was measured for each of the dried fractions. Of these, 100% methanol elution fractions that showed strong COX-2 inhibitory activity were further subjected to silica gel column chromatography (elution solvents were chloroform: methanol: water = 95: 5: 0, 90: 10: 0, 78: 20: 2, 53: 40: 7) and fractionated into 15 fractions (fractions 1-15). When the COX-2 activity inhibition rate was measured for the fraction, strong COX-2 inhibitory activity was observed in fraction 2-6.

The fractions 5 and 6 were purified by reverse phase chromatography (column: COSMOSIL Cholester (manufactured by Nacalai Tesque), elution solvent: 50% acetonitrile in 0.1% trifluoroacetic acid aqueous solution) several times, and compound I-III Isolated (Figures 1, 2). The compound I-III was measured for NMR spectrum and HR-MS spectrum to identify the chemical structure. As a result, Compound I was (10E, 12Z, 15Z) -9-hydroxy-10,12,15-octadecatrienoic acid (Compound 5), and Compound II was (10E, 12Z) -9-hydroxy-10, 12-octadecadienoic acid (compound 1) and (9Z, 11E) -13-hydroxy-9,11-octadecadienoic acid (compound 2), compound III is (10E, 12E) -9-hydroxy-10, It was found to be 12-octadecadienoic acid (compound 3) and (9E, 11E) -13-hydroxy-9,11-octadecadienoic acid (compound 4) (Table 5).
Since compounds 1 and 2 have the same physical property values measured by NMR and have tautomerism, compound I (Example 21) is considered to be an equimolar mixture of compounds 1 and 2. For the same reason, compound II (Example 22) is also considered an equimolar mixture of compounds 3 and 4.
The analysis conditions by the reverse phase chromatography and the analysis results of the NMR and HR-MS (High resolution-mass spectrometry) are shown below.

<HPLC analysis conditions>
Apparatus: Prominence UFLC system (manufactured by Shimadzu Corporation), software: LabSolutions v.5.57, column: COSMOSIL 2.5Cholester (2.5 μm) diameter 3.0 × 100 mm (manufactured by Nacalai Tesque), mobile phase: 0.1% trifluoroacetic acid Aqueous solution: acetonitrile = 50: 50, flow rate: 0.75 ml / min, column temperature 40 ° C., detection: UV 230 nm

<NMR and HR-MS property data of Compound I>
Hydroxyoctadecatrienoic acid (double bond is trans, cis, cis): Compound 5
¹ H-NMR (CD ₃ OD) δ: 0.91 (3H, t, J = 7.5 Hz), 1.32 (8H, s), 1.51 (2H, m), 1.60 (2H, m), 2.08 (2H, q, J = 6.5 Hz), 2.29 (2H, t, J = 7.5 Hz), 2.93 (2H, t, J = 7.5 Hz), 4. 01 (1H, m), 5.3-5.4 (3H, m), 5.64 (1H, dd, J = 15, 6.5 Hz), 5.98 (1H, t, J = 11 Hz), 6.53 (1H, dd, J = 15, 11 Hz). HR-ESI-TOF-MS: m / z 293.2116 [M−H] ⁻ (calcd.forC18H29O3, 293.2122), 329.184 [M + Cl] ⁻ (calcd.for C18H30O3Cl, 329.1889).

<NMR and HR-MS property data of Compound II>
Hydroxyoctadecadienoic acid (double bond is in cis and trans positions): Compound 1 and / or Compound 2
¹ H-NMR (CD ₃ OD) δ: 0.88 (3H, t, J = 6.5 Hz), 1.30-1.53 (16H, m), 1.59 (2H, t, J = 7) .5Hz), 2.19 (2H, dt, J = 7.5, 7.5Hz), 2.26 (2H, t, J = 7.5Hz), 4.07 (1H, dt, J = 6. 5, 6.5 Hz), 5.41 (1H, dt, J = 11.3, 7.5 Hz), 5.61 (1H, dd, J = 15.3, 7 Hz), 5.97 (1H, dd , J = 11, 11 Hz), 6.49 (1H, dd, J = 15.5, 11 Hz). HR-ESI-TOF-MS: m / z 295.2273 [M−H] ⁻ (calcd. For C18H31O3, 255.2279), 331.2041 [M + Cl] ⁻ (calcd. For C18H32O3Cl, 331.02045).

<NMR and HR-MS property data of Compound III>
Hydroxyoctadecadienoic acid (double bonds are all cis positions): Compound 3 and / or Compound 4
¹ H-NMR (CD ₃ OD) δ: 0.90 (3H, t, J = 7 Hz), 1.28-1.52 (16H, m), 1.59 (2H, t, J = 7 Hz), 2.08 (2H, dt, J = 7, 7 Hz), 2.26 (2H, t, J = 7.5 Hz), 4.01 (1H, dt, J = 6.5, 6.5 Hz), 5 .51 (1H, dd, J = 15, 7 Hz), 5.68 (1H, dt, J = 14.5, 7 Hz), 6.02 (1H, dd, J = 15.5, 10.5 Hz), 6.14 (1H, dd, J = 15, 10.5 Hz). HR-ESI-TOF-MS: m / z 295.2272 [M−H] ⁻ (calcd. For C18H31O3, 255.2279), 331.239 [M + Cl] ⁻ (calcd. For C18H32O3Cl, 331.02045).

The fraction 2-3 contained linoleic acid, α-linolenic acid, and caffeine, and it was confirmed that the COX-2 inhibitory activity of the fraction was due to linoleic acid and / or α-linolenic acid.
In addition, as a comparative example, a neutral extraction using ion-exchanged water (pH 7.0) as an extraction solvent is performed on the dried tea leaves for green tea, and the neutral extract is separated by the same method as the alkaline extract. Fracturing and purification were performed, and COX-2 activity-inhibiting components contained in the neutral extract were identified (Table 5).

  As shown in Table 5, the COX-2 activity inhibitory component contained in the neutral extract of green tea leaves is the most (-)-epigallocatechin gallate (about 5.08%), followed by (-)- Epigallocatechin (about 1.30%), gallic acid (about 0.26%), and linoleic acid (about 0.04%).

  In contrast, (-)-epigallocatechin gallate and (-)-epigallocatechin were not detected in the alkaline extract of tea leaves for green tea, and gallic acid (about approx. 0.64%), α-linolenic acid (about 0.48%), linoleic acid (about 0.26%), hydroxyoctadecadienoic acid (about 0.022%), and hydroxyoctadecatrienoic acid (about 0.018%) were identified. Although gallic acid and linoleic acid are also contained in the neutral extract, the content of the alkaline extract increased to about 2.5 times (gallic acid) and about 6.5 times (linoleic acid). Gallic acid is known to be produced by the degradation of tea polyphenols having a galloyl group, such as (-)-epigallocatechin gallate, but both (-)-epigallocatechin gallate and purified gallic acid preparations are the same as above. When it is exposed to alkaline extraction conditions (heat treatment at 80 ° C for 30 minutes in an aqueous solution at pH 13.5), it completely decomposes (data not disclosed). Therefore, in the alkaline extraction process of the present invention, there is a possibility that gallic acid generated from the tea polyphenol or the like may be decomposed by forming a complex with other components.

  The most noticeable result in Table 5 is the presence of α-linolenic acid, hydroxyoctadecadienoic acid, and hydroxyoctadecatrienoic acid contained in the alkaline extract. Since the amount of unsaturated fatty acids in tea leaves is extremely small (12.3-20.0mg / 100g around tea leaves) regardless of tea season and maturity (Non-patent Document 4), these free fatty acids extract tea leaves alkaline. It is thought that it was generated in the process. Although α-linolenic acid and linoleic acid have been known to have COX-2 inhibitory activity (Non-patent Document 5), hydroxyoctadecadienoic acid and hydroxyoctadecatrienoic acid have COX-2 inhibitory activity. This is the first time revealed in this application.

  In the alkaline extract, hydroxyoctadecadienoic acid (Example 21) in which the double bond is in the cis-position and trans-position is about 0.018%, and hydroxyoctadecadienoic acid in which all the double bonds are in the cis position (implementation) Example 22) contains about 0.004%, hydroxyoctadecatrienoic acid (Example 23) having hydroxyl group / double bond at trans position, cis position, and cis position at 9th position (Example 23). All fatty acids had significant COX-2 inhibitory activity. In particular, hydroxyoctadecadienoic acid (Example 22) in which all the double bonds are in the cis position and hydroxyoctadecatrienoic acid having a hydroxyl group in the 9 position and the double bonds in the trans, cis and cis positions The COX-2 inhibitory activity of (Example 23) was very high, indicating that COX-2 activity could be suppressed by 74.6% (Example 22) and 94.5% (Example 23), respectively. This is a much stronger COX-2 inhibitory activity than acetylsalicylic acid (positive control) and also stronger than (−)-epigallocatechin gallate and (−)-epigallocatechin.

  From the above results, when tea leaves are treated at 40-100 ° C. in an aqueous solvent having a pH of 12.5 or higher, gallic acid and linoleic acid, which are COX-2 inhibitory active ingredients, increase, and further, α having COX-2 inhibitory activity. -It was found that linolenic acid, hydroxyoctadecadienoic acid, and hydroxyoctadecatrienoic acid were produced. Although data is omitted, the hydroxyoctadecadienoic acid (compound 1-4) and hydroxyoctadecatrienoic acid (compound 5) are produced even when the neutral extraction residue of green tea leaves is subjected to the alkali heat treatment. It was also confirmed.

  Examples of foods and drinks and quasi drugs containing the COX-2 inhibitor according to the present invention will be given below, but the present invention is not limited thereto. The following compounding amounts all represent% by weight, and these foods and drinks and quasi drugs were produced according to a conventional method.

[Example 24: Cookies]
Ingredient Mixing amount Soft flour 40.0
Chocolate 20.0
Butter 13.0
Granulated sugar 13.0
Cocoa powder 8.0
Tea extract for green tea (dried extract of Example 14) 5.0
Milk residue
Total 100.0

[Example 25: Tea flavored beverage]
Ingredients Blending amount Roasted tea extract 0.1
Vitamin C 0.02
Tea extract for black tea (dried extract of Example 18) 0.2
Perfume
Purified water residue
Total 100.0

[Example 26: Mouthwash (mouth wash)]
Ingredient Blending amount Ethanol 8.0
Glycerin 5.0
Polyoxyethylene hydrogenated castor oil 0.7
Sodium benzoate 0.1
Hydroxyoctadecadienoic acid (Example 22) 0.01
Sodium lauryl sulfate 0.3
Perfume
Purified water remaining total 100.0

[Example 27: Mouth freshener]
Ingredient Compounding amount Ethanol
Glycerin 3.0
Polyoxyethylene hydrogenated castor oil 0.5
Saccharin sodium 0.1
Tea extract for green tea (dried extract of Example 14) 1.0
Perfume
Purified water remaining total 100.0

[Example 28: Gel for oral cavity]
Ingredient Blending amount Carboxymethylcellulose 0.2
Glycerin 40.0
Tea extract for oolong tea (dried extract of Example 16) 1.0
Sodium lauryl sulfate 0.3
Perfume
Purified water remaining total 100.0

[Example 29: Toothpaste]
Ingredient Blending amount Dibasic calcium phosphate 30.0
Glycerin 10.0
Sorbitol 20.0
Sodium carboxymethylcellulose 1.0
Sodium lauryl sulfate 1.5
Carrageenan 0.5
Saccharin sodium 0.1
Polyoxyethylene hydrogenated castor oil 0.5
Saccharin sodium 0.1
Alkaline extract of neutral extract residue of tea leaves for green tea (dried extract of Example 13) 4.0
Perfume Appropriate amount Sodium benzoate Appropriate amount
Purified water remaining total 100.0

[Example 30: Gargle solution]
Component Blending amount Ethanol 20.0
Glycerin 5.0
Saccharin sodium 0.2
Chlorhexidine 0.005
Hydroxyoctadecatrienoic acid (Example 23) 0.02
Perfume
Purified water remaining total 100.0

The COX-2 inhibitor according to the present invention is simply and rapidly produced by merely subjecting tea leaves to an alkali heat treatment (treatment at 40 to 100 ° C. at pH 12.5 or higher). As the raw material, inexpensive tea leaves with low market value and tea husks produced after the production of tea beverages (that is, the extraction residue after being heated and extracted with a neutral solvent) can be used, which is very cheap and safe to manufacture. can do. It also provides an effective utilization method for tea leaves that have been treated as industrial waste.

Claims (9)

  A cyclooxygenase-2 inhibitor comprising hydroxyoctadecadienoic acid and / or hydroxyoctadecatrienoic acid as an active ingredient.   The hydroxyoctadecadienoic acid is (10E, 12E) -9-hydroxy-10,12-octadecadienoic acid, (9E, 11E) -13-hydroxy-9,11-octadecadienoic acid, (10E, 12Z 2. The compound according to claim 1, which is) -9-hydroxy-10,12-octadecadienoic acid, or (9Z, 11E) -13-hydroxy-9,11-octadecadienoic acid, or a mixture of one or more thereof. Cyclooxygenase-2 inhibitor.   The cyclooxygenase-2 inhibitor according to claim 1, wherein the hydroxyoctadecatrienoic acid is (10E, 12Z, 15Z) -9-hydroxy-10,12,15-octadecatrienoic acid.   The foodstuff containing the cyclooxygenase-2 inhibitor in any one of Claims 1-3.   A tea beverage containing the cyclooxygenase-2 inhibitor according to any one of claims 1-3.   A quasi-drug containing the cyclooxygenase-2 inhibitor according to any one of claims 1-3.   A method for producing a cyclooxygenase-2 inhibitor from tea leaves, comprising a step of extracting tea leaves in an aqueous solvent having a pH of 12.5 or higher kept at 40-100 ° C.   A method for producing a tea beverage having enhanced cyclooxygenase-2 inhibitory activity from tea leaves, comprising a step of extracting tea leaves in an aqueous solvent having a pH of 12.5 or higher kept at 40-100 ° C.   The method according to claim 7 or 8, wherein the tea leaves are tea leaves heated and extracted with an aqueous solvent having a pH of 12.0 or less.