JP2012144532A - Hypotensive agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hypotensive agent and an angiotensin-converting enzyme inhibitor utilizing a new functionality inherent in catechin metabolites, and to provide an object containing the same for applying to the oral cavity.SOLUTION: The hypotensive agent and angiotensin-converting enzyme inhibitor are characterized by including, as active ingredient, at least one compound among a phenylcarboxylic acid having been reported as an enterobacterial metabolite of tea catechins and represented by formula (I); 5-phenyl-γ-valerolactone represented by formula (II); and 5-phenyl-4-hydroxyvaleric acid represented by formula (III).

Description

The present invention relates to a novel antihypertensive agent comprising a metabolite derived from tea catechin as an active ingredient. More specifically, the present invention relates to a phenylcarboxylic acid represented by formula (I) and 5-phenyl-γ-formula described by formula (II). An antihypertensive agent, an angiotensin converting enzyme inhibitor characterized by containing valerolactone and 5-phenyl-4-hydroxyvaleric acid described in formula (III) as active ingredients, and preventing or improving hypertension The present invention relates to an oral application object used for the purpose.

Tea-derived catechins, a kind of plant polyphenols, have antibacterial, antiviral, antioxidant, anticancer, anticariogenic, antimutation, platelet aggregation suppression, blood cholesterol lowering, blood sugar elevation suppression, antiallergic, deodorant It is known to have a very wide range of physiological activities such as action, and is used in various fields.
Studies on in vivo kinetics after ingestion of catechins have also been widely conducted. Although catechins have excellent bioregulatory effects, the amount absorbed into the living body (bioavailability) is considered to be very low, and catechins are absorbed into the living body after ingestion of catechins. It is thought that the metabolites produced by intestinal bacteria are more dominant than the metabolites themselves. In a test in which tea is actually orally administered to rats or humans, it has been reported that 5-phenyl-γ-valerolactone, phenylacetic acid, etc. are detected in urine (Non-patent Documents 1, 2, 10, 11).
There are many reports that catechins are degraded by enterobacteria in the intestine. Catechin is mainly converted into 5-phenyl-γ-valerolactone or 5-phenyl-4-hydroxy-valeric acid by intestinal bacteria. Converted to 5-phenyl-valeric acid, 3-phenyl-propionic acid, phenylacetic acid, benzoic acid, etc. It has also been reported (Non-Patent Documents 3, 4, and 5). Thus, since catechin is susceptible to microbial degradation, it is considered that the possibility of catechin being absorbed in vivo after ingestion is more likely to be a metabolite produced by microbial degradation than tea catechin.
However, regarding catechin metabolites, little is known about the functionality in vivo. Nitric oxide (NO), 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone, which is one of the compounds represented by formula (II) which is the only metabolite of catechins Only a production inhibitory effect has been reported (Non-patent Document 6).

J. et al. Agric. Food Chem. 49, 4102-4112, 2001. Chem. Res. Toxicol. 13, 177-184, 2000. J. et al. Agric. Food Chem. , 51, 5561-5566 2003. J. et al. Agric. Food Chem. 51, 6893-6898 2003. J. et al. Agric. Food Chem. , 58, 1313-1321, 2010. J. et al. Agric. Food Chem. , 58, 1296-1304, 2010. Chem. Pharm. Bull. 45, 888-893, 1997. Bioor. Med. Chem. Lett. 15, 873-876, 2005.

An object of the present invention is to provide an antihypertensive agent and an angiotensin converting enzyme inhibitor utilizing the new functionality of a catechin metabolite, and to provide an oral application object containing the same.

The inventors of the present invention have reported phenylcarboxylic acids described in formula (I), 5-phenyl-γ-valerolactone described in formula (II), and 5 described in formula (III) as the main metabolites of catechins. The physiological activity of -phenyl-4-hydroxyvaleric acid was examined. As a result, these catechin metabolites were found to have an effect of suppressing the action of angiotensin converting enzyme (ACE) involved in blood pressure increase, and the present invention was completed. The fact that tea catechin metabolites have an antihypertensive effect has not been reported so far and is a completely new finding.

That is, the invention of claim 1 of the present application is a phenylcarboxylic acid represented by the formula (I), 5-phenyl-γ-valerolactone represented by the formula (II) and 5-phenyl represented by the formula (III). An antihypertensive agent comprising at least one of phenyl-4-hydroxyvaleric acid as an active ingredient.

Formula (I)
(In formula (I), n represents an integer of 1-7, and R1, R2, and R3 each independently represent a hydroxyl group (OH) or hydrogen (H).)

Formula (II)
(R1 and R2 in the formula (II) each independently represent a hydroxyl group (OH) or hydrogen (H).)

Formula (III)
(R1 and R2 in the formula (III) each independently represent a hydroxyl group (OH) or hydrogen (H).)

The invention according to claim 2 of the present application is the blood pressure lowering agent according to claim 1, wherein n of the phenylcarboxylic acid represented by the formula (I) is an integer of 3 to 6.
The invention according to claim 3 of the present application includes a phenylcarboxylic acid represented by formula (I), 5-phenyl-γ-valerolactone represented by formula (II), and 5-phenyl- represented by formula (III). An angiotensin-converting enzyme inhibitor comprising at least one 4-hydroxyvaleric acid as an active ingredient.
Further, the invention according to claim 4 of the present application is an oral application object containing the blood pressure lowering agent according to claim 1 or 2, or the angiotensin converting enzyme inhibitor according to claim 3.
The present invention according to claim 5 is an oral application object according to claim 4 which is a food or drink.
The present invention according to claim 6 is the object to be applied to the oral cavity according to claim 4 which is a quasi-drug or a pharmaceutical product.

The present invention provides a catechin metabolite, a phenylcarboxylic acid described by formula (I), a 5-phenyl-γ-valerolactone described by formula (II), and a 5-phenyl-4-hydroxyvaleric acid described by formula (III). The present invention provides a novel antihypertensive agent and an angiotensin converting enzyme inhibitor as active ingredients. The present invention is highly versatile and can be widely applied to foods and drinks, pharmaceuticals, and the like, and can be used to prevent and / or improve hypertension.

The blood pressure lowering action shown in the present invention includes not only an effect of lowering blood pressure but also an action of suppressing an increase in blood pressure.

The catechin metabolite in the present invention can be obtained by a known organic chemical synthesis method (Non-patent Documents 6 and 7) or by combining organic synthesis methods as necessary. Moreover, it is also possible to manufacture by the microbial conversion method by intestinal bacteria (nonpatent literature 3, 4, 5).
Synthesis 2010, No. 1 9, pp1512-1520

The catechin metabolite obtained by the organic chemical synthesis method or the microbial conversion method described above may be a crude product as long as it conforms to a pharmaceutically or food acceptable standard and exhibits the effects of the present invention. Moreover, the purity of the obtained synthesized product or extract may be increased by appropriately combining known separation and purification methods.

When the target compound is produced by an organic synthesis method, various known purification means can be selected and combined. For example, a solvent extraction method using a polar solvent such as water, hot water, alcohol or the like, or a nonpolar solvent, and purification means such as centrifugation, high performance liquid chromatograph, column chromatograph, etc.

When producing catechin metabolites by microbial conversion, after incubating feces and caecal contents containing rat intestinal bacteria and growing intestinal bacteria, the cultured cells are buffered, physiological saline, water, etc. A tea catechin metabolite can be produced by adding tea catechin as a starting material (substrate) to the solution suspended in the solution and incubating under anaerobic conditions. As another method, the incubation treatment may be performed by combining several strains having the ability to convert catechins. In this case, the ability to convert tea catechins refers to the ability to convert catechins represented by formula (IV) into catechin derivatives represented by formula (V), and catechin derivatives represented by formula (V) represented by formula (II) The ability to convert to 5-phenyl-γ-valerolactone and / or formula (III) 5-phenyl-4-hydroxyvaleric acid as described in 1).
Formula (IV)
(Wherein R represents a hydroxyl group (OH) or hydrogen (H))

Formula (V)
(Wherein R1 and R2 each independently represent a hydroxyl group (OH) or hydrogen (H))

Preferred examples of microorganisms having the ability to convert the catechins represented by the formula (IV) into the catechin derivatives represented by the formula (V) include Egerterra lenta: Egerthella lenta JCM9979 strain and Adler Cruzia equolifaciens : Adlercreutzia equifaciens MT4s-5 strain (Accession No. FERM P-21738).

Further, the ability to convert the catechin derivative represented by the formula (V) into 5-phenyl-γ-valerolactone represented by the formula (II) and / or 5-phenyl-4-hydroxyvaleric acid represented by the formula (III) Preferable examples of the microorganisms include Eubacterium bacteria and Clostridium bacteria (bacteria belonging to Flavonifractor bacteria as a new scientific name), and more preferably, Eubacterium plautii (new). Scientific name; Flavoni fractor pluti: ATCC 29863 strain, Eubacterium pluti: Eubacterium plutii (new scientific name: Flavoni fractor plutii) MT42 strain (FERM P-21765) and Clostridium orbiviruses thin dense: Clostridium orbiscindens (new names; Flavobacterium two Hula restrictor-Purauti: Flavonifractor plautii) can be mentioned ATCC49531 shares.

A microorganism capable of converting a catechin represented by formula (IV) into a catechin derivative represented by formula (V) described in paragraph [0013], and a catechin represented by formula (V) described in paragraph [0014] Cultured cells in which a microorganism having the ability to convert a derivative-containing material into 5-phenyl-γ-valerolactone described in formula (II) and / or 5-phenyl-4-hydroxyvaleric acid described in formula (III) coexists By adding the catechins represented by the formula (IV) and / or a catechin-containing material as a starting material (substrate) to the suspension or culture solution and incubating under anaerobic conditions, the above-mentioned 5 -Phenyl-4-hydroxyvaleric acid and / or 5-phenyl-γ-valerolactone-containing product can be obtained. In this incubation treatment, after culturing the microorganism, the cultured cells are collected and suspended in a buffer solution, physiological saline, water, etc., and then a substrate is added or the microorganism is cultured. Or it can carry out by adding a substrate to the culture solution which passed for a fixed period after the culture | cultivation start. Alternatively, by adding a catechin and / or a catechin-containing material represented by the formula (IV), which is a starting material (substrate), to a culture solution in which the microorganism grows, an incubation treatment is performed under anaerobic conditions. It is also possible to obtain compounds of formula (II) and / or formula (III).

Of the catechins represented by formula (IV), in order to microbially convert catechins in which R is hydrogen into catechin derivatives in which R1 and R2 in formula (V) are hydrogen, hydrogen and / or Or it is desirable to add formic acid. In addition, in order to microbially convert catechins of formula (IV) in which R is a hydroxyl group into catechin derivatives in which R1 of formula (V) is a hydroxyl group and R2 is hydrogen, hydrogen and / or formic acid is added to the medium. There is a need to. Furthermore, when hydrogen and formic acid are not added to the medium, hydrogen and / or formic acid-producing microorganisms can coexist. Examples of such microorganisms include Escherichia coli.

When culturing the microorganisms described above, they are inoculated into a nutrient source-containing medium in which the microorganisms can grow and cultured under anaerobic conditions. A general anaerobic microorganism culture method can be employed for culturing microorganisms for obtaining cultured cells and culturing microorganisms in the presence of a substrate. In addition, it is desirable to carry out the incubation under anaerobic conditions also when incubation is performed in the presence of the substrate after collecting the cultured cells. The medium used for the culture is not particularly limited as long as the microorganism can grow therein. For example, GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) can be used.
The culture conditions can be appropriately selected within the range in which the microorganism can grow. Usually, it is pH 6.0-7.5, 35-40 degreeC, Preferably it is pH 6.5-7.3, 37-39 degreeC. The culture time is usually 24 to 120 hours, preferably 48 to 72 hours. The various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, and the like, and optimal conditions can be selected.

In addition, when producing a tea catechin metabolite by microbial conversion, an extract containing several types of catechin metabolites is obtained at the time of preparation, but a highly purified tea catechin metabolite can be obtained by repeated purification. The degree of purification can be adjusted by selecting and combining various known purification means in the same manner as in the organic synthesis method. For example, separation / purification by solvent extraction using ethyl acetate, ether, butanol, method using desorption of synthetic resin adsorbent, column chromatography such as silica gel or high performance liquid chromatography alone or in combination as appropriate. The content of tea catechin metabolites in the extract can be adjusted.

In the case of producing a catechin metabolite by microbial conversion, the type of catechin metabolite contained in the extract can be adjusted by adjusting the degree of purification. From the viewpoint of enhancing the blood pressure lowering action, an extract containing several types of catechin metabolites may be used as a blood pressure lowering agent.

The form of use of the antihypertensive agent or angiotensin converting enzyme inhibitor of the present invention is not limited, and examples thereof include liquids, powders, granules, etc., and forms usually used as foods or nutritional supplements such as liquids, suspensions, etc. It can be used for suspensions, powders, granules, fine granules, tablets, capsules, syrups, elixirs and spirits. In the present invention, the oral application target may be in any form as long as it can be contained in the mouth.

The antihypertensive agent or angiotensin converting enzyme inhibitor of the present invention may be used as another antihypertensive agent (for example, α blocker, β blocker, αβ blocker, ACE inhibitor, angiotensin II receptor antagonist, Ca blocker, diuresis). Drugs, antipsychotics, etc.) and various vitamin agents (for example, vitamin A, vitamin B ₁ , B ₂ , B ₆ , B ₁₂ , vitamin C, vitamin D, vitamin E, etc.) .

In addition, when the antihypertensive agent or angiotensin converting enzyme inhibitor of the present invention is used as a medicine, it is not particularly limited as long as it can be contained in the mouth with a medicine stored in the Japanese Pharmacopoeia. Orally acceptable carriers can be added to make oral preparations. Examples of the dosage form include tablets, granules, fine granules, pills, powders, capsules, troches, chewables, liquids (drinks) and the like.

The quasi-drug is not particularly limited as long as it is a quasi-drug specified by the Minister of Health, Labor and Welfare and can be contained in the mouth. Examples thereof include oral liquids, health drinks, vitamin-containing health agents, and the like.

The food containing the antihypertensive agent or angiotensin converting enzyme inhibitor of the present invention may be in any form, for example, a liquid form such as an aqueous solution, a turbid substance or an emulsion, a gel form or a paste form. Or a solid form such as a supplement such as powder, granule, capsule or tablet.

Examples of the food and drink containing the blood pressure lowering agent or angiotensin converting enzyme inhibitor of the present invention include instant foods (immediate noodles, cup noodles, retort / cooked foods, cooked canned foods, microwave foods, instant miso soup and sucked foods, soup canned foods) , Freeze-dried foods, etc.), carbonated drinks, citrus fruits (grapefruits, oranges, lemons, etc.), juices, fruit drinks, soft drinks with fruit juices, citrus fruit drinks, fruit drinks with fruits, tomatoes, peppers, celery, cucumbers, carrots , Vegetable drinks including vegetables such as potatoes, asparagus, soy milk and soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutrition drinks, alcoholic drinks and tobacco, etc., macaroni・ Spaghetti, noodles, cake mix, deep-fried flour, bread crumbs, gyoza skin Flour products, caramel candy, chewing gum, chocolate, cookies and biscuits, cakes and pie, snacks and crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, soy sauce, miso, sauces, tomato processed seasoning , Basic seasonings such as mirins, vinegars, sweeteners, flavor seasonings, cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices and other complex seasonings and foods, butter, margarine , Mayonnaise, oils and fats such as vegetable oil, milk / processed milk, milk beverages, yogurt, lactic acid bacteria beverages, cheese, ice creams, prepared powdered milk, milk and dairy products such as cream, frozen material, semi-cooked frozen Food, frozen food such as cooked frozen food, canned fish, canned fruits and pastes, fish ham and sausage Marine products, marine products such as marine products, marine products, canned fish, canned fish, canned meat, canned fruits, jams, marmalades, pickles, boiled beans, dried agricultural products, cereals ) And other processed agricultural products, baby foods, and commercial foods such as sprinkles and green tea paste.

Although the compounding quantity of the composition of this invention with respect to food / beverage products is not restrict | limited in particular, A compounding quantity is suitably set with the food / beverage products used as object. Generally, it is preferably 0.01 to 20% by weight in the final product, more preferably 0.05 to 10% by weight, and even more preferably 0.1 to 5%.

Hereinafter, the present invention will be described in more detail based on production examples and test examples, but the present invention is not limited to these examples.

Production Example 1: 5- (3 ′, 4 ′, 5′trihydroxyphenyl) valeric acid [5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -valeric acid] and 5- (3 ′, Method for Producing 5′-Dihydroxyphenylvaleric Acid [5- (3 ′, 5′-dihydroxyphenyl) -valeric acid] Feces (3.6 g) were collected from three Wistar rats ( Nada , Charles River Japan Co., Ltd.). GAM bouillon (composition (in 1 L)): 10 g peptone, 3 g soybean peptone, 10 g proteose peptone, 13.5 g digested serum powder, 5 g yeast extract, 2.2 g meat extract, 1.2 g liver extract, 3 g glucose, phosphorus Potassium dihydrogen 2.5 g, sodium chloride 3 g, soluble starch 5 g, L-cysteine hydrochloride 0.3 g Sodium thioglycolate 0.3 g, pH 7.1, manufactured by Nissui Pharmaceutical Co., Ltd.) was subjected to anaerobic culture for 2 days at 37 ° C. The culture solution was placed in a sterilized centrifuge tube at 9000 rpm for 20 minutes. The supernatant was discarded, 200 ml of sterilized water was added to the cells, the cells were washed, and then centrifuged again, and 200 mg of epigallocatechin (EGC) was added to the obtained cells. The resulting suspension was suspended in 150 ml of 0.2 M phosphate buffer (pH 7.2) and incubated under anaerobic conditions at 37 ° C. 50 ml of the solution was recovered on the second day of culture, and 2 M hydrochloric acid water was added. 5 ml was added to adjust the pH to 5.5, and once frozen, and after 4 days of culture, 5 ml of 2M hydrochloric acid was added to the remaining 100 ml of the solution to adjust the pH to 5.5. 15000 × g, 20 minutes), and the cells were removed, extracted three times with 100 ml of ethyl acetate, and the organic solvent phases were combined and used in an evaporator to remove the organic solvent and dry. The dried solid was dissolved in a 10% aqueous methanol solution and subjected to preparative HPLC, and the preparative HPLC conditions are described below.

Column used: CAPCELLPAK MG (20 × 150 mm, 5 μm, manufactured by Shiseido Co., Ltd.), flow rate: 9.8 ml / min, column temperature: 40 ° C., solvent A; methanol: water: acetic acid (10: 90: 1 volume ratio ( v / v / v)), solvent B; methanol: acetic acid (100: 1 volume ratio (v / v)), gradient; 0 min: A95% B5%, 30 min: A60% B40%, 40 min: A20% B80%, 43 minutes: A10% B90%, 45 minutes: A95% B5%, detector: UV 230 nm.
The fractionated fractions were each subjected to LC / MS analysis, and fractions containing metabolites were confirmed. The LC / MS analysis conditions are described below.

Column used: CAPCELLPAK C18 MG (2.0 × 100.0 mm, 5 μm, manufactured by Shiseido Co., Ltd.), column temperature: 40 ° C., flow rate: 0.2 ml / min, mobile phase A (water / acetonitrile / acetic acid = 100 / 2.5 / 0.1 volume ratio (v / v / v)), B (water / acetonitrile / methanol / acetic acid = 50 / 2.5 / 50 / 0.1 volume ratio (v / v / v / v) ), Gradient: 0-2 minutes Isocratic A 100%, 2-25 minutes Linear gradient A 100-0%, B0-100%, 25.1-33 minutes Isocratic A 100%, detector: UV 270 nm, interface: ESI, polarity: negative.

From the results of LC / MS analysis, a fraction containing a metabolite was confirmed and concentrated to dryness with an evaporator. 5 ml of pure water was added to the dried product to dissolve it, and the solution was again concentrated to dryness under reduced pressure. This operation was repeated three times to completely remove the acid contained in the organic solvent phase. A small amount of pure water was added to the dried product to dissolve it and subjected to lyophilization. Thereby, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -valeric acid [5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -valeric acid]: 15 mg (HPLC area percentage 98.0%) ), 5- (3 ′, 5′-dihydroxyphenyl) -valeric acid [5- (3 ′, 5′-dihydrox)
yphenyl) -valeric acid]: 68.6 mg (HPLC area percentage: 98.0% or more).

Production Example 2: Process for producing 5- (3′-hydroxyphenyl) -valeric acid [5- (3′-hydroxyphenyl) -valeric acid], 3-hydroxyphenylacetic acid [3-hydroxyphenyl acid acid] by microbial conversion Wistar system Cecal contents (2.0 g) were collected from 3 rats (Nada, Charles River Japan Co., Ltd.), placed in 200 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.), and subjected to anaerobic culture at 37 ° C. for 4 days. . The culture solution was put into a sterilized centrifuge tube and centrifuged at 9000 rpm for 20 minutes. The supernatant was discarded, and 100 ml of sterilized water was added to the cells to wash the cells, and then centrifuged again. The obtained microbial cells were suspended in 150 ml of 0.2 M phosphate buffer (pH 7.2) containing 100 mg of (−)-epicatechin (EC). Incubation treatment was performed under anaerobic conditions at 37 ° C. After 10 days of culture, hydrochloric acid was added to adjust the pH to 4.0. The culture solution was subjected to centrifugation (15000 × g, 20 minutes) to remove the cells. Extraction was performed 3 times with 100 ml of ethyl acetate, and then the organic solvent phases were combined and subjected to an evaporator to remove the organic solvent and dry. The dried solid was dissolved in a 10% aqueous methanol solution and subjected to preparative HPLC. Preparative HPLC conditions are described below.

Column used: YMC-Pack ODS-A (20 × 150 mm, 5 μm, manufactured by YMC), flow rate: 9.8 ml / min, column temperature: 40 ° C., solvent A; methanol: water: acetic acid (10:90: 1 volume ratio (v / v / v)), solvent B; methanol: acetic acid (100: 1 volume ratio (v / v)), gradient; 0 min: A80% B20%, 15 min: A60% B40%, 30 Minute: A30% B70%, 35 minutes: A80% B20%, detector: UV 230 nm.
The fractionated fractions were analyzed under the same conditions as the LC / MS analysis method described in paragraph [0030] of Production Example 1 to confirm metabolites. The fraction containing metabolites was treated and freeze-dried in the same manner as in the method described in paragraph [0031] of Production Example 1. Thereby, 5- (3′-hydroxyphenyl) -valeric acid [5- (3′-hydroxyphenyl) -valeric acid]: 14.8 mg (HPLC area percentage: 98.0% or more), 3-hydroxyphenylacetic acid [3- Hydroxyphenyl acid acid]: 5.0 mg (HPLC area percentage 98.0% or more) was obtained.

Production Example 3: 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) from epigallocatechin in the presence of Egerterra Renta JCM9979 strain and Clostridium orubicindens (Flavonifracter plowty) ATCC 49931 strain -4-Hydroxyvaleric acid and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone production method Egerterra Renta JCM9979 strain and Clostridium orubicindens (flavonifracter -Prouty: ATCC49531 strain was inoculated into 30 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) and anaerobically cultured at 37 ° C for 48 hours. The culture solution of the two strains was added to 100 ml of the same medium containing 221.4 mg of epigallocatechin. This medium was anaerobically cultured at 37 ° C. for 3 days. After removing the cells by high-speed centrifugation (15000 × g, 10 minutes, 10 ° C.), phosphoric acid was added to the obtained supernatant to adjust the pH to 1.5. To this solution, 100 ml of ethyl acetate: butanol (1: 1, v / v) was added and mixed well. After separating into two phases with a centrifuge (5000 × g, 5 minutes), the organic solvent phase was recovered. This extraction operation was repeated three times. To an organic solvent phase (300 ml), an equal amount (300 ml) of a 0.1 M aqueous sodium carbonate solution (containing 0.1% sodium ascorbate) was added and mixed well, and then organically separated by a centrifuge (5000 × g, 5 minutes). It was divided into a solvent phase and an aqueous phase. The organic solvent phase is treated in the same manner as described in Production Example 1, and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone [5- (3 ′, 4 ′, 5 ′). -Trihydroxyphenyl) -γ-valerolactone] was obtained in an amount of 31 mg (HPLC area percentage: 98.0% or more). The aqueous phase (300 ml) was adjusted to pH 7.0 by adding acid, and then concentrated under reduced pressure until it reached about 30 ml. Five times the amount of ethanol was added to this concentrated solution, and insoluble matters were removed by high-speed centrifugation (15000 × g, 15 minutes, 4 ° C.). The obtained supernatant was further concentrated to about 1 ml under reduced pressure, and adjusted to pH 2.0-3.0 with 2M HCl. This solution was subjected to preparative HPLC. Preparative HPLC conditions are described below.

Column: CAPCELLPAK MG (20 × 150 mm, 5 μm, manufactured by Shiseido Co., Ltd.), flow rate 9.5 ml / min, temperature 40 ° C., solvent A: acetonitrile: methanol: water (5: 5: 90 volume ratio (v / v / v)), solvent B: acetonitrile: methanol: water (5:60:35 volume ratio (v / v / v)), gradient; A80% B20% isocratic, detector: UV 230 nm.

After fractionation, the fraction containing 5- (3 ', 4', 5'-trihydroxyphenyl) -4-hydroxyvaleric acid was further subjected to cation exchange treatment. To a fraction (50 ml) containing 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid, ½ amount of pure water (25 ml) was added and concentrated under reduced pressure. The solvent in the solution was removed to make an aqueous solution. This aqueous solution was passed through a cation exchange resin (diaion SK1B sodium type, 10 × 65 mm) equilibrated with pure water, and further eluted with pure water. The resulting solution was concentrated under reduced pressure and freeze-dried. As a result, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid [5- (3 ′, 4 ′, 5′-trihydroxyphenyl) was obtained. -4-hydroxyvaleric acid] sodium salt (65 mg, HPLC area percentage: 98.0% or more) was obtained.

Production Example 4: 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyx in the presence of Egerterra Renta JCM9979, Eubacterium pluti (Flavonifracter pruti) ATCC 29863, and Escherichia coli K12 Method for producing valeric acid and 5- (3 ', 5'- dihydroxyphenyl )-[gamma] -valerolactone-containing material Egertera Renta JCM9979 strain into 30 ml GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) Inoculated and anaerobically cultured at 37 ° C. for 48 hours to obtain a preculture solution. Escherichia coli K12 and Eubacterium pluti (Flavonifracter pruti) ATCC 29863 were anaerobically cultured in 10 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) for 24 hours as a preculture. (-)-Pre-culture solution of the above three strains was added to 100 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 290 mg of epigallocatechin and subjected to a conversion reaction by anaerobic culture at 37 ° C. for 48 hours. (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 5′-dihydroxyphenyl) -γ-valerolactone-containing product were obtained. The culture solution was centrifuged at high speed (15000 × g, 10 minutes, 10 ° C.) to remove the cells. Hydrochloric acid was added to the supernatant to adjust the pH to 3.5, followed by extraction with 120 ml of ethyl acetate three times. To the ethyl acetate phase (about 350 ml), 100 ml of 40 mM sodium carbonate aqueous solution was added and mixed well. The aqueous phase was recovered by separating into two phases by centrifugation (5000 × g, 5 minutes). Again, 100 ml of 40 mM aqueous sodium carbonate solution was added to the ethyl acetate phase, and the two phases were similarly separated. After adjusting the pH of the collected aqueous sodium carbonate solution to around 7.0, the solution was concentrated under reduced pressure to about 5 ml. Further, hydrochloric acid was added to adjust the pH to 2.0 to 5.0, and after high speed centrifugation (15000 × g, 20 minutes, 4 ° C.), the supernatant was subjected to preparative HPLC. Preparative HPLC was performed according to the method described in paragraph 3 of Production Example 3. However, the gradient is gradient; 0 min: A80% B20%, 5 min: A80% B20%, 15 min: A20% B80%, 18 min: A20% B80%, 19 min: A80% B20%, 24 min: A80% B20%.

After fractionation, the fraction containing 5- (3 ′, 5′-dihydroxyphenyl) -γ-valerolactone was subjected to concentration and lyophilization according to the method described in paragraph [0031] of Production Example 1 to obtain 45 mg. It was. Moreover, the fraction containing 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid was subjected to cation exchange treatment described in paragraph [0036] of Production Example 3 and then subjected to lyophilization. . As a result, 103 mg (HPLC area percentage 98) of sodium salt of 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid [5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid] was obtained. 0.0% or more).

Production Example 5: 5- (3 ′, 4′-Dihydroxyphenyl) -γ in the Coexistence of Egerterra Renta JCM9979 Strain and Eubacterium Pluti (Flavonifracter Pruti) MT42 (FERM P-21765) Strain -Method for producing valerolactone [5- (3 ', 4'-dihydroxyphenyl) -gamma-valerolactone] Inoculated Egerterra Renta JCM9979 strain in 30 ml GAM bouillon (Nissui Pharmaceutical Co., Ltd.) And anaerobic culture at 37 ° C. for 48 hours. Further, Eubacterium pluti (Flavonifracter pruti) MT42 strain was anaerobically cultured with 10 ml of GAM bouillon (Nissui Pharmaceutical Co., Ltd.) for 24 hours. (-)-Epicatechin 215 mg containing 100 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) is added with the above two strains of pre-cultured solution, anaerobically cultured at 37 ° C. for 48 hours, and converted to 5- ( 3 ′, 4′-Dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) -γ-valerolactone were produced. The culture solution was centrifuged at high speed (15000 × g, 10 minutes, 10 ° C.) to remove the cells, and then hydrochloric acid was added to the supernatant to adjust the pH to 3.5. This supernatant was extracted three times with 120 ml of ethyl acetate, and the ethyl acetate phase was concentrated to dryness under reduced pressure. As a result of dissolving the precipitate in a small amount of pure water and freeze-drying, 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid, 5- (3 ′, 4′-dihydroxyphenyl) -γ -506 mg of valerolactone-containing material was obtained.

The obtained content was dissolved in 30 ml of pure water, and then adjusted to pH 1.5 by adding 2M hydrochloric acid. This aqueous solution was allowed to stand at room temperature for 24 hours to convert 5- (3 ', 4'-hydroxyphenyl) -4-hydroxyvaleric acid into 5- (3', 4'-hydroxyphenyl) -γ-valerolactone. After adjusting the pH of the aqueous solution to 4.0 with 1 M sodium carbonate, preparative HPLC and lyophilization were performed by the method described in Production Example 1. As a result, 73 mg (HPLC area percentage: 98.0% or more) of 5- (3 ′, 4′-dihydroxyphenyl) -γ-valerolactone [5- (3 ′, 4′-dihydroxyphenyl) -γ-valerolactone] was obtained. Obtained.

Test Example 1: Angiotensin converting enzyme (ACE) inhibitory activity test for catechin metabolites The ACE activity inhibitory test is described below.
Among the catechin metabolites used for the test, 5- (3 ′, 4′-dihydroxyphenyl) -γ-valerolactone [5- (3 ′, 4′-dihydroxyphenyl) -γ-valelalactone], 5- (3 ', 5'-dihydroxyphenyl) -4-hydroxyvaleric acid [5- (3', 5'-dihydroxyphenyl) -4-hydroxyvaleric acid], 5- (3 ', 5'-dihydroxyphenyl) -γ- Valerolactone [5- (3 ′, 5′-dihydroxyphenyl) -γ-valelalactone], 5- (3′-hydroxyphenyl) -valeric acid [5- (3′-hydroxyphenyl) -valeric acid], 5- (3 ', 4', 5'-trihydroxyphenyl) -valeric acid [5- (3 ', 4', 5'-t rihydroxyphenyl) -valeric acid], 5- (3 ′, 5′-dihydroxyphenyl) -valeric acid [5- (3 ′, 5′-dihydroxyphenyl) -valeric acid], 5- (3 ′, 4 ′, 5 '-Trihydroxyphenyl) -γ-valerolactone [5- (3', 4 ', 5'-trihydroxyphenyl) -γ-valeractrone], 5- (3', 4 ', 5'-trihydroxyphenyl) -4 —Hydroxyvaleric acid [5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid] prepared by the method described in Production Examples 1 to 5 was used.
5-phenyl-valeric acid [6-phenyl-valeric acid], 6-phenylhexanoic acid [6-phenyl-hexanoic acid], 2-hydroxyphenyl acetic acid [2-hydroxyphenyl acetic acid], 4-hydroxyphenyl acetic acid [4-hydroxyphenyl] [Acetic acid], 3- (4′-hydroxyphenyl) -propionic acid [3- (4′-hydroxyphenyl) -propionic acid], 3-hydroxyphenyl acetic acid [3-hydroxyphenyl acetic acid] used by ACROS ORGANICS did.
The metabolites used as inhibitors were dried under heat at 40 ° C. under reduced pressure overnight and then weighed. Each substance was dissolved in 1 mM phosphate-citrate buffer (pH 4.0) containing 5% methanol to prepare a 90-20 mM inhibitor solution. Each inhibitor solution was further diluted in 5 steps with 1 mM phosphate-citrate buffer (pH 4.0) containing 5% methanol, and inhibitors having different concentrations stepwise within a concentration range of 3 to 20 mM. A solution was prepared.
To 150 mM HEPES (4- (2-hydroxyethyl) -1-piperazine etheric acid) buffer (pH 8.3), 450 mM sodium chloride was added and dissolved. To this solution, 4 mM Hippuryl-L-histidyl-L-leucine (HHL) as a reaction substrate was added and dissolved to obtain a substrate solution.
Add 200 μl of substrate solution, 40 μl of 0.1 U / ml Angiotensin Converting Enzyme (ACE) (manufactured by SIGMA-ALDRICH), 60 μl of inhibitor solution (metabolite solution) to a microtube, and in a 37 ° C. water bath for 30 minutes Incubated. The reaction was stopped by adding 200 μl of 1M acetate buffer (pH 4.0), and then subjected to LC / MS analysis. Analysis was performed by a selected ion mode (SIM), and hippuric acid produced by the reaction was measured. The LC / MS analysis conditions for this hippuric acid measurement are described below.

Column: CAPCELLPAK MG (2.0 × 100.0 mm, 5 μm, manufactured by Shiseido Co., Ltd.), flow rate: 0.2 ml / ml, temperature: 40 ° C., mobile phase A: 10% acetonitrile, 0.2% formic acid, mobile phase B: 40% acetonitrile, 0.2% formic acid gradient conditions: 0-3 min A100% B0%, 3-15 min A50% B50%, 15-16 min A100% B0%, 16-20 min A100% B0%
Detector: Polarity: Positive, Selected ion mode (SIM) m / z 180 The measurement results were plotted on a graph, an approximate curve by exponential approximation was created, and IC50 value was calculated therefrom.

(result)

As shown in Table 1, ACE inhibitory activity was confirmed for all metabolites tested. In particular, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -valeric acid (R1, R2, and R3 are hydroxyl groups, n is an integer 4) and 5- (3 ′) which are phenylcarboxylic acids described in formula (I) , 5′-dihydroxyphenyl) -valeric acid (R1, R3 are hydroxyl groups, R2 is hydrogen, n is an integer 4), 5- (3′-hydroxyphenyl) -valeric acid (R1 is hydroxyl group, R2, R3 are hydrogen, n Is an integer 4), and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valelalactone (R1 and R2 are hydroxyl groups) which is 5-phenyl-γ-valerolactone described in formula (II) High ACE inhibitory activity was confirmed.

Test Example 2: Blood pressure lowering test using SHR rats (100 mg / kg)
(A) Animals and methods of use 10-week-old male spontaneously hypertensive rats (SHR) were purchased and raised for 4 weeks in order to acclimate to the breeding environment. During the rearing period, blood pressure was measured discontinuously three times or more using a commercially available non-invasive automatic blood pressure measuring device for rats BP-98A (manufactured by Softron), and evaluation was performed after acclimatization to blood pressure measurement operation. The test was conducted. All rats were housed under conditions of room temperature 23 ° C. ± 3 ° C., relative humidity 50 ± 5%, and irradiation time 12 hours (rat room breeding room).

(B) Administration method and measurement method 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone [5- (3 ′, 4 ′, 5 ′) prepared according to the method described in Production Example 3. -Trihydroxyphenyl) -γ-valerolactone] was dissolved in physiological saline, and orally administered to SHR rats using a metal gastric sonde so as to be 100 mg (100 mg / kg) per kg body weight of the rats. The dose was 1 ml / animal, and orally administered to 8 rats as an administration group. As a control group, 1 ml of physiological saline was administered to 8 rats. At 2 hours and 4 hours after administration, systolic blood pressure was measured using a non-invasive automatic blood pressure measuring device for rats BP-98A (manufactured by Softron). The blood pressure before administration was also measured in advance.

(C) Statistical processing method The difference of systolic blood pressure before and after administration was calculated from the obtained measurement results. The obtained result was shown by the mean value (mean) and standard error (SE), and the significant difference before and after administration was examined by welch's t-test. Significance levels were set to * p <0.05 and ** p <0.01.

(result)

When 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone was orally administered to SHR rats at a dose of 100 mg / kg, it was administered to the systolic blood pressure before administration in the administration group. It was observed that the systolic blood pressure for the next 4 hours was significantly reduced.

Test Example 3: Blood pressure lowering test using SHR rats (150 mg / kg)
(A) Animal and method to be used It was carried out according to the animal and method to be used described in paragraph [0045].

(B) Administration method and measurement method The test was conducted according to the administration method described in paragraph [0046]. However, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone [5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valelalactone] is 150 mg per 1 kg body weight of the rat. (150 mg / kg) was dissolved in physiological saline and orally administered to SHR rats. The number of rats tested was 7 in both the administration group and the control group.

(C) Statistical processing method This is the same method as described in paragraph [0047].

(result)

When 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone was orally administered to rats at a dose of 150 mg / kg, the post-administration blood pressure was compared with the systolic blood pressure before administration in the administration group. Significant reductions in 2 and 4 hour systolic blood pressure were observed.

Production Examples: Candy sugar: 33% by weight, starch syrup: 66% by weight, citric acid: 0.67% by weight, flavoring: 0.21% by weight, coloring: 0.07% by weight and 5- (3 ′, 4 ′) 0.05 ′% of 5′-trihydroxyphenyl) -γ-valerolactone was prepared by a conventional method using a candy formulation, and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone was prepared. A candy for the purpose of prevention and improvement of contained hypertension was obtained.

Production Example: Soft Drink Fructose Glucose Liquid Sugar 13%, Citric Acid 0.3%, Ascorbic Acid 0.03%, Sodium Citrate 0.02%, Fragrance (Grapefruit Flavor) 0.1%, 5- (3 ′ 4 ', 5'-trihydroxyphenyl) -4-hydroxy-valeric acid was dissolved by adding water to 0.1% to prepare a 5-liter beverage. Dissolve 100 ml of the solution into a glass bottle container, sterilize by a conventional method, and prevent and improve hypertension containing 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxy-valeric acid A soft drink aimed at.

Production example: Sugar-free tea beverage As a commercially available sugar-free tea beverage, 0.1 g of 2-hydroxyphenylacetic acid [0.1 g] was added to 500 ml of green tea (manufactured by Suntory Ltd.), dissolved and then sterilized by a conventional method. -A sugar-free tea beverage containing hydroxyphenylacetic acid for the purpose of preventing and improving hypertension was obtained.

Production example: chewable tablet 5-phenyl valeric acid 0.5 parts by weight, xylitol 33.8 parts by weight, mannitol 30.6 parts by weight, microcrystalline cellulose 6.1 parts by weight, flavoring 14.1 parts by weight, stearic acid A powder containing 4.3 parts by weight, 0.6 parts by weight of talc, and 10.0 parts by weight of sorbitol was compressed by a tablet press to obtain a tablet containing 5-phenylvaleric acid.

Production Example: Sports beverage 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -γ-valerolactone 0.1 mg, vitamin B1 hydrochloride 0.45 mg, vitamin B20.2 mg, vitamin C 10 mg, niacin 0.8 mg , Pantothenic acid Ca 0.22 mg, ammonium iron citrate 12.57 mg, citric acid 100 mg and fructose 2.5 g were added with ion-exchanged water to make a total volume of 200 ml, and 5- (3 ′, 4 ′, 5′-trihydroxy A sports drink containing phenyl) -γ-valerolactone was prepared.

The blood pressure lowering agent or angiotensin converting enzyme inhibitor of the present invention can be used in health foods, nutritional supplements, foods for specified health use, ingredient preparation foods, etc. as health-oriented foods in addition to pharmaceuticals and quasi drugs. Is something you can do.

Claims (6)

At least one of phenylcarboxylic acid represented by formula (I), 5-phenyl-γ-valerolactone represented by formula (II) and 5-phenyl-4-hydroxyvaleric acid represented by formula (III) An antihypertensive agent comprising the above as an active ingredient.

Formula (I)
(In formula (I), n represents an integer of 1-7, and R1, R2, and R3 each independently represent a hydroxyl group (OH) or hydrogen (H).)

Formula (II)
(R1 and R2 in the formula (II) each independently represent a hydroxyl group (OH) or hydrogen (H).)
Formula (III)
(R1 and R2 in the formula (III) each independently represent a hydroxyl group (OH) or hydrogen (H).)
2. The blood pressure lowering agent according to claim 1, wherein n of the phenylcarboxylic acid represented by the formula (I) is an integer of 3 to 6. At least one of phenylcarboxylic acid represented by formula (I), 5-phenyl-γ-valerolactone represented by formula (II) and 5-phenyl-4-hydroxyvaleric acid represented by formula (III) The angiotensin converting enzyme inhibitor characterized by including the above as an active ingredient. An oral application target comprising the blood pressure lowering agent according to claim 1 or 2, or the angiotensin converting enzyme inhibitor according to claim 3.   The object to be applied to the oral cavity according to claim 4, which is a food or drink. The object to be applied to the oral cavity according to claim 4, which is a quasi-drug or a pharmaceutical product.