TRPA1 and TRPV1 activators

Abstract

The present invention aims to discover substances having a TRPA1 activating effect or a TRPV1 activating effect, and to provide a TRPA1 activator or a TRPV1 activator containing the same as an active ingredient. [Solution] By using one or more active ingredients selected from the group consisting of 3-(4-hydroxy-3-methoxyphenyl)propionic acid, 3,4-dihydroxyhydrocinnamic acid, 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, 4-hydroxy-3-methoxycinnamic acid, and 3,4-dihydroxycinnamic acid, the present invention provides TRPA1 activators and TRPV1 activators with excellent effects.

Description

[Technical Field]

[0001] The present invention relates to a TRPA1 activator and a TRPV1 activator. [Background technology]

[0002] Transient receptor potential (TRP) channels are Ca channels in which one subunit has a six-transmembrane structure. 2+ TRP channels are highly permeable cation channels that sense and open various changes in the intracellular and extracellular environments, and are known to be involved in many biological reactions and pathologies. They are thought to function not only as "sensor" proteins in cells, but also as "scaffolds" for the formation of signaling complexes by interacting with various proteins (Non-Patent Document 1). TRP channels constitute a superfamily in mammals, with more than 20 genes, and their activation mechanisms are diverse.

[0003] TRPA1 (Transient receptor potential ankyrin 1) is a TRP channel with many ankyrin repeat structures at the N-terminus and is expressed in the dorsal root ganglion, trigeminal ganglion, nodose ganglion, brain, inner ear, tongue, heart, lung, intestine, pancreas, smooth muscle, etc. TRPA1 is involved in the regulation of intracellular Ca2+ by depolarization and intracellular Ca2+. 2+ It is known to be activated by various stimuli and substances such as increased concentration, pH, mechanical stimulation, and cold sensation (below 17°C) (Non-Patent Documents 1-2).

[0004] Activation of TRPA1 is thought to cause thermogenesis, increased appetite, intestinal contraction, etc. (Non-Patent Documents 3 to 7). Components known to activate TRPA1 include allyl isothiocyanate, cinnamaldehyde (Non-Patent Document 4), pepper-derived components such as piperine (Non-Patent Document 5), voacangin (Non-Patent Document 6), and β-eudesmol (Non-Patent Document 7).

[0005] Meanwhile, TRPV1 (Transient receptor potential vanilloid 1) is a TRP channel also known as a capsaicin receptor, and is expressed in the dorsal root ganglion, trigeminal ganglion, nodose ganglion, brain, lung, stomach, liver, intestine, kidney, pancreas, spleen, placenta, testis, uterus, etc. TRPV1 is known to be activated by capsaicin, acid, heat (43°C or higher), etc. (Non-Patent Documents 1 and 2).

[0006] Activation of TRPV1 is thought to impart a pungent taste to the tongue and the like, as well as promote energy metabolism and heat production, and cause vasodilation, etc. (Non-Patent Documents 3 to 6). In addition to capsaicin, other known components that activate TRPV1 include allicin, gingerol (see Non-Patent Document 2), and pepper-derived components such as piperine (Non-Patent Document 5). [Prior art documents] [Patent documents]

[0007] [Non-Patent Document 1] Biochemistry, November 2009, Vol. 81, No. 11, pp. 962-983 [Non-patent document 2] Pharmaceutical Journal, 2010, Vol. 130, No. 3, pp. 289-294 [Non-patent document 3] EMBO reports,2011,Vol.12,No.11,pp.1094-1101 [Non-patent document 4] Biosci. Biotechnol. Biochem.,2008,Vol.72,Issue 10,pp.2608-2614 [Non-Patent Document 5] Biosci. Biotechnol. Biochem.,2010,Vol.74,Issue 5,pp.1068-1072 [Non-patent document 6] J. Nat. Prod.,2014,Vol.77,Issue 2,pp.285-297 [Non-Patent Document 7] Sci. Rep., 2017, Vol.7, Article number 15785 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to find substances that have a TRPA1 activating effect or a TRPV1 activating effect, and to provide TRPA1 activators and TRPV1 activators that contain them as active ingredients. [Means for solving the problem]

[0009] In order to solve the above problems, the TRPA1 activator of the present invention is characterized in that it contains as an active ingredient one or more compounds selected from the group consisting of compounds a to e represented by the following general formula (I): Furthermore, the TRPV1 activator of the present invention is characterized in that it contains, as an active ingredient, one or more compounds selected from the group consisting of compounds a to f represented by the following general formula (I):

[0010] [ka] [Effects of the Invention]

[0011] According to the present invention, by using the above compounds a to f as active ingredients, it is possible to provide a TRPA1 activator or TRPV1 activator with excellent effects. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment of the present invention will be described. The TRPA1 activator according to this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of compounds a to e represented by the following general formula (I): Furthermore, the TRPV1 activator according to this embodiment is characterized in that it contains, as an active ingredient, one or more compounds selected from the group consisting of compounds a to f represented by the following general formula (I):

[0013] [ka]

[0014] [Phenylpropionic acids] The compounds a to f represented by the above general formula (I) are all phenylpropionic acid analogs.

[0015] Compound a is 3-(4-hydroxy-3-methoxyphenyl)propionic acid (hereinafter sometimes abbreviated as "HMPA") represented by the following formula (a).

[0016] [ka]

[0017] Compound b is 3,4-dihydroxyhydrocinnamic acid represented by the following formula (b).

[0018] [ka]

[0019] Compound c is 3-(4-hydroxyphenyl)propionic acid represented by the following formula (c).

[0020] [ka]

[0021] Compound d is 3-phenylpropionic acid represented by the following formula (d).

[0022] [ka]

[0023] Compound e is 4-hydroxy-3-methoxycinnamic acid (hereinafter sometimes abbreviated as "HMCA") represented by the following formula (e).

[0024] [ka]

[0025] Compound f is 3,4-dihydroxycinnamic acid represented by the following formula (f).

[0026] [ka]

[0027] The carboxyl groups (-COOH) in the compounds a to f can dissociate hydrogen ions and exchange them with other cations to form salts of the compounds a to f with the cations. In this embodiment, salts of the compounds a to f may be used as the compounds a to f. Examples of cations capable of forming salts with compounds a to f include sodium ions, potassium ions, ammonium ions, magnesium ions, and calcium ions, and among these, sodium ions, potassium ions, and ammonium ions are preferred.

[0028] Compounds a to f can be produced, for example, by synthesis, or by purification and isolation from a plant extract containing compounds a to f. In this case, the plant extract containing compounds a to f can be obtained by a method commonly used for plant extraction. Examples of plants containing compounds a to f include rice, barley, wheat, soybean, adzuki bean, and corn.

[0029] Of the above compounds, compound a can also be produced by, for example, fermenting compound e or a derivative thereof, or a composition containing either or both (e.g., a crushed plant material or extract, etc.) with a microorganism having phenolic acid reductase, converting compound e to compound a, and then extracting, purifying, and isolating the resulting fermentation product. Similarly, compounds b, c, and d can also be produced by fermenting compositions containing compound f, 4-hydroxycinnamic acid, and cinnamic acid with a microorganism having phenolic acid reductase, converting them to compounds b to d, respectively, and then extracting, purifying, and isolating the resulting fermentation product. Examples of compositions containing compound e, compound f, 4-hydroxycinnamic acid, or cinnamic acid include crushed materials and extracts of the plants exemplified above, as well as crushed materials and extracts of plants such as coffee, wheat, corn, tomato, yerba mate, mugwort, and burdock. Note that compounds e and f are components or intermediates of lignin in woody plants and herbaceous plants, and therefore lignin or a composition containing it may be used as the fermentation raw material. On the other hand, examples of microorganisms having phenolic acid reductase include lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus delbrueckii, Lactobacillus buchneri, Lactobacillus kefiranofaciens, Lactobacillus gallinarum, Enterococcus faecalis, Weissella cibaria, and Weissella confusa.

[0030] The method for extracting, purifying, and isolating compounds a to f from the above-mentioned plants or fermented products is not particularly limited, and can be carried out according to conventional methods. For example, the extraction process can be carried out by drying the above-mentioned plants or fermented products as the extraction raw material, and then subjecting them to extraction with an extraction solvent either directly or after pulverization using a crusher. Drying can be carried out in the sun or using a commonly used dryer. In addition, the raw material can be used after pretreatment such as degreasing with a nonpolar solvent such as hexane. Pretreatment such as degreasing allows for efficient extraction with a polar solvent.

[0031] As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent, which is preferably used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent.

[0032] Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has undergone various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, water that can be used as an extraction solvent in this embodiment also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0033] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin; and lower aliphatic ketones having 3 to 5 carbon atoms, such as acetone and methyl ethyl ketone.

[0034] When a mixture of two or more polar solvents is used as an extraction solvent, the mixture ratio can be any and can be adjusted appropriately. For example, when a mixture of water and a hydrophilic organic solvent is used as an extraction solvent, the mixture can be mixed at any ratio, i.e., between more than 0:100 and less than 100:0 (volume ratio, hereinafter the same), and can be adjusted appropriately. For example, when a mixture of water and a lower aliphatic alcohol is used as the extraction solvent, the mixing ratio (volume ratio) of water to lower aliphatic alcohol can be 9:1 or more, or even 7:3 or more, or the mixing ratio of water to lower aliphatic alcohol can be 1:9 or less, or even 2:8 or less. When a mixture of water and a polyhydric alcohol is used, the mixing ratio of water to polyhydric alcohol can be 8:2 or more, or 1:9 or less, and when a mixture of water and a lower aliphatic ketone is used, the mixing ratio of water to lower aliphatic ketone can be 9:1 or more, or 2:8 or less.

[0035] The extraction process is not particularly limited as long as it can dissolve the soluble components contained in the extraction raw material into the extraction solvent, and can be carried out according to conventional methods. For example, the extraction raw material is immersed in an extraction solvent in an amount (mass ratio) 5 to 15 times the amount of the extraction raw material, and the soluble components are extracted at room temperature or under reflux heating, followed by filtration to remove the extraction residue, to obtain an extract. The solvent is distilled off from the obtained extract to obtain a paste-like concentrate, which is then further dried to obtain a dried product.

[0036] The method for purifying and isolating compounds a to f from the extract obtained as described above, a concentrate of the extract, or a dried product of the extract is not particularly limited, and can be carried out by a conventional method.

[0037] [TRPA1 activators, TRPV1 activators] Of the compounds a to f obtained as described above, compounds a to e have excellent TRPA1 activation activity, and compounds a to f have excellent TRPV1 activation activity. Therefore, compounds a to f can be used as TRPA1 activators or active ingredients of TRPV1 activators, respectively. In other words, compounds a to e can be used to produce TRPA1 activators, and compounds a to f can be used to produce TRPV1 activators.

[0038] The TRPA1 activator and TRPV1 activator of this embodiment can be used in a wide range of applications, such as pharmaceuticals, quasi-drugs, cosmetics, oral compositions, etc. Here, the TRPA1 activator and TRPV1 activator of this embodiment are preferably agents labeled with information regarding the TRPA1 activating activity or TRPV1 activating activity of compounds a to f.

[0039] When compounds a to f are used as the active ingredient of the TRPA1 activator or TRPV1 activator of this embodiment, a composition containing compounds a to f may be used instead of isolated compounds a to f. Here, the "composition containing compounds a to f" in this embodiment includes an extract obtained by using a natural product containing one or more of compounds a to f as an extraction raw material, a processed natural product such as a fermented natural product containing one or more of compounds a to f, and an extract obtained by using the processed natural product as an extraction raw material.

[0040] In this embodiment, the term "extract" includes an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, or a dried product obtained by drying the extract.

[0041] The TRPA1 activator or TRPV1 activator of this embodiment may consist of only compounds a to f, or may be a formulation of compounds a to f. The TRPA1 activator or TRPV1 activator of this embodiment can be formulated into any dosage form, such as powder, granules, tablets, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to a conventional method. In this case, examples of auxiliary agents that can be used include excipients, binders, disintegrants, lubricants, stabilizers, and flavoring agents. The TRPA1 activator and TRPV1 activator can be used, for example, as oral compositions, topical skin preparations, ointments, topical liquids, patches, and the like.

[0042] When the TRPA1 activator or TRPV1 activator of this embodiment is formulated into a formulation, the contents of compounds a to f are not particularly limited and can be set appropriately depending on the purpose.

[0043] In addition, the TRPA1 activator or TRPV1 activator of this embodiment may use only compounds a to f as the active ingredient having TRPA1 activating activity or TRPV1 activating activity, or, if necessary, other ingredients having TRPA1 activating activity or TRPV1 activating activity may be combined with compounds a to f and used as active ingredients.

[0044] The TRPA1 activator or TRPV1 activator of this embodiment can be administered to patients by oral administration, transdermal administration, etc., and a method suitable for prevention, treatment, etc. can be selected appropriately depending on the type of disease. The dose of the TRPA1 activator or TRPV1 activator of this embodiment can also be increased or decreased appropriately depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0045] The TRPA1 activator of this embodiment can, for example, promote thermogenesis, increase appetite, improve digestion by intestinal contraction, etc. through the TRPA1 activating activity of the active ingredients, compounds a to e. However, in addition to these uses, the TRPA1 activator of this embodiment can also be used for any other uses in which exerting TRPA1 activating activity is significant.

[0046] The TRPV1 activator of this embodiment can, for example, promote energy metabolism and heat production, reduce blood pressure and promote blood circulation by dilating blood vessels, etc. through the TRPV1 activating activity of the active ingredients, compounds a to f. However, in addition to these uses, the TRPV1 activator of this embodiment can also be used for any other uses in which exerting TRPV1 activating activity is meaningful.

[0047] Furthermore, since the TRPA1 activator and TRPV1 activator of the present embodiment have excellent TRPA1 activating activity and TRPV1 activating activity, they are suitable for incorporation into, for example, oral compositions and topical skin preparations described below. In this case, compounds a to f may be incorporated as they are, or may be formulated and then incorporated.

[0048] Furthermore, the TRPA1 activator and TRPV1 activator of this embodiment have excellent TRPA1 activating activity and TRPV1 activating activity, and therefore can be suitably used as reagents for research into the mechanisms of these actions.

[0049] Incorporation into oral compositions Compounds a to e have excellent TRPA1 activation activity, and compounds a to f have excellent TRPV1 activation activity, so they are suitable for incorporation into oral compositions. In this case, compounds a to f may be incorporated as they are, or may be formulated and then incorporated. This allows for the preparation of oral compositions suitable for TRPA1 activation and TRPV1 activation. In other words, incorporation into oral compositions is one of the suitable uses of TRPA1 activators and TRPV1 activators.

[0050] Here, oral compositions refer to compositions that are unlikely to be harmful to human health and that are taken orally or by administration through the gastrointestinal tract in normal social life, and are not limited to administrative classifications such as foods, medicines, and quasi-drugs. Therefore, the "oral composition" in this embodiment broadly encompasses orally taken general foods, health foods, health functional foods (foods for specified health uses, foods with nutrient claims, and foods with functional claims), quasi-drugs, medicines, and the like. The oral composition according to this embodiment is preferably an oral composition that can display the favorable effects of compounds a to f on the oral composition or its packaging, and is particularly preferably a health functional food (foods for specified health uses, foods with functional claims, foods with nutrient claims), quasi-drugs, and medicines.

[0051] When compounds a to f are incorporated into an oral composition, the amount of active ingredient therein can be appropriately changed taking into consideration the purpose of use, symptoms, gender, etc., but taking into consideration the general intake of the oral composition to be added, it is preferable to adjust the daily intake of compounds a to f (compounds a to f converted into mass) per adult to about 1 to 1000 mg. When the oral composition to be added is in the form of granules, tablets, or capsules, the amount of compounds a to f added (compounds a to f converted into mass) is usually 0.1 to 100% by mass, preferably 5 to 100% by mass, of the oral composition to be added.

[0052] The oral composition of this embodiment may be prepared by incorporating compounds a to f into any oral composition that does not interfere with their activity, or may be a nutritional supplement containing compounds a to f as the main ingredients.

[0053] When producing the oral composition of this embodiment, any auxiliary agent can be added, such as sugars such as dextrin and starch; proteins such as gelatin, soy protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; and fats and oils such as soybean oil and medium-chain fatty acid triglycerides, to form an oral composition in any shape.

[0054]

[0039] Oral compositions that can incorporate compounds a to f are not particularly limited, and specific examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated concentrates and powders for adjusting these beverages); frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba noodles, udon noodles, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; sweets such as candy, chewing gum, candies, gum, chocolate, candy tablets, snacks, biscuits, jelly, jam, cream, and baked goods; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-based foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; condiments such as sauces and dressings; soups, stews, salads, side dishes, and pickles; and various other forms of health and nutritional supplements; tablets, capsules, and energy drinks. When compounds a to f are formulated into these oral compositions, commonly used auxiliary raw materials and additives can be used in combination.

[0055] [Incorporation into topical skin preparations] Compounds a to f have excellent TRPA1 activating activity and TRPV1 activating activity, and are therefore suitable for incorporation into topical skin preparations. In this case, compounds a to f may be incorporated as they are, or may be formulated and then incorporated. This allows for the creation of topical skin preparations suitable for TRPA1 activation and TRPV1 activation. In other words, incorporation into topical skin preparations is one of the suitable uses of TRPA1 activators and TRPV1 activators.

[0056] Here, topical skin preparations are not limited to specific categories and include a wide range of transdermal medicines, quasi-drugs, cosmetics, etc., and specific examples include ointments, creams, emulsions, skin lotions, beauty serums, lotions, gels, beauty oils, packs, foundations, lip balms, bath additives, hair tonics, hair lotions, soaps, body shampoos, etc.

[0057] The amount of the active ingredient in the topical skin preparation can be adjusted as appropriate depending on the type of topical skin preparation. For example, the preferred content ratio of compounds a to f (calculated as the mass of compounds a to f) is 0.0001 to 10% by mass, and the most preferred content ratio is 0.001 to 1% by mass.

[0058] The topical skin preparation of this embodiment can be used in combination with a main ingredient, auxiliary agent, or other ingredient typically used in the manufacture of topical skin preparations, such as astringents, bactericides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / antiallergic agents, antioxidants / active oxygen scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, etc., so long as the combination does not interfere with the TRPA1 activation or TRPV1 activation activity of compounds a to f. Such combinations can result in a more general-purpose product, and the synergistic effect with the other active ingredients used in combination can sometimes bring about superior effects beyond those normally expected.

[0059] The TRPA1 activator and TRPV1 activator of this embodiment are suitable for use in humans, but can also be used in animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective effects are achieved. [Example]

[0060] The present invention will be specifically explained below by showing test examples, formulation examples, etc., but the present invention is not limited to the following examples. In the test examples and formulation examples, the following samples 1 to 6 were used as compounds a to f.

[0061] [Table 1]

[0062] [Test Example 1] TRPA1 activation test (1) TRPA1-transfected HEK cells TRPA1-transfected HEK cells were used as described in Non-Patent Document 6 (J. Nat. Prod., 2014, Vol. 77, Issue 2, pp. 285-297). Briefly, hTRPA1 cDNA was amplified by RT-PCR from single-stranded cDNA derived from human WI38 cells and cloned into pcDNA4 / TO (Invitrogen). The resulting plasmid was transfected into HEK T-REx cells using Lipofectamine 2000 (Invitrogen). Selection was performed with 500 μg / mL Zeocin and 10 μg / mL Blasticidin to establish HEK T-REx cells stably expressing hTRPA1. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS and antibiotics.

[0063] (2) Intracellular Ca 2+ Concentration measurement Intracellular Ca 2+ The concentration is Ca 2+ Measurement was performed according to the method described in Non-Patent Document 6, except that the sensitive fluorescent dye was changed to Fluo-8 (manufactured by AAT Bioquest). Specifically, TRPA1-transfected HEK cells were seeded in a 96-well plate and cultured for 24 hours in medium supplemented with 1 μg / mL tetracycline to induce hTRPA1 expression. After incubation, the medium was replaced with 50 μL of assay buffer (5.37 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 0.34 mM Na2HPO4·7H2O, 5.56 mM D-glucose, 20 mM HEPES, 1 mM CaCl2, 0.1% BSA, and 2.5 mM probenecid, pH 7.4) containing 3 μM Fluo-8. The cells were then incubated at 37°C for 1 hour to allow for Fluo-8 uptake. The assay buffer containing Fluo-8 was then discarded and washed with 100 μL of assay buffer per well. Again, 180 μL / well of measurement buffer was added, and the plate was set in a plate reader FlexStation II system (Molecular Devices).

[0064] Then, 20 μL of measurement buffer containing test samples (samples 1 to 5, final concentrations listed in Tables 2 to 6) was added to the cells, and the change in fluorescence intensity due to the addition of the test samples was measured using a plate reader FlexStation II system (Molecular Devices). Measurements were performed over time before and after the addition of the samples, and the fluorescence intensity F0 (baseline) before the addition of the samples and the maximum fluorescence intensity F1 after the addition were recorded. max obtained.

[0065] In this study, activation of TRPA1 increased intracellular Ca 2+ The level of TRPA1 activation was evaluated using the TRPA1 response value calculated using the following formula. A positive response value indicates that TRPA1 has been activated, and the higher the response value, the greater the level of activation.

[0066] TRPA1 response value = ΔF / F0 =(F max -F0) / F0 F0: Fluorescence intensity F0 (baseline) before adding sample F max : Maximum fluorescence after addition The results are shown in Tables 2 to 6.

[0067] [Table 2]

[0068] [Table 3]

[0069] [Table 4]

[0070] [Table 5]

[0071] [Table 6]

[0072] (3) Confirmation of TRPA1 activation To confirm that the increase in fluorescence intensity due to the addition of the sample described above was due to the activation of TRPA1, an inhibitor test was conducted in which the cells were simultaneously treated with HC-030031, a selective TRPA1 inhibitor. Specifically, 20 μL of measurement buffer containing test samples (samples 1 to 5, final concentrations indicated with * in Tables 2 to 6) mixed with HC-030031 (ChemBridge) at a final concentration of 30 μM was added to TRPA1-introduced HEK cells that had been loaded with Fluo-8 as described above (2), and the change in fluorescence intensity was measured. The increase in fluorescence intensity confirmed in Tables 2 to 6 was suppressed to approximately F0 (baseline) by the addition of the inhibitor (data not shown).

[0073] (4) Results As shown in Tables 2 to 6, compound a (HMPA, Sample 1), compound b (3,4-dihydroxyhydrocinnamic acid, Sample 2), compound c (3-(4-hydroxyphenyl)propionic acid, Sample 3), compound d (3-phenylpropionic acid, Sample 4), and compound e (HMCA, Sample 5) all induced concentration-dependent responses. Furthermore, inhibitor tests confirmed that these responses were due to TRPA1 activation. Therefore, compounds a to e were all confirmed to have excellent TRPA1 activation activity.

[0074] [Test Example 2] TRPV1 activation test (1) TRPV1-introduced HEK cells TRPV1-transfected HEK cells were established in the same manner as the TRPA1-transfected HEK cells in Test Example 1. Briefly, hTRPV1 cDNA was amplified by RT-PCR from single-stranded cDNA derived from human brain and cloned into pcDNA4 / TO (Invitrogen). The resulting plasmid was transfected into HEK T-REx cells using Lipofectamine 2000 (Invitrogen), and selection was performed with 500 μg / mL Zeocin and 10 μg / mL Blasticidin to establish HEK T-REx cells stably expressing hTRPV1. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS and antibiotics.

[0075] (2) Intracellular Ca 2+ Concentration measurement Intracellular Ca 2+ The concentration is Ca 2+ Measurement was performed according to the method described in Non-Patent Document 6, except that the sensitive fluorescent dye was changed to Fluo-8 (manufactured by AAT Bioquest). Specifically, TRPV1-transfected HEK cells were seeded in a 96-well plate and cultured for 24 hours. After incubation, the medium was replaced with 50 μL of assay buffer (5.37 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 0.34 mM Na2HPO4·7H2O, 5.56 mM D-glucose, 20 mM HEPES, 1 mM CaCl2, 0.1% BSA, and 2.5 mM probenecid, pH 7.4) containing 3 μM Fluo-8 and incubated at 37°C for 1 hour to allow the cells to incorporate Fluo-8. The assay buffer containing Fluo-8 was then discarded, and 100 μL / well of assay buffer was added for washing. 180 μL / well of assay buffer was added again, and the plate was loaded onto a FlexStation II plate reader (Molecular Devices).

[0076] Then, 20 μL of measurement buffer containing test samples (samples 1 to 6, final concentrations shown in Tables 7 to 12) was added to the cells, and the change in fluorescence intensity due to the addition of the test samples was measured using a plate reader FlexStation II system (Molecular Devices). Measurements were performed in the same manner as in Test Example 1, and the degree of TRPV1 activation was evaluated as the TRPV1 response value using the following formula.

[0077] TRPV1 response value = ΔF / F0 =(F max -F0) / F0 F0: Fluorescence intensity F0 (baseline) before adding sample F max : Maximum fluorescence after addition The results are shown in Tables 7 to 12.

[0078] [Table 7]

[0079] [Table 8]

[0080] [Table 9]

[0081] [Table 10]

[0082] [Table 11]

[0083] [Table 12]

[0084] (3) Confirmation of TRPV1 activation To confirm that the increase in fluorescence intensity upon sample addition was due to TRPV1 activation, we performed an inhibitor test in which the TRPV1-selective inhibitor BCTC (N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide) was simultaneously treated. Specifically, 20 μL of measurement buffer containing the test samples (samples 1 to 6, final concentrations indicated with * in Tables 7 to 12) and BCTC (manufactured by Tocris BioScience) at a final concentration of 1 μM was added to TRPV1-introduced HEK cells that had been loaded with Fluo-8 as described above (2), and the change in fluorescence intensity was measured. The increase in fluorescence intensity confirmed in Tables 7 to 12 was suppressed to approximately F0 (baseline) by the addition of the inhibitor (data not shown).

[0085] (4) Results As shown in Tables 7 to 12, compound a (HMPA, Sample 1), compound b (3,4-dihydroxyhydrocinnamic acid, Sample 2), compound c (3-(4-hydroxyphenyl)propionic acid, Sample 3), compound d (3-phenylpropionic acid, Sample 4), compound e (HMCA, Sample 5), and compound f (3,4-dihydroxycinnamic acid, Sample 6) all induced concentration-dependent responses. Furthermore, inhibitor tests confirmed that these responses were due to TRPV1 activation. Therefore, compounds a to e were confirmed to have excellent TRPV1 activation activity.

[0086] [Formulation example 1] Tablets were produced according to the following formulation in a conventional manner. Tablets were also produced in which compound a was replaced with compounds b to f. Compound a 5.0mg Dolomite (contains 20% calcium and 10% magnesium) 83.4mg Casein phosphopeptide 16.7mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid ester 12.0mg

[0087] [Formulation example 2] Oral liquid preparations were prepared according to the following recipes using a conventional method. Compounds b to f were also prepared in place of compound a. <Composition in 1 ampoule (100 mL)> Compound a 0.3% by mass Sorbitol 12.0% by mass Sodium benzoate 0.1% by mass Fragrance 1.0% by mass Calcium sulfate 0.5% by mass Purified water remainder (100% by mass)

[0088] [Formulation example 3] Emulsions were prepared by a conventional method according to the following formulation. Compounds b to f were also prepared by replacing compound a. Compound a 0.01g Jojoba oil 4.00g 1,3-butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20E.O.) 2.50g 2.00g olive oil Squalane 2.00g Cetyl alcohol 2.00g Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhetinate 0.10g Phellodendron bark extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g Phellodendron bark extract 0.10g Chamomile extract 0.10g Fragrance 0.05g Purified water Remainder (total amount is 100g)

[0089] [Formulation example 4] Creams having the following formulations were produced by a conventional method. Compounds b to f were also produced in place of compound a. Compound a 0.05g Sophora root extract 0.1g Scutellaria root extract 0.1g Liquid paraffin 5.0g White beeswax 4.0g Squalane 10.0g Cetanol 3.0g Lanolin 2.0g Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g Fragrance 0.1g Purified water Remainder (total amount is 100g)

Claims

1. A TRPA1 activator characterized by comprising, as an active ingredient, one or more compounds selected from the group consisting of compounds a to e represented by the following general formula (I): 【Chemistry 1】

2. A TRPV1 activator characterized by comprising, as an active ingredient, one or more compounds selected from the group consisting of compounds a to f represented by the following general formula (I): 【Chemistry 2】

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