Trp channel activity inhibitor

Abstract

To provide a TRPA1 and / or TRPV1 activity inhibitor having high safety and suitable for clinical application.SOLUTION: It has been found that an extract of Alisma orientale is effective in inhibiting the activity of TRPA1 and TRPV1, and also has high safety. Accordingly, the TRPA1 and / or TRPV1 activity inhibitor of the present invention has potential for improving diseases involving TRPA1 and / or TRPV1, such as cough and asthma, and is applicable to foods, cosmetics, quasi-drugs, and pharmaceuticals.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a TRPA1 and / or TRPV1 activity inhibitor characterized by containing an extract of Saccio modesta.

Background Art

[0002] Transient Receptor Potential (TRP) channels are six-transmembrane ion channels that sense temperature, pain, osmotic pressure, mechanical stimuli, etc., and in humans, they are composed of six subfamilies and 27 channels. Among these, it has been clarified that nine types of TRP channels have temperature sensitivity, and each has a different activation temperature threshold. Among the TRP channels with temperature sensitivity, TRPA1 is an ion channel that is sensitive to various exogenous factors such as AITC (allyl isothiocyanate), which is the pungent component of wasabi, and cinnamaldehyde, which is a cinnamon component, in addition to cold stimuli below 17°C. TRPV1 is an ion channel that is sensitive to capsaicin, which is the pungent component of chili peppers, acids, inflammatory substances, etc., in addition to heat stimuli above 43°C (Non-Patent Document 1). TRPA1 and TRPV1 are known to be closely related not only to sensations such as pain, pungency, itching, and numbness, but also to diseases such as cough and asthma.

[0003] Regarding the relationship between TRPA1 and diseases, it is known that TRPA1 is involved in the induction of cough because inhalation of a TRPA1 agonist in guinea pigs and humans induces cough, and cough is suppressed by a selective antagonist (Non-Patent Documents 2 and 3). Also, since airway inflammation is not observed in asthmatic model mice in which TRPA1 has been knocked out, it is known that TRPA1 is involved in the onset of asthma (Non-Patent Document 3).

[0004] Regarding the relationship between TRPV1 and diseases, it is known that TRPV1 is involved in the induction of coughing, since capsaicin, an agonist of TRPV1, induces coughing, and TRPV1 blockers suppress coughing induced by citric acid or capsaicin (Non-Patent Document 4). In addition, it is known that TRPV1 is involved in the onset of asthma, since bradykinin and the like released from mast cells activate TRPV1 and enhance allergic airway inflammation, and the expression of TRPV1 in the airway epithelium is enhanced in patients with intractable asthma (Non-Patent Document 3).

[0005] As described above, TRPA1 and TRPV1 are known to be associated with diseases such as cough, asthma, etc. Therefore, clinical trials of substances that inhibit the activity of TRPA1 and / or TRPV1 are being conducted as drugs for improving cough and asthma, but there is a demand for TRPA1 and / or TRPV1 activity inhibitors that are safer and more suitable for clinical application.

[0006] Alisma orientale (scientific name: Alisma orientale) is a perennial plant belonging to the genus Alisma of the family Alismaceae, and the herbal medicine Taksha is made by drying the rhizome of Alisma orientale, and contains alisol (triterpene) and is used in Chinese medicine with diuretic and thirst-quenching effects. It has been known that Alisma orientale extracts have anti-inflammatory effects such as treating contact dermatitis caused by type IV allergy (Patent Document 1), inhibiting the production of interleukin 4 (Patent Document 2), inhibiting the production of TNF-α (Patent Document 3), and inhibiting the production of prostaglandin E2 (Patent Document 4), as well as promoting the production of ceramide (Patent Document 5). However, there has been no known TRP channel activity inhibitor or symptom-improving agent for cough, asthma, etc., which is characterized by containing Alisma orientale extract as an active ingredient. [Prior art documents] [Patent documents]

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Document

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0009] The problem of the present invention is to provide a TRPA1 and / or TRPV1 activity inhibitor having excellent safety.

Means for Solving the Problems

[0010] That is, the present invention comprises the following (1) to (3).

[0011] (1) A TRPA1 and / or TRPV1 activity inhibitor characterized by containing an extract of Salsola soda. An agent for improving cough and / or asthma, characterized by containing the TRPA1 and / or TRPV1 activity inhibitor described in (2)(1).

Mode for Carrying Out the Invention

[0012] The Alisma orientale (scientific name: Alisma orientale, Japanese name: Shimen-taka, crude drug name: Alisma rhizome (Takusha)) used in the present invention is a perennial herb belonging to the genus Alisma in the Alismataceae family of the Alismatales order. It mainly inhabits northern Japan, East Asia, Central Asia, etc., but the production area is not particularly limited. In the present invention, the extract of Alisma orientale refers to an extract of a part or the whole of the plant body such as its rhizome (tuber), flower, fruit, seed, leaf, stem, or a mixture thereof. However, the part used as the extraction raw material in the present invention is preferably the rhizome used as the crude drug (Alisma rhizome). Further, for extraction, the plant body may be used as it is, or treatments such as drying, pulverization, and fine cutting may be performed.

[0013] The extraction method is not particularly limited, but water or hot water, or a mixed solvent of water and an organic solvent can be used, and it can be carried out by a method such as stirring or column extraction. Examples of the extraction solvent include For example, water, lower alcohols (such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (such as 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (such as acetone, methyl ethyl ketone, etc.), acetonitrile, esters (such as ethyl acetate, butyl acetate, etc.), hydrocarbons (such as hexane, heptane, liquid paraffin, etc.), ethers (such as ethyl ether, tetrahydrofuran, propyl ether, etc.) and the like can be mentioned. Preferably, polar solvents such as water, lower alcohols and liquid polyhydric alcohols are good, and particularly preferably, water, ethanol, 1,3-butylene glycol and propylene glycol are good. These solvents may be used alone or in combination of two or more. There is no particular limitation on the amount of the solvent used. For example, it may be 10 times or more, preferably 20 times or more, based on the rhizome of Sagittaria trifolia (dry weight), but it is preferably 100 times or less for the convenience of operations when concentrating or isolating after extraction. Further, the extraction temperature and time can be appropriately selected depending on the type of the solvent used, the pressure during extraction, and the like.

[0014] The above extract may be used as the extracted solution as it is, but if necessary, within the range where the effects of the present invention are exhibited, treatments such as concentration (vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc. may be performed before use. Further, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0015] The present invention may use the above extract as it is, and within the range not impairing the effects of the extract, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, ultraviolet absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, film-forming agents, sweeteners, acidulants and other components which are components used in cosmetics, quasi-drugs, pharmaceuticals or foods, etc. may be contained.

[0016] The present invention can be used in any of cosmetics, quasi-drugs, pharmaceuticals, and foods. As its dosage forms, for example, bath agents, ointments, poultices, tablets, capsules, chocolates, gums, candies, beverages, powders, granules, tablets, sugar-coated tablets, syrups, pills, suspensions, solutions, emulsions, suppositories, injection solutions, etc. can be mentioned.

[0017] In the case of external use, the content of the above extract used in the present invention is preferably 0.00001% by weight or more, more preferably 0.0001 - 10% by weight, in terms of solids. Further, 0.0001 - 1% by weight is most preferable. If it is less than 0.00001% by weight, sufficient effects may not be obtained, and if it exceeds 10% by weight, it is difficult to recognize an enhancement of the effects and it is uneconomical.

[0018] In the case of internal use, the intake amount varies depending on age, body weight, symptoms, treatment effects, administration methods, treatment time, etc. Usually, as the daily intake amount per adult, 5 mg or more is preferable, 10 mg - 5 g is more preferable. Further, 20 mg - 2 g is most preferable.

[0019] Next, in order to explain the present invention in detail, production examples, experimental examples, and formulation examples of the extract used in the present invention are given as examples, but the present invention is not limited thereto. The % shown in the production examples indicates % by weight, and the parts of the content shown in the formulation examples indicate parts by weight.

Examples

[0020] An extract of Sargassum muticum was produced as follows.

[0021] (Production Example 1) Preparation of a hot water extract of Sargassum muticum 200 mL of water was added to 10 g of the dried product of Sargassum muticum, and extraction was carried out at 95 - 100 °C for 2 hours. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 2.0 g of a hot water extract of Sargassum muticum.

[0022] (Production Example 2) Preparation of a 50% ethanol extract of Sargassum muticum 10 g of dried Sagittaria trifolia was immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness with an evaporator to obtain 1.5 g of 50% ethanol extract of Sagittaria trifolia.

[0023] (Production Example 3) Preparation of ethanol extract of Sagittaria trifolia 10 g of dried Sagittaria trifolia was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness with an evaporator to obtain 0.5 g of ethanol extract of Sagittaria trifolia.

[0024] (Production Example 4) Preparation of 1,3-butylene glycol extract of Sagittaria trifolia 10 g of dried Sagittaria trifolia was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 195 g of 1,3-butylene glycol extract of Sagittaria trifolia.

Examples

[0025] (Formulation Example 1) Bath agent Formulation Content (parts) 1. Ethanol extract of Sagittaria trifolia (Production Example 3) 1.0 2. Sodium hydrogen carbonate 50.0 3. Yellow No. 202 (1) Appropriate amount 4. Perfume Appropriate amount 5. Make the total amount 100 with sodium sulfate [Production method] Mix Components 1 to 5 uniformly to obtain a product.

[0026] (Formulation Example 2) Ointment Formulation Content (parts) 1. 1,3-Butylene glycol extract of Sagittaria trifolia (Production Example 4) 5.0 2. Polyoxyethylene cetyl ether (30 E.O.) 2.0 3. Glyceryl monostearate 10.0 4. Liquid paraffin 5.0 5. Cetyl alcohol 6.0 6. Methyl paraoxybenzoate 0.1 7. Propylene glycol 10.0 8. Make the total volume 100 with purified water [Manufacturing method] Heat and dissolve components 2 - 5 and mix them, keep at 70°C to form an oil phase. Heat and dissolve components 1 and 6 - 8 and mix them, keep at 75°C to form an aqueous phase. Add the aqueous phase to the oil phase for emulsification, and cool to 30°C while stirring to obtain the product.

[0027] (Formulation Example 3) Powder Formulation Content (parts) 1. Hot water extract of Sargassum horneri (Production Example 1) 1.0 2. Dry corn starch 39.0 3. Microcrystalline cellulose 60.0 [Manufacturing method] Mix components 1 - 3 to obtain a powder.

[0028] (Formulation Example 4) Tablets Formulation Content (parts) 1. 50% ethanol extract of Sargassum horneri (Production Example 2) 5.0 2. Dry corn starch 25.0 3. Calcium carboxymethyl cellulose 20.0 4. Microcrystalline cellulose 40.0 5. Polyvinylpyrrolidone 7.0 6. Talc 3.0 [Manufacturing method] Mix components 1 - 4, then add an aqueous solution of component 5 as a binder and granulate. Add component 6 to the formed granules and tableting. Each tablet is 0.52 g.

[0029] (Formulation Example 5) Chewable tablets Formulation Content (parts) 1. Hot water extract of Sargassum horneri (Production Example 1) 1.0 2. Ethanol extract of Sargassum horneri (Production Example 3) 1.0 3. Dry corn starch 49.8 4. Erythritol 40.0 5. Citric acid 5.0 6. Sucrose fatty acid ester 3.0 7. Spice 0.1 8. Purified water 0.1 [Manufacturing method] Mix components 1 to 5 and 8, and perform granule molding. Add components 6 and 7 to the molded granules and tableting. Make it 1.0 g per tablet.

[0030] (Formulation Example 6) Beverage Formulation Content (parts) 1. Hot water extract of Sajimo daka (Production Example 1) 0.05 2. Stevia 0.05 3. Malic acid 5.0 4. Spice 0.1 5. Purified water 94.8 [Manufacturing method] Dissolve components 2 and 3 in a small amount of component 5. Then, add components 1, 4 and the remaining component 5 and mix.

[0031] (Comparative Example 1) Conventional beverage In Formulation Example 6, a conventional beverage was prepared by replacing 1. Hot water extract of Sajimo daka (Production Example 1) with purified water.

[0032] Next, in order to explain the effects of the present invention in detail, experimental examples will be given.

Example

[0033] Experimental Example 1 Evaluation of the effect of the extract of Sajimo daka on the sensory stimulus activation of human TRPA1 or TRPV1-expressing HEK293 cells First, the cDNA encoding human TRPA1 (polynucleotide at positions 176 to 3535 of the nucleotide sequence shown in NM_007332.3) or the cDNA encoding human TRPV1 (polynucleotide at positions 276 to 2795 of the nucleotide sequence shown in NM_080704.4) was inserted into the multiple cloning site of pcDNA3.1(+) (manufactured by Thermo Fisher Scientific) to construct human TRPA1 or TRPV1 expression vectors, respectively. The constructed human TRPA1 or TRPV1 expression vectors were introduced into HEK293 cells by transfection using Lipofectamine 3000 Reagent (manufactured by Thermo Fisher Scientific). HEK293 cells were cultured using DMEM (manufactured by nacalai) containing 10% FBS. Stable expression strains were established by drug selection, and human TRPA1 or TRPV1-expressing cells were established, respectively.

[0034] Next, the cells were seeded at 100,000 cells per well in a 96-well plate (black, transparent bottom), cultured in DMEM containing 10% FBS for 24 hours, then the medium in the well was removed, and HHBS (Hanks′ Buffer with 20 mM Hepes) containing 10 μM calcium indicator Cal-520 and 0.04% Pluronic F-127 (manufactured by sigma-aldrich) was added, incubated at 37°C and 5% CO2 for 60 minutes, and then further incubated at room temperature for 30 minutes.

[0035] Before exposure to AITC (manufactured by Tokyo Chemical Industry Co., Ltd.), which is a TRPA1 agonist, or capsaicin (manufactured by Fujifilm Wako Pure Chemical Corporation), which is a TRPV1 agonist, the fluorescence intensity was measured using a plate reader under the conditions of an excitation wavelength of 490 nm and an emission wavelength of 525 nm. Subsequently, AITC as a TRPA1 agonist at a final concentration of 200 μM or capsaicin as a TRPV1 agonist at a final concentration of 20 μM and a hot water extract of Sargassum horneri as a test substance at final concentrations of 0, 0.000072, 0.00072, and 0.0024% were dissolved in HHBS and exposed respectively, and the fluorescence intensity every 1 second was measured for 5 minutes. For the measured series of fluorescence values, the value obtained by subtracting the minimum value before the addition of the test substance from the maximum value after the addition of the test substance (maximum value - minimum value) was calculated for each, and this was defined as the TRPA1 or TRPV1 activity. The TRPA1 activity inhibition rate or TRPV1 activity inhibition rate was calculated by dividing the difference in TRPA1 or TRPV1 activity between the untreated group and the treated group of the hot water extract of Sargassum horneri by the TRPA1 or TRPV1 activity of the untreated group. As a result, treatment with the hot water extract of Sargassum horneri suppressed both TRPA1 and TRPV1 in a concentration-dependent manner (Tables 1 and 2).

[0036]

Table 1

[0037]

Table 2

[0038] From the above results, the extract of Sargassum horneri of the present invention had the effect of effectively suppressing the activities of TRPA1 and TRPV1. Therefore, by applying the TRPA1 and / or TRPV1 activity inhibitor of the present invention to cough, asthma, etc. in which the activation of TRPA1 and / or TRPV1 is involved, a higher improvement effect can be expected.

Example

[0039] Experimental Example 2 Influence of the extract of Sargassum horneri on cough Thirty subjects who had been concerned about coughing on a regular basis were each given 100 mL of the beverage of Formulation Example 6 and 100 mL of the beverage of Comparative Example 1 to drink for 3 months, with 15 subjects for each beverage. After 3 months of drinking, a questionnaire survey was conducted on the improvement of coughing, and the degree of improvement of coughing was evaluated. As items of the questionnaire, the subjects were asked to answer whether there was an "obvious improvement effect", a "slight improvement effect", or "no improvement effect", and the numbers of such answers were tabulated.

[0040]

Table 3

[0041] As shown in Table 3, in the subjects who drank the beverage of Formulation Example 6, an obvious improvement effect on coughing was observed as compared with the subjects who drank the beverage of Comparative Example 1. From the above, it was considered that the extract of Salsola soda has an expected improvement effect on coughing.

Industrial Applicability

[0042] The extract of Salsola soda of the present invention had an effect of effectively suppressing the activities of TRPA1 and TRPV1. Therefore, the TRPA1 and / or TRPV1 activity inhibitor of the present invention can be expected to be applied to foods, cosmetics, quasi-drugs, pharmaceuticals, etc. as a relaxant for conditions related to the activation of TRPA1 and / or TRPV1 such as coughing and asthma.

Claims

1. A TRPA1 and / or TRPV1 activity inhibitor characterized by containing an extract of Sardinella zunasi.

2. A cough and / or asthma ameliorant characterized by containing the TRPA1 and / or TRPV1 activity inhibitor according to Claim 1.

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