JP2021088527A - TRPV1 activity inhibitor - Google Patents

Abstract

To discover that gardenia extract has TRPV1 activity inhibitory action and provide a TRPV1 activity inhibitor that is safe and has excellent stability.SOLUTION: It has been discovered that gardenia extract has excellent TRPV1 activity inhibitory action and also has excellent stability. The gardenia extract can be expected to be applied to an analgesic topical preparation and a sensory stimulation reducer, a frequent urination improver or the like, a TRPV1 activation-associated condition mitigating agent or the like, and food, cosmetics, quasi drugs and pharmaceuticals or the like.SELECTED DRAWING: None

Description

The present invention provides an external preparation and an internal preparation having an excellent effect of suppressing TRPV1 activity by containing an extract of cuttlefish.

TRPV1 is an ion channel sensitive to capsaicin, heat and the like (Non-Patent Document 1). In addition, activation of TRPV1 is known to be associated with inflammatory pain, sensory stimulation, and pollakiuria. Therefore, clinical trials of substances that inhibit the activity of TRPV1 are underway as analgesics for inflammatory pain, reduction agents for sensory irritation, and improvement agents for pollakiuria, but TRPV1 is safer and more suitable for clinical application. There is a demand for activity inhibitors.

Gardenia (Gardenia jasmines, Rubiaceae, scientific name) is a plant that blooms pure white flowers with a sweet scent similar to jasmine during the rainy season. So far, collagenase inhibitory action (Patent Document 1), ATP production promoting action (Patent Document 2), and ceramide production promoting action (Patent Document 3) have been reported for cutinashi. However, it has not been known so far that it has a TRPV1 activity inhibitory effect.

Japanese Unexamined Patent Publication No. 2003-2813 JP-A-2009-256272 JP-A-2002-370998

Takayuki Nakagawa, "Pain Receiving Mechanism and Possibility of Creating New Analgesics", Biochemistry, The Japanese Biochemical Society, July 25, 2013, Vol. 85, No. 7, p. 561-565

It is an object of the present invention to provide a TRPV1 activity inhibitor which is safe and has excellent stability by finding a TRPV1 activity inhibitory effect in an extract of cuttlefish.

That is, the present invention comprises the following (1) to (5).

(1) A TRPV1 activity inhibitor characterized by containing an extract of cuttlefish.
(2) An analgesic characterized by containing an extract of cuttlefish.
(3) A sensory irritation reducing agent characterized by containing an extract of cuttlefish.
(4) A sensible temperature reducing agent containing an extract of cuttlefish.
(5) A pollakiuria improving agent characterized by containing an extract of cuttlefish.

The gardenia used in the present invention is a gardenia of the genus Gardenia of the family Rubiaceae (scientific name: Gardenia jasminedes), which has a height of about 30 to 60 cm, blooms white flowers in summer, and has a jasmine-like fragrance. The fruits are used in crude drugs and are called "gardenia".

The extract of Kuchinashi in the present invention is an extract of a part or whole plant of a plant such as a flower, a fruit (fruit), a seed, a leaf, a stem, and a root of the Kuchinashi. Preferably, the fruit is good. The extraction temperature is not particularly limited, and for example, it may be extracted by heating or may be extracted at room temperature. Further, the plant body may be used as it is for extraction, or may be subjected to treatments such as drying, crushing, and shredding.

The extraction method is not particularly limited, but can be carried out by a method of stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.) and liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, etc.). , Glycerin, etc.), Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Etc.). Polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferred, and water, ethanol, 1,3-butylene glycol and propylene glycol are particularly preferred. These solvents may be used alone or in admixture of two or more. Particularly preferable extraction solvents include water and water-ethanol mixed polar solvents. The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, based on the whole plant (dry weight) of Kuchinashi, but may be concentrated or isolated after extraction. It is preferably 100 times or less for convenience of operation in the case. The extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure at the time of extraction, and the like.

The above extract may be used as it is in the extracted solution, but if necessary, concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc. is performed within the range in which the effect of the present invention is exhibited. , Deodorization, ethanol precipitation, etc. may be performed before use. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze drying and the like, and used as a dried product.

In the present invention, the above-mentioned extract may be used as it is, and fats and oils, waxes, and carbonized components used in cosmetics, non-medicinal products, pharmaceuticals, foods, etc., as long as the effects of the extracts are not impaired. Hydrogens, fatty acids, alcohols, esters, surfactants, metal soaps, pH regulators, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents , Chelating agents, excipients, filming agents, sweeteners, acidity agents and the like may be contained.

The TRPV1 activity inhibitor of the present invention is characterized by containing an extract of cuttlefish as an active ingredient for suppressing the activity of TRPV1.

The TRPV1 is one of the transient receptor potential channels. Factors that activate TRPV1 include chemical stimuli such as capsaicin, camphor, and protons, thermal stimuli (stimulations around 43 ° C), pain stimuli, and mechanical stimuli that cause sodium ions and calcium from outside the cell to the inside of the cell. Permeation of cations such as ions can be mentioned.

The present invention can be used for any of cosmetics, non-pharmaceutical products, pharmaceuticals, and foods, and the dosage forms thereof include, for example, cosmetics, creams, emulsions, gels, aerosols, essences, packs, and cleaning agents. , Bathing agents, foundations, dusting powders, lipsticks, ointments, poultices and other external preparations, tablets, capsules, chocolates, gums, candy, beverages, powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills Examples thereof include internal preparations such as preparations, suspensions, liquids, emulsions, suppositories, and injection solutions.

In the case of external use, the content of the extract used in the present invention is not particularly limited, but is preferably 0.1% by weight or more, more preferably 0.1 to 1% by weight in terms of solid matter.

For internal use, the intake varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, and the like. Usually, the daily intake per adult is preferably 5 mg or more, and more preferably 10 mg to 5 g. Further, 20 mg to 2 g is most preferable.

The amount of change in the TRPV1 activity-suppressing effect of the cuttlefish extract can be calculated by exposing TRPV1-expressing cells to the TRPV1 agonist in the presence of the cutlet extract and measuring the intracellular calcium concentration before and after the exposure. Furthermore, it can be evaluated by dividing by the amount of change in intracellular calcium concentration when the TRPV1 agonist is exposed in the absence of the extract of cuttlefish.

The TRPV1-expressing cells may be wild-type cells such as sensory nerve cells expressing endogenous TRPV1, brain cells, and bladder epithelial cells, and the nucleic acid encoding TRPV1 is a human fetal kidney cell. It may be an exogenous TRPV1-expressing cell introduced into a cultured cell such as HEK293 cell.

The change in intracellular calcium concentration of TRPV1-expressing cells before and after contact with the test substance is, for example, TRPV1-expressing cells in which TRPV1-expressing cells are treated with the calcium indicator Cal-520 (manufactured by AAT Bioquest) and Cal-520 is incorporated. It can be measured by measuring the amount of the calcium indicator bound to the calcium ion in the intracellular calcium ion over time. In this case, the change in intracellular calcium concentration can be measured based on the fluorescence intensity at 525 nm at the excitation wavelength (490 nm).

Next, in order to explain the present invention in detail, examples of production, experiment, and formulation of the extract used in the present invention will be given as examples, but the present invention is not limited thereto. The% shown in the examples means% by weight.

Example of production of extract of cuttlefish The extract of cuttlefish was produced as follows. In Production Examples 1 to 4, pear nuts were used as the extraction material.

(Production Example 1) Preparation of hot water extract of Kuchinashi 200 mL of water was added to 10 g of a dried product of Kuchinashi, and the mixture was extracted at 95 to 100 ° C. for 2 hours. The obtained extract was filtered, and the filtrate was concentrated to obtain 1.4 g of a lyophilized hot water extract of Kuchinashi.

(Production Example 2) Preparation of 50% Ethanol Extract of Kuchinashi Extraction was carried out by immersing 10 g of a dried product of Kuchinashi in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days. After filtering the obtained extract, 1.1 g of a 50% ethanol extract of Kuchinashi concentrated and dried by an evaporator was obtained.

(Production Example 3) Preparation of Ethanol Extract of Kuchinashi 10 g of a dried product of Kuchinashi was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, 0.6 g of an ethanol extract of Kuchinashi, which was concentrated and dried by an evaporator, was obtained.

(Production Example 4) Preparation of Hexane Extract of Kuchinashi 10 g of a dried product of Kuchinashi was immersed in 200 mL of hexane at room temperature for 7 days for extraction. After filtering the obtained extract, 0.4 g of a hexane extract of Kuchinashi, which was concentrated and dried by an evaporator, was obtained.

(Prescription example 1) Toner prescription content (%)
1. 1. Hot water extract of cuttlefish (Production Example 1) 2.0
2.1,3-butylene glycol 8.0
3. 3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40EO) 0.1
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water to make the total amount 100, and both are mixed and filtered to obtain a product.

(Prescription example 2) Cream prescription content (%)
1. 1. 50% ethanol extract of cuttlefish (Production Example 2) 1.0
2. Squalene 5.5
3. 3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Behenyl alcohol 1.5
9. Glycerin monostearate 2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12.1,3-butylene glycol 8.5
13. [Manufacturing method] Ingredients 1 to 9 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 11 to 13 are heated and dissolved, mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 10 is added at 45 ° C., and the mixture is further cooled to 30 ° C. to obtain a product.

(Prescription example 3) Emulsion prescription content (%)
1. 1. 50% ethanol extract of cuttlefish (Production Example 2) 0.01
2. Squalene 5.0
3. 3. Olive oil 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glycerin monostearate 2.0
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Polyoxyethylene sorbitan monooleate (20EO) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Ingredients 1 to 8 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 10 to 13 are heated and dissolved, mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 9 is added at 45 ° C., and the mixture is further cooled to 30 ° C. to obtain a product.

(Prescription example 4) Gel preparation Prescription content (%)
1. 1. Ethanol extract of cuttlefish (Production Example 3) 1.0
2. Ethanol 5.0
3. 3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60EO) 0.1
5. Appropriate amount of fragrance 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Manufacturing method] Ingredients 1 to 5 and components 6 to 11 each having a total amount of 100 in purified water are uniformly dissolved, and both are mixed to obtain a product.

(Prescription example 5) Pack prescription content (%)
1. 1. Hexane extract of cuttlefish (Production Example 4) 1.0
2. 50% ethanol extract of cuttlefish (Production Example 2) 5.0
3. 3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5.1,3-butylene glycol 8.0
6. Methyl paraoxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20EO) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 11 having a total amount of 100 in purified water are uniformly dissolved to prepare a product.

(Prescription example 6) Foundation prescription content (%)
1. 1. Hexane extract of cuttlefish (Production Example 4) 1.0
2. Stearic acid 2.4
3. 3. Polyoxyethylene sorbitan monostearate (20EO) 1.0
4. Polyoxyethylene cetyl ether (20EO) 2.0
5. Cetanol 1.0
6. Liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium Carboxymethyl Cellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Appropriate amount of fragrance 19. [Manufacturing method] Ingredients 1 to 8 having a total amount of 100 in purified water are dissolved by heating and kept at 80 ° C. to prepare an oil phase. Component 9 is well swollen with component 19, followed by addition of components 10 to 13 and uniformly mixed. Ingredients 14 to 17 pulverized and mixed by a pulverizer are added thereto, and the mixture is stirred with a homomixer and kept at 75 ° C. to prepare an aqueous phase. Add the aqueous phase to the oil phase while stirring to emulsify. Then, the mixture is cooled, component 18 is added at 45 ° C., and the product is cooled to 30 ° C. with stirring to obtain a product.

(Prescription example 7) Prescription content for bath (%)
1. 1. Ethanol extract of cuttlefish (Production Example 3) 1.0
2. Sodium bicarbonate 50.0
3. 3. Yellow No. 202 (1) Appropriate amount 4. Appropriate amount of fragrance 5. [Manufacturing method] Ingredients 1 to 5 having a total amount of 100 with sodium sulfate are uniformly mixed to prepare a product.

(Prescription example 8) Ointment prescription content (%)
1. 1. Hot water extract of cuttlefish (Production Example 1) 5.0
2. Hexane extract of cuttlefish (Production Example 4) 1.0
3. 3. Polyoxyethylene cetyl ether (30EO) 2.0
4. Glycerin monostearate 10.0
5. Liquid paraffin 5.0
6. Cetanol 6.0
7. Methyl paraoxybenzoate 0.1
8. Propylene glycol 10.0
9. [Manufacturing method] Ingredients 3 to 6 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1, 2 and 7 to 9 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring.

(Prescription example 9) Powder prescription content (%)
1. 1. Ethanol extract of cuttlefish (Production Example 3) 1.0
2. Dried cornstarch 39.0
3. 3. Microcrystalline Cellulose 60.0
[Manufacturing method] Ingredients 1 to 3 are mixed to prepare a powder.

(Prescription example 10) Tablet prescription content (%)
1. 1. 50% ethanol extract of cuttlefish (Production Example 2) 5.0
2. Dried cornstarch 25.0
3. 3. Carboxymethyl Cellulose Calcium 20.0
4. Microcrystalline Cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6. Talc 3.0
[Manufacturing method] Ingredients 1 to 4 are mixed, and then an aqueous solution of ingredient 5 is added as a binder to form granules. Ingredient 6 is added to the molded granules and the mixture is locked. One tablet weighs 0.52 g.

(Prescription example 11) Tablet confectionery prescription content (% by weight)
1. 1. 50% ethanol extract of cuttlefish (Production Example 2) 2.0
2. Dried cornstarch 49.8
3. 3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance 0.1
7. Set the total amount to 100 with purified water
[Manufacturing method] Ingredients 1 to 4 and 7 are mixed and granulated. Ingredients 5 and 6 are added to the molded granules and the mixture is locked. One grain is 1.0 g.

(Prescription example 12) Beverage prescription content (% by weight)
1. 1. Hot water extract of cuttlefish (Production Example 1) 0.05
2. Stevia 0.05
3. 3. Malic acid 5.0
4. Fragrance 0.1
5. [Manufacturing method] Components 2 and 3 having a total amount of 100 in purified water are dissolved in a small amount of water. Then, ingredients 1, 4 and 5 are added and mixed.

Experimental Example Evaluation of the effect of a test substance on sensory stimulation activation using human TRPV1-expressing HEK293 cells First, a cDNA encoding human TRPV1 (polynucleotide at positions 276 to 2795 of the nucleotide sequence shown in MN_0800704.4) was used. , PCDNA3.1 (+) (manufactured by Thermo Fisher Scientific) was inserted into a multicloning site to construct a human TRPV1 expression vector. The constructed human TRPV1 expression vector was gene-introduced into HEK293 cells by a transfection method using a Lipofectamine 3000 Reagent (manufactured by Thermo Fisher Scientific). HEK293 cells were cultured in DMEM (manufactured by nacalai) containing 10% FBS. A stable expression strain was established by drug selection, and human TRPV1-expressing cells were established.

Next, 100,000 cells per well were seeded in 96-well plate (black, transparent bottom) and cultured in DMEM containing 10% FBS for 24 hours (the amount of culture medium was 100 μL in the well). Remove the medium in the wells, add HHBS (Hanks' Buffer with 20 mM Hepes) containing 10 μM calcium indicator Cal-520 and 0.04% Pluronic F-127 (manufactured by sigma-Aldrich), and add 37 ° C., 5%. After incubating under CO _{2 for} 60 minutes, the cells were incubated at room temperature for another 30 minutes. Then, the calcium indicator mixture in the well is removed, and the extract of Kuchinashi as a test substance is added to HHBS so as to have a final concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.0%. Was dissolved in and added.

Prior to exposure to the TRPV1 agonist capsaicin (manufactured by sigma-aldrich), the fluorescence intensity was measured with a plate reader under the conditions of an excitation wavelength of 490 nm and a fluorescence wavelength of 525 nm. After the pre-exposure measurement, the final concentration of capsaicin as a TRPV1 agonist was 20 μM, and the final concentration of the extract of cutinashi as a test substance was 0, 0.1, 0.2, 0.4, 0.8, 1.0%. Each was dissolved in HHBS and exposed, and the fluorescence intensity was measured every 4 seconds for 10 minutes. As a result of analyzing the measurement results, a remarkable enhancement of the fluorescence intensity was observed in the capsaicin-exposed group, and further, the fluorescence intensity concentration-dependent in the kuchinashi extract-containing group as compared with the kuchinashi extract-free group. Suppression was observed (Table 1).

From the above results, the extract of Kuchinashi of the present invention had an effect of effectively suppressing the activity of TRPV1 and was also excellent in stability. From this, it is expected that a higher analgesic effect can be obtained by applying the TRPV1 activity inhibitor of the present invention to a site where pain or the like associated with TRPV1 activation occurs.

The extract of Kuchinashi of the present invention had an effect of effectively suppressing the activity of TRPV1 and was also excellent in stability. Therefore, the TRPV1 activity inhibitor of the present invention can be expected to be applied to foods, cosmetics, quasi-drugs, pharmaceuticals, etc., in addition to external preparations having an analgesic effect, palliative agents for conditions related to TRPV1 activation, and the like. ..

Claims (5)

A TRPV1 activity inhibitor characterized by containing an extract of cuttlefish. An analgesic characterized by containing an extract of cuttlefish. A sensory irritation reducing agent characterized by containing an extract of cuttlefish. A sensible temperature reducing agent characterized by containing an extract of cuttlefish. A pollakiuria improving agent characterized by containing an extract of cuttlefish.