JP2013091631A - Involucrin expression inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an involucrin expression inhibitor capable of inhibiting expression of involucrin, a kinky hair ameliorant and a skin care external preparation.SOLUTION: In an involucrin expression inhibitor, a kinky hair ameliorant and a skin care external preparation, one or more kinds of plants selected from Halenia corniculate, Begonia pedatifida Levl., Dichondra micrantha, China forget-me-not and Veronica anagallis-aquatica or extracts therefrom are used as active ingredients.

Description

  The present invention relates to an involucrin expression inhibitor, a comb hair improving agent, and a skin external preparation.

  Involucrin is a protein produced in spinous cells according to the differentiation of epidermal keratinocytes, and is a strong insoluble membrane that lines the cell membrane of keratinocytes (cornified envelope; hereinafter also abbreviated as CE). ) Is one of the main components (Non-Patent Document 1). CE is formed by cross-linking and insolubilizing multiple CE precursor proteins including involucrin with the enzyme transglutaminase. Furthermore, involucrin that constitutes CE contributes to the formation of an intercellular lipid lamellar structure by covalently binding a lipid such as ceramide to a part of the involucrin (Non-patent Document 2). Such CE formation / maturation stabilizes the lamellar structure of the intercellular lipid, and the barrier function of the stratum corneum works normally, thereby enhancing the moisture retention function of the skin and the resistance to external stimuli.

  Recently, the present applicant has searched for the relationship between the hair shape and the gene expression of various genes in the hair root, and found that involcrine (IVL) in the hair root of a non-hairy person compared to the hair root part of the hairless person. It has been reported that the expression level of the gene is significantly increased, and it is possible to suppress or promote comb hair by controlling the expression of involucrin (Patent Document 1).

  On the other hand, the genus Hanakari of Gentianaceae is used for the treatment of jaundice hepatitis, gastritis and cholecystitis, Begonia pedatifida Levl. Is used for the treatment of hemorrhoids and hemostasis, and the treatment of the genus Convolvulaceae is the diuresis, analgesia, For the detoxification and nephritis treatment, the urchinaceae of the genus Orrisoaceae is used for private use for hemostasis, antitussives and bleeding wounds, and the common stag beetle is used for diuresis, sore throat and antitussive. However, it is not known that these plants have an involucrin expression-inhibiting action and a comb hair improving action.

International Publication No. 2011/043330 Pamphlet

Steinert, PM. Et al., J. Biol. Chem., 270 (30), p17702-17711 (1995) Nemes, Z., Proc. Natl. Acad. Sci. USA, 96 (15), p8402-8407 (1999)

  The present invention relates to providing an involucrin expression inhibitor, a hair ameliorating agent, and a skin external preparation, which suppress the expression of involucrin.

  The present inventors examined involucrin expression suppression and found that a specific plant extract has an excellent involucrin expression suppression effect, which is useful as a component or material for suppressing involucrin expression and improving comb hair. I found it.

That is, the present invention relates to the following 1) to 3).
1) An involucrin expression inhibitor comprising as an active ingredient at least one plant selected from Hanaikari, Begonia pedatifida Levl., Green moss, Shinawasuna Nagasa and Okawajisha.
2) A comb hair improving agent comprising, as an active ingredient, one or more plants selected from Hanaikari, Begonia pedatifida Levl., Aoiike, Shinawasunagusa and Okawajisha.
3) A topical skin preparation containing one or more plants selected from Hanaikari, Begonia pedatifida Levl., Blue Mushrooms, Shinawasuna Nagasa and Okawajisha or extracts thereof.

  According to the present invention, it is possible to provide cosmetics, quasi-drugs and the like that have an involucrin expression inhibitory action and improve comb hair.

The graph which shows inboliclin expression suppression effect.

In the present invention, “Hanaikari” means “Halenia elliptica D.Don” of Gentianaceae, “Begonia pedatifida Levl.” Means “Begonia pedatifida Levl.” Of “Begoniaceae”, “Aoigoke” means “Aoigoke” Forst.), Sinawas renagusa means Cynoglossum amabile Staf et Drum., And Okawajisha means Veronica angalis-aquatica L.
The plant can use parts such as leaves, stems, buds, flowers, seeds, fruits, whole grasses, etc., but it is preferable to use whole plants as they are or after pulverization.

  Such a plant can be used as it is or as a squeezed juice obtained by squeezing it, a dried product obtained by drying the plant itself or a pulverized product thereof, or an extract extracted from these, but it can be used as an extract. preferable.

As the extract, various solvent extracts obtained by a known extraction method such as extraction of the plant at room temperature or under heating or extraction using an extraction device such as a Soxhlet extractor, dilutions thereof, A concentrated solution or a dry powder thereof may be mentioned.
Known extraction methods include, for example, immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, and microwave extraction.

  As the extraction solvent used for obtaining the extract, either a polar solvent or a nonpolar solvent can be used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran and diethyl ether Linear and cyclic ethers such as polyethylene; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; aroma such as benzene and toluene Group hydrocarbons; pyridines and the like, and these can be used alone or as a mixture. Among these, it is particularly preferable to use water, alcohol-based (alcohols and / or polyhydric alcohols (V / V)) solvents, and water-alcohol mixed solvents, and water-alcohol mixed solvents (V / V). Is preferred. Furthermore, alcohols are preferably alcohols, and among alcohols, methanol, ethanol, and propanol are more preferable, and ethanol is particularly preferable.

  The plant extract of this invention can be performed by using 1-50 mass parts extraction solvent with respect to 1 mass part of plants, for example, and extracting at 4-100 degreeC for 0.5 hour-30 days. More specifically, when water is used as the extraction solvent, it is preferably 5 to 30 parts by mass and 40 to 100 ° C. for 1 hour to 1 day with respect to 1 part by mass of the plant. Moreover, when 40-60% ethanol aqueous solution is used as an extraction solvent, it is 3-20 mass parts with respect to 1 mass part of plants, Room temperature-under reflux, Preferably it is 20-50 degreeC, It is 1 hour-20 Days are preferred. Moreover, when using 75-100% ethanol aqueous solution, 3-20 mass parts with respect to 1 mass part of plants, 20 hours-under reflux, 1 hour-20 days are preferable.

  The above extract can be used as it is, but it can also be used by diluting, concentrating or lyophilizing the extract and preparing it in a powder or paste form.

  Moreover, you may use the plant of this invention, or its extract in mixture of 2 or more types. Moreover, you may use a commercial item other than the said extraction processed material.

  The extract is preferably used after removing inactive impurities by a known technique such as liquid-liquid distribution, solid-liquid distribution, filtration membrane, activated carbon, absorption resin, ion exchange resin and the like. As the solvent used at this time, those exemplified for the extraction solvent may be used. Moreover, you may use these, after giving processes, such as a deodorizing and a decoloring, by a well-known method as needed. Moreover, when the extract is further purified, the known techniques and methods may be used.

  As will be described later in Examples, the plant extract of the present invention suppresses the expression of involucrin. As described above, the expression level of involucrin (IVL) gene is significantly increased in the hair root part of the hairless person compared to the hair root part of the non-hairy hair person, and by suppressing the expression of involucrin, It is considered that this can be improved (see Patent Document 1).

  Therefore, the plant of the present invention or an extract thereof can be an involucrin expression inhibitor and a comb hair improving agent (hereinafter referred to as “involucrin expression inhibitor etc.”), and can also produce an involucrin expression inhibitor or a comb hair improving agent. Can be used to That is, it can be applied to humans and used to suppress the expression of involucrin or to improve comb hair.

  The involucrin expression inhibitor may itself be a cosmetic, pharmaceutical, quasi-drug, or used in combination with the cosmetic, pharmaceutical, quasi-drug, etc. It may be a material or a preparation.

  Here, unless otherwise specified, “comb hair” generally refers to shapes other than straight hair when compared to straight hair, and “improves comb hair” means curl radius and curvature of the hair. To reduce traits and bring traits closer to straight hair.

When the involucrin expression inhibitor of the present invention is used as cosmetics, quasi drugs or pharmaceuticals, it is in the form of an external preparation for skin, specifically, ointment, emulsified cosmetic, cream, emulsion, lotion, gel, It can be used in various forms such as aerosol, but it should be in the form of hair rinse, hair conditioner, hair treatment, hair lotion, hair pack, hair cream, conditioning mousse, hair mousse, hair spray, shampoo, leave-on treatment, etc. Is preferred.
Such preparations are prepared according to general production methods, respectively, directly or pharmaceutically acceptable carriers such as various oils, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohol, water, chelate. It can be obtained by mixing and dispersing together with an agent, a thickener, an ultraviolet absorber, an emulsion stabilizer, a pH adjuster, a dye, a fragrance and the like, and then processing into a desired form. In addition, for these cosmetics, quasi-drugs, pharmaceuticals, etc., the plant extract of the present invention or an extract thereof, and a plant extract, a bactericide, a moisturizer, an anti-inflammatory agent, an antibacterial agent, as appropriate, depending on the respective preparation. A refreshing agent, an antiseborrheic agent, and the like can be appropriately blended within a range that does not interfere with the effects of the present invention.

  In general, the content of the plant of the present invention or the extract thereof in the cosmetic, quasi-drug, or pharmaceutical product is preferably 0.0005 to 50% by mass as the solid content concentration, and 0.001 to 20%. It is more preferable to set it as the mass%.

The dosage of the plant of the present invention or the extract thereof in the above-mentioned pharmaceuticals and quasi drugs is not particularly limited as long as the effect is obtained, and varies according to the condition, weight, sex, age or other factors of the subject. However, it is preferably 0.01 to 1500 mg, more preferably 0.03 to 600 mg, for example, as a plant or an extract thereof (in terms of dry matter) per day for an adult (60 kg). The preparation can be ingested / administered according to any ingestion / administration plan, but is preferably divided into once to several times a day and continuously administered for several weeks to several months.
In addition, the application target of the cosmetic, pharmaceutical product, or quasi drug is not particularly limited as long as it is required, but a human who aims to suppress involcrine expression and improve hair loss is preferable.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Production Example 1 Production of plant extract (1) Production of Hanaikari extract Add 200 mL of 50% ethanol to 200 g of dry extract of Hanaikari elliptica D. Don (whole plant) from Sichuan, China, and heat at 85 ° C. Then, after 2 hours of immersion, it was filtered. To the residue after filtration, 1000 mL of 50% ethanol was added, and extraction was performed again at 85 ° C. for 1 hour. After filtration, the two filtrates were combined, concentrated under reduced pressure, and dried to obtain 15 g of 50% ethanol extract. This was made into a 50 vol% ethanol solution with a solid content concentration of 1.0 w / v%, and used as an evaluation extract.

(2) Manufacture of Begonia pedatifida Levl. Extract To 200 g of a dry extract of Begonia pedatifida Levl. (Whole plant) from Sichuan, China, 1000 mL of 50% ethanol was added, immersed for 2 hours under heating at 85 ° C, and then filtered. To the residue after filtration, 1000 mL of 50% ethanol was added, and extraction was performed again at 85 ° C. for 1 hour. After filtration, the two filtrates were combined, concentrated under reduced pressure, and dried to obtain 39 g of 50% ethanol extract. This was made into a 50 vol% ethanol solution with a solid content concentration of 1.0 w / v%, and used as an evaluation extract.

(3) Manufacture of aoi moss extract To 200 g of a dry extract of Dichondra repens Forst. (Whole plant) from Sichuan, China, 1000 mL of 50% ethanol was added, and immersed for 2 hours under heating at 85 ° C., followed by filtration. To the residue after filtration, 1000 mL of 50% ethanol was added, and extraction was performed again at 85 ° C. for 1 hour. After filtration, the two filtrates were combined, concentrated under reduced pressure, and dried to obtain 41 g of 50% ethanol extract. This was made into a 50 vol% ethanol solution with a solid content concentration of 1.0 w / v%, and used as an evaluation extract.

(4) Manufacture of extract of cinnamon moth (Cynoglossum amabile Staf et Drum.) From whole Sichuan Province, China And filtered. To the residue after filtration, 1000 mL of 50% ethanol was added, and extraction was performed again at 85 ° C. for 1 hour. After filtration, the two filtrates were combined, concentrated under reduced pressure, and dried to obtain 16.1 g of a 50% ethanol extract. This was made into a 50 vol% ethanol solution with a solid content concentration of 1.0 w / v%, and used as an evaluation extract.

(5) Manufacture of extract of Okawajisha Add 1000mL of 50% ethanol to 200g of dry extract of Veronica angalis-aquatica L. (whole plant) from Sichuan, China, soak for 2 hours under heating at 85 ° C, and filter did. To the residue after filtration, 1000 mL of 50% ethanol was added, and extraction was performed again at 85 ° C. for 1 hour. After filtration, the two filtrates were combined, concentrated under reduced pressure, and dried to obtain 25 g of 50% ethanol extract. This was made into a 50 vol% ethanol solution with a solid content concentration of 1.0 w / v%, and used as an evaluation extract.

Example 1 Involucrin Expression Inhibitory Action Involucrin ELISA Assay Kit (BT-650, BTI) was used to determine the amount of involucrin protein.
A 24-well plate was seeded with HaCaT cells at 5 × 10 ⁴ cells / well and cultured. On the next day, each plant extract obtained in Production Example 1 was replaced with a medium containing a final concentration of 0.5% v / v and further cultured for 24 hours. 24 hours after the addition of the extract, the cells were washed twice with PBS (Gibco), and 400 μl of a buffer containing Complete Mini Protease Inhibitor Cocktail (Roche) in 20 mM Tris-HCl (pH: 7.5) and 2 mM EDTA was added to the cells. did. One Protease Inhibitor Cocktail was added to 10 ml of buffer. Cells were detached and collected with a cell scraper in the presence of this buffer, and then sonicated with a sonicator. The supernatant collected by centrifugation of this solution was used as an lysate for evaluation.

  The total protein concentration of the lysate was quantified using BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's instructions. The lysate was diluted with buffer B (2 mM EDTA, 5 g / L Tween 20, 2.5 g / L Gelatin in PBS) to a total well of 3 µg per well of a 96-well plate and added to the 96-well plate Then, an anti-involucrin antibody (kit accessory, dilution ratio 1/300) was added and a primary antibody reaction was performed overnight at 4 ° C.

In addition, human involucrin protein (supplied with the kit) is added to another 96-well plate (Narugenunk) at a rate of 1 ng / well, and EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, Kanto Chemical) 10 μg as a condensing agent Along with / well, antigen was adsorbed overnight at 4 ° C. On the next day, the solution of the plate on which the antigen was adsorbed was removed by aspiration, and 100 μl of 0.1M NH ₄ Cl was added and allowed to react at room temperature for 30 minutes. After the reaction, the plate was washed 4 times with distilled H ₂ O (Gibco) and once with buffer B, and 100 μl of the primary antibody reaction solution of the previous day was added and reacted at room temperature for 30 minutes. After this reaction, the plate was washed 5 times with buffer B, and the anti-rabbit-AP Conjugate antibody (dilution ratio 1/2000) attached to the kit was added and reacted at room temperature for 1 hour. After the reaction, 4 times with buffer B, then washed once with _{buffer D (1mM MgCl 2 .6H 2} O in 0.05M carbonate / bicarbonate ( Sigma), p-nitrophenylphosphate (1mg / ml in buffer D, Sigma) solution 100μl After the color development was confirmed, the absorbance at 405 nm was measured to determine the amount of involucrin protein.The expression level relative to the involucrin expression level in the control (no extract added) was 1.0. Values were determined and the results are shown in FIG.

  As is clear from FIG. 1, each plant extract had an excellent inboliclin expression suppression effect.

Claims (3)

  An involucrin expression inhibitor comprising as an active ingredient at least one plant selected from Hanaikari, Begonia pedatifida Levl., Aoiike, Shinawasunagusa and Okawajisha.   A comb hair improving agent comprising, as an active ingredient, one or more plants selected from Hanaikari, Begonia pedatifida Levl., Aoiike, Shinawasunagusa and Okawajisha.   An external preparation for skin containing at least one plant selected from Hanaikari, Begonia pedatifida Levl., Aoioke, Shinawasunagusa and Okawajisha or an extract thereof.

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