Formulation containing callicarpa japonica extract and topical skin preparation

Abstract

To provide a formulation containing an active ingredient exhibiting superior effect in enhancing skin elasticity, as well as a topical skin preparation containing the same.SOLUTION: An agent containing an extract derived from the fruit of Callicarpa japonica as an active ingredient promotes the expression of the oxytocin receptor, thereby enhancing the expression-promoting effect of type I collagen and the expression-promoting effect of Fibulin-4 and MFAP-4 genes. This agent can be added to compositions such as anti-aging cosmetics as a topical skin preparation.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a preparation containing purple perilla extract. Specifically, it relates to an oxytocin receptor activator, a type I collagen expression promoter, a Fibulin-4 expression promoter, and an MFAP-4 expression promoter having purple perilla extract as an active ingredient, and a topical skin preparation containing them.

Background Art

[0002] Aging symptoms of the skin are prominently manifested in the appearance such as wrinkles and sagging due to a decrease in skin elasticity. Therefore, the problem of how to prevent / suppress the decrease in skin elasticity as age increases, or how to maintain / improve skin elasticity, has been a major concern in a wide range of ages from the younger generation to the older generation.

[0003] Regarding the improvement of skin elasticity, various proposals have been made so far. Among them, in recent years, the correlation between oxytocin (a peptide hormone) known as the "happiness hormone" or "love hormone" and the skin improvement effect has been known.

[0004] For example, Japanese Patent Application Laid-Open No. 2023-032206 (Patent Document 1) states that "it can be presumed that enhancing the action of oxytocin in the skin brings about skin improvement", and it is described that oxytocin can be a component that affects skin elasticity and the like. Therefore, an effect of improving the skin by enhancing the action of oxytocin is expected.

[0005] As a means of enhancing the action of oxytocin, Patent Document 1 discloses enhancing the reaction (signal) of oxytocin. Further, Japanese Patent Application Laid-Open No. 2020-200240 (Patent Document 2) discloses increasing the number of oxytocin receptors using cinnamon extract.

[0006] On the one hand, purple gromwell extract is known as a whitening ingredient, a moisturizing ingredient, and a bioactive ingredient, and is formulated in topical skin preparations and cosmetics (Japanese Patent Application Laid-Open No. 2022-181296; Patent Document 3, etc.). In addition, topical skin preparations listing many plant extracts such as purple gromwell extract as active ingredients have also been proposed (Japanese Patent Application Laid-Open No. 2023-510236; Patent Document 4, etc.).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a preparation containing an active ingredient having a high effect of enhancing skin elasticity and a topical skin preparation containing the same.

Means for Solving the Problems

[0009] The present inventors measured the oxytocin concentration in saliva and skin properties of dozens of women, and investigated the correlation. As a result, it was found that among women aged 35 and above, the higher the oxytocin concentration in saliva, the more elastic the skin, and it was confirmed that there is a correlation between the oxytocin concentration and skin elasticity.

[0010] On the other hand, as a result of searching for an active ingredient that activates the oxytocin receptor in cells using a cultured cell line (cultured human skin fibroblasts), it was found that purple gromwell extract has an effect of promoting the expression of the oxytocin receptor.

[0011] Furthermore, when indigo naturalis extract and oxytocin were simultaneously administered to a cultured cell line (cultured human dermal fibroblasts), the inventors found efficacy in promoting the expression of type I collagen related to skin elasticity and increasing the gene expression of Fibulin-4 and MFAP-4, which are essential for elastin formation. The present invention has been completed under such circumstances.

[0012] That is, the present invention relates to a preparation containing indigo naturalis extract as described below. (1) An oxytocin receptor expression promoter containing indigo naturalis extract as an active ingredient. (2) The oxytocin receptor expression promoter according to (1), which is a type I collagen expression promoter. (3) The oxytocin receptor expression promoter according to (1), which is a Fibulin-4 expression promoter. (4) The oxytocin receptor expression promoter according to (1), which is an MFAP-4 expression promoter. (5) A topical skin preparation containing the oxytocin receptor activator according to any one of (1) to (4).

[0013] (6) A cosmetic containing the topical skin preparation according to (5). (7) A skin care method including the step of applying the cosmetic according to (6) to the skin.

Advantages of the Invention

[0014] The oxytocin receptor expression promoter containing indigo naturalis extract of the present invention (hereinafter sometimes referred to as "the agent of the present invention") has the effect of promoting the expression of oxytocin receptors in dermal fibroblasts.

[0015] By promoting the expression of oxytocin receptor, the number of oxytocin receptors increases and the sensitivity to oxytocin rises. By enhancing the sensitivity to oxytocin, the expression of type I collagen is promoted. Furthermore, the gene expression of Fibulin-4 and MFAP-4, which are essential for elastin formation, is promoted.

[0016] Fibulin-4 is a protein required for increasing the strength by cross-linking collagen and elastin, which are components of the extracellular matrix in the skin dermis. MFAP-4 is a protein essential for elastin formation in the skin dermis. When the amount of these proteins increases in the skin, the formation of collagen and elastin (elastic fibers) is promoted, the elasticity of the skin is improved, and it is possible to prevent the decrease in the elasticity of the skin due to aging and ultraviolet rays. Therefore, it has a particularly high anti-aging effect.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0018] 1. Purple perilla extract The purple beautyberry extract in the present invention is an extract of the whole herb or flowers, fruits, stems, roots, or leaves of the plant "purple beautyberry" (scientific name: Callicarpa japonica) of the Lamiaceae family, preferably an extract of the fruit. Examples of the extract include extracts of fruits, fractions of the extract, purified fractions, and solvent-removed products thereof. Also, plants of the same genus such as large purple beautyberry (scientific name: Callicarpa japonica var. luxurians), common beautyberry (scientific name: Callicarpa dichotoma), Formosan beautyberry (scientific name: Callicarpa formosana), soft beautyberry (scientific name: Callicarpa mollis), and Shikoku beautyberry (scientific name: Callicarpa shikokiana) can also be used.

[0019] (1) Extraction target The purple beautyberry extract can be obtained by drying, purifying, and extracting from the fruits of the above-mentioned plant (purple beautyberry) by known methods.

[0020] The raw material can be used raw or dried, but in order to facilitate the extraction of the extract, fruits and their dried products can be juiced, pulverized, shredded, etc. into a size that is easy to extract in advance and then used. The form to be used can be appropriately selected, and treatments such as sterilization can be performed as necessary.

[0021] (2) Extraction method Examples of the extraction method of the extract of the present invention include solvent extraction. In solvent extraction, the whole or part of the fruit is dried as necessary, and further shredded or pulverized as necessary, and then extracted by appropriately selecting a preferred solvent (extraction solvent) according to the nature of the raw material and the use of the composition.

[0022] (3) Solvent Examples of the extraction solvent include water, anhydrous or hydrous organic solvents, etc. As water, cold water, warm water, or hot water with a boiling point or lower can be appropriately selected.

[0023] Examples of anhydrous or water-containing organic solvents include one or more selected from polar solvents such as lower alcohols having about C1 to C5, such as methanol, ethanol, isopropyl alcohol, and butanol; lower polyhydric alcohols, such as 1,3-butanediol and polypropylene glycol; ketones, such as acetone and methyl ethyl ketone; and ethers, such as diethyl ether and tetrahydrofuran.

[0024] The lower alcohol may be, for example, a water-containing lower alcohol, in which case the water content may be, for example, 0 to 10 v / v%, 10 to 40 v / v%, 20 to 30 v / v%, 30 to 50 v / v%, 50 to 80 v / v%, 80 to 99.5 v / v%, etc.

[0025] Among the above extraction solvents, polar solvents such as water, lower alcohols, and polyhydric alcohols are preferred, with water, methanol, ethanol, 1,3-butylene glycol, and the like being particularly preferred.

[0026] The extraction method is not limited to solvent extraction, and any commonly used method known in the art may be used. The extraction method and form of the extract used in the present invention are arbitrary as long as they do not impair the effects of the present invention.

[0027] The form of the extract may be not only the extract itself, but also an extract that has been appropriately diluted or concentrated by a conventional method, or a powder or lump solid obtained by drying the extract.

[0028] A specific extraction method in the present invention includes a method in which approximately 1 to 30 parts by mass of a solvent is added to 1 part by mass of the material to be extracted, followed by immersion for several days at room temperature or several hours at a temperature near the boiling point, cooling to room temperature, and then removing insoluble matter and / or the solvent as desired, and fractionating and purifying the material by column chromatography or the like.

[0029] 2. Benefits of Veronica Extract (1) Promoting oxytocin receptor expression The inventors of the present invention have discovered that purple gromwell extract has a high oxytocin receptor expression promoting effect. Based on this discovery, the present invention provides an oxytocin receptor expression promoter containing purple gromwell extract as an active ingredient.

[0030] Oxytocin (CAS No.: 50-56-6) is a peptide hormone consisting of 9 amino acids (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly) and is one of the posterior pituitary hormones.

[0031] Normally, oxytocin receptors are present in fibroblasts, and purple gromwell extract has the effect of promoting this expression. Here, the promotion of the expression of the receptor in fibroblasts can be confirmed by measuring the amount of the receptor protein and the expression level of the gene encoding the protein by conventional methods, or by measuring the sensitivity of fibroblasts to oxytocin. The "expression promoter" referred to here is not limited to medical components such as pharmaceuticals as long as it is a component that promotes the expression of oxytocin receptors.

[0032] (2) Effect of promoting type I collagen expression The inventors of the present invention have discovered that purple gromwell extract has a high type I collagen expression promoting effect by promoting the expression of oxytocin receptors and enhancing the sensitivity to oxytocin. Based on this discovery, the present invention provides a type I collagen expression promoter comprising an oxytocin receptor expression promoter containing purple gromwell extract as an active ingredient.

[0033] (3) Effect of promoting the expression of Fibulin-4 and MAPF-4 Regarding Fibulin-4 and MFAP-4, which are proteins important for elastin (elastic fiber) formation, the inventors of the present invention discovered that purple gromwell extract exhibits a high effect of increasing their gene expression. Based on this discovery, the present invention provides a Fibulin-4 expression promoter and an MFAP-4 expression promoter containing purple gromwell extract as an active ingredient.

[0034] (5) Use The oxytocin receptor expression promoter of the present invention (the agent of the present invention), the type I collagen expression promoter, Fibulin-4 expression promoter, and MFAP-4 expression promoter composed of the same can select various dosage forms in any form such as a transdermal agent or an oral agent. Considering the effect of improving skin elasticity, a transdermal agent is preferred, but not limited thereto. That is, the agent of the present invention is useful as an active ingredient of a skin external preparation.

[0035] In addition, the agent of the present invention can also be used as a composition containing the same. The agent or composition of the present invention can be administered by any route such as topical administration or oral administration. As the form of topical administration, for example, creams, emulsions, liquids, sheets, sprays, gels, etc. can be arbitrarily selected. As the form of oral administration, for example, tablets, supplements, beverages, powders, etc. can be arbitrarily selected.

[0036] 4. Skin external preparation The agent of the present invention is useful as an active ingredient of a skin external preparation. When the agent of the present invention is contained in a skin external preparation, the content of purple gromwell extract is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, still more preferably 0.1% by mass or more, and preferably 10.0% by mass or less, more preferably 5.0% by mass or less, still more preferably 1.0% by mass or less in total with respect to the total amount of the skin external preparation. Within this range, the desired effect can be easily obtained.

[0037] Examples of the dosage forms of the external preparation for skin of the present invention include, but are not particularly limited to, lotion dosage forms, emulsified dosage forms such as emulsions and creams, oil dosage forms, oil gel dosage forms, gel dosage forms, packs, cleansing agents, etc. Further, the external preparation for skin of the present invention is suitable for the forms of cosmetics and quasi-drugs.

[0038] In the external preparation for skin of the present invention, in addition to the purple gromwell extract, optional components used in ordinary external preparations for skin can be arbitrarily contained as long as the effects of the present invention are not impaired. Such optional components include various other active ingredients, oily components, surfactants, polyhydric alcohols, thickeners, powders, ultraviolet absorbers, ultraviolet scatterers, excipients, coloring agents, preservatives, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, pH adjusters, etc. For these, known ones can be appropriately selected and used. The external preparation for skin of the present invention can be produced by a conventional method with a formulation in which the above additives are arbitrarily selected and appropriately combined according to the dosage form as needed.

[0039] Examples of other active ingredients include whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, extracts derived from other animals and plants, etc. The whitening ingredients are not particularly limited as long as they are generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbyl glucoside, 3-O-ethylascorbic acid, arbutin, ellagic acid, kojic acid, linoleic acid, niacinamide, 5,5'-dipropylbiphenyl-2,2'-diol, disodium 5'-adenylate, potassium 4-methoxysalicylate, hydroquinone, pantothenic acid, tranexamic acid, etc. can be mentioned.

[0040] There are no particular limitations on the wrinkle-improving ingredients as long as they are generally used in cosmetics. For example, sodium isopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate, nicotinamide, vitamin A or its derivatives (retinol, retinal, retinoic acid, tretinoin, isotretinoin, retinoic acid tocopherol, retinol palmitate, retinol acetate, etc.), benzyl ursoate, phosphate ester of ursoic acid, benzyl ester of betulinic acid, phosphate ester of benzoic acid can be mentioned.

[0041] As the anti-inflammatory ingredients, glycyrrhizic acid, glycyrrhetinic acid, allantoin, isopropylmethylphenol, clarinone, glabridin, salicylic acid, tocopherol acetate, tocopherol nicotinate, nicotinamide, pantothenic acid, pantothenyl alcohol, tranexamic acid and their salts or derivatives, etc. can be mentioned. Preferably, they are glycyrrhizic acid and its salts, glycyrrhetinic acid and its salts, pantothenyl alcohol, and pantothenic acid and its salts.

[0042] There are no particular limitations on other extracts derived from animals and plants as long as they are generally used in cosmetics, etc. For example, Akebia extract, Asclepias extract, Asparagus extract, Avocado extract, Amacha, Coptis extract, Peppermint extract, Sedum extract, Rosa multiflora extract, Lavender extract, Bottle gourd extract, Barley fermentation extract, Hydrolyzed black bean extract, Bupleurum extract, Hydrolyzed conchiolin solution (pearl protein decomposition extract), etc. can be mentioned.

[0043] Examples of surfactants include anionic surfactants such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and triethanolamine alkyl sulfate ethers; cationic surfactants such as stearyl trimethyl ammonium chloride, benzalkonium chloride, and lauryl amine oxide; betaine surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.); imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.); amphoteric surfactants such as acyl methyl taurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.); glycerin fatty acid esters (glyceryl monostearate, etc.); propylene glycol fatty acid esters (propylene glycol monostearate, etc.); hydrogenated castor oil derivatives; glycerin alkyl ethers; POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.); POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.); POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.); POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.); POE alkyl ethers (POE 2-octyldodecyl ether, etc.); POE alkyl phenyl ethers (POE nonyl phenyl ether, etc.); pluronic types; POE·POP alkyl ethers (POE·POP 2-decyltetradecyl ether, etc.); tetronic types; POE castor oil·hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.); sucrose fatty acid esters; nonionic surfactants such as alkyl glucoside, etc.

[0044] Examples of the polyhydric alcohol include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol and the like.

[0045] Examples of the thickener include guar gum, quince seed, carrageenan, galactan, gum arabic, pectin, mannan, starch, xanthan gum, curdlan, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, methyl hydroxypropyl cellulose, chondroitin sulfate, dermatan sulfate, glycogen, heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, mucitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, carroninic acid, chitin, chitosan, carboxymethyl chitin, agar, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, sodium polyacrylate, polyethylene glycol, bentonite and the like.

[0046] Examples of the ultraviolet absorber include para-aminobenzoic acid-based ultraviolet absorbers, anthranilic acid-based ultraviolet absorbers, salicylic acid-based ultraviolet absorbers, cinnamic acid-based ultraviolet absorbers, benzophenone-based ultraviolet absorbers, sugar-based ultraviolet absorbers, ultraviolet absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane and the like.

[0047] 5. Use (1) Composition such as cosmetics The agent of the present invention may be added as an active ingredient to a cosmetic composition or a food composition. For example, the agent of the present invention may be blended with ingredients that can be used in ordinary cosmetics to form a cosmetic for improving skin conditions. In that case, the cosmetic ingredients that can be blended with the agent of the present invention can be selected from the above-described skin-external preparation ingredients. For example, whitening ingredients, anti-inflammatory ingredients, surfactants, polyhydric alcohols, antioxidant ingredients, anti-wrinkle ingredients, etc. may be mentioned. Among them, whitening ingredients, anti-wrinkle ingredients, etc. are particularly preferable.

[0048] (2) Skin care method In the present invention, it is possible to provide a skin care method or a beauty method for improving skin conditions, including administering the agent or composition of the present invention. For example, a skin care method including a step of applying the agent of the present invention or a composition such as a cosmetic containing the same to the skin may be mentioned. Examples of the method of applying to the skin include a method of directly applying to the skin and a method of applying while massaging with the hands.

[0049] In the present invention, by increasing the expression of oxytocin receptor with the purple perilla extract and increasing the sensitivity to oxytocin, the action of enhancing the effect of oxytocin secreted in the body is exerted. Such action effects can increase the oxytocin concentration in the body, for example, by applying the agent or composition of the present invention to the cheeks and wrapping the cheeks with both hands, gently pressing the skin after applying to the skin, or gently stroking the skin at a speed of about 5 cm per second for example.

[0050] The cosmetic of the present invention may be various cosmetics such as emulsions, creams, essences, lotions, packs, facial washes, soaps, body soaps, shampoos, etc., and may be in various forms such as liquid, emulsion, cream, solid, sheet, spray, gel, foam, powder, etc. Further, the food composition of the present invention may be a powder, a beverage, or a tablet, and may be in various forms such as powdery, liquid, solid, granular, granule, paste, gel, etc.

[0051] The external preparation for skin of the present invention is useful as a cosmetic composition such as an anti-aging cosmetic. It can also be used for a beauty method of applying it to human skin to increase the oxytocin concentration and the like.

Example

[0052] The present invention will be described below with reference to examples, but the present invention is not limited by these examples at all.

[0053] <Example 1> (1) Preparation of sample 1 g of dried fruits of Prunus mume was pulverized in liquid nitrogen, 20 mL of an 80% ethanol aqueous solution was added, and the mixture was allowed to stand for 24 hours. After 24 hours, it was filtered, and the 80% ethanol aqueous solution was added to make 50 mL to obtain a sample solution.

[0054] (2) Cell culture Normal human skin fibroblasts (Kurabo) were cultured under conditions of 37 ° C and 5% CO2 using DMEM (manufactured by Sigma) containing 5% FBS. The cells were detached using PBS (-) containing 0.25% trypsin and 0.02% EDTA, and 1.0 × 10 4 cells / well or 1.5 × 10 4 cells / well were seeded at a density and cultured for 24 hours. After culturing, the medium was removed, the cells were washed with PBS, and then 100 μl of a 0.5% FBS-containing DMEM medium containing Prunus mume extract or a solvent control was added, and the cells were cultured for 48 hours in an environment of 37 ° C and 5% CO2.

[0055] (3) Measurement of the expression level of oxytocin receptor activity The expression level of the oxytocin receptor was measured by the following method.

[0056] After culturing for 48 hours in a 0.5% FBS-containing DMEM medium containing Prunus mume extract (final concentration 1%) or a solvent control, the medium was removed, 100 μl / well of methanol at -20 ° C was added, and the mixture was allowed to stand at 4 ° C for 15 minutes to fix the cells.

[0057] After cell fixation, 50 μl / well of 0.1% Triton X-100 was added, and the mixture was allowed to stand at room temperature for 5 minutes for permeabilization treatment. After the permeabilization treatment, 100 μl / well of a 4 μM Hoechst 33342 / PBS solution was added, and the mixture was allowed to stand at room temperature for 30 minutes in the dark for nuclear staining and fluorescence measurement (Ex / Em = 360 / 465).

[0058] After measurement, 150 μl / well of 1% BSA / PBS solution was added, and the mixture was allowed to stand at 37 °C for 1 hour for blocking. 100 μl / well of the primary antibody (oxytocin receptor antibody / 0.3% BSA (1:5,000)) was added and allowed to stand overnight at 4 °C.

[0059] The next day, 100 μl / well of the secondary antibody Goat Anti-Rabbit IgG (Biotin) / 0.3% BSA (1:10,000) was added and reacted at room temperature for 2 hours. Then, 100 μl / well of Streptavidin-HRP / 0.3% BSA (1:500) was added and reacted at 37 °C for 30 minutes. Then, 150 μl / well of ABTS solution was added and reacted at room temperature in the dark for 20 minutes, and the absorbance at 405 nm was measured to determine the relative expression level of the oxytocin receptor. The results are shown in Figure 1. In the graph of Figure 1, the vertical axis represents the "relative expression level of oxytocin receptor", the left side of the horizontal axis is the control, and the right side is the result when containing 1% purple perilla extract. In the figure, "**" represents p < 0.01 (vs control: number of experimental replicates n = 4) (p is the probability that a rarer event occurs than the observed event when assuming that the null hypothesis is correct: P value).

[0060] <Example 2> Using the purple perilla extract prepared in the same manner as in Example 1, the expression level of type I collagen was measured by the following method.

[0061] Using DMEM medium, normal human dermal fibroblasts were seeded in a 96-well plate at 1.0×10 4Cells were seeded at a density of

[0062] After culturing for 48 hours, oxytocin-containing medium adjusted to 3 nM was added, and after culturing for 24 hours, protein quantification of the cells was performed by BCA assay, and quantification of type I collagen was performed on the culture supernatant.

[0063] 100 μl of the culture supernatant diluted 3-fold was added to an ELISA plate and coated at 37 °C for 1 hour. After coating, 150 μl / well of 1% BSA / PBS solution was added and allowed to stand at 37 °C for 1 hour for blocking. 100 μl / well of the primary antibody (type I collagen antibody / 0.3% BSA (1:4,500)) was added and allowed to stand overnight at 4 °C.

[0064] The next day, 100 μl / well of Streptavidin-HRP / 0.3% BSA (1:500) was added and reacted at 37 °C for 1 hour. Then, 150 μl / well of ABTS solution was added and reacted at room temperature in the dark for 20 minutes, and the absorbance at 405 nm was measured to measure the expression level of type I collagen. The results are shown in Figure 2. In the graph of Figure 2, the vertical axis represents "type I collagen expression level", the left end of the horizontal axis is the control, the second from the left is the case containing 1% purple perilla extract, the second from the right is the case containing 3 nM oxytocin, and the right end is the result of the case containing 1% purple perilla extract and 3 nM oxytocin. In the figure, "**" represents p < 0.01 (vs control: n = 4).

[0065] <Example 3> Using the purple perilla extract prepared in the same manner as in Example 1, the gene expression levels of Fibulin-4 and MFAP-4 were measured by the following method.

[0066] Using DMEM medium, normal human dermal fibroblasts were seeded at a density of 1.5 × 104 Cells were seeded at cells / well and cultured for 24 hours in an environment of 37°C and 5% CO₂. After culturing, the culture medium was removed, and the cells were washed with PBS. Then, 100 μl of DMEM medium containing 0.5% FBS with either purple perilla extract (final concentration 1%) or the solvent control was added, and the cells were cultured at 37°C and 5% CO 2 for 48 hours in an environment. After culturing for 48 hours, oxytocin-containing medium adjusted to 1 nM was added and cultured for 24 hours.

[0067] After culturing, total RNA extraction and cDNA synthesis were performed using the Power SYBR TM Green Cells-to-CT TM Kit. Subsequently, the expression levels of Fibulin-4 and MFAP-4 were measured by real-time PCR (Step One TM SYBR TM Green Master Mix using an Applied Biosystems instrument. The primer sequences for Fibulin-4, MFAP-4, and GAPDH used as an internal standard are shown in Table 1. The primer sequences used are shown in Table 1. TM ; Applied Biosystems).

[0068]

Table 1

[0069] The measurement results are shown in FIGS. 3 and 4. In the graph of FIG. 3, the vertical axis represents the "relative expression level of the Fibulin-4 gene", the left end of the horizontal axis is the control, the second from the left is the case containing 1% purple perilla extract, the second from the right is the case containing 1 nM oxytocin, and the right end is the result of the case containing 1% purple perilla extract and 1 nM oxytocin. In the graph of FIG. 4, the vertical axis represents the "relative expression level of the MFAP-4 gene", the left end of the horizontal axis is the control, the second from the left is the case containing 1% purple perilla extract, the second from the right is the case containing 1 nM oxytocin, and the right end is the result of the case containing 1% purple perilla extract and 1 nM oxytocin. In the figure, "*" represents p < 0.05, and "**" represents p < 0.01 (vs control: n = 4).

Industrial Applicability

[0070] The agent of the present invention promotes the expression of oxytocin receptor, and can enhance the elasticity of the skin by promoting the expression of type I collagen related to skin elasticity and promoting the gene expression of Fibulin-4 and MFAP-4 important for elastin formation. Therefore, the agent of the present invention is useful as an active ingredient of various cosmetics such as anti-aging cosmetics that can enhance the firmness and elasticity of the skin, and external skin preparations such as beauty agents.

Explanation of Signs

[0071] *; p < 0.05 (vs control: n = 4) **; p < 0.01 (vs control: n = 4)

Claims

1. An oxytocin receptor expression promoter containing indigo woad root extract as an active ingredient.

2. The oxytocin receptor expression promoter according to Claim 1, which is a type I collagen expression promoter.

3. The oxytocin receptor expression promoter according to Claim 1, which is a Fibulin-4 gene expression promoter.

4. The oxytocin receptor expression promoter according to Claim 1, which is an MFAP-4 gene production promoter.

5. A topical skin preparation containing the oxytocin receptor expression promoter according to any one of Claims 1 to 4.

6. A cosmetic containing at least the topical skin preparation according to Claim 5.

7. A skin care method including the step of applying the cosmetic according to Claim 6 to the skin.

Images