JP7281156B2 - Skin topical agent - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act JOURNAL OF INVESTIGATE DERMATOLOGY website

TECHNICAL FIELD The present invention relates to an external skin preparation containing plant-derived isoflavones as an active ingredient.

Conventionally, with aging, inactivation of cell proliferation and differentiation, decrease in hormone secretion, quantitative decrease in extracellular matrix components (collagen, laminin, etc.) that make up the skin, and muscle strength decrease due to decrease in muscle fibers, etc. It is known that skin aging phenomena (wrinkles, sagging, etc.) occur for the following reasons.

Various active ingredients have been proposed for the purpose of preventing and ameliorating unhealthy cells and aging, and cosmetics, foods and drinks, and pharmaceuticals containing these active ingredients have been put on the market. Examples include cell activating components such as α-hydroxycarboxylic acid, placental extract, and γ-amino-β-hydroxybutyric acid; extracellular matrix components such as collagen, elastin, hyaluronic acid or salts thereof; moisturizing agents such as urea; be done.

Conventionally, moisturizing agents and anti-inflammatory agents containing plant extracts as active ingredients have been proposed based on the mechanisms of cell aging and unhealthy cells (Patent Documents 1 and 2).

Japanese Patent Application Laid-Open No. 2001-064192 JP 2001-122730

BACKGROUND ART Conventionally, there has been a demand for active ingredients for external use on the skin that are derived from natural products such as plants and exhibit new efficacy.

In order to solve the above problems, the present applicant focused on the phenomenon of thinning of the skin (thinning) due to ultraviolet rays, aging, decreased hormone secretion, etc., and by preventing and improving thinning of the skin. , with the aim of finding active ingredients for improving the barrier function of the skin.
That is, the present invention is an external preparation for skin containing plant-derived isoflavones (formonetin and biochanin A) or plant extracts containing these isoflavones as active ingredients.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an external skin preparation capable of preventing and improving thinning of the skin using the active ingredient of the present invention.

Preferred embodiments of the present invention are described in detail below.
The isoflavones that are the active ingredients of the present invention are formononetin and Biochanin A. In addition to these two isoflavones, the active ingredients of the present invention include other isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, platenthein, pseudobaptigenin, irilon, or prunetin). may contain

Plants containing two kinds of isoflavones, which are the active ingredients of the present invention, include leguminous plants. In the present invention, plants belonging to the genus Trifolium of the Fabaceae family are preferred, but plants containing the above two types of isoflavones can also be used. Plants belonging to the genus Chara include, for example, Trifolium pratense (also known as Trifolium pratense), Trifolium repens, Trifolium dubium, Trifolium campestre, and Trifolium repens L. form. Peterm.), safflower date (Trifolium incarnatum L.) and the like. Parts that can be used for extraction include whole plants, leaves, flowers, stems or roots.

The plant extract containing the active ingredient of the present invention is prepared by first washing the plant to be extracted with water to remove foreign substances, if necessary, then drying it as it is or drying it, and if necessary, finely chopping or pulverizing it. After that, it can be carried out by contacting with an extraction solvent according to a conventional method such as an immersion method, a countercurrent extraction method, a steam distillation method, or the like. Moreover, in the present invention, a supercritical extraction method may be employed.

Extraction solvents used for extract treatment include water; lower alcohols such as methanol, ethanol and propanol; polyhydric alcohols such as ethylene glycol, propylene glycol, 1,3-butylene glycol and glycerin; ethyl acetate and butyl acetate. , esters such as methyl propionate; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether and isopropyl ether; Two or more kinds are mixed and used.

In preparation of the extract, there is no particular limitation on the pH of the extract, but it is generally preferred to be in the range of pH 3-9. The pH is adjusted by adding an alkalinity adjuster such as sodium hydroxide, sodium carbonate, or potassium hydroxide, or an acidity adjuster such as citric acid, hydrochloric acid, phosphoric acid, or sulfuric acid to the extraction solvent. be able to.

Extraction conditions such as extraction temperature and time vary depending on the type and pH of the solvent used, or the size of the plant material. In the case of the immersion method, the extraction temperature is in the range of 0 to 80°C. The extraction time is preferably in the range of 1 hour to 7 days in the case of cold extraction at 4°C, and in the range of 1 hour to 3 days in the case of medium temperature extraction around 40°C. In the case of high temperature extraction at 80° C., the time should be in the range of 1 hour to 24 hours. In the case of the immersion method, the bath ratio is generally 1 to 200 times, preferably 1 to 100 times the weight of the plant material.

The plant extract prepared as described above is further subjected to one or more of activated carbon treatment, ion exchange resin treatment, synthetic adsorbent, silica gel, and recrystallization treatment in combination to obtain cyclic adenosine monophosphate. It is preferable to prepare an extract containing a high concentration of Activated charcoal can be made from plant materials such as pine, bamboo, coconut shells, walnut shells, etc., as well as coal, petroleum, etc., and high-temperature carbonization using gases such as steam, carbon dioxide, and air for these raw materials. Activated carbon can be obtained by a physical method such as a chemical method such as a method, or by a chemical method such as treatment with zinc chloride and the like followed by heating to make it porous. Examples of ion exchange resins include weakly basic anion exchange resins, strongly acidic cation exchange resins, strongly basic ion exchange resins, and weakly acidic cation exchange resins. Examples of synthetic adsorbents include nonionic resins such as chelate resins, strongly acidic cation-adsorbing resins, styrene/divinylbenzene copolymers, and methacrylic acid ester polymers.

In the case of formononetin, the amount of isoflavones contained in the extract obtained by the extraction treatment as described above is preferably at least 40 ppm or more, more preferably 50 ppm or more, and still more preferably 80 ppm or more. In the case of Biochanin A, it is preferably 20 ppm or more, more preferably 30 ppm or more, and even more preferably 80 ppm or more. Furthermore, in the present invention, the total amount of formononetin and biochanin A contained in the extract is preferably 60 ppm or more, more preferably 80 ppm or more, and still more preferably 100 ppm or more.

In the present invention, external preparations for skin include cosmetics, quasi-drugs, and pharmaceuticals for external use. , white powder, facial cleanser, body shampoo, slimming agent, hair shampoo, soap, etc., and bath agents, etc., but are of course not limited to these.

In addition to the extract according to the present invention, external skin preparations include components used in external skin preparations, such as oily components, surfactants (synthetic and natural products), moisturizers, thickeners, antiseptics and sterilizers. Agents, powder components, ultraviolet absorbers, antioxidants, pigments, perfumes, etc. can be appropriately blended as needed. In addition, as long as the effectiveness and characteristics of the extract, hydrolyzate, or fermented product according to the present invention are not impaired, other physiologically active ingredients may be used in combination.

When the fermented product according to the present invention is used for external skin preparations, ingredients that can be blended in them, such as oily ingredients, surfactants (synthetic, natural products), emulsifiers, moisturizers, thickeners, preservatives, Bactericides, antifoaming agents, powder components, antioxidants, chelating agents, pH adjusters, pigments, perfumes and the like can be appropriately blended as needed. Moreover, as long as the effectiveness and characteristics of the fermented product according to the present invention are not impaired, it may be blended in combination with other physiologically active ingredients.

Examples of oily components include lotus oil, olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice bran oil, rice germ oil, coconut oil, chamomile oil, palm oil, cacao oil, meadowfoam oil, Bergamot oil, rosehip oil, Arabic coffee tree seed oil, lavender oil, shea butter, tea tree oil, avocado oil, macadamia nut oil, vanilla oil, plant-derived oils such as squalane; mink oil, turtle oil, etc. Animal-derived oils and fats; Waxes such as beeswax, carnauba wax, rice wax, and lanolin; Hydrocarbons such as liquid paraffin, petrolatum, paraffin wax, and squalane; Myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, eicosene Fatty acids such as acids; Higher alcohols such as lauryl alcohol, cetanol, and stearyl alcohol; ) and synthetic triglycerides.

Examples of surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene glycerin fatty acid esters, poly Nonionic surfactants such as oxyethylene hydrogenated castor oil and polyoxyethylene sorbitol fatty acid ester; Anionic surfactants such as ethylene alkylphenyl ether sulfates, polyoxyethylene alkyl ether phosphates, α-sulfonated fatty acid alkyl ester salts, polyoxyethylene alkylphenyl ether phosphates; quaternary ammonium salts, primary to secondary Tertiary fatty amine salts, trialkylbenzylammonium salts, alkylpyridinium salts, 2-alkyl-1-alkyl-1-hydroxyethylimidazolinium salts, N,N-dialkylmorphonium salts, polyethylenepolyamine fatty acid amide salts, etc. Cationic surfactant; N,N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine, N,N,N-trialkyl-N-alkyleneammoniocarboxybetaine, N-acylamidopropyl-N',N Amphoteric surfactants such as '-dimethyl-N'-β-hydroxypropylammoniosulfobetaine can be used.

Examples of emulsifiers or emulsifying aids include stevia derivatives such as enzyme-treated stevia, saponin or its derivatives, casein or its salts (sodium, etc.), sugar-protein complexes, sucrose or its esters, lactose, soybean-derived Water-soluble polysaccharide, complex of soybean-derived protein and polysaccharide, lanolin or its derivative, cholesterol, stevia derivative (stevia enzyme fermented product, etc.), silicate (aluminum, magnesium, etc.), carbonate (calcium, sodium, etc.), saponin and derivatives thereof, lecithin and its derivatives (hydrogenated lecithin, etc.), lactic acid bacteria fermented rice, lactic acid bacteria fermented germinated rice, lactic acid bacteria fermented grains (wheat, beans, cereals, etc.), etc. can also be blended.

Examples of moisturizing agents include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidone carboxylate, and 2-methacryloyloxyethylphosphorylcholine/butyl methacrylate copolymer liquid. etc., and further sugars such as trehalose, mucopolysaccharides (e.g., hyaluronic acid and its derivatives, chondroitin and its derivatives, heparin and its derivatives, etc.), tuberose polysaccharides, elastin and its derivatives, collagen and its derivatives, NMF-related Substances, hydrolyzed conchiolin, hydrolyzed silk, sphingomonas culture, glycosphingolipid, ceramide, lactic acid, urea, higher fatty acid octyldodecyl, seaweed extract, sylvatica root extract, various amino acids and their derivatives mentioned.

Examples of thickeners include alginic acid, agar, carrageenan, fucoidan, and other brown algae, green algae, or red algae-derived components; Birlan root (white) extract; pectin, locust bean gum, aloe polysaccharide, and Alcaligenes-producing polysaccharide. Polysaccharides such as polysaccharides; Gums such as xanthan gum, tragacanth gum, roasted bean gum, guar gum; Cellulose derivatives such as carboxymethyl cellulose and hydroxyethyl cellulose; Synthetic polymers such as conjugates; hyaluronic acid and its derivatives; polyglutamic acid and its derivatives;

Examples of antiseptic/bactericidal agents include urea; paraoxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate; phenoxyethanol, dichlorophen, hexachlorophene, and chlorhexidine hydrochloride. , benzalkonium chloride, salicylic acid, ethanol, undecylenic acid, phenols, jamal (imidazodenyl urea), polyphosphoric acid, propanediol, 1,2-pentanediol, various essential oils, dry distillation of bark, fermented radish, sugar cane plant-derived ethanol or 1,3-butylene glycol.

Examples of powder components include sericite, titanium oxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, silicic anhydride, mica, nylon powder, polyethylene powder, and silk. There are powders, cellulose-based powders, powders of grains (rice, wheat, corn, millet, etc.), and powders of beans (soybeans, adzuki beans, etc.).

Examples of UV absorbers include ethyl para-aminobenzoate, ethylhexyl paradimethylaminobenzoate, amyl salicylate and derivatives thereof, 2-ethylhexyl para-methoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2- Hydroxy-4-methoxybenzophenone-5-sulfonate, 4-tert-butyl-4-methoxybenzoylmethane, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, ethyl urocanate, aloe extract etc.

Antifoaming agents include, for example, ethanol, isopropanol, disiloxane, dimethylpolycyclosan, silica dimethicone silicate, trisiloxane, silica silylate, dimethicone, trimethylsiloxysilicate, and DPG isobornyl ether.

Antioxidants include, for example, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, purslane extract, sycamore root extract, peony extract, vitamin E and derivatives thereof (e.g., vitamin E nicotinate, vitamin E linoleate, etc.).

Examples of chelating agents include trisodium ethylenediaminehydroxyethyltriacetate, edetic acid or salts thereof, gluconic acid, phytic acid, sodium polyphosphate, sodium metaphosphate, tetrasodium hydroxyethanediphosphonate, and the like.

Examples of pH adjusters include citric acid or salts thereof, lactic acid or salts thereof, glycolic acid, succinic acid, hydrochloric acid, monoethanolamine, diethanolamine, triethanolamine, sodium hydroxide and potassium hydroxide.

As whitening agents, t-cycloamino acid derivatives, kojic acid and its derivatives, ascorbic acid and its derivatives, hydroquinone and its derivatives, ellagic acid and its derivatives, nicotinic acid and its derivatives, resorcinol derivatives, tranexamic acid and its derivatives , 4-methoxysalicylic acid potassium salt, magnolignan (5,5'-dipropyl-biphenyl-2,2'-diol), hydroxybenzoic acid and its derivatives, vitamin E and its derivatives, α-hydroxy acid, AMP (adenosine mono phosphate, adenosine monophosphate), and these may be blended singly or in combination.

Examples of the above kojic acid derivatives include kojic acid esters such as kojic acid monobutyrate, kojic acid monocaprate, kojic acid monopalmitate and kojic acid dibutyrate; kojic acid sugars such as kojic acid ethers and kojic acid glucoside; Ascorbic acid derivatives such as L-ascorbic acid-2-phosphate sodium, L-ascorbic acid-2-phosphate magnesium, L-ascorbic acid-2-sulfate sodium, L-ascorbic acid -Ascorbic acid ester salts such as 2-sulfate magnesium, L-ascorbic acid-2-glucoside, L-ascorbic acid-5-glucoside, ascorbyl tocopheryl maleate, ascorbyl tocopheryl potassium phosphate, myristyl 3-glyceryl ascorbate , ascorbic acid sugar derivatives such as caprylyl 2-glyceryl ascorbic acid, 6-position acylated products of these ascorbic acid sugar derivatives (acyl group is hexanoyl group, octanoyl group, decanoyl group, etc.), L-ascorbic acid tetraisopalmitate, L-ascorbic acid tetra-fatty acid esters such as L-ascorbic acid tetralaurate, 3-O-ethylascorbic acid, L-ascorbic acid-2-phosphate-6-O-palmitate sodium, glyceryl ascorbic acid or its Acylated derivatives, ascorbic acid glycerol derivatives such as bisglyceryl ascorbic acid, aminopropyl L-ascorbate phosphate, hyaluronic acid derivatives of L-ascorbic acid, 3-OD lactose-L-ascorbic acid, isostearyl ascorbyl phosphate Salts, hydroquinone derivatives such as arbutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside), etc., and tranexamic acid derivatives such as tranexamic acid esters (eg, lauryl tranexamic acid) ester, tranexamic acid hexadecyl ester, tranexamic acid cetyl ester or a salt thereof), amides of tranexamic acid (e.g., tranexamic acid methylamide), etc., and resorcinol derivatives include, for example, 4-n-butylresorcinol, 4- isoamylresorcinol and the like, and 2,5-dihydroxybenzoic acid derivatives such as 2,5-diacetoxybenzoic acid, 2-acetoxy-5-hydroxybenzoic acid, 2-hydroxy-5-propionyloxybenzoic acid, and nicotinic acid. Examples of derivatives include nicotinamide and benzyl nicotinate. Examples of α-hydroxy acids include lactic acid, malic acid, succinic acid, citric acid and α-hydroxyoctanoic acid.

Examples of physiologically active ingredients include placenta extract, saxifrage extract, saxifrage extract, perilla extract, rice bran extract or its hydrolyzate, white mustard extract or its hydrolyzate, and fermented white mustard. , peony extract or its hydrolyzate, lactic acid bacteria fermented rice, purple shikibu extract, lotus seed extract or its hydrolyzate, lotus seed fermented product, pearl barley hydrolyzate, pearl barley seed fermented product, royal jelly fermented product, sake lees extract Or ceramide contained therein, fermented sake lees, Pandanus amaryllifolius Roxb. extract, Arcangelicia flava Merrilli extract, chamomile extract and the like. In addition, coral grass extract, rice leaf extract or its hydrolyzate, eggplant (lotus, long eggplant, Kamo eggplant, rice eggplant, etc.) extract or its hydrolyzate, apricot fruit extract, eucheuma cornflower, etc. seaweed extract, marine flowering plant extract such as eelgrass, fermented soymilk, jellyfish water, rice extract or its hydrolyzate, fermented rice extract, germinated rice extract or its hydrolyzate, fermented germinated rice , black soybean extract or its hydrolyzate, brown sugar extract or its fermented product, damask rose flower extract, ylang ylang flower extract, bamboo shoot skin extract, linoleic acid and its derivatives or processed products (e.g. liposomal linoleic acid, etc.), animal- or fish-derived collagen and its derivatives, elastin and its derivatives, glycyrrhizic acid and its derivatives (dipotassium salt, etc.), t-cycloamino acid derivatives, vitamin A and its derivatives, allantoin, diisopropylamine dichloro Acetate, γ-amino-β-hydroxybutyric acid, gentian extract, licorice extract, carrot extract, panax ginseng extract or its fermented product, camellia extract, red ginseng extract, honey kelp extract, loofah extract, sea lettuce extract peach extract, plum extract, peach kernel extract, kiwi extract, sunflower extract, Zizyphus joazeiro extract, Pau d'Arco bark extract, daylily extract or ferment, hibiscus flower extract Or fermented product, red wormwood extract, cherimoya extract, mango extract, mangosteen extract, funori extract, oolong tea extract, red fuki extract, lilac extract, sweet potato extract, Japanese pepper peel or seed coat extract or water Decomposition product, safflower flower extract, Casablanca extract, sweet potato extract or its fermented product, guava leaf extract, olive leaf extract, horse chestnut extract, ginger extract, vanilla extract, Houttuynia cordata extract, Banpeiyu extract, aloe extract, aloe vera extract, fig flower extract, apple extract, white asparagus extract, and the like.

Next, the present invention will be described in more detail with production examples, formulation examples, and test examples, but the present invention is not limited thereto. In the following, all parts mean parts by weight, and all % means weight %.

A method for preparing a plant extract containing two isoflavones, which are active ingredients of the present invention, is shown below.

Production example 1. Extract preparation (1)
1000 g of a mixture of purified water and ethanol (1:1) was added to 100 g of dried red clover flower pulverized product, extracted at 40 ° C. for 2 hours, filtered, and brown transparent red clover flower extract (solid content 947 g (concentration 1.28%) were obtained.

Production example 2. Extract preparation (2)
1000 g of a mixture of purified water and ethanol (7:3) was added to 100 g of dried red clover flower pulverized product, extracted at 40 ° C. for 2 hours, filtered, and brown transparent red clover flower extract (solid content 950 g (concentration 1.30%) were obtained.

Production example 3. Extract preparation (3)
1000 g of a mixture of purified water and ethanol (1:1) was added to 100 g of the washed dried red clover flower pulverized product, extracted at room temperature for 4 days, and the extract was filtered to obtain a brown transparent red clover flower extract ( 962 g of solids concentration 1.43%) were obtained.

Production example 4. Extract preparation (4)
1000 g of a mixture of purified water and 1,3-propanediol (3:7) was added to 100 g of the pulverized red clover flower dried product, and the mixture was extracted at room temperature for 4 days. 918 g of extract (solids concentration 1.31%) was obtained.

Comparative production example 1. Preparation of extract 1000 g of purified water was added to 100 g of dried red clover flower pulverized product, extracted at 40 ° C. for 2 hours, filtered, and brown transparent red clover flower extract (solid content concentration 1.66%) 938 g was obtained.

Test example 1. Determination of isoflavones [determination method]
2.0 g of the extracts of Production Examples 1 to 3 and Comparative Production Example 1 were accurately weighed and dissolved in a 70% ethanol aqueous solution to make exactly 10 mL to prepare a sample solution. Exactly 10 μL of the sample solution was taken and tested by liquid chromatography (HPLC) under the following test conditions. The content (%) of formononetin and biochanin A in the product was calculated from a calibration curve prepared in advance.
Content (ppm) of formononetin or biochanin A in this product = content (ppm) of component amount in the sample solution obtained from the calibration curve x (2/amount of sample (g) of this product) x 5
[Preparation of calibration curve]
About 10.0 mg of formononetin for quantitative determination and biocanin A were accurately weighed and dissolved in a 70% ethanol aqueous solution to make exactly 100 mL, which was used as a standard undiluted solution. Next, 1 mL and 2 mL were each accurately measured from the standard undiluted solution, and a 70% ethanol aqueous solution was added to make exactly 10 mL, which was used as a standard solution. Test the standard stock solution by liquid chromatography under the same conditions as the sample solution. From the obtained chromatogram, a calibration curve was prepared with the peak area on the horizontal axis and the concentration (w/v%) of the standard in the standard solution on the vertical axis.
[Test condition]
(1) Detector: UV absorption photometer (measurement wavelength: 255 nm)
(2) Column: A stainless steel tube having an inner diameter of 4.6 mm and a length of 150 mm was filled with octadecylsilylated silica gel for liquid chromatography having an average particle size of 5 μm.
(3) column temperature: constant temperature around 30 ° C. (4) mobile phase A: acetonitrile / formic acid (100) mixture (99.9: 0.1)
(5) Mobile phase B: water/formic acid (100) mixture (99.9:0.1)
(6) Liquid transfer of mobile phase: The concentration gradient is controlled by changing the mixing ratio of mobile phase A and mobile phase B as follows.
(i) Time after injection: 0 to 40 minutes, Mobile phase A (Vol%): 5→40, Mobile phase B (Vol%): 95→60
(ii) Time after injection: 40 to 55 minutes, mobile phase A (Vol%): 40, mobile phase B (Vol%): 60
(8) Flow rate: 1.0 mL per minute
(9) Area measurement range: 55 minutes after sample injection

Table 1 shows the measurement results of formononetin and biochanin A contained in the extracts of Production Examples 1 to 3 and the extract of Comparative Production Example 1.
[Table 1]

It was confirmed that the amount of formononetin and biochanin A contained in the extract of Comparative Production Example 1 was extremely small, and that the extract did not contain a substantial amount of the effect of the present invention.

Hereinafter, the active ingredient according to the present invention and the evaluation of the effect of preventing and improving thinning of the skin will be described. In the present embodiment, attention is focused on epidermal stem cells, which are cells that differentiate into epidermal cells and are involved in skin barrier function and turnover. was evaluated for its effect on

In the present embodiment, hypoxia-inducible factor (HIF)-1α (Seikagaku, Vol. 85, No. 3, pp. 187-195, 2013), the effect of promoting the transcriptional activity of HIF-1α by the active ingredient of the present invention was evaluated.

Test example 1. Evaluation of Transcriptional Activity of HIF-1α Normal human epidermal cells were prepared at 1×10 ⁵ cells/mL with growth additive-containing Humedia-KG2 [manufactured by Kurabo Industries], 100 μL of which was seeded in a 96-well microplate, and subjected to 5% CO2. _2. Cultured at 37°C under saturated steam. After culturing for 24 hours, a firefly luciferase reporter vector incorporating a nucleotide sequence (HRE) that binds to HIF-1α and a vector incorporating Renilla luciferase as an internal standard (Cignal HIF Pathway Reporter Assay Kit) [manufactured by QIAGEN] were added to cells. was transfected using ViaFect transfection reagent (Promega). After another 24 hours, the extracts of Production Examples 1 to 3 were added to the medium as sample solutions. Here, the sample solution was added so that the final concentration as a solution in the medium was 0.5% and 1.0%. After 24 hours, the cell contents were extracted, and the luciferase activity of the cell extract was measured using a luciferase assay system (Promega) with a luminometer (Promega GloMax-Multi+Detection System). The value obtained by dividing the measured value of firefly luciferase (HRE transcription amount) by the measured value of Renilla luciferase (cell amount) was defined as the HIF-1α transcriptional activity per cell, and 50% was used as a control instead of the sample solution. The relative value was calculated with the HIF-1α transcription activity in the group (control) to which -butylene glycol (1.0%) was added as 100. In addition, in order to compare the HIF-1α transcriptional activity with the extract of Comparative Production Example 1, the extract was tested at concentrations of 0.5% and 1.0% at the same time.

Table 2 shows the results of Test Example 2.
[Table 2]

As shown in Table 2, it was confirmed that the extract containing the active ingredients (formonetin and biochanin A) according to the present invention has a concentration-dependent HIF-1α transcriptional activity-enhancing effect. On the other hand, Comparative Production Example 1, which does not substantially contain formononetin and biochanin A, did not exhibit an HIF-1α transcriptional activity enhancing effect.

Next, MCSP (Melanoma-associated chondroitin sulfate proteoglycan), which is a marker protein of epidermal stem cells, was used to evaluate the MCSP expression promoting effect of the active ingredient according to the present invention.

Test example 3. Evaluation of MCSP Expression Promotion Effect Normal epidermal keratinocytes (NHEK(F)) were seeded at 8×10 ³ cells/well in a 96-well plate, and then incubated at 37° C. using HuMedia-KG2 medium (Kurashiki Boseki Co., Ltd.). Incubated for 24 hours. After culturing, the extracts of Production Examples 1 and 2 were added as sample solutions to the medium, and cultured for 48 hours. Here, the sample solution was added so that the final concentration as a solution in the test medium was 0.5% and 1.0%, respectively. After culturing, immunodetection was performed using an MCSP antibody. That is, after washing with PBS (-), the cells were treated with 15% neutral buffered formalin for 30 minutes to fix, permeabilized with 0.5% Triton X-100 solution for 1 hour, and 5-fold diluted Blocking One P (Nacalai After blocking by treatment with a Tesque solution for 2 hours, MCSP antibody was added and allowed to stand at room temperature for 1 hour. Next, the plate was washed with PBS(-), a fluorescently labeled secondary antibody was added, and the plate was allowed to stand in the dark for a certain period of time. After washing with PBS(-), fluorescence intensity was measured. First, the fluorescent label of the secondary antibody (Alexa Fluor488) was measured at Ex = 485 nm and Em = 520 nm using a fluorescence microplate reader (Fluoroscan Ascent, Thermo Fisher Scientific), followed by DNA staining with Hoechst33342. and measured at Ex = 355 nm and Em = 460 nm. The MCSP expression level was determined by dividing the fluorescence intensity of Alexa Fluor488 in each test group by the fluorescence intensity of Hoechst33342. The same operation as above was performed for the case of no sample addition (control) in which 50% 1,3-butylene glycol (1.0%) was added instead of the sample solution. The relative value of the MCSP expression level at the time of addition of each sample to the MCSP expression level obtained here was determined and used as the MCSP expression promotion level (%).

Table 3 shows the results of Test Example 3.
[Table 3]

As shown in Table 3, it was confirmed that the extract containing the active ingredients (formonetin and biochanin A) according to the present invention has an effect of promoting MCSP expression in a concentration-dependent manner. On the other hand, in Comparative Production Example 1, which does not substantially contain formononetin and biochanin A, no MCSP expression promoting effect was observed.

Test example 4. Effect of maintaining undifferentiated epidermis three-dimensional model Epidermal cells were seeded in a culture cup using a human epidermis model preparation kit, and a medium containing 1.0% each of the extracts of Production Examples 1 and 2 as a sample solution was added. Cultured for 3 weeks. Medium was changed every two days. After completion of the culture, sections were prepared, HE staining and MSCP fluorescent immunostaining were performed, and the sections were observed using an optical microscope and a fluorescence microscope, respectively. In addition, instead of the sample solution, cells cultured in a medium (control) containing 50% 1,3-butylene glycol (1.0%) and cultured in a medium (comparative) containing the extract of Comparative Production Example 1 Similar observations were also made for the cells that had been treated.

According to the observation results of Test Example 4, the extract containing the active ingredients (formonetin and biochanin A) according to the present invention maintains undifferentiated epidermal stem cells even after 3 weeks, compared with the control and the extract for comparison. confirmed that it is possible.

The results of Test Examples 1 to 4 suggested that the active ingredient of the present invention promotes HIF-1α transcriptional activity and participates in the maintenance of epidermal stem cells. From this, the active ingredient according to the present invention improves the skin barrier function by preventing and improving the thinning of the skin, and also prevents skin aging, unhealthy skin, and skin tone (redness, etc.). Prevention and improvement are expected.

Formulation example 1. Lotion [Ingredients] Part Olive oil 1.0
Polyoxyethylene (5.5) cetyl alcohol 5.0
Butylparaben 0.1
Extract of Production Example 1 5.0
Ethanol 5.0
Glycerin 5.0
1,3-butylene glycol 5.0
Potassium hydroxide Appropriate amount Purified water Amount to make the total amount 100 parts Perfume Appropriate amount

Formulation example 2. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 5.0 parts of the extract of Production Example 2 was used in place of the extract of Production Example 1 contained in Formulation Example 1.

Formulation example 3. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 5.0 parts of the extract of Production Example 3 was used in place of the extract of Production Example 1 contained in Formulation Example 1.

Formulation example 4. Lotion A lotion was obtained in the same manner as in Formulation Example 1, except that 5.0 parts of the extract of Production Example 4 was used in place of the extract of Production Example 1 contained in Formulation Example 1.

Formulation example 5. Emulsion [Ingredients] Part Liquid paraffin 6.0
Hexalane 4.0
Jojoba oil 1.0
Polyoxyethylene (20) sorbitan monostearate 1.0
Lipophilic glyceryl stearate 1.0
Soy lecithin 1.5
Extract of Production Example 3 3.0
L-ascorbic acid-2-glucoside 2.0
Potassium hydroxide 0.5
Glycerin 3.0
1,3-butylene glycol 2.0
Carboxymethyl cellulose 0.3
Sodium hyaluronate 0.01
Purified water Amount that makes the total amount 100 parts Fragrance Appropriate amount

Formulation example 6. Milky Lotion A milky lotion was obtained in the same manner as in Formulation Example 5, except that 3.0 parts of the extract of Production Example 1 was used in place of the 3.0 parts of the extract of Production Example 3.

Formulation example 7. Milky lotion A milky lotion is obtained in the same manner as in Formulation Example 5, except that 2.0 parts of L-ascorbic acid-2-glucoside and 3.0 parts of arbutin are used in place of 0.5 parts of potassium hydroxide. rice field.

Formulation example 8. Milky lotion A milky lotion is prepared in the same manner as in Formulation Example 5, except that 2.0 parts of L-ascorbic acid-2-glucoside and 2.0 parts of tranexamic acid are used instead of 0.5 parts of potassium hydroxide. Obtained.

Formulation example 9. Milky lotion Milky lotion in the same manner as in Formulation Example 5, except that 2.0 parts of L-ascorbic acid-2-glucoside and 3.0 parts of nicotinamide are used instead of 0.5 parts of potassium hydroxide. got

Formulation example 10. Lotion [Ingredients] Part Extract of Production Example 1 10.0
Ethanol 10.0
Glycerin 3.0
1,3-butylene glycol 2.0
Methylparaben 0.2
Citric acid 0.1
Sodium citrate 0.3
Carboxyvinyl polymer 0.1
Xanthan gum 0.1
Fragrance Appropriate amount Potassium hydroxide Appropriate amount Purified water Amount to make the total amount 100 parts

Formulation example 11. Essence [ingredient] part ethanol 2.0
Glycerin 5.0
1,3-butylene glycol 5.0
Methylparaben 0.1
Hyaluronic acid 0.1
Extract of Production Example 3 5.0
Citric acid 0.3
Sodium citrate 0.6
Purified water Amount that makes the total amount 100 parts

Formulation example 12. Essence An essence was obtained in the same manner as in Formulation Example 11, except that 5.0 parts of the extract of Production Example 2 was used instead of 5.0 parts of the extract of Production Example 3.

Example 13. Liquid foundation [ingredients] part stearic acid 2.4
Propylene glycol monostearate 2.0
Cetostearyl alcohol 0.2
Liquid Lanolin 2.0
Liquid paraffin 3.0
Isopropyl myristate 8.5
Propylparaben 0.05
Extract of Production Example 1 5.0
Carboxymethylcellulose sodium 0.2
Bentonite 0.5
Propylene glycol 4.0
Triethanolamine 1.1
Methylparaben 0.1
Purified water Amount to make the total amount 100 parts Titanium oxide 8.0
Talc 4.0
Appropriate amount of coloring pigment

Formulation example 14. Body Shampoo [Ingredients] Part N-lauroylmethylalanine sodium 25.0
Coconut fatty acid potassium liquid (40%) 26.0
Coconut fatty acid diethanolamide 3.0
Methylparaben 0.1
Extract of Production Example 2 5.0
1,3-butylene glycol 2.0
Purified water Amount that makes the total amount 100 parts

Formulation example 15. Hair Shampoo [Ingredient] Part Sodium N-coconut oil fatty acid methyl taurate 10.0
Sodium polyoxyethylene (3) alkyl ether sulfate 20.0
Lauryldimethylaminoacetate betaine 10.0
Coconut fatty acid diethanolamide 4.0
Methylparaben 0.1
Citric acid 0.1
Extract of Production Example 3 2.0
1,3-butylene glycol 2.0
Purified water Amount that makes the total amount 100 parts

Example 16. Hair conditioner [Ingredients] Part polyoxyethylene (10) hydrogenated castor oil 1.0
Distearyldimethylammonium chloride 1.5
Stearyltrimethylammonium chloride 2.0
Glyceryl 2-ethylhexanoate 1.0
Cetanol 3.2
Stearyl alcohol 1.0
Methylparaben 0.1
Extract of Production Example 3 2.0
1,3-butylene glycol 5.0
Purified water Amount that makes the total amount 100 parts

Claims (2)

An agent for improving thinning of the skin containing formononetin and biochanin A as active ingredients and containing water and a lower alcohol extract or water and a polyhydric alcohol extract of red clover flowers belonging to the genus Trifolium of the family Leguminosae. 4. An agent for improving skin thinning, wherein the extract contains formononetin of 40 ppm or more and biochanin A of 20 ppm or more. An external preparation for skin containing formononetin and biochanin A as active ingredients, containing water and a lower alcohol extract or water and a polyhydric alcohol extract of red clover flowers of the genus Trifolium of the legume family, wherein the extract contains 40 ppm or more of formononetin and 20 ppm or more of biochanin A are contained in the external preparation for skin.