Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/34—Alcohols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

• the present disclosure relates to, for example, an agent for exerting a retinol-like effect on human skin and its uses.
• Retinoic acid which is retinol
• Retinoic acid is known to regulate the expression of retinoic acid receptors (RAR) in epidermal keratinocytes and the like when applied to the skin.
• RAR retinoic acid receptors
• Non-Patent Document 1 describes that retinoic acid weakly induces (enhances) the expression of RAR ⁇ gene and RAR ⁇ gene among RARs.
• the retinoic acid activates skin metabolism, it suppresses the differentiation of epidermal keratinocytes, which poses a problem in that it causes side effects such as skin erythema and desquamation.
• the present disclosure aims to provide an agent that can reduce inhibition of epidermal keratinocyte differentiation, induce retinol-like effects, and is mainly applicable to human skin and the like.
• the agent used for inducing retinol-like action of the present disclosure (hereinafter also referred to as "retinol-like action inducing agent”) is bakuchiol (4-[(1E,3S)-3 -ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).
• the agent of the present disclosure for use in inducing the expression of a maturation-promoting gene in epidermal keratinocytes contains bakuchiol.
• CERS1 gene expression inducing agent The agent used for inducing expression of the ceramide synthase 1 (CERS1) gene (hereinafter also referred to as “CERS1 gene expression inducing agent”) of the present disclosure includes bakuchiol.
• the agent for use in promoting proliferation and/or maturation of epidermal keratinocytes includes bakuchiol.
• an agent that can reduce inhibition of epidermal keratinocyte differentiation, induce retinol-like effects, and is mainly applicable to human skin.
• FIGS. 1(A) and (B) are graphs showing the expression of the RAR ⁇ gene in Example 1.
• FIG. 1(A) is the result after 48 hours of culture
• FIG. 1(B) is the result after 72 hours of culture.
• the results are shown below.
• 2(A) and (B) are graphs showing the expression of the RAR ⁇ gene in Example 1.
• FIG. 2(A) is the result after 48 hours of culture
• FIG. 2(B) is the result after 72 hours of culture.
• 3(A) and (B) are graphs showing the expression of the RAR ⁇ gene in Example 1.
• FIG. 3(A) is the result after 48 hours of culture
• FIG. 3(B) is the result after 72 hours of culture. The results are shown below.
• FIG. 4(A) and (B) are graphs showing the expression of the filaggrin (FLG) gene in Example 1.
• FIG. 4(A) is the result after 48 hours of culture
• FIG. 4(B) is the result after 48 hours of culture.
• the results are shown after 72 hours.
• 5(A) and (B) are graphs showing the expression of the involucrin (IVL) gene in Example 1.
• FIG. 5(A) is the result after 48 hours of culture
• FIG. 5(B) is the result after 48 hours of culture.
• the results are shown after 72 hours.
• 6(A) and (B) are graphs showing the expression of the CERS1 gene in Example 1.
• FIG. 6 (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• FIG. 7 is a graph showing the amount of melanin produced in Example 2.
• the present disclosure provides agents or compositions capable of inducing retinol-like effects.
• Agents for use in inducing retinol-like effects of the present disclosure include bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).
• composition for use in inducing retinol-like action of the present disclosure is a composition for use in inducing retinol-like action (hereinafter also referred to as “retinol-like action inducing composition”). ,7-dimethylocta-1,6-dienyl]phenol).
• retinol-like action inducing composition a composition for use in inducing retinol-like action.
• bakuchiol has an effect on retinol (vitamin A) ATRA (all-trans retinoic acid (all- It was found that it has a retinol-like effect, that is, a retinol-like effect.
• bakuchiol reduced the effect of suppressing the differentiation of epidermal keratinocytes observed with ATRA, while the effect of inducing expression of RAR ⁇ gene and RAR ⁇ gene observed with ATRA.
• the present inventors have discovered that the present invention exhibits an inhibitory effect on the expression of .
• ATRA is presumed to induce side effects such as skin erythema and desquamation by inducing expression of the RAR ⁇ gene. Therefore, according to the retinol-like effect inducer of the present disclosure, for example, while suppressing the occurrence of side effects such as skin erythema and desquamation due to retinol, which are caused by retinol's effect of inhibiting differentiation of epidermal keratinocytes, It is expected that this product will have the same wrinkle-improving effects as observed with retinol.
• the above “bakuchiol” is 4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol, for example, a compound represented by the following formula (1). You can also do it.
• the bakuchiol may be, for example, a phenoxide ion.
• the bakuchiol may be esterified, for example.
• the bakuchiol may be an isolated or purified compound, or a composition containing bakuchiol.
• the composition containing bakuchiol is, for example, an extract containing bakuchiol; a crudely purified product of the extract, a dried product of the extract, a freeze-dried product of the extract, a treatment of the extract such as spray drying, etc. things; etc.
• the extract containing bakuchiol can be produced, for example, by performing solvent extraction on a plant containing bakuchiol.
• plants containing bakuchiol include Psoralea corylifolia and Otholobium pubescens .
• One type or two or more types of plants may be used.
• the plant material to be subjected to the extraction may be an individual plant or a part of a plant.
• the plant parts include, for example, roots, rhizomes, leaves, stems, whole flowers, or mixtures thereof.
• the material may be the collected plant itself, or may be a processed product obtained by drying and/or crushing.
• the solvent used for the extraction of bakuchiol is, for example, an aqueous solvent such as water or a buffer solution; a lower alcohol or hydrous lower alcohol such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, or isobutanol; propylene glycol, 1,3 -Butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,3, Polyhydric alcohol or hydrous polyhydric alcohol such as 5-pentanetriol, glycerin, polyethylene glycol (e.g.
• acetone ethyl acetate, diethyl ether, dimethyl ether, ethyl methyl ether, dioxane, hexane, acetonitrile, xylene , benzene, chloroform, carbon tetrachloride, phenol, toluene, etc.
• acids hydroochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.
• alkalis sodium hydroxide, potassium hydroxide, Calcium hydroxide, ammonia, etc.
• the solvents may be used alone or in combination of two or more.
• the treatment of the treated product includes, for example, decomposition by addition of acid (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or alkali (sodium hydroxide, calcium hydroxide, ammonia, etc.); fermentation or metabolic conversion by microorganisms; ion Adsorption of components using exchange resins, activated carbon, diatomaceous earth, etc.; Fractionation using chromatography with various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.); Examples include filtration using a filter paper, membrane filter, ultrafiltration membrane, etc.; pressurization or reduced pressure; heating or cooling: drying or freeze-drying; pH adjustment; deodorization; decolorization; long-term stationary storage; and the like.
• the above-mentioned processing may be performed by one type alone or by two or more types.
• retinol-like effect refers to retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6, 8-tetraenoic acid), particularly a compound or composition that exhibits at least one of the effects exhibited by ATRA.
• the retinol-like effect may be, for example, an effect on the skin or cells constituting the skin, or may be an effect on gene expression in the cells, that is, induction or suppression of gene expression.
• the retinol-like action inducer of the present disclosure exhibits one or more types of retinol-like action.
• the retinol-like effect is an effect on the skin or cells constituting the skin
• the retinol-like effect is, for example, strengthening the basal layer of the skin, stabilizing the basal layer of the skin, and promoting proliferation of epidermal keratinocytes. etc. can be mentioned.
• the above-mentioned “strengthening of the basal layer of the skin” means that cells in the basal layer are less likely to be destroyed when external stimulation is applied to the skin (resistance to external stimulation).
• the reinforcement of the basal layer of the skin against the external stimulus can be evaluated, for example, by a marker expressed in the basal layer (basal layer marker).
• the basal layer markers include hyaluronic acid synthase 3 (HAS3), integrin ⁇ 6 (ITG ⁇ 6), and integrin ⁇ 1 (ITG ⁇ 1).
• stabilization of the basal layer of the skin means to suppress the rupture of the basal layer that occurs with aging.
• the stabilization of the basal layer of the skin can be evaluated, for example, using the basal layer marker.
• proliferation promotion of epidermal keratinocytes means that the proliferation of epidermal keratinocytes, particularly human epidermal keratinocytes, is promoted or enhanced.
• the proliferation of the epidermal keratinocytes can be promoted, for example, by evaluating the proliferation of the epidermal keratinocytes.
• epidermal keratinocytes refer to keratinocytes produced by division of basal layer cells of the epidermis. It is known that the epidermal keratinocytes further differentiate in the epidermis and eventually differentiate into corneocytes that form the stratum corneum.
• the retinol-like effect is an effect on gene expression in the cell
• examples of the retinol-like effect include induction of retinoic acid receptor (RAR) ⁇ gene expression, suppression of RAR ⁇ gene expression, etc., and preferably , induction of RAR ⁇ gene expression and suppression of RAR ⁇ gene expression.
• RAR retinoic acid receptor
• the mRNA encoded by the human RAR ⁇ gene includes, for example, a polynucleotide consisting of a base sequence registered in Genbank with accession number: NM_000965.5.
• the mRNA encoded by the human RAR ⁇ gene includes, for example, a polynucleotide consisting of a base sequence registered in Genbank with accession number: NM_000966.6.
• RAR ⁇ gene expression induction means that the expression level of the RAR ⁇ gene is increased or enhanced, and may mean a change from a state where the RAR ⁇ gene is not expressed to a state where it is expressed.
• the expression of the RAR ⁇ gene can be evaluated, for example, by measuring the expression level of RAR ⁇ gene mRNA according to Example 1 described below.
• the expression level of the RAR ⁇ gene the expression level of any one or more isoforms of the RAR ⁇ gene may be measured, or the expression of all isoforms may be measured, but preferably, The latter.
• “Suppression of RAR ⁇ gene expression” means that the expression level of the RAR ⁇ gene is suppressed or decreased, and may mean a change from a state in which the RAR ⁇ gene is expressed to a state in which it is not expressed.
• the expression of the RAR ⁇ gene can be evaluated, for example, by measuring the expression level of RAR ⁇ gene mRNA according to Example 1 described below. As the expression level of the RAR ⁇ gene, the expression level of any one or more isoforms of the RAR ⁇ gene may be measured, or the expression of all isoforms may be measured, but preferably, The latter.
• the “gene” may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA).
• the DNA may be double-stranded or single-stranded.
• the “gene” may include additional sequences such as untranslated region (UTR) sequences.
• the retinol-like action inducer of the present disclosure can induce retinol-like action, for example, by using it in a subject.
• the usage conditions (administration conditions) of the retinol-like effect inducer of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set, for example, depending on the type of administration target.
• the retinol-like action inducers of the present disclosure may be used, for example, in vivo or in vitro .
• the recipient of the retinol-like effect inducer of the present disclosure is not particularly limited.
• the subject to be administered may be, for example, a human or a non-human animal other than humans.
• the non-human animals include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs, and birds.
• the administration target may be, for example, a cell, tissue, organ, etc.
• the cell may be, for example, a cell collected from a living body, a cultured cell, etc.
• the tissue or organ include tissue (living tissue) or organ collected from a living body.
• the amount of bakuchiol blended may be within a range that exhibits the retinol-like effect, that is, an effective amount.
• the blending amount (upper limit and lower limit) of the bakuchiol is, for example, as follows.
• the upper limit value and the lower limit value can be arbitrarily combined.
• ⁇ Lower limit 0.1 ⁇ mol/l, 1 ⁇ mol/l, preferably 2.5 ⁇ mol/l - Upper limit: 200 mmol/l, preferably 100 mmol/l, preferably 50 mmol/l, 10 mmol/l, 1 mmol/l, preferably 500 ⁇ mol/l, more preferably 100 ⁇ mol/l
• “mol/l” is sometimes referred to as "M” in this specification. Further, in the Examples described below, the contents of experiments in which 5 ⁇ M (0.000125% (w/v%)) or 10 ⁇ M (0.00025% (w/v%)) of bakuchiol was added are described.
• the administration form of the retinol-like effect inducer of the present disclosure includes oral administration or parenteral administration.
• parenteral administration include transdermal administration, application to the skin (contact), and the like.
• Application to the skin may also include application to the oral mucosa, ie application to or contact with epithelial cells within the oral cavity.
• the application to the skin may include, in addition to or instead of application to the skin surface, intradermal or subcutaneous administration or injection through the skin surface.
• the administration or injection into the skin via the skin surface can be performed using, for example, microneedles.
• the dosage form of the retinol-like effect inducer of the present disclosure is not particularly limited, and can be determined as appropriate depending on, for example, the dosage form.
• the dosage form include liquid and solid forms.
• the dosage form is, for example, in the form of an oil gel (for example, a form in which bakuchiol is contained in an oil gel), in the form of oil gel particles (for example, in a form in which bakuchiol is contained in an oil gel particle), or in the form of a liposome (for example, in a form in which bakuchiol is contained in a liposome).
• a form containing bakuchiol can also be mentioned.
• examples of the dosage form include tablets, pills, capsules, granules, powders, and liquids.
• oil gel means a gel-like oil (oil component) thickened with a lipophilic gelling agent.
• the oil gel can be prepared, for example, by adding an oil gelling agent to an oil agent.
• oil gel particles refer to one or more particles composed of oil gel and having desired components dissolved or dispersed in the oil agent of the oil gel.
• liposome refers to a vesicle having a lipid bilayer membrane dispersed in an aqueous medium.
• the retinol-like action inducer of the present disclosure may, for example, contain an additive if necessary, and when used as a composition, the additive may be a pharmaceutically acceptable additive or a pharmaceutically acceptable It is preferable to include a carrier.
• the additives are not particularly limited, and include, for example, base raw materials, excipients, coloring agents, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring agents such as fragrances, etc. can be given.
• the amount of the additive is not particularly limited as long as it does not interfere with the function of bakuchiol.
• the excipients include, for example, sugar derivatives such as lactose, lactose hydrate, white sugar, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; Rubber; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; calcium carbonate, etc.
• Carbonate Examples include inorganic excipients such as sulfates such as calcium sulfate. Examples of the coloring agent include yellow iron sesquioxide.
• Examples of the lubricant include stearic acid metal salts such as stearic acid, calcium stearate, and magnesium stearate; talc; polyethylene glycol; silica; and hydrogenated vegetable oil.
• Examples of the flavoring agent include flavoring agents such as cocoa powder, peppermint, aromatic powder, peppermint oil, licorice, and cinnamon powder, sweeteners, and acidulants.
• Examples of the binder include hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and macrogol.
• the disintegrant examples include cellulose derivatives such as carboxymethyl cellulose and carboxymethyl cellulose calcium; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, and sodium starch glycolate.
• the stabilizers include, for example, paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid. ; Examples include sorbic acid.
• the coating agent examples include hypromellose, macrogol such as macrogol 6000, talc, and titanium oxide.
• the retinol-like effect-inducing composition of the present disclosure is an orally administered composition
• specific examples of the orally administered composition include beverages, foods, pharmaceuticals (classes), quasi-drugs (classes), etc. .
• the form of the skin external preparation depends on the usage form. Depending on the situation, ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mist, sprays, etc. can be mentioned.
• the forms of use include, for example, pharmaceuticals (class); quasi-drugs (class); topical or systemic skin preparations; medicinal and/or cosmetic preparations applied to the scalp and hair; Examples include bath preparations that are used by pouring; other preparations; and the like.
• the topical or systemic skin preparations include, for example, basic cosmetics such as lotions, milky lotions, creams, ointments, lotions, oils, and packs, facial cleansers or skin cleansers such as bar soaps, liquid soaps, and hand washes.
• Make-up cosmetics such as cosmetics, massage agents, cleansing agents, hair removal agents, depilatory agents, shaving agents, aftershave lotions, pre-show lotions, shaving creams, foundations, lipsticks, blushers, eyeshadows, eyeliners, mascara, etc. gargles, perfumes, nail polishes, nail enamel, nail enamel removers, poultices, plasters, tapes, sheets, patches, aerosols, toothpastes, mouthwashes, etc. ) etc.
• the medicinal and/or cosmetic preparations applied to the scalp and hair include, for example, shampoos, conditioners, hair treatment agents, pre-hair treatment agents, permanent solutions, hair dyes, hair styling agents, hair tonic agents, hair growth agents, etc.
• hair nourishing agents include poultices, plasters, tapes, sheets, aerosols, etc.
• the other preparations include armpit odor preventive agents or deodorants, antiperspirants, sanitary products, sanitary cottons, wet tissues, and the like.
• the skin external preparation can be produced by arbitrarily selecting and/or using in combination the following components and/or additives as long as they do not interfere with the action of bakuchiol.
• oils and fats avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange laffa oil, sesame oil, cacao butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil , shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japanese oak, palm oil Oil, beef tallow, lard, squalene, squalane, pristane, or hydrogenated products of these fats and oils (hardened oil, etc.), etc.
• Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol, etc. .
• Esters Isopropyl myristate, Isopropyl palmitate, Butyl stearate, Hexyl laurate, Myristyl myristate, Oleyl oleate, Decyl oleate, Octyldodecyl myristate, Hexyldecyl dimethyloctoate, Cetyl lactate, Myristyl lactate, Diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.
• Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.
• Gummies sugars or water-soluble polymer compounds Gum arabic, benzoin gum, dammar gum, guaiac butter, Irish moss, karaya gum, tragacanth gum, carob gum, quinseed, agar, casein, lactose, fructose, sucrose or its ester, Trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxide such as ethylene oxide is added, low-molecular-weight chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salts, hyaluronic acid or its salts, chondroitin sulf
• Surfactant Anionic surfactant (alkyl carboxylate, alkyl sulfonate, alkyl sulfate, alkyl phosphate), cationic surfactant (alkyl amine salt, alkyl quaternary ammonium salt), amphoteric Surfactants: Carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate ester type amphoteric surfactants, nonionic surfactants ( Ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants ( natural surfactants, protein hydrolyzate derivatives, polymer surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), etc.
• Vitamin B group thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol, vitamin E group: vitamin E or its derivatives Derivatives, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), others, essential fatty acids (vitamin F), carnitine, ferulic acid, ⁇ -oryzanol , orotic acid
• the skin external preparation may further contain various additives derived from animals or plants.
• the additives can be arbitrarily selected from various materials and added, for example, by performing conventional processing depending on the type and form of the product to be added.
• the processing may optionally include, for example, crushing, milling, washing, hydrolysis, fermentation, purification, squeezing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc. Selective and/or combined processing is possible.
• the solvent used for the extraction can be selected in consideration of the purpose and type of the product to be used, the processing to be performed later, etc.
• the extraction solvent is, for example, lower alcohol or water-containing lower alcohol such as water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, polyhydric alcohol or water-containing alcohol such as propylene glycol, 1,3-butylene glycol, glycerin, etc. It is desirable to use one type or a mixture of two or more types selected from various organic solvents such as polyhydric alcohols, acetone, and ethyl acetate. However, as the extraction solvent, if, for example, it is not desirable to contain an organic solvent depending on the purpose, water alone or ethanol, which is easily removed after extraction, may be used alone or in any mixture with water. Alternatively, exploited extracts may be used.
• the skin external preparations may, for example, protect the skin and hair, moisturize the skin, and improve the feel and texture of the skin. , imparting flexibility, alleviating irritation, alleviating stress with aroma, cell activation (prevention of cell aging), suppression of inflammation, improvement of skin and hair quality, prevention and improvement of rough skin, hair growth, hair growth, prevention of hair loss,
• cosmetic effects such as adding luster, cleaning effects, alleviating fatigue, promoting blood flow, and warming bath effects
• effects such as scenting, deodorizing, thickening, preservative, and buffering.
• the skin external preparation is expected to have various cosmetic and pharmaceutical effects of each of the raw materials known so far, and by combining these, the desired effects of the present disclosure can be achieved. It is also possible to create a product that is expected to have multifunctional effects by increasing the
• the present disclosure provides agents or compositions capable of inducing maturation-promoting genes in epidermal keratinocytes, particularly human epidermal keratinocytes.
• the agent for use in inducing the expression of the epidermal keratinocyte maturation-promoting gene of the present disclosure includes bakuchiol.
• the composition of the present disclosure for use in inducing expression of a gene that promotes maturation of epidermal keratinocytes contains bakuchiol.
• the maturation-promoting gene expression inducer or composition of the present disclosure it is possible to promote the maturation of epidermal keratinocytes, so that effects such as strengthening of the skin barrier function and moisturizing effect can be obtained. Furthermore, the maturation-promoting gene expression inducer or composition of the present disclosure is expected to suppress, for example, the side effects of ATRA, and therefore suppress the side effects caused by retinol while suppressing the wrinkle-improving effect observed with retinol. expected to be obtained.
• the epidermal keratinocyte maturation-promoting gene refers to a gene that, when expressed, promotes the maturation of epidermal keratinocytes.
• the maturation-promoting gene include the FLG gene and the IVL gene, and preferably the FLG gene or a combination of the FLG gene and the IVL gene.
• the FLG gene is a gene that is expressed during the differentiation and maturation of epidermal keratinocytes, and is known to, for example, induce keratinization of epidermal keratinocytes and contribute to the maturation of epidermal keratinocytes.
• the IVL gene is a gene expressed in the early stages of differentiation and maturation of epidermal keratinocytes, and is known to, for example, contribute to the differentiation of epidermal keratinocytes.
• the mRNA encoded by the human FLG gene includes, for example, a polynucleotide consisting of a base sequence registered in Genbank with accession number: NM_002016.2.
• the mRNA encoded by the human IVL gene includes, for example, a polynucleotide consisting of a base sequence registered in Genbank with accession number: NM_005547.4.
• induction of expression of the FLG gene means that the expression level of the FLG gene is increased or enhanced, and may mean a change from a state where the FLG gene is not expressed to a state where it is expressed.
• the expression of the FLG gene can be evaluated, for example, by measuring the expression level of mRNA of the FLG gene according to Example 1 described below.
• “Induction of expression of the IVL gene” means that the expression level of the IVL gene is increased or enhanced, and may also mean a change from a state where the IVL gene is not expressed to a state where it is expressed.
• the expression of the FLG gene can be evaluated, for example, by measuring the expression level of IVL gene mRNA according to Example 1 described below.
• the expression-inducing agent for a maturation-promoting gene of the present disclosure can induce the expression of a maturation-promoting gene, for example, by using it in an administration subject.
• the maturation-promoting gene expression inducer of the present disclosure can achieve effects such as anti-wrinkle effects and skin barrier function strengthening effects.
• the conditions for use (administration conditions) of the maturation-promoting gene expression inducer of the present disclosure can refer to the description of the conditions for use of the retinol-like effect inducer of the present disclosure.
• the present disclosure provides agents or compositions capable of inducing the CERS1 gene.
• Agents for use in inducing expression of the CERS1 gene of the present disclosure include bakuchiol.
• the composition for use in inducing expression of the CERS1 gene of the present disclosure contains bakuchiol. According to the CERS1 gene expression inducer or composition of the present disclosure, it is possible to promote the differentiation of epidermal keratinocytes, so that effects such as strengthening of the skin barrier function and moisturizing effect can be obtained.
• the CERS1 gene expression inducer or composition of the present disclosure is expected to suppress the side effects of ATRA, for example, it can suppress the side effects caused by retinol while achieving the wrinkle-improving effect observed with retinol. It is expected that
• the CERS1 gene is a gene that is expressed in the early stages of differentiation of epidermal keratinocytes, and is known to contribute to the differentiation of epidermal keratinocytes, strengthening of barrier function, etc. by synthesizing ceramide, for example. .
• the mRNA encoded by the human CERS1 gene includes, for example, a polynucleotide consisting of a base sequence registered in Genbank with accession number: NM_021267.5.
• the expression level of the CERS1 gene the expression level of any one or more isoforms of the CERS1 gene may be measured, or the expression of all isoforms may be measured.
• “Induction of expression of the CERS1 gene” means that the expression level of the CERS1 gene is increased or enhanced, and may mean a change from a state where the CERS1 gene is not expressed to a state where it is expressed.
• the expression of the FLG gene can be evaluated, for example, by measuring the expression level of mRNA of the CERS1 gene according to Example 1 described below.
• the CERS1 gene expression inducer of the present disclosure can induce the expression of the CERS1 gene, for example, by using it in a subject.
• the CERS1 gene expression inducer of the present disclosure can, for example, induce the differentiation of the epidermal keratinocytes, and can obtain effects such as anti-wrinkle effects and skin barrier function strengthening effects.
• the usage conditions (administration conditions) of the CERS1 gene expression inducer of the present disclosure the above description of the usage conditions of the retinol-like effect inducer of the present disclosure can be referred to.
• the present disclosure provides agents or compositions capable of promoting proliferation and/or maturation of epidermal keratinocytes, particularly human epidermal keratinocytes.
• the agent for use in promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure includes bakuchiol.
• the composition for use in promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure includes bakuchiol.
• the maturation promoter or composition of the present disclosure proliferation and/or maturation of epidermal keratinocytes can be promoted, so effects such as wrinkle-improving effects and skin barrier function strengthening effects can be obtained. Furthermore, since the maturation accelerator or composition of the present disclosure is expected to suppress the side effects of ATRA, for example, it is expected that the wrinkle-improving effect observed with retinol can be obtained while suppressing the side effects caused by retinol. be done.
• the maturation promoter of the present disclosure can promote the proliferation and/or maturation of epidermal keratinocytes, for example, by using it on a subject to whom it is administered.
• the maturation accelerator of the present disclosure can obtain effects such as anti-wrinkle effects and skin barrier function strengthening effects.
• the conditions for use (administration conditions) of the maturation accelerator of the present disclosure can refer to the above description of the conditions for use of the inducer of retinol-like action of the present disclosure.
• the present disclosure provides agents or compositions capable of inducing melanin production.
• the agent for use in suppressing melanin production (hereinafter also referred to as "melanin suppressant") of the present disclosure includes bakuchiol.
• the composition for use in suppressing melanin production of the present disclosure includes bakuchiol.
• the melanin suppressant or composition of the present disclosure can suppress melanin production. Therefore, the melanin suppressor or composition of the present disclosure can be suitably used, for example, as a whitening agent.
• the melanin suppressant of the present disclosure can suppress melanin production, particularly melanin production in the skin, by using it on a subject, for example.
• the usage conditions (administration conditions) of the melanin suppressant of the present disclosure the explanation of the usage conditions of the retinol-like effect inducer of the present disclosure can be referred to.
• the present disclosure discloses methods capable of inducing retinol-like effects.
• the method for inducing retinol-like action of the present disclosure uses the retinol-like action inducer or composition of the present disclosure.
• the method for inducing a retinol-like effect of the present disclosure for example, while suppressing the occurrence of side effects such as skin erythema and desquamation caused by retinol, which are caused by retinol's effect of inhibiting differentiation of epidermal keratinocytes, retinol can It is expected that the observed wrinkle-improving effect and other effects will be obtained.
• the method of inducing retinol-like action of the present disclosure includes the step of using the retinol-like action inducer or composition of the present disclosure in a subject.
• the use may be, for example, contact with the skin or the like, or administration.
• the step of using may be performed, for example, in vitro or in vivo .
• the subject (administration subject) and administration conditions of the method for inducing retinol-like action of the present disclosure for example, the explanation of the subject and administration conditions of the inducer or composition of retinol-like action of the present disclosure can be referred to.
• the present disclosure discloses a method capable of inducing expression of a maturation-promoting gene in epidermal keratinocytes.
• the method for inducing expression of a gene that promotes maturation of epidermal keratinocytes of the present disclosure uses the agent or composition for inducing expression of a gene that promotes maturation of epidermal keratinocytes of the present disclosure.
• the method of inducing the expression of a gene that promotes the maturation of epidermal keratinocytes of the present disclosure for example, the maturation of epidermal keratinocytes can be promoted, so that effects such as strengthening of the skin barrier function and moisturizing effect can be obtained.
• the method of inducing the expression of maturation-promoting genes of the present disclosure is expected to suppress the side effects of ATRA, for example, so it is expected that the wrinkle-improving effect observed with retinol can be obtained while suppressing the side effects of retinol. Be expected.
• the method of inducing the expression of a gene that promotes maturation of epidermal keratinocytes of the present disclosure includes the step of using the expression inducer or composition of the gene that promotes maturation of epidermal keratinocytes of the present disclosure in a subject.
• the use may be, for example, contact with the skin or the like, or administration.
• the step of using may be performed, for example, in vitro or in vivo .
• target administration target
• administration conditions of the method for inducing expression of a gene that promotes maturation of epidermal keratinocytes of the present disclosure for example, refer to the description of the target and administration conditions of the retinol-like action inducer or composition of the present disclosure. can.
• the present disclosure discloses a method capable of inducing expression of the CERS1 gene.
• the CERS1 gene expression induction method of the present disclosure uses the CERS1 gene expression inducer or composition of the present disclosure.
• the method of inducing expression of the CERS1 gene of the present disclosure differentiation of epidermal keratinocytes can be promoted, so effects such as strengthening of skin barrier function and moisturizing effect can be obtained.
• the method of inducing expression of the CERS1 gene of the present disclosure is expected to suppress the side effects of ATRA, for example, it is expected that the wrinkle-improving effect observed with retinol can be obtained while suppressing the side effects caused by retinol. be done.
• the method for inducing expression of the CERS1 gene of the present disclosure includes the step of using the CERS1 gene expression inducer or composition of the present disclosure in a subject.
• the use may be, for example, contact with the skin or the like, or administration.
• the step of using may be performed, for example, in vitro or in vivo .
• the subject (administration subject) and administration conditions of the method for inducing expression of the CERS1 gene of the present disclosure for example, the description of the subject and administration conditions of the inducer or composition of retinol-like action of the present disclosure can be referred to.
• the present disclosure discloses a method capable of promoting proliferation and/or maturation of epidermal keratinocytes, particularly human epidermal keratinocytes.
• the epidermal keratinocyte proliferation and/or maturation promoting method of the present disclosure uses the epidermal keratinocyte proliferation and/or maturation promoting agent or composition of the present disclosure. According to the method for promoting the proliferation and/or maturation of epidermal keratinocytes of the present disclosure, it is possible to promote the proliferation and/or maturation of epidermal keratinocytes, thereby obtaining effects such as wrinkle improvement effects and skin barrier function strengthening effects. Can be done.
• the method of promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure is expected to suppress the side effects of ATRA, for example, and therefore improve wrinkles observed with retinol while suppressing side effects caused by retinol. It is expected that the effect will be obtained.
• the method for promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure includes the step of using the agent or composition for promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure in a subject.
• the use may be, for example, contact with the skin or the like, or administration.
• the step of using may be performed, for example, in vitro or in vivo .
• target (administration target) and administration conditions of the method for promoting proliferation and/or maturation of epidermal keratinocytes of the present disclosure for example, refer to the description of the target and administration conditions of the retinol-like action inducer or composition of the present disclosure. can.
• the present disclosure discloses a method capable of suppressing melanin production.
• the method for inhibiting melanin production of the present disclosure uses the melanin production inhibitor or composition of the present disclosure. According to the method for suppressing melanin production of the present disclosure, melanin production can be suppressed. Therefore, the method for suppressing melanin production of the present disclosure can be suitably used, for example, to induce a whitening effect.
• the method for inhibiting melanin production of the present disclosure includes the step of using the melanin production inhibitor or composition of the present disclosure on a subject.
• the use may be, for example, contact with the skin or the like, or administration.
• the step of using may be performed, for example, in vitro or in vivo .
• the subject (administration subject) and administration conditions of the method for inducing melanin production of the present disclosure for example, the explanation of the subject and administration conditions of the inducer or composition of retinol-like action of the present disclosure can be referred to.
• the present disclosure is the use of bakuchiol, the retinol-like effect-inducing agent, or the retinol-like effect-inducing composition for use in inducing retinol-like effect.
• the present disclosure provides bakuchiol, an expression inducer of the epidermal keratinocyte maturation-promoting gene, or an expression inducer of the epidermal keratinocyte maturation-promoting gene, for use in inducing the expression of the epidermal keratinocyte maturation-promoting gene.
• Use of the composition is the use of the composition.
• the present disclosure is the use of bakuchiol, the CERS1 gene expression inducer, or the CERS1 gene expression induction composition for use in inducing the expression of the CERS1 gene.
• the present disclosure provides bakuchiol, an agent for promoting proliferation and/or maturation of epidermal keratinocytes, or a promoter for promoting proliferation and/or maturation of epidermal keratinocytes, for use in promoting proliferation and/or maturation of epidermal keratinocytes.
• Use of the composition is the use of bakuchiol, the aforementioned melanin production inhibitor, or the aforementioned melanin production inhibitory composition for use in inhibiting melanin production.
• the present disclosure is the use of bakuchiol to produce an agent for use in inducing a retinol-like effect, or a composition for use in inducing a retinol-like effect.
• the present disclosure relates to the use of bakuchiol for producing an agent for inducing the expression of a gene that promotes the maturation of epidermal keratinocytes, or a composition for inducing the expression of a gene that promotes the maturation of epidermal keratinocytes. be.
• the present disclosure is the use of bakuchiol for producing an agent for use in inducing expression of the CERS1 gene or a composition for use in inducing expression of the CERS1 gene.
• the present disclosure relates to the use of bakuchiol for producing an agent for use in promoting proliferation and/or maturation of epidermal keratinocytes, or a composition for use in promoting proliferation and/or maturation of epidermal keratinocytes.
• the present disclosure is the use of bakuchiol to produce an agent for use in suppressing melanin production, or a composition for use in suppressing melanin production.
• Example 1 It was confirmed that bakuchiol exhibits a retinol-like effect in regulating RAR expression. It was also confirmed that bakuchiol has a reduced effect of inhibiting differentiation of epidermal keratinocytes compared to retinol, and has an effect of promoting differentiation and maturation of epidermal keratinocytes.
• Sample Preparation ATRA was prepared by dissolving it in DMSO to a concentration of 1 mmol/l.
• Bakuchiol manufactured by BIB Corporation was prepared by dissolving it in DMSO to a concentration of 20 mmol/l.
• NBNHEK normal human epidermal keratinocytes
• the culture medium was replaced with a sample-added medium (ATRA: final concentration 1 ⁇ mol/l, bakuchiol: 5 or 10 ⁇ mol/l) prepared at a predetermined concentration with KGM EGF+/BPE+.
• ATRA sample-added medium
• the control group (nt) without sample addition was replaced with a medium supplemented with DMSO so that the final concentration was equal to that of the sample addition group.
• RNA was extracted using QIAshredder (manufactured by QIAGEN) and RNeasy mini kit (manufactured by QIAGEN). Based on the measured total RNA concentration, cDNA was synthesized using PrimeScript (manufactured by Takara). Furthermore, quantitative analysis of gene expression level was performed using Realtime PCR from the obtained cDNA using SYBR Premix Ex Taq (manufactured by Takara).
• the target genes were RAR ⁇ gene, RAR ⁇ gene, and RAR ⁇ gene.
• FIG. 1 is a graph showing the expression of the RAR ⁇ gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the RAR ⁇ gene.
• the numerical values on the vertical axis in FIGS. 1A and 1B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis in FIG. 1(A) are as follows.
• ⁇ ATRA addition group 1.52
• the numerical value on the vertical axis in Fig. 1(B) is. It is as follows.
• FIGS. 1(A) and (B) expression of the RAR ⁇ gene was induced in the ATRA addition group.
• FIGS. 1(A) and 1(B) the expression of the RAR ⁇ gene did not change significantly in the bakuchiol-added group.
• FIG. 2 is a graph showing the expression of the RAR ⁇ gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the RAR ⁇ gene.
• the numerical values on the vertical axis in FIGS. 2A and 2B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis of FIG. 2(A) are as follows.
• ⁇ ATRA addition group 3.98, bakuchiol 5 ⁇ M addition group: 1.76, bakuchiol 10 ⁇ M addition group: 2.20
• the numerical values on the vertical axis in FIG. 2(B) are as follows.
• FIGS. 2(A) and 2(B) the expression of the RAR ⁇ gene was induced in the ATRA addition group.
• RAR ⁇ gene expression was also significantly induced in the bakuchiol addition group.
• FIG. 3 is a graph showing the expression of the RAR ⁇ gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the RAR ⁇ gene.
• the numerical values on the vertical axis in FIGS. 3A and 3B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis of FIG. 3(A) are as follows.
• ⁇ ATRA addition group 0.89, Bakuchiol 5 ⁇ M addition group: 0.83, Bakuchiol 10 ⁇ M addition group: 0.54
• the numerical values on the vertical axis in FIG. 3(B) are as follows. ⁇ ATRA addition group: 0.70, Bakuchiol 5 ⁇ M addition group: 0.95, Bakuchiol 10 ⁇ M addition group: 0.71
• the expression of the RAR ⁇ gene was suppressed in the ATRA addition group.
• FIGS. 3(A) and 3(B) the expression of the RAR ⁇ gene was also significantly suppressed in the bakuchiol addition group.
• FIG. 4 is a graph showing the expression of the FLG gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the FLG gene.
• the numerical values on the vertical axis in FIGS. 4A and 4B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis in FIG. 4(A) are as follows.
• ⁇ ATRA addition group 0.42, Bakuchiol 5 ⁇ M addition group: 2.69, Bakuchiol 10 ⁇ M addition group: 2.07
• the numerical values on the vertical axis in FIG. 4(B) are as follows.
• FIGS. 4(A) and (B) the expression of the FLG gene was significantly suppressed in the ATRA-added group.
• FIGS. 4(A) and 4(B) the expression of the FLG gene significantly increased in the bakuchiol-added group.
• FIG. 5 is a graph showing the expression of the IVL gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the IVL gene.
• the numerical values on the vertical axis in FIGS. 5A and 5B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis of FIG. 5(A) are as follows.
• ⁇ ATRA addition group 1.69, Bakuchiol 5 ⁇ M addition group: 2.72, Bakuchiol 10 ⁇ M addition group: 3.22
• the numerical values on the vertical axis in FIG. 5(B) are as follows.
• the expression of the IVL gene was increased (after 48 hours) or maintained (after 72 hours).
• FIGS. 5(A) and 5(B) the expression of the IVL gene was significantly increased in the bakuchiol-added group.
• FIG. 6 is a graph showing the expression of the CERS1 gene.
• (A) shows the results after 48 hours of culture
• (B) shows the results after 72 hours of culture.
• the horizontal axis indicates the type of sample
• the vertical axis indicates the expression level of the CERS1 gene.
• the numerical values on the vertical axis in FIGS. 6A and 6B indicate the values of each group, with the value of the nt group being 1.0 (1.00).
• the numerical values on the vertical axis in FIG. 6(A) are as follows.
• ⁇ ATRA addition group 4.79, bakuchiol 5 ⁇ M addition group: 3.07, bakuchiol 10 ⁇ M addition group: 9.74
• the numerical values on the vertical axis in FIG. 6(B) are as follows.
• the expression of the CERS1 gene increased in the ATRA-added group.
• FIGS. 6(A) and (B) the expression of the CERS1 gene significantly increased in the bakuchiol-added group.
• the expression control behavior of the RAR ⁇ gene and RAR ⁇ gene among the RAR ⁇ gene, RAR ⁇ gene, and RAR ⁇ gene was similar to that in the ATRA group, confirming that bakuchiol exerts a retinol-like effect.
• Tazarotene a selective retinoid, acts on the RAR ⁇ and RAR ⁇ genes and suppresses the aforementioned side reactions compared to retinoic acid. Therefore, it has been suggested that the side reactions of retinoic acid depend on the induction of RAR ⁇ gene expression.
• bakuchiol exhibits a retinol-like effect by regulating the expression of the RAR ⁇ gene and RAR ⁇ gene, but does not enhance the expression of the RAR ⁇ gene, so it was expected that the side reactions observed with retinoic acid would be suppressed.
• the expression of the FLG gene which is a maturation marker of epidermal keratinocytes, was significantly suppressed, confirming suppression of differentiation of epidermal keratinocytes, which is the cause of the above-mentioned side effects.
• the expression of marker genes associated with differentiation and maturation of epidermal keratinocytes such as the FLG gene, IVL gene, and CERS1 gene, was induced. Based on these findings, bakuchiol was considered to exhibit wrinkle-improving effects and skin barrier function-strengthening effects while suppressing side reactions caused by retinoic acid by promoting the differentiation and maturation of epidermal keratinocytes.
• Sample Preparation Bakuchiol (manufactured by BIB Corporation) was prepared by dissolving it in 100% ethanol so as to have a predetermined final concentration (5 ⁇ mol/l or 10 ⁇ mol/l) upon addition to the medium.
• B16 cells (B16 mouse melanoma cells, provided by RIKEN BRC, Lot. RCB1283, passage number 20) were seeded at 1.75 x 10 5 cells/dish in a 60 mm dish. , 5% CO 2 and 37° C. overnight.
• the medium used was E-MEM medium (5% FBS/E-MEM medium, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) containing 5% fetal bovine serum (FBS, manufactured by Sigma-Aldrich). After removing the medium in the dish by suction, bakuchiol prepared to the above-mentioned final concentration was added, and the B16 cells were cultured under conditions of 5% CO 2 and 37° C.
• a non-treated (NT) group was cultured with only 5% FBS/E-MEM medium
• a positive control (PC) group was cultured with the addition of arbutin (manufactured by LKT Laboratories, Inc.) (final concentration 200 ⁇ g/ml).
• a negative control (NC) group in which cells were cultured with the addition of 100% ethanol.
• the ethanol concentration was set to 0.05% in all treatment groups except the NT group. After removing the medium in the dish by suction, B16 cells were collected by trypsin treatment, centrifuged, and the supernatant was removed.
• FIG. 7 is a graph showing the amount of melanin produced.
• the horizontal axis shows the type of sample
• the vertical axis shows the relative value of melanin production.
• the numerical values on the vertical axis in FIG. 7 indicate the values of each group, with the value of the NC group being 100 (%).
• the numerical values on the vertical axis in FIG. 7 are as follows. ⁇ NT group: 32.63, PC group: 64.27, bakuchiol 5 ⁇ M addition group: 75.90, bakuchiol 10 ⁇ M addition group: 17.78
• melanin production decreased in a bakuchiol concentration-dependent manner.
• Cytotoxicity B16 cells were seeded in a 96-well plate at 7 ⁇ 10 3 cells/well and cultured overnight at 37° C. and 5% CO 2 .
• the culture medium used in the culture was the same as in Example 2 (2).
• a sample was added in the same manner as in the melanin production suppression test, and cultured under conditions of 5% CO 2 and 37° C. for 72 hours.
• MTT assay was performed on each well using Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich). The cell survival rate was evaluated by measuring the absorbance of the sample with the target absorbance at 570 nm and the reference at 620 nm.
• bakuchiol can suppress the amount of melanin produced in melanin-producing cells and can suppress melanin production in the skin.
• Example 3 Through cell tests (tests using epidermal keratinocytes), it was confirmed whether bakuchiol has the effect of promoting hyaluronic acid synthesis in the epidermal layer and strengthening the basement membrane.
• Sample Preparation ATRA was prepared by dissolving it in DMSO to a concentration of 1 mmol/l.
• Bakuchiol manufactured by BIB Corporation was prepared by dissolving it in DMSO to a concentration of 20 mmol/l.
• NBNHEK normal human epidermal keratinocytes
• KGM EGF+/BPE+ KGM EGF+/BPE+
• ATRA final concentration 1 ⁇ mol/l
• bakuchiol final concentration 2.5, 5, or 10 ⁇ mol/l
• the control group (n.t.) without sample addition was replaced with a medium supplemented with DMSO so that the final concentration was equal to that of the sample addition group.
• the target gene for epidermal layer hyaluronic acid synthesis was the HAS3 gene.
• the target genes for strengthening the epidermal basement membrane were the ITG ⁇ 6 gene and the ITG ⁇ 1 gene.
• the RPS18 gene was used as a housekeeping gene (internal standard gene).
• the expression level of each gene was a relative value to the gene expression level of the RPS18 gene.
• the measurement results of the expression levels of the genes are shown in Tables 2 to 4 below. Note that the results shown in Tables 2 to 4 below are shown by calculating the average value of three samples in each group, and setting the value (average value) of the control group (nt.) as 1.00.
• the results shown in Tables 2 to 4 below show ***: p ⁇ 0.001, **: p ⁇ 0.01, *: p ⁇ 0.05 compared to the values of the control group.
• ⁇ Agent for inducing retinol-like action> An agent containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in inducing retinol-like effects.
• bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in inducing retinol-like effects.
• the agent according to Supplementary Note 1 wherein the induction of retinol-like action is strengthening of the basal layer of the skin, stabilization of the basal layer of the skin, and/or promotion of proliferation of epidermal keratinocytes.
• composition for use in inducing retinol-like action A composition containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in inducing a retinol-like effect.
• Appendix 8 The composition according to appendix 7, wherein the induction of the retinol-like effect is strengthening the basal layer of the skin, stabilizing the basal layer of the skin, and/or promoting proliferation of epidermal keratinocytes.
• Appendix 9 9.
• Appendix 10 The composition according to any one of appendices 7 to 9, wherein the bakuchiol is an extract containing bakuchiol.
• Appendix 11 A composition for application to the skin, comprising the composition according to any one of appendices 7 to 10.
• Appendix 12 A composition for oral administration, comprising the composition according to any one of Supplementary Notes 7 to 10.
• Agent for inducing the expression of a gene that promotes maturation of epidermal keratinocytes > (Appendix 14) An agent containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in inducing the expression of genes that promote the maturation of epidermal keratinocytes.
• compositions for use in inducing expression of a gene that promotes maturation of epidermal keratinocytes A composition containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in inducing the expression of a gene that promotes the maturation of epidermal keratinocytes. thing. (Additional note 21) 21. The composition according to appendix 20, wherein the maturation-promoting gene includes a filaggrin gene. (Additional note 22) 22. The composition according to appendix 21, wherein the maturation-promoting gene further includes an involucrin gene.
• composition according to any one of appendices 20 to 22, wherein the bakuchiol is an extract containing bakuchiol is an extract containing bakuchiol.
• a composition for application to the skin comprising the composition according to any one of appendices 20 to 23.
• a composition for oral administration comprising the composition according to any one of Supplementary Notes 20 to 23.
• composition for use in inducing expression of CERS1 gene A composition for use in inducing expression of ceramide synthase 1 (CERS1) gene, which contains bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).
• Appendix 32 The composition according to appendix 31, wherein the bakuchiol is an extract containing bakuchiol.
• Appendix 33 A composition for application to the skin, comprising the composition according to appendix 31 or 32.
• Appendix 34 A composition for oral administration, comprising the composition according to appendix 31 or 32.
• composition according to any one of appendices 31 to 34 comprising the agent according to any one of appendices 27 to 30.
• Agent for promoting proliferation and maturation of epidermal keratinocytes An agent containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in promoting proliferation and/or maturation of epidermal keratinocytes.
• Appendix 37 The agent according to appendix 36, wherein the bakuchiol is an extract containing bakuchiol.
• composition for use in promoting proliferation and maturation of epidermal keratinocytes (Additional note 40) A composition containing bakuchiol for use in promoting proliferation and/or maturation of epidermal keratinocytes. (Additional note 41) The composition according to appendix 40, wherein the bakuchiol is an extract containing bakuchiol. (Additional note 42) A composition for application to the skin, comprising the composition according to appendix 40 or 41.
• compositions for oral administration comprising the composition according to appendix 40 or 41.
• composition according to any one of attachments 40 to 43 comprising the agent according to any one of attachments 36 to 39.
• Agent for suppressing melanin production An agent containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in suppressing melanin production.
• bakuchiol is an extract containing bakuchiol.
• composition for use in suppressing melanin production A composition containing bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol) for use in suppressing melanin production.
• bakuchiol is an extract containing bakuchiol.
• (Appendix 51) A composition for application to the skin, comprising the composition according to appendix 49 or 50.
• Appendix 52 A composition for oral administration, comprising the composition according to appendix 49 or 50.
• Appendix 53 The composition according to any one of appendices 49 to 52, comprising the agent for use in suppressing melanin production according to any one of appendices 45 to 48.
• Method 54 Retinol using an agent for inducing a retinol-like effect according to any one of Supplementary notes 1 to 6 and/or a composition for inducing a retinol-like effect according to any one of Supplementary notes 7 to 13.
• Appendix 55 55.
• the method of induction according to appendix 54 which includes the step of using the agent for inducing the retinol-like effect and/or the composition for inducing the retinol-like effect in a subject.
• Appendix 56 The induction method according to appendix 54 or 55, which is used in vitro or in vivo .
• (Appendix 58) A step of using an agent for inducing the expression of the epidermal keratinocyte maturation-promoting gene and/or a composition for inducing the expression of the epidermal keratinocyte maturation-promoting gene in the subject; The guidance method according to appendix 57. (Appendix 59) The induction method according to appendix 57 or 58, which is used in vitro or in vivo .
• ⁇ Method for inducing expression of CERS1 gene> CERS1 using an agent for inducing expression of the CERS1 gene according to any one of Supplementary notes 27 to 30 and/or a composition for inducing expression of the CERS1 gene according to any one of Supplementary notes 31 to 35.
• Appendix 61 Method for inducing gene expression.
• Appendix 61 The induction method according to appendix 60, comprising the step of using, in a subject, an agent for inducing expression of the CERS1 gene and/or a composition for inducing expression of the CERS1 gene.
• Additional note 62 The induction method according to appendix 60 or 61, which is used in vitro or in vivo .
• the induction method according to supplementary note 66 comprising the step of using the agent for suppressing melanin production and/or the composition for suppressing melanin production in a subject.
• Appendix 68 The induction method according to appendix 66 or 67, which is used in vitro or in vivo .
• Appendix 69 An agent for use in inducing a retinol-like effect according to any one of Supplementary Notes 1 to 6 or an agent for use in inducing a retinol-like effect according to any one of Supplementary Notes 7 to 13. Use of the composition.
• the present disclosure it is possible to provide an agent that can reduce inhibition of epidermal keratinocyte differentiation, induce retinol-like effects, and is mainly applicable to human skin. Therefore, the present invention can be said to be extremely useful, for example, in the fields of pharmaceuticals, quasi-drugs, external skin preparations, and the like.

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