Agent for use to induce retinol-like action, and application thereof

Abstract

Provided is an agent that can induce a retinol-like action while reducing the suppression of epidermal keratinocyte differentiation. The agent is applicable to human skin and the like.　The agent for use to induce retinol-like action according to the present disclosure contains bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).

Description

Agents for use in inducing retinol-like effects and uses thereof

[0001] The present disclosure relates to an agent for exerting a retinol-like action on the skin of, for example, a human, and uses thereof.

[0002] It is known that retinoic acid, which is retinol, when applied to the skin, controls the expression of retinoic acid receptors (RAR) in epidermal keratinocytes, etc. Non-Patent Document 1 describes that retinoic acid weakly induces (enhances) the expression of the RARβ gene and the RARγ gene among RARs.

[0003] While retinoic acid activates skin metabolism, it also inhibits differentiation of epidermal keratinocytes, which causes side effects such as erythema and desquamation of the skin, which has become a problem.

[0004] Ding-Dar Lee et.al., “Retinoid-Responsive Transcriptional Changes in Epidermal Keratinocytes”, J Cell Physiol. 2009 August; 220(2): pp.427-439

[0005] Therefore, an object of the present disclosure is to provide an agent that reduces the inhibition of epidermal keratinocyte differentiation, can induce retinol-like effects, and is applicable primarily to human skin, etc.

[0006] In order to achieve the above-mentioned objective, the agent for use in inducing retinol-like action of the present disclosure (hereinafter also referred to as "retinol-like action inducer") contains bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).

[0007] The agent for use in inducing the expression of a maturation-promoting gene in epidermal keratinocytes of the present disclosure (hereinafter also referred to as "maturation-promoting gene expression inducer") contains bakuchiol.

[0008] The agent for use in inducing expression of the ceramide synthase 1 (CERS1) gene of the present disclosure (hereinafter also referred to as "CERS1 gene expression inducer") contains bakuchiol.

[0009] The agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes (hereinafter also referred to as "maturation promoter") of the present disclosure contains bakuchiol.

[0010] According to the present disclosure, an agent can be provided that can reduce the inhibition of differentiation of epidermal keratinocytes, induce retinol-like effects, and is applicable mainly to human skin, etc.

[0011] Figures 1(A) and (B) are graphs showing the expression of the RARα gene in Example 1, with Figure 1(A) showing the results after 48 hours of culture and Figure 1(B) showing the results after 72 hours of culture. Figures 2(A) and (B) are graphs showing the expression of the RARβ gene in Example 1, with Figure 2(A) showing the results after 48 hours of culture and Figure 2(B) showing the results after 72 hours of culture. Figures 3(A) and (B) are graphs showing the expression of the RARγ gene in Example 1, with Figure 3(A) showing the results after 48 hours of culture and Figure 3(B) showing the results after 72 hours of culture. Figures 4(A) and (B) are graphs showing the expression of the filaggrin (FLG) gene in Example 1, with Figure 4(A) showing the results after 48 hours of culture and Figure 4(B) showing the results after 72 hours of culture. Figures 5(A) and (B) are graphs showing the expression of the involucrin (IVL) gene in Example 1, with Figure 5(A) showing the results after 48 hours of culture and Figure 5(B) showing the results after 72 hours of culture. Figures 6(A) and (B) are graphs showing the expression of the CERS1 gene in Example 1. In Figure 6, (A) shows the results after 48 hours of culture and (B) shows the results after 72 hours of culture. Figure 7 is a graph showing the amount of melanin produced in Example 2.

[0012] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0013] <Agent or Composition for Use in Inducing Retinol-Mimetic Action> In certain embodiments, the present disclosure provides an agent or composition capable of inducing retinol-mimetic action. The agent for use in inducing retinol-mimetic action of the present disclosure comprises bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). Furthermore, the composition for use in inducing retinol-mimetic action of the present disclosure (hereinafter also referred to as a "retinol-mimetic action-inducing composition") comprises bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). In the following description, unless otherwise specified, the description of each agent of the present disclosure can be incorporated into the description of the corresponding composition.

[0014] As a result of extensive research, the present inventors have discovered that bakuchiol has an effect similar to that of ATRA (all-trans retinoic acid), a retinol (vitamin A), in regulating the expression of retinoic acid receptors (RAR) in epidermal keratinocytes, i.e., a retinol-like effect. Further research led the present inventors to find that bakuchiol reduces the inhibitory effect on epidermal keratinocyte differentiation observed with ATRA, while also exhibiting the same RARβ gene expression-inducing and RARγ gene expression-inhibiting effects observed with ATRA, thereby establishing the present invention. As described below, it is presumed that ATRA induces side effects such as skin erythema and desquamation by inducing RARα gene expression. Therefore, the retinol-like action inducer and the like of the present disclosure are expected to provide the effects observed with retinol, such as wrinkle improvement, while suppressing the occurrence of side effects of retinol, such as erythema and desquamation of the skin, which are caused by the inhibitory effect of retinol on the differentiation of epidermal keratinocytes.

[0015] The "bakuchiol" is 4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol, and can be, for example, a compound represented by the following formula (1). The bakuchiol may be, for example, a phenoxide ion. The bakuchiol may also be, for example, esterified.

[0016] The bakuchiol may be an isolated or purified compound, or a composition containing bakuchiol. Examples of the composition containing bakuchiol include an extract containing bakuchiol, a crude extract, a dried extract, a freeze-dried extract, a spray-dried extract, or other processed product of the extract.

[0017] The extract containing bakuchiol can be produced, for example, by solvent extraction of a plant containing bakuchiol. Examples of plants containing bakuchiol include Psoralea corylifolia and Otholobium pubescens. One or more types of plants may be used. The plant material used for the extraction may be an entire plant or a part of a plant. Examples of plant parts include roots, rhizomes, leaves, stems, whole flowers, or a mixture thereof. The material may be the collected plant itself or a processed product obtained by drying and / or pulverizing.

[0018] Examples of the solvent used for extracting bakuchiol include aqueous solvents such as water and buffer solutions; lower alcohols or hydrous lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and 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, 1,3,5-pentanetriol, and glycerin. and polyethylene glycol (for example, molecular weight 100 to 100,000); organic solvents such as acetone, ethyl acetate, diethyl ether, dimethyl ether, ethyl methyl ether, dioxane, hexane, acetonitrile, xylene, benzene, chloroform, carbon tetrachloride, phenol, and toluene; acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) or alkalis (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) whose normality has been adjusted appropriately; etc. The above-mentioned solvents may be used alone or in combination of two or more.

[0019] Examples of treatments for the treated product include decomposition by adding an acid (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or an alkali (sodium hydroxide, calcium hydroxide, ammonia, etc.); fermentation or metabolic conversion by a microorganism; component adsorption using an ion exchange resin, activated carbon, diatomaceous earth, etc.; fractionation using chromatography with various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.); filtration using filter paper, membrane filter, ultrafiltration membrane, etc.; pressurization or decompression; heating or cooling; drying or freeze-drying; pH adjustment; deodorization; decolorization; prolonged static storage; etc. One type of treatment may be performed alone, or two or more types may be performed.

[0020] The "retinol-mimetic action" refers to a compound or composition that exhibits at least one of the actions exhibited by retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), particularly ATRA. The retinol-mimetic action may be, for example, an action on the skin or cells that constitute the skin, or an action on gene expression in the cells, i.e., induction or suppression of gene expression. The retinol-mimetic action inducer of the present disclosure exhibits one or more types of retinol-mimetic action.

[0021] When the retinol-like action is an action on the skin or cells that constitute the skin, examples of the retinol-like action include strengthening the basal layer of the skin, stabilizing the basal layer of the skin, and promoting the proliferation of epidermal keratinocytes.

[0022] The "strengthening of the basal layer of the skin" means that the cells of the basal layer are less likely to be destroyed when an external stimulus is applied to the skin (resistance to external stimuli). Strengthening 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). Examples of the basal layer marker include hyaluronic acid synthase 3 (HAS3), integrin α6 (ITGα6), and integrin β1 (ITGβ1).

[0023] The "stabilization of the basal layer of the skin" means suppressing the breakdown of the basal layer that occurs with aging. The stabilization of the basal layer of the skin can be evaluated, for example, by the basal layer marker.

[0024] The "promotion of epidermal keratinocyte proliferation" means promoting or enhancing the proliferation of epidermal keratinocytes, particularly human epidermal keratinocytes. The promotion of epidermal keratinocyte proliferation can be carried out, for example, by evaluating the proliferation of the epidermal keratinocytes.

[0025] The term "epidermal keratinocytes" refers to keratinocytes generated by the division of cells in the basal layer of the epidermis. It is known that the epidermal keratinocytes further differentiate in the epidermis and ultimately differentiate into corneocytes that form the stratum corneum.

[0026] When the retinol-like effect is an effect on gene expression in the cells, the retinol-like effect can be, for example, induction of retinoic acid receptor (RAR) β gene expression, inhibition of RARγ gene expression, etc., preferably induction of RARβ gene expression and inhibition of RARγ gene expression.

[0027] An example of the RARβ gene is mRNA encoded by the human RARβ gene, such as a polynucleotide consisting of the base sequence registered in Genbank under accession number NM — 000965.5.

[0028] As an example of the RARγ gene, mRNA encoded by the human RARγ gene is, for example, a polynucleotide consisting of the base sequence registered in Genbank under accession number NM — 000966.6.

[0029] The "induction of RARβ gene expression" means that the expression level of the RARβ gene is increased or enhanced, and may also mean that the expression level of the RARβ gene changes from an unexpressed state to an expressed state. The expression of the RARβ gene can be evaluated, for example, by measuring the expression level of the mRNA of the RARβ gene according to Example 1 described below. The expression level of the RARβ gene may be measured by measuring the expression level of one or more isoforms of the RARβ gene, or by measuring the expression of all isoforms, although the latter is preferred.

[0030] The "suppression of RARγ gene expression" means that the expression level of the RARγ gene is suppressed or reduced, and may also mean that the expression of the RARγ gene changes from a state in which it is expressed to a state in which it is not expressed. The expression of the RARγ gene can be evaluated, for example, by measuring the expression level of the mRNA of the RARγ gene according to Example 1 described below. The expression level of the RARγ gene may be measured by measuring the expression level of any one or more isoforms of the RARγ gene, or by measuring the expression of all isoforms, but the latter is preferred.

[0031] The "gene" may exist in the form of RNA (e.g., mRNA) or DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. As used herein, the "gene" may include additional sequences such as sequences of untranslated regions (UTRs).

[0032] The retinol-mimetic action inducer of the present disclosure can induce retinol-mimetic action, for example, by administering it to a subject. The conditions for use (administration conditions) of the retinol-mimetic action inducer of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of subject.

[0033] The retinol-like effect inducers of the present disclosure may be used, for example, in vivo or in vitro.

[0034] The subject to which the retinol-mimetic action inducer of the present disclosure is administered is not particularly limited. When the retinol-mimetic action inducer of the present disclosure is used in vivo, the subject to which the retinol-mimetic action inducer of the present disclosure is administered can be, for example, a human or a non-human animal other than a human. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs, as well as birds. When the retinol-mimetic action inducer of the present disclosure is used in vitro, the subject to which the retinol-mimetic action inducer of the present disclosure is administered can be, for example, a cell, tissue, or organ. Examples of the cell include cells collected from a living organism, cultured cells, and the like, and examples of the tissue or organ include tissue (biological tissue) or organ collected from a living organism.

[0035] In the following topical skin preparations (e.g., transdermal or skin application preparations or compositions) or orally administered preparations or compositions containing the retinol-like activity inducer of the present disclosure, the amount of bakuchiol may be within the range that exhibits the retinol-like activity, i.e., an effective amount. The amount of bakuchiol (upper and lower limits) is, for example, as follows. In the examples below, the upper and lower limits can be arbitrarily combined. Lower limit: 0.1 μmol / L, 1 μmol / L, preferably 2.5 μmol / L; Upper limit: 200 mmol / L, preferably 100 mmol / L, preferably 50 mmol / L, 10 mmol / L, 1 mmol / L, preferably 500 μmol / L, and more preferably 100 μmol / L. Note that "mol / L" may also be abbreviated as "M" in this specification. In addition, the Examples below describe the details of experiments in which bakuchiol was added at 5 μM (0.000125% (w / v%)) or 10 μM (0.00025% (w / v%)).

[0036] The administration form of the retinol-like activity inducer of the present disclosure can be oral or parenteral. Examples of parenteral administration include transdermal administration and application (contact) to the skin. Application to the skin can also mean application to the oral mucosa, i.e., application to or contact with epithelial cells in the oral cavity. Furthermore, application to the skin can also mean administration or injection into the skin or subcutaneously via the skin surface, in addition to or instead of application to the skin surface. Administration or injection into the skin via the skin surface can be performed, for example, using a microneedle.

[0037] The dosage form of the retinol-like activity inducer of the present disclosure is not particularly limited and can be appropriately determined depending on, for example, the administration form. Examples of the dosage form include liquid and solid forms. Examples of the dosage form include oil gels (e.g., a form in which bakuchiol is contained in an oil gel), oil gel particles (e.g., a form in which bakuchiol is contained in an oil gel particle), and liposomes (e.g., a form in which bakuchiol is contained in a liposome). When the administration form is oral administration, examples of the dosage form include tablets, pills, capsules, granules, powders, and liquids.

[0038] The "oil gel" refers to a gel-like oil agent (oily component) thickened with a lipophilic gelling agent. The oil gel can be prepared, for example, by adding an oily gelling agent to an oil agent. The "oil gel particle" refers to one or more particles composed of oil gel, in which a desired component is dissolved or dispersed in the oil agent of the oil gel. The "liposome" refers to a vesicle having a lipid bilayer membrane dispersed in an aqueous solvent.

[0039] The retinol-like activity inducer of the present disclosure may contain additives, if necessary. When used as a composition, the additives preferably include pharmaceutically acceptable additives or pharmaceutically acceptable carriers. The additives are not particularly limited, and examples thereof include base materials, excipients, colorants, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring agents such as fragrances, etc. In the present disclosure, the amount of the additives is not particularly limited as long as they do not interfere with the function of bakuchiol.

[0040] Examples of the excipient include sugar derivatives such as lactose, lactose hydrate, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic; dextran; and pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate. Examples of the colorant include yellow ferric oxide. Examples of the lubricant include metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; polyethylene glycol; silica; and hydrogenated vegetable oil. Examples of the flavoring agent include flavorings such as cocoa powder, peppermint, aromatic powder, peppermint oil, borneol, and cinnamon powder, as well as sweeteners and acidulants. Examples of the binder include hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, etc. Examples of the disintegrant include cellulose derivatives such as carboxymethyl cellulose and carboxymethyl cellulose calcium; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, carboxymethyl starch sodium, cross-linked polyvinylpyrrolidone, and sodium starch glycolate; examples of the stabilizer include parahydroxybenzoic 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; sorbic acid; and examples of the coating agent include hypromellose, macrogol such as Macrogol 6000, talc, titanium oxide, etc.

[0041] When the retinol-like action-inducing composition of the present disclosure is an orally administered composition, specific examples of the orally administered composition include beverages, foods, pharmaceutical products (drugs), and quasi-drug products (drugs).

[0042] When the retinol-like action inducer or composition of the present disclosure is used for transdermal administration or application to the skin (hereinafter also referred to as "topical skin preparation"), the form of the topical skin preparation may be, depending on the form of use, an ampoule, capsule, powder, granule, liquid, gel, foam, emulsion, sheet, mist, spray, etc. Examples of the form of use include pharmaceutical product(s), quasi-drug product(s), topical or systemic topical skin preparations, medicinal and / or cosmetic preparations applied to the scalp and hair, bath additives used by adding them to bathwater, other preparations, etc. Examples of the topical or systemic skin preparations include basic cosmetics such as lotions, milky lotions, creams, ointments, lotions, oils, and packs; face washes or skin cleansers such as solid soaps, liquid soaps, and hand washes; massage agents, cleansing agents, hair removers, depilatories, shaving treatments, aftershave lotions, pre-shave lotions, shaving creams; makeup cosmetics such as foundations, lipsticks, blushers, eye shadows, eyeliners, and mascaras; perfumes; nail polish, nail enamel, nail enamel removers; poultices, plasters, tapes, sheets, patches, aerosols, toothpaste, and mouthwashes. Examples of the medicinal and / or cosmetic preparations to be applied to the scalp and hair include shampoos, rinses, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and care products, poultices, plasters, tapes, sheets, aerosols, etc. Examples of the other preparations include underarm odor inhibitors or deodorants, antiperspirants, sanitary products, sanitary cotton products, wet tissues, etc.

[0043] The topical skin preparation can be prepared by optionally selecting and / or combining the following components and / or additives as needed, provided that they do not interfere with the action of bakuchiol.

[0044] (1) Various oils and fats: avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange roughage 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, Japan wax, coconut oil, beef tallow, lard, squalene, squalane, pristane, and hydrogenated products of these oils and fats (hardened oils, etc.).

[0045] (2) Waxes: beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0046] (3) Mineral oils: liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystalline wax, etc.

[0047] (4) Fatty Acids Natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acids; and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

[0048] (5) Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, and phenoxyethanol; and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.

[0049] (6) Polyhydric Alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, and the like.

[0050] (7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, and the like.

[0051] (8) Metallic soaps: aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0052] (9) Gums, sugars or water-soluble polymer compounds Gum arabic, gum benzoin, gum dammar, guaiac butter, Irish moss, gum karaya, gum tragacanth, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its ester, trehalose or its derivative, 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 salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salt, Hyaluronic acid or a salt thereof, chondroitin sulfate or a salt thereof, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylates, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or crosslinked polymers thereof, carboxyvinyl polymers, polyethyleneimine, etc.

[0053] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfate ester salts, alkyl phosphate ester salts), cationic surfactants (alkylamine salts, alkyl quaternary ammonium salts), 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, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium or silicon, fluorocarbon surfactants), etc.

[0054] (11) Various Vitamins Vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group: vitamin C acid or a derivative thereof, vitamin D group: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol, vitamin E group: vitamin E or a derivative thereof, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), other essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin P group (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0055] (12) Various Amino Acids: valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, and the like, as well as their sulfates, phosphates, nitrates, citrates, and amino acid derivatives such as pyrrolidone carboxylic acid.

[0056] (13) Additives The topical skin preparation may further contain various additives of animal or plant origin. The additives can be added by, for example, performing conventional processing depending on the type and form of the product to which they are to be added, and optionally selecting from a variety of materials. The processing can be, for example, any of the following processes selected and / or combined: crushing, milling, washing, hydrolysis, fermentation, refining, squeezing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, bleaching, etc.

[0057] The solvent used for the extraction can be selected taking into consideration the intended use and type of the product, as well as subsequent processing. The extraction solvent is preferably one or a mixture of two or more selected from the following: water; lower alcohols or hydrous lower alcohols such as water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol; polyhydric alcohols or hydrous polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, and glycerin; and various organic solvents such as acetone and ethyl acetate. However, when the inclusion of an organic solvent is undesirable depending on the intended use, water alone or ethanol, which is easily removed after extraction, may be used alone or in any mixture with water, or a product obtained by squeezing and extracting the extract may be used.

[0058] When the additives derived from plant or animal raw materials are used in external preparations or cosmetics for systemic or local use, the external preparations for skin can be expected to have cosmetic effects such as protection of the skin and hair, moisturizing, improving feel and texture, imparting softness, easing irritation, relieving stress through fragrance, activating cells (preventing cell aging), suppressing inflammation, improving skin and hair quality, preventing and improving rough skin, promoting hair growth, preventing hair loss, imparting shine, cleansing effects, relieving fatigue, promoting blood flow, and providing a warm bath effect, as well as fragrance, deodorizing, thickening, antiseptic, buffering, and other effects.

[0059] For example, the topical skin preparation can be made into a product that is expected to have multifunctional effects by combining the various cosmetic and pharmaceutical effects of each raw material material that has been known up to now, thereby enhancing the effects aimed at by the present disclosure.

[0060] <Agent or composition for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes> In another aspect, the present disclosure provides an agent or composition capable of inducing a maturation-promoting gene in epidermal keratinocytes, particularly in human epidermal keratinocytes. The agent for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes of the present disclosure comprises bakuchiol. Furthermore, the composition for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes of the present disclosure comprises bakuchiol. The maturation-promoting gene expression inducer or composition of the present disclosure can promote the maturation of epidermal keratinocytes, thereby providing effects such as strengthening skin barrier function and moisturizing effects. Furthermore, the maturation-promoting gene expression inducer or composition of the present disclosure is expected to suppress the side effects of, for example, ATRA, and is therefore expected to provide the wrinkle-improving effect observed with retinol while suppressing the side effects of retinol.

[0061] The epidermal keratinocyte maturation-promoting gene refers to a gene whose expression promotes the maturation of epidermal keratinocytes. Examples of 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 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 stage of differentiation and maturation of epidermal keratinocytes, and is known to, for example, contribute to the differentiation of epidermal keratinocytes.

[0062] An example of the FLG gene is mRNA encoded by the human FLG gene, such as a polynucleotide consisting of the base sequence registered in Genbank under accession number NM_002016.2.

[0063] An example of the IVL gene is mRNA encoded by the human IVL gene, such as a polynucleotide consisting of the base sequence registered in Genbank under accession number NM_005547.4.

[0064] The "induction of FLG gene expression" means that the expression level of the FLG gene is increased or enhanced, and may also mean that the expression level of the FLG gene changes from an absent state to an expressed state. The expression of the FLG gene can be evaluated, for example, by measuring the expression level of FLG gene mRNA according to Example 1 described below.

[0065] The "induction of IVL gene expression" means that the expression level of the IVL gene is increased or enhanced, and may also mean that the expression level of the IVL gene changes from an absent state to an expressed state. 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.

[0066] The expression inducer of the maturation-promoting gene of the present disclosure can induce expression of the maturation-promoting gene, for example, by administering it to a subject. As a result, the expression inducer of the maturation-promoting gene of the present disclosure can achieve effects such as anti-wrinkle action and strengthening of skin barrier function. The use conditions (administration conditions) of the expression inducer of the maturation-promoting gene of the present disclosure can be determined by reference to the explanation of the use conditions of the inducer of retinol-like action of the present disclosure.

[0067] <Agent or composition for use in inducing expression of the CERS1 gene> In another aspect, the present disclosure provides an agent or composition capable of inducing the CERS1 gene. The agent for use in inducing expression of the CERS1 gene of the present disclosure comprises bakuchiol. The composition for use in inducing expression of the CERS1 gene of the present disclosure comprises bakuchiol. The CERS1 gene expression inducer or composition of the present disclosure can promote differentiation of epidermal keratinocytes, thereby providing effects such as strengthening of skin barrier function and moisturizing effects. Furthermore, the CERS1 gene expression inducer or composition of the present disclosure is expected to suppress the side effects of, for example, ATRA, and is therefore expected to provide the wrinkle-improving effect observed with retinol while suppressing the side effects of retinol.

[0068] The CERS1 gene is a gene that is expressed in the early stage of differentiation of epidermal keratinocytes, and is known to contribute to the differentiation of epidermal keratinocytes, strengthening of barrier function, and the like, for example, by synthesizing ceramide.

[0069] An example of the CERS1 gene is mRNA encoded by the human CERS1 gene, such as a polynucleotide consisting of the base sequence registered in Genbank under accession number NM_021267.5. The expression level of the CERS1 gene may be measured by measuring the expression levels of one or more isoforms of the CERS1 gene, or by measuring the expression of all isoforms.

[0070] The "induction of CERS1 gene expression" means that the expression level of the CERS1 gene is increased or enhanced, and may mean that the CERS1 gene is changed from a state in which it is not expressed to a state in which it is expressed. The expression of the FLG gene can be evaluated by measuring the expression level of the mRNA of the CERS1 gene, for example, in accordance with Example 1 described below.

[0071] The CERS1 gene expression inducer of the present disclosure can induce CERS1 gene expression, for example, by administering it to a subject. This allows the CERS1 gene expression inducer of the present disclosure to induce differentiation of epidermal keratinocytes, thereby achieving effects such as anti-wrinkle effects and strengthening of skin barrier function. The use conditions (administration conditions) for the CERS1 gene expression inducer of the present disclosure can be determined from the same explanation as for the use conditions for the retinol-like activity inducer of the present disclosure.

[0072] <Agent or composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes> In another aspect, the present disclosure provides an agent or composition capable of promoting the proliferation and / or maturation of epidermal keratinocytes, particularly human epidermal keratinocytes. The agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure comprises bakuchiol. The composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure comprises bakuchiol. The maturation promoter or composition of the present disclosure can promote the proliferation and / or maturation of epidermal keratinocytes, thereby achieving effects such as wrinkle improvement and strengthening of skin barrier function. Furthermore, the maturation promoter or composition of the present disclosure is expected to suppress the side effects of, for example, ATRA, and is therefore expected to achieve the wrinkle improvement effect observed with retinol while suppressing the side effects of retinol.

[0073] The maturation accelerator of the present disclosure can promote the proliferation and / or maturation of epidermal keratinocytes, for example, by administering it to a subject. As a result, the maturation accelerator of the present disclosure can achieve effects such as anti-wrinkle action and strengthening of skin barrier function. The use conditions (administration conditions) of the maturation accelerator of the present disclosure can be determined from the explanation of the use conditions of the retinol-like activity inducer of the present disclosure.

[0074] <Agent or composition for use in inhibiting melanin production> In another aspect, the present disclosure provides an agent or composition capable of inducing melanin production. The agent for use in inhibiting melanin production (hereinafter also referred to as a "melanin inhibitor") of the present disclosure comprises bakuchiol. The composition for use in inhibiting melanin production of the present disclosure comprises bakuchiol. The melanin inhibitor or composition of the present disclosure can inhibit melanin production. Therefore, the melanin inhibitor or composition of the present disclosure can be suitably used, for example, as a skin whitening agent.

[0075] The melanin inhibitor of the present disclosure can suppress melanin production, particularly melanin production in the skin, by administering it to a subject. The conditions for use (administration conditions) of the melanin inhibitor of the present disclosure can be the same as those for use of the retinol-like activity inducer of the present disclosure.

[0076] <Method for Inducing Retinol-Mimetic Action> In another aspect, the present disclosure discloses a method capable of inducing retinol-mimetic action. The method for inducing retinol-mimetic action of the present disclosure uses the retinol-mimetic action inducer or composition of the present disclosure. The method for inducing retinol-mimetic action of the present disclosure is expected to achieve the effects observed with retinol, such as wrinkle improvement, while suppressing the occurrence of side effects caused by retinol, such as skin erythema and desquamation, which are caused by the inhibitory effect of retinol on epidermal keratinocyte differentiation.

[0077] The method for inducing retinol-like action of the present disclosure includes a step of using the retinol-like action inducer or composition of the present disclosure in a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0078] In the method for inducing retinol-like action of the present disclosure, the using step may be carried out, 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 can be determined by reference to the explanation of the administration subject and administration conditions of the agent or composition for inducing retinol-like action of the present disclosure.

[0079] <Method for inducing expression of a maturation-promoting gene in epidermal keratinocytes> In another aspect, 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 maturation-promoting gene in epidermal keratinocytes disclosed herein uses the expression inducer or composition of the maturation-promoting gene in epidermal keratinocytes disclosed herein. The method for inducing expression of a maturation-promoting gene in epidermal keratinocytes disclosed herein can promote the maturation of epidermal keratinocytes, thereby achieving effects such as strengthening skin barrier function and moisturizing effects. Furthermore, the method for inducing expression of a maturation-promoting gene disclosed herein is expected to suppress the side effects of, for example, ATRA, and is therefore expected to achieve the wrinkle-improving effect observed with retinol while suppressing the side effects of retinol.

[0080] The method for inducing the expression of a maturation-promoting gene in epidermal keratinocytes disclosed herein includes a step of using the agent or composition for inducing the expression of a maturation-promoting gene in epidermal keratinocytes disclosed herein in a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0081] In the method for inducing expression of a maturation-promoting gene in epidermal keratinocytes disclosed herein, the using step may be carried out, for example, in vitro or in vivo. The subject (administration subject) and administration conditions of the method for inducing expression of a maturation-promoting gene in epidermal keratinocytes disclosed herein can be determined by reference to the explanation of the administration subject and administration conditions for the inducer or composition of retinol-like activity disclosed herein.

[0082] <Method for inducing expression of CERS1 gene> In another aspect, the present disclosure discloses a method capable of inducing expression of the CERS1 gene. The method for inducing expression of the CERS1 gene of the present disclosure uses the CERS1 gene expression inducer or composition of the present disclosure. The method for inducing expression of the CERS1 gene of the present disclosure can promote differentiation of epidermal keratinocytes, thereby achieving effects such as strengthening skin barrier function and moisturizing effects. Furthermore, the method for inducing expression of the CERS1 gene of the present disclosure is expected to suppress, for example, the side effects of ATRA, and therefore is expected to achieve the wrinkle-improving effect observed with retinol while suppressing the side effects of retinol.

[0083] The method for inducing expression of the CERS1 gene according to the present disclosure includes a step of using the CERS1 gene expression inducer or composition according to the present disclosure in a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0084] In the method for inducing expression of the CERS1 gene disclosed herein, the using step may be carried out, for example, in vitro or in vivo. The subject (administration subject) and administration conditions of the method for inducing expression of the CERS1 gene disclosed herein can be determined by reference to the explanation of the administration subject and administration conditions for the inducer or composition of retinol-like action disclosed herein.

[0085] <Method for Promoting Proliferation and / or Maturation of Epidermal Keratinocytes> In another aspect, the present disclosure discloses a method capable of promoting the proliferation and / or maturation of epidermal keratinocytes, particularly human epidermal keratinocytes. The method for promoting proliferation and / or maturation of epidermal keratinocytes of the present disclosure uses the epidermal keratinocyte proliferation and / or maturation promoter or composition disclosed herein. The method for promoting proliferation and / or maturation of epidermal keratinocytes of the present disclosure can promote the proliferation and / or maturation of epidermal keratinocytes, thereby achieving effects such as wrinkle improvement and strengthening of skin barrier function. Furthermore, the method for promoting proliferation and / or maturation of epidermal keratinocytes of the present disclosure is expected to suppress the side effects of, for example, ATRA, and is therefore expected to achieve the wrinkle improvement effect observed with retinol while suppressing the side effects of retinol.

[0086] The method for promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure includes a step of using the agent or composition for promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure in a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0087] In the method for promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure, the using step may be carried out, for example, in vitro or in vivo. The subject (administration subject) and administration conditions of the method for promoting the proliferation and / or maturation of epidermal keratinocytes of the present disclosure can be determined by reference to the explanation of the administration subject and administration conditions for the inducer of retinol-like action or composition of the present disclosure, for example.

[0088] <Method for inhibiting melanin production> In another aspect, the present disclosure discloses a method capable of inhibiting 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 inhibiting melanin production of the present disclosure, melanin production can be inhibited. Therefore, the method for inhibiting melanin production of the present disclosure can be suitably used to induce, for example, a whitening effect.

[0089] The method for inhibiting melanin production according to the present disclosure includes a step of using the melanin production inhibitor or composition according to the present disclosure in a subject. The use may be, for example, by contact with the skin or the like or by administration.

[0090] In the method for inducing melanin production of the present disclosure, the using step may be carried out, 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 can be determined by reference to the explanation of the administration subject and administration conditions of the inducer or composition of retinol-like action of the present disclosure.

[0091] <Use of Bakuchiol> The present disclosure relates to the use of bakuchiol, the retinol-like activity inducer, or the retinol-like activity inducer composition for use in inducing retinol-like activity. The present disclosure relates to the use of bakuchiol, the expression inducer of the epidermal keratinocyte maturation-promoting gene, or the composition for inducing expression of the epidermal keratinocyte maturation-promoting gene for use in inducing expression of an epidermal keratinocyte maturation-promoting gene. The present disclosure relates to the use of bakuchiol, the CERS1 gene expression inducer, or the composition for inducing expression of the CERS1 gene for use in inducing expression of the CERS1 gene. The present disclosure relates to the use of bakuchiol, the epidermal keratinocyte proliferation and / or maturation promoter, or the composition for promoting proliferation and / or maturation of epidermal keratinocytes for use in promoting proliferation and / or maturation of epidermal keratinocytes. The present disclosure relates to the use of bakuchiol, the melanin production inhibitor, or the composition for inhibiting melanin production for use in inhibiting melanin production.

[0092] The present disclosure relates to the use of bakuchiol for producing an agent for inducing retinol-like activity or a composition for use in inducing retinol-like activity. The present disclosure relates to the use of bakuchiol for producing an agent for inducing the expression of a gene that promotes maturation in epidermal keratinocytes or a composition for use in inducing the expression of a gene that promotes maturation in epidermal keratinocytes. The present disclosure relates to the use of bakuchiol for producing an agent for inducing the expression of the CERS1 gene or a composition for use in inducing the expression of the CERS1 gene. The present disclosure relates to the use of bakuchiol for producing an agent for promoting the proliferation and / or maturation of epidermal keratinocytes or a composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes. The present disclosure relates to the use of bakuchiol for producing an agent for inhibiting melanin production or a composition for use in inhibiting melanin production.

[0093] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0094] Example 1: We confirmed that bakuchiol exhibits retinol-like effects in regulating RAR expression, and that bakuchiol has a reduced inhibitory effect on epidermal keratinocyte differentiation compared to retinol, and promotes the differentiation and maturation of epidermal keratinocytes.

[0095] (1) 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.

[0096] (2) Culturing of epidermal keratinocytes 2.1 × 10 cells were cultured in a 12-well plate. ４Neonatal-derived normal human epidermal keratinocytes (NBNHEK, purchased from Kurabo Industries, Ltd.) were seeded at 100 cells / well. KBM™ Gold Keratinocyte Growth Medium BulletKit™ (KGM all+, manufactured by Lonza) was used as the medium. After seeding, the NBNHEK cells were cultured until confluent. After the culture, the medium in each well was replaced with KBM™ Gold Basal Medium supplemented with only EGF (epidermal growth factor), BPE (bovine pituitary extract), and the included antibiotic (GA-1000) (KGM EGF+ / BPE+). After culturing the NBNHEK cells for 6 hours, the medium was replaced with sample-supplemented medium prepared with KGM EGF+ / BPE+ at the specified concentrations (ATRA: final concentration 1 μmol / L, bakuchiol: 5 or 10 μmol / L). For the control group (nt) to which no sample was added, the medium was replaced with one to which DMSO had been added so that the final concentration was equal to that of the sample-added group.

[0097] (3) Expression of RAR Gene and Differentiation Marker Genes After culturing for a predetermined period (48 or 72 hours) in the culture medium, total RNA was extracted using QIAshredder (QIAGEN) and RNeasy mini kit (QIAGEN). Based on the measured total RNA concentration, cDNA was synthesized using PrimeScript (Takara). Quantitative analysis of gene expression levels was performed by real-time PCR from the resulting cDNA using SYBR Premix Ex Taq (Takara). The target genes were RARα, RARβ, and RARγ. The target genes for epidermal keratinocyte differentiation and maturation markers were FLG, CERS1, and IVL. The RPS18 gene was used as a housekeeping gene (internal control gene). The expression level of each gene was expressed relative to that of RPS18. Each group was run with three samples (n = 3). Statistical analysis was performed using Dunnett's test. The results are shown in Figures 1 to 6.

[0098] Figure 1 is a graph showing the expression of the RARα gene. In Figure 1, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 1(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the RARα gene. The values ​​on the vertical axis in Figures 1(A) and (B) indicate the value for each group, with the value for the nt group set at 1.0 (1.00). The values ​​on the vertical axis in Figure 1(A) are as follows: ATRA-added group: 1.52, 5 μM bakuchiol-added group: 1.16, 10 μM bakuchiol-added group: 1.11. The values ​​on the vertical axis in Figure 1(B) are as follows. ATRA-added group: 1.61, bakuchiol 5 μM added group: 1.03, bakuchiol 10 μM added group: 1.06. As shown in Figures 1(A) and 1(B), RARα gene expression was induced in the ATRA-added group. On the other hand, as shown in Figures 1(A) and 1(B), RARα gene expression did not change significantly in the bakuchiol-added group.

[0099] Next, Figure 2 is a graph showing the expression of the RARβ gene. In Figure 2, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 2(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the RARβ gene. The values ​​on the vertical axis in Figures 2(A) and (B) indicate the value for each group, with the value for the nt group set to 1.0 (1.00). The values ​​on the vertical axis in Figure 2(A) are as follows: ATRA-added group: 3.98, 5 μM bakuchiol-added group: 1.76, 10 μM bakuchiol-added group: 2.20. The values ​​on the vertical axis in Figure 2(B) are as follows. ATRA-added group: 3.48, bakuchiol 5 μM added group: 1.31, bakuchiol 10 μM added group: 1.69. As shown in Figures 2(A) and (B), RARβ gene expression was induced in the ATRA-added group. Also, as shown in Figures 2(A) and (B), RARβ gene expression was significantly induced in the bakuchiol-added group.

[0100] Next, Figure 3 is a graph showing the expression of the RARγ gene. In Figure 3, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 3(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the RARγ gene. The values ​​on the vertical axis in Figures 3(A) and (B) indicate the values ​​for each group, with the value for the nt group being 1.0 (1.00). The values ​​on the vertical axis in Figure 3(A) are as follows: ATRA-added group: 0.89, 5 μM bakuchiol-added group: 0.83, 10 μM bakuchiol-added group: 0.54. The values ​​on the vertical axis in Figure 3(B) are as follows. ATRA-added group: 0.70, 5 μM bakuchiol-added group: 0.95, 10 μM bakuchiol-added group: 0.71. As shown in Figures 3(A) and 3(B), RARγ gene expression was suppressed in the ATRA-added group. Also, as shown in Figures 3(A) and 3(B), RARγ gene expression was significantly suppressed in the bakuchiol-added group.

[0101] Next, Figure 4 is a graph showing the expression of the FLG gene. In Figure 4, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 4(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the FLG gene. The values ​​on the vertical axis in Figures 4(A) and (B) indicate the values ​​for each group, with the value for the nt group being 1.0 (1.00). The values ​​on the vertical axis in Figure 4(A) are as follows: ATRA-added group: 0.42, 5 μM bakuchiol-added group: 2.69, 10 μM bakuchiol-added group: 2.07. The values ​​on the vertical axis in Figure 4(B) are as follows. ATRA-added group: 0.03, 5 μM bakuchiol-added group: 1.59, 10 μM bakuchiol-added group: 1.19. As shown in Figures 4(A) and (B), FLG gene expression was significantly suppressed in the ATRA-added group. On the other hand, as shown in Figures 4(A) and (B), FLG gene expression was significantly increased in the bakuchiol-added group.

[0102] Next, Figure 5 is a graph showing the expression of the IVL gene. In Figure 5, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 5(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the IVL gene. The values ​​on the vertical axis in Figures 5(A) and (B) indicate the values ​​for each group, with the value for the nt group set to 1.0 (1.00). The values ​​on the vertical axis in Figure 5(A) are as follows: ATRA-added group: 1.69, 5 μM bakuchiol-added group: 2.72, 10 μM bakuchiol-added group: 3.22. The values ​​on the vertical axis in Figure 5(B) are as follows. ATRA-added group: 0.72, 5 μM bakuchiol-added group: 2.72, 10 μM bakuchiol-added group: 3.29. As shown in Figures 5(A) and (B), IVL gene expression increased (after 48 hours) or maintained (after 72 hours) in the ATRA-added group. On the other hand, IVL gene expression significantly increased in the bakuchiol-added group.

[0103] Next, Figure 6 is a graph showing the expression of the CERS1 gene. In Figure 6, (A) shows the results after 48 hours of culture, and (B) shows the results after 72 hours of culture. In Figures 6(A) and (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the CERS1 gene. The values ​​on the vertical axis in Figures 6(A) and (B) indicate the values ​​for each group, with the value for the nt group set to 1.0 (1.00). The values ​​on the vertical axis in Figure 6(A) are as follows: ATRA-added group: 4.79, 5 μM bakuchiol-added group: 3.07, 10 μM bakuchiol-added group: 9.74. The values ​​on the vertical axis in Figure 6(B) are as follows. ATRA-added group: 2.36, bakuchiol 5 μM added group: 1.71, bakuchiol 10 μM added group: 3.21 As shown in Figures 6(A) and (B), CERS1 gene expression increased in the ATRA-added group. On the other hand, CERS1 gene expression significantly increased in the bakuchiol-added group.

[0104] (4) Cytotoxicity: 1.8 × 10 cells were placed in a 96-well plate. ３NBNHEK cells were seeded at 100 cells / well and cultured in the same manner as in Example 1(2). After changing to the medium containing the sample, the cells were further cultured for 24 or 48 hours. After the culture, cells were collected from each well, and the viable cell count was evaluated using Cell Counting Kit-8 (DOJINDO). Each group consisted of six samples (n = 6). Statistical analysis was performed using Dunnett's test. As a result, no difference in the percentage of viable cells was observed between the control and any of the samples used. In other words, the addition of bakuchiol did not result in cytotoxicity.

[0105] Furthermore, in the bakuchiol group, among the RARα gene, RARβ gene, and RARγ gene, the expression regulation behavior of the RARβ gene and RARγ gene was similar to that of the ATRA group, confirming that bakuchiol exerts a retinol-like effect.It has been reported that the selective retinoid Tazarotene acts on the RARβ gene and RARγ gene, and suppresses the above-mentioned side reactions compared to retinoic acid.Therefore, it has been suggested that the side reactions of retinoic acid depend on the induction of RARα gene expression.Therefore, bakuchiol exerts a retinol-like effect by regulating the expression of the RARβ gene and RARγ gene, while not enhancing the expression of the RARα gene, and is therefore expected to suppress the side reactions observed with retinoic acid.

[0106] In addition, in the ATRA group, the expression of FLG gene, which is a maturation marker of epidermal keratinocytes, was significantly suppressed, and the suppression of differentiation of epidermal keratinocytes, which is the cause of the above-mentioned side effects, was confirmed.On the other hand, in the bakuchiol group, the expression of marker genes related to the differentiation and maturation of epidermal keratinocytes, such as FLG gene, IVL gene, and CERS1 gene, was induced.From these results, it is believed that bakuchiol promotes the differentiation and maturation of epidermal keratinocytes, suppresses the side effects of retinoic acid, and shows the effect of improving wrinkles and strengthening skin barrier function.

[0107] Example 2 It was confirmed that bakuchiol has an inhibitory effect on melanin production.

[0108] (1) Sample Preparation Bakuchiol (BIB Corporation) was dissolved in 100% ethanol to a predetermined final concentration (5 μmol / L or 10 μmol / L) at the time of addition to the medium.

[0109] (2) Melanoma cell culture: 1.75 × 10 cells were cultured in a 60 mm dish. ５ B16 cells (B16 mouse melanoma cells, provided by RIKEN BRC, Lot. RCB1283, passage number 20) were seeded at 100 cells / dish and incubated in 5% CO ２ The cells were cultured overnight at 37°C in a 5% CO atmosphere. The culture medium used was E-MEM medium (5% FBS / E-MEM medium, Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% fetal bovine serum (FBS, Sigma-Aldrich). After removing the medium from the dish by suction, bakuchiol adjusted to the aforementioned final concentration was added, and the B16 cells were incubated in a 5% CO atmosphere. ２ The cells were cultured at 37°C for 72 hours. The following groups were included: a non-treated (NT) group cultured in 5% FBS / E-MEM medium alone; a positive control (PC) group cultured in the presence of arbutin (LKT Laboratories, Inc.) (final concentration 200 μg / ml); and a negative control (NC) group cultured in the presence of 100% ethanol. To account for the influence of 100% ethanol used for sample dilution, the ethanol concentration was set to 0.05% in all treatment groups except the NT group. After removing the medium from the dish by aspiration, B16 cells were harvested by trypsinization, centrifuged, and the supernatant was removed.

[0110] The resulting precipitate was added with a 3:1 (volume ratio) mixture of ethanol and diethyl ether, and centrifuged. The supernatant was removed to obtain a melanin precipitate. The precipitate was dried overnight, and the resulting melanin was dissolved in 400 μL of a 1N NaOH / 10% DMSO mixture. Absorbance at 420 nm was measured to determine the melanin production level. Each group consisted of three samples (n = 3). Statistical analysis was performed using Dunnett's test (*: p<0.05, ***: p<0.001 vs. NC). These results are shown in Figure 7. Figure 7 is a graph showing the melanin production level. In Figure 7, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative melanin production level. The values ​​on the vertical axis in Figure 7 represent the values ​​for each group, with the value for the NC group set at 100 (%). The values ​​on the vertical axis in Figure 7 are as follows: NT group: 32.63, PC group: 64.27, 5 μM bakuchiol added group: 75.90, 10 μM bakuchiol added group: 17.78 As shown in Figure 7, the amount of melanin production decreased in a bakuchiol concentration-dependent manner.

[0111] (3) Cytotoxicity: 7 × 10 cells were placed in a 96-well plate. ３ B16 cells were seeded at 100 cells / well and incubated in 5% CO ２ The medium used in the culture was the same as in Example 2(2). After removing the medium from each well, a sample was added in the same manner as in the melanin production inhibition test, and the cells were incubated overnight under the conditions of 5% CO ２ The cells were cultured at 37°C for 72 hours. After the culture, an MTT assay was performed on each well using Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich). Cell viability was assessed by measuring absorbance at 570 nm and 620 nm as the reference. The melanin production in each group was expressed relative to the NC value of 100. Six samples were used for each group (n = 6). Statistical analysis was performed using Dunnett's test (***: p < 0.001 vs. NC). No significant difference was observed in the percentage of viable cells compared to the control for any sample used. In other words, the addition of bakuchiol did not result in cytotoxicity.

[0112] These results suggest that bakuchiol can suppress the amount of melanin produced in melanin-producing cells and thus suppress melanin production in the skin.

[0113] The sequences and other information of the primers used in Examples 1 and 2 are shown in Table 1 below.

[0114]

[0115] Example 3 A cell test (a test using epidermal keratinocytes) was conducted to confirm whether bakuchiol has the effect of promoting hyaluronic acid synthesis in the epidermal layer and strengthening the basement membrane.

[0116] (1) 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.

[0117] (2) Culturing of epidermal keratinocytes 2.1 × 10 cells were cultured in a 12-well plate. ４ Neonatal normal human epidermal keratinocytes (NBNHEK, purchased from Kurabo Industries, Ltd.) were seeded at 100 cells / well. KBM™ Gold Keratinocyte Growth Medium BulletKit™ (KGM all+, manufactured by Lonza) was used as the medium. After seeding, the NBNHEK cells were cultured until confluent. After the culture, the medium in each well was replaced with KBM™ Gold Basal Medium supplemented with only EGF (epidermal growth factor), BPE (bovine pituitary extract), and the included antibiotic (GA-1000) (KGM EGF+ / BPE+). After culturing the NBNHEK for 6 hours, the medium was replaced with sample-supplemented medium prepared with KGM EGF+ / BPE+ at the specified concentrations (ATRA: final concentration 1 μmol / L, bakuchiol: final concentrations 2.5, 5, or 10 μmol / L). In the control group (nt) to which no sample was added, the medium was replaced with one to which DMSO had been added so that the final concentration was equal to that of the sample-added group.

[0118] (3) Expression of Marker Genes After culturing for a predetermined period of time (48 hours for the HAS3 gene expression level measurement sample and the ITGβ gene expression level measurement sample, and 72 hours for the ITGα6 gene expression level measurement sample), total RNA was extracted using QIAshredder (QIAGEN) and RNeasy mini kit (QIAGEN). Based on the measured total RNA concentration, cDNA was synthesized using PrimeScript (Takara). Furthermore, quantitative analysis of gene expression levels was performed by real-time PCR from the obtained cDNA using SYBR Premix Ex Taq (Takara). The target gene for epidermal layer hyaluronic acid synthesis was the HAS3 gene. The target genes for epidermal layer basement membrane reinforcement were the ITGα6 gene and the ITGβ1 gene. The RPS18 gene was used as a housekeeping gene (internal standard gene). The expression level of each gene was expressed relative to the expression level of the RPS18 gene. Three samples were used for each group (n=3). Statistical analysis was performed using Dunnett's test. The measurement results of the expression levels of the genes are shown in Tables 2 to 4 below. The results shown in Tables 2 to 4 below were calculated by average value of three samples for each group, and the value (average value) of the control group (n.t.) is shown as 1.00. The results shown in Tables 2 to 4 below indicate ***: p<0.001, **: p<0.01, *: p<0.05 compared to the value of the control group.

[0119]

[0120]

[0121]

[0122] As shown in Tables 2 to 4 above, the expression of epidermal layer hyaluronic acid synthesis genes and epidermal layer basement membrane strengthening genes was found to be increased in the group with the addition of a predetermined amount of ATRA and the group with the addition of a predetermined amount of bakuchiol compared to the control group. From these results, it is thought that the addition of bakuchiol exerts an effect similar to that of ATRA, which is retinol, namely, the effect of promoting hyaluronic acid synthesis in the epidermal layer and strengthening the basement membrane.

[0123] The sequences and other information of the primers used in Example 3 above are shown in Table 5 below.

[0124]

[0125] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0126] This application claims priority based on Japanese Patent Application No. 2022-136384 filed on August 29, 2022, and Japanese Patent Application No. 2023-049423 filed on March 27, 2023, the disclosures of which are incorporated herein in their entireties.

[0127] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendices. <Agents for use in inducing retinol-like activity> (Appendix 1) An agent for use in inducing retinol-like activity, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 2) The agent according to Appendix 1, wherein the induction of retinol-like activity is strengthening of the basal layer of the skin, stabilization of the basal layer of the skin, and / or promotion of epidermal keratinocyte proliferation. (Appendix 3) The agent according to Appendix 1 or 2, wherein the retinol-like activity is enhancement of retinoic acid receptor beta (RAR beta) gene expression and / or suppression of retinoic acid receptor gamma (RAR gamma) gene expression. (Appendix 4) The agent according to any of Appendices 1 to 3, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 5) An agent for application to skin, comprising the agent according to any one of Appendices 1 to 4. (Appendix 6) An agent for oral administration, comprising the agent according to any one of Appendices 1 to 4. <Compositions for use in inducing retinol-like action> (Appendix 7) A composition for use in inducing retinol-like action, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 8) The composition according to Appendices 7, 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. (Appendix 9) The composition according to Appendices 7 or 8, wherein the retinol-like action is enhancement of retinoic acid receptor beta (RAR beta) gene expression and / or suppression of retinoic acid receptor gamma (RAR gamma) gene expression. (Appendix 10) The composition of any one of Appendices 7 to 9, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 11) A composition for application to skin, comprising the composition of any one of Appendices 7 to 10. (Appendix 12) A composition for oral administration, comprising the composition of any one of Appendices 7 to 10. (Appendix 13) The composition of any one of Appendices 7 to 12, comprising an agent for inducing retinol-like action as described in any one of Appendices 1 to 6.<Agent for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes> (Appendix 14) An agent for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 15) The agent according to Appendix 14, wherein the maturation-promoting gene comprises a filaggrin gene. (Appendix 16) The agent according to Appendix 15, wherein the maturation-promoting gene further comprises an involucrin gene. (Appendix 17) The agent according to any one of Appendices 14 to 16, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 18) An agent for application to skin, comprising the agent according to any one of Appendices 14 to 17. (Appendix 19) An agent for oral administration, comprising the agent according to any one of Appendices 14 to 17. <Composition for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes> (Appendix 20) A composition for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 21) The composition according to Appendix 20, wherein the maturation-promoting gene comprises a filaggrin gene. (Appendix 22) The composition according to Appendix 21, wherein the maturation-promoting gene further comprises an involucrin gene. (Appendix 23) The composition according to any one of Appendixes 20 to 22, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 24) A composition for application to skin, comprising the composition according to any one of Appendixes 20 to 23. (Appendix 25) A composition for oral administration, comprising the composition according to any one of Appendixes 20 to 23. (Appendix 26) The composition according to any one of Appendices 20 to 25, comprising the agent according to any one of Appendices 14 to 19. <Agent for use in inducing expression of CERS1 gene> (Appendix 27) An agent for use in inducing expression of ceramide synthase 1 (CERS1) gene, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 28) The agent according to Appendices 27, wherein the bakuchiol is an extract containing bakuchiol.(Appendix 29) An agent for application to the skin, comprising the agent according to Appendix 27 or 28. (Appendix 30) An agent for oral administration, comprising the agent according to Appendix 27 or 28. <Composition for use in inducing expression of the CERS1 gene> (Appendix 31) A composition for use in inducing expression of the ceramide synthase 1 (CERS1) gene, comprising 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. (Appendix 35) A composition according to any one of Appendices 31 to 34, comprising an agent according to any one of Appendices 27 to 30. <Agent for use in promoting the proliferation and maturation of epidermal keratinocytes> (Appendix 36) An agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 37) The agent according to Appendix 36, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 38) An agent for application to skin, comprising the agent according to Appendix 36 or 37. (Appendix 39) An agent for oral administration, comprising the agent according to Appendix 36 or 37. <Composition for use in promoting the proliferation and maturation of epidermal keratinocytes> (Appendix 40) A composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes, comprising bakuchiol. (Appendix 41) The composition of Appendices 40, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 42) A composition for application to skin, comprising the composition of Appendices 40 or 41. (Appendix 43) A composition for oral administration, comprising the composition of Appendices 40 or 41. (Appendix 44) The composition of any of Appendices 40 to 43, comprising an agent of any of Appendices 36 to 39.<Agent for use in inhibiting melanin production> (Appendix 45) An agent for use in inhibiting melanin production, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 46) The agent according to Appendix 45, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 47) An agent for application to skin, comprising the agent according to Appendix 45 or 46. (Appendix 48) An agent for oral administration, comprising the agent according to Appendix 45 or 46. <Composition for use in inhibiting melanin production> (Appendix 49) A composition for use in inhibiting melanin production, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol). (Appendix 50) The composition of Appendix 49, wherein the bakuchiol is an extract containing bakuchiol. (Appendix 51) A composition for application to skin, comprising the composition of Appendix 49 or 50. (Appendix 52) A composition for oral administration, comprising the composition of Appendix 49 or 50. (Appendix 53) The composition of any of Appendixes 49 to 52, comprising an agent for use in suppressing melanin production, according to any of Appendixes 45 to 48. <Method for inducing retinol-like action> (Appendix 54) A method for inducing retinol-like action, using the agent for use in inducing retinol-like action, according to any of Appendixes 1 to 6, and / or the composition for use in inducing retinol-like action, according to any of Appendixes 7 to 13. (Appendix 55) The method for induction according to Appendix 54, comprising a step of using the agent for use in inducing retinol-like action and / or the composition for use in inducing retinol-like action in a subject. (Appendix 56) The induction method according to Appendices 54 or 55, which is used in vitro or in vivo. <Method for inducing expression of a maturation-promoting gene in epidermal keratinocytes> (Appendix 57) A method for inducing expression of a maturation-promoting gene in epidermal keratinocytes, using an agent for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes according to any of Appendices 14 to 19, and / or a composition for use in inducing expression of a maturation-promoting gene in epidermal keratinocytes according to any of Appendices 20 to 26.(Appendix 58) The method for induction according to Appendix 57, comprising a use step of using, in a subject, an agent for use in inducing expression of the maturation-promoting gene in epidermal keratinocytes and / or a composition for use in inducing expression of the maturation-promoting gene in epidermal keratinocytes. (Appendix 59) The method for induction according to Appendix 57 or 58, which is used in vitro or in vivo. <Method for inducing expression of CERS1 gene> (Appendix 60) A method for inducing expression of the CERS1 gene, comprising using, in a subject, an agent for use in inducing expression of the CERS1 gene according to any of Appendices 27 to 30 and / or a composition for use in inducing expression of the CERS1 gene according to any of Appendices 31 to 35. (Appendix 61) The method for induction according to Appendix 60, comprising a use step of using, in a subject, the agent for use in inducing expression of the CERS1 gene and / or the composition for use in inducing expression of the CERS1 gene. (Appendix 62) The induction method according to Appendix 60 or 61, which is used in vitro or in vivo. <Method for promoting the proliferation and / or maturation of epidermal keratinocytes> (Appendix 63) A method for promoting the proliferation and / or maturation of epidermal keratinocytes, using an agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes according to any of Appendices 36 to 39, and / or a composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes according to any of Appendices 40 to 44. (Appendix 64) The induction method according to Appendix 63, which comprises a use step of using the agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes and / or the composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes in a subject. (Appendix 65) The induction method according to Appendix 63 or 64, used in vitro or in vivo <Method of suppressing melanin production> (Appendix 66) A method of suppressing melanin production, using an agent for use in suppressing melanin production according to any of Appendices 45 to 48, and / or a composition for use in suppressing melanin production according to any of Appendices 49 to 53. (Appendix 67) The induction method according to Appendix 66, comprising a use step of using the agent for use in suppressing melanin production and / or the composition for use in suppressing melanin production in a subject.(Appendix 68) The method for induction according to Appendices 66 or 67, used in vitro or in vivo. <Use> (Appendix 69) Use of an agent for use in inducing a retinol-like effect according to any of Appendices 1 to 6, or a composition for use in inducing a retinol-like effect according to any of Appendices 7 to 13, for use in inducing a retinol-like effect. (Appendix 70) Use of an agent for use in inducing the expression of a maturation-promoting gene in epidermal keratinocytes according to any of Appendices 14 to 19, or a composition for use in inducing the expression of a maturation-promoting gene in epidermal keratinocytes according to any of Appendices 20 to 26, for use in inducing the expression of a maturation-promoting gene in epidermal keratinocytes. (Appendix 71) Use of an agent for use in inducing the expression of the CERS1 gene according to any of Appendices 27 to 30, or a composition for use in inducing the expression of the CERS1 gene according to any of Appendices 31 to 35, for use in inducing the expression of the ceramide synthase 1 (CERS1) gene. (Appendix 72) Use of an agent for use in promoting the proliferation and / or maturation of epidermal keratinocytes according to any of Appendices 36 to 39 or a composition for use in promoting the proliferation and / or maturation of epidermal keratinocytes according to any of Appendices 40 to 44. (Appendix 73) Use of an agent for use in suppressing melanin production according to any of Appendices 45 to 48 or a composition for use in suppressing melanin production according to any of Appendices 53, for use in suppressing melanin production.

[0128] As described above, the present disclosure provides an agent that reduces the inhibition of differentiation of epidermal keratinocytes and induces retinol-like activity, and that is applicable primarily to human skin, etc. Therefore, the present invention can be said to be extremely useful, for example, in the fields of pharmaceuticals, quasi-drugs, topical skin preparations, etc.

Claims

1. An agent for use in inducing the expression of the filaggrin gene, the involucrin gene, and / or the ceramide synthase 1 (CERS1) gene in epidermal keratinocytes, comprising bakuchiol (4-[(1E,3S)-3-ethenyl-3,7-dimethylocta-1,6-dienyl]phenol).

2. An agent as described in claim 1 for use in suppressing melanin production.