Agent for promoting keratinization of epidermal keratinocyte and applications thereof

Abstract

To provide a new agent capable of inducing the effect of promoting the expression of genes related to keratinization in epidermal keratinocytes, applicable mainly for human skin.SOLUTION: An agent for promoting the keratinization of epidermal keratinocytes, disclosed herein, includes shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to an agent for promoting keratinization of epidermal keratinocytes and uses thereof. [Background technology]

[0002] Lithospermum Root (Shikon), the root of the murasaki plant, has long been used as a raw material for herbal medicines such as shiunko and medicines, and as a natural dye. Lithospermum Root and its extracts contain shikonin and shikonin derivatives (hereinafter also referred to as "shikonins"), and it has been reported that the shikonins have anti-inflammatory and antibacterial effects. For this reason, various cosmetics and external preparations that have the above effects, focusing on the shikonins, have been studied (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2004-075613 A Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, an object of the present disclosure is to provide a new agent that can induce the effect of promoting the expression of keratinization-related genes in epidermal keratinocytes and is primarily applicable to human skin. [Means for solving the problem]

[0005] In order to achieve the above-mentioned objective, the agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure (hereinafter also referred to as "keratinization promoter") contains shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.

[0006] The agent for use in inducing expression of the Transglutaminase 1 (TGM1) gene of the present disclosure (hereinafter also referred to as "TGM1 gene expression promoter") includes shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.

[0007] The agent for use in inducing expression of the Involucrin (IVL) gene of the present disclosure (hereinafter also referred to as "IVL gene expression promoter") includes shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.

[0008] The agent for use in inducing expression of the Filaggrin (FLG) gene of the present disclosure (hereinafter also referred to as "FLG gene expression promoter") includes shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.

[0009] The agent for use in inducing expression of the ceramide synthase 1 (CERS1) gene disclosed herein (hereinafter also referred to as the "CERS1 gene expression promoter") includes shikonin, β-hydroxyisovaleryl shikonin, acetylshikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. Effect of the Invention

[0010] According to the present disclosure, it is possible to provide a new agent that can induce the effect of promoting the expression of keratinization-related genes in epidermal keratinocytes and is primarily applicable to human skin. [Brief description of the drawings]

[0011]

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

[0013] <Agent for use in promoting keratinization of epidermal keratinocytes> In one embodiment, the present disclosure provides an agent or composition for use in promoting keratinization of epidermal keratinocytes. The agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure includes shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. In addition, the composition for use in promoting keratinization of epidermal keratinocytes of the present disclosure contains shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin. In the following description, unless otherwise specified, the description of each agent of the present disclosure can be used to refer to the description of the corresponding composition.

[0014] As a result of intensive research, the present inventors have found that shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin, which are types of shikonin contained in extracts of Lithospermum radix, exhibit an effect of promoting the expression of genes related to keratinization (keratinization-related genes) in epidermal keratinocytes, and have established the present disclosure. Therefore, according to the agent or composition for use in promoting keratinization of epidermal keratinocytes of the present disclosure, for example, it is possible to promote the expression of genes that have been shown to be related to keratinization, and therefore it is expected that keratinization in the skin can be promoted.

[0015] In this disclosure, "shikonins" refers to shikonin (formula (1) below) which has a naphthoquinone skeleton, and shikonin derivatives which are compounds in which the hydroxyl group of the side chain of shikonin is replaced with an organic group. The shikonins are known to be extracted, for example, from the roots of the Boraginaceae plant Lithospermum erythrorhizon Siebold & Zuccarini. The shikonin derivatives can be represented, for example, by the following formula (2). In the following formula (2), R forms an ester together with an oxygen atom. Specific examples of the shikonin derivatives include β-hydroxyisovaleryl shikonin (R=COCH2C(CH3)2OH), acetyl shikonin (R=COCH3), isobutyryl shikonin (R=COCH(CH3)2), β,β-dimethyl acryl shikonin (R=COCH=C(CH3)2), isovaleryl shikonin (R=COCH2CH(CH3)2), and α-methyl-n-butyryl shikonin (R=COCH(CH3)CH2CH3) (formulas (3) to (8)). The shikonins are preferably shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin, as they can more strongly promote the expression of the keratinization-related genes, and more preferably acetylshikonin and β,β-dimethylacrylshikonin. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0016] The shikonins may be isolated or purified compounds, artificially synthesized compounds, or compositions containing shikonins. The shikonins may be self-prepared or commercially available. The composition containing shikonins may be, for example, an extract containing shikonins; a crude product of the extract, a dried product of the extract, a freeze-dried product of the extract, a processed product of the extract such as spray drying, etc.

[0017] The extract containing shikonins can be produced, for example, by performing solvent extraction on a plant containing shikonins. Examples of plants containing shikonins include plants of the Boraginaceae family (e.g., Lithospermum erythrorhizon, Lithospermum officinale L.). One type of plant may be used, or two or more types may be used. The plant material to be subjected to the extraction may be an individual plant or a part of the plant. Examples of the part of the plant include roots, rhizomes, or a mixture thereof. The material may be the plant itself as it is harvested, or may be a processed product that has been dried and / or crushed.

[0018] Examples of solvents used for extracting the shikonins include aqueous solvents such as water and buffer solutions; lower alcohols or water-containing lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol; polyhydric alcohols or water-containing polyhydric alcohols such as 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, glycerin, and polyethylene glycol (e.g., 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; and acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) or alkalis (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) with appropriate normality adjustment. The solvent is preferably ethanol, 1,3-butylene glycol, or a mixed solvent thereof. The solvent may be used alone or in combination of two or more kinds.

[0019] Examples of the treatment of the treated product include 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; component adsorption by ion exchange resins, activated carbon, diatomaceous earth, etc.; fractionation using chromatography having 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; long-term static storage; etc. The above treatments may be performed alone or in combination of two or more.

[0020] The "epidermal keratinization" refers to the process in the epidermis in which basal cells change into spinous cells, granular cells, and keratinocytes. The epidermal keratinization refers to a series of processes in which keratinocytes (epidermal keratinocytes or keratinocytes) undergo cell division in the basal layer, one of which remains in the basal layer, and the other passes through the spinous layer and granular layer to the stratum corneum, and finally becomes dirt and falls off. The "promotion of keratinization of epidermal keratinocytes" refers to the promotion of the process. The promotion of keratinization of epidermal keratinocytes can be evaluated, for example, directly or indirectly. In the case of the direct evaluation, the promotion of keratinization of epidermal keratinocytes can be carried out, for example, by evaluating the keratinization of the epidermal keratinocytes. In the case of the indirect evaluation, the promotion of keratinization of epidermal keratinocytes can be evaluated, for example, by measuring the degree of expression of keratinization-related genes, specifically, by the promotion of expression of the keratinization-related genes.

[0021] The term "epidermal keratinocytes" refers to cells generated by the division of basal layer cells of the epidermis. The epidermal keratinocytes have the ability to differentiate into basal cells, spinous cells, granular cells, and keratinocytes that constitute the epidermis. It is known that the epidermal keratinocytes further differentiate in the epidermis and finally differentiate into keratinocytes that form the stratum corneum, and this process is called keratinization.

[0022] The "keratinization-related gene" refers to a gene that contributes to the differentiation of epidermal keratinocytes, etc. Examples of the keratinization-related gene include the TGM1 gene, the IVL gene, the FLG gene, and the CERS1 gene.

[0023] The "TGM1 (Transglutaminase 1)" is a type of protein cross-linking enzyme. The expression of the TGM1 gene is induced during the final differentiation of keratinized squamous epithelium, and it is known that the gene contributes to, for example, the promotion of the formation of the cornified envelope of the epidermis (cornified envelope). The cornified envelope is considered to fulfill the barrier function by forming a strong lining structure in the stratum corneum. It is also known that a large number of immature cells of the cornified envelope are observed in the skin of atopic dermatitis, etc., in which the barrier function is reduced. Therefore, it can be said that the TGM1 gene, which has the function of promoting the formation of the cornified envelope, contributes to, for example, the strengthening of the barrier function.

[0024] An example of the TGM1 gene is an mRNA encoded by the human TGM1 gene, such as a polynucleotide consisting of the base sequence registered in Genbank under the accession number NM_000359.3. As the expression level of the TGM1 gene, the expression levels of any one or more isoforms of the TGM1 gene may be measured, or the expression of all isoforms may be measured.

[0025] The "IVL (Involucrin)" is a type of protein that constitutes the cornified envelope. The expression of the IVL gene is induced in the early stages of differentiation and maturation of epidermal keratinocytes, and it is known that the IVL gene contributes to, for example, the differentiation of epidermal keratinocytes. In addition, since the IVL is a type of protein that constitutes the cornified envelope, the IVL gene can be said to contribute to, for example, the strengthening of the barrier function.

[0026] An example of the IVL gene is an mRNA encoded by a human IVL gene, such as a polynucleotide consisting of a base sequence registered in Genbank under the accession number NM_005547.4. As the expression level of the TGM1 gene, the expression levels of any one or more isoforms of the IVL gene may be measured, or the expression of all isoforms may be measured.

[0027] The "FLG (filagrin)" is an interfibrillar condensing substance that binds to and condenses keratin fibers, and is a protein that is subsequently decomposed to become a natural moisturizing factor. The FLG gene is a gene that is expressed during the differentiation and maturation of epidermal keratinocytes, and is known to contribute to, for example, the induction of keratinization of epidermal keratinocytes, the maturation of epidermal keratinocytes, and the strengthening of barrier function.

[0028] An example of the FLG gene is an mRNA encoded by a human FLG gene, such as a polynucleotide consisting of the base sequence registered in Genbank under the accession number NM_002016.2. Regarding the expression level of the FLG gene, the expression levels of any one or more isoforms of the FLG gene may be measured, or the expression of all isoforms may be measured.

[0029] The "CERS1 (ceramide synthase 1)" is an enzyme that catalyzes the synthesis of ceramide. 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, by synthesizing ceramide, for example.

[0030] An example of the CERS1 gene is an mRNA encoded by the human CERS1 gene, such as a polynucleotide consisting of a base sequence registered in Genbank under the accession number NM_021267.5. As the expression level of the CERS1 gene, the expression levels of any one or more isoforms of the CERS1 gene may be measured, or the expression of all isoforms may be measured.

[0031] The "promotion of gene expression" means that the expression level of a target gene is promoted or increased, and may mean that the expression level of the target gene changes from a state in which the target gene is not expressed to a state in which the target gene is expressed. The expression of the target gene can be evaluated, for example, by measuring the expression level of the mRNA of the target gene using quantitative PCR in accordance with Example 1 described below. When the target gene has multiple isoforms, the expression level of the target gene may be measured by measuring the expression level of any one or more isoforms of the target gene, or may be measured for the expression of all isoforms, but the latter is preferred.

[0032] 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).

[0033] The agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure can promote keratinization of epidermal keratinocytes, for example, by administering it to a subject. The conditions for use (administration conditions) of the agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure are not particularly limited, and the administration form, administration time, administration amount, etc. can be appropriately set depending on, for example, the type of subject to be administered.

[0034] The agent for use in promoting keratinization of epidermal keratinocytes according to the present disclosure includes, for example, in vivo You can also use in vitromay be used.

[0035] There are no particular limitations on the subjects to which the agent for promoting keratinization of epidermal keratinocytes of the present disclosure is administered. in vivo In the case of use in the above, the subject of administration 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, and birds. in vitro When used in the above, the subject to be administered can be, for example, a cell, tissue, organ, etc., the cell can be, for example, a cell collected from a living body, a cultured cell, etc., and the tissue or organ can be, for example, a tissue (living tissue) or an organ, etc. collected from a living body.

[0036] In the following topical skin preparations (e.g., transdermal or skin application preparations or compositions) or the following orally administered preparations or compositions containing the agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure, the amount of the shikonins may be within the range that exhibits the effect of promoting keratinization of epidermal keratinocytes, i.e., an effective amount. The amount of the shikonins (upper and lower limits) is, for example, as follows. The amount below may be the amount of one type of shikonin alone, or the total amount of two or more types of shikonins. In the following examples, the upper and lower limits can be combined in any combination. Lower limit: 2.0×10 -2 μg / ml, preferably 2.0×10 -1 μg / ml Upper limit: 1.0×10 2 μg / ml, preferably 2.0×10 1 μg / ml As a specific example of the blend amounts, when Shikonix Liquid (BG) (manufactured by Ichimaru Pharcos Co., Ltd.) is used at a concentration of 1% (v / v), the blend amount of the shikonins is approximately 20.32 μg / ml, and the blend amounts of each shikonin are, for example, as follows: Shikonin: 0.39μg / ml β-Hydroxyisovalerylshikonin: 3.75μg / ml Acetylshikonin: 6.15μg / ml Isobutyrylshikonin: 3.53μg / ml β,β-dimethylacryl shikonin: 0.66μg / ml Isovaleryl shikonin: 2.92μg / ml α-Methyl-n-butyrylshikonin: 2.92μg / ml

[0037] When the shikonin is shikonin, the amount of shikonin is, for example, 3ng / ml to 15ng / ml, preferably 5ng / ml to 13ng / ml, more preferably 5.9ng / ml to 11.8ng / ml. When the shikonin is β-hydroxyisovaleryl shikonin, the amount of β-hydroxyisovaleryl shikonin is, for example, 40ng / ml to 130ng / ml, preferably 50ng / ml to 120ng / ml, more preferably 56.3ng / ml to 112.6ng / ml. When the shikonin is acetyl shikonin, the amount of acetyl shikonin is, for example, 80ng / ml to 200ng / ml, preferably 90ng / ml to 190ng / ml, more preferably 92.2ng / ml to 184.4ng / ml. When the shikonin is isobutyryl shikonin, the amount of isobutyryl shikonin is, for example, 40ng / ml to 120ng / ml, preferably 50ng / ml to 110ng / ml, more preferably 53ng / ml to 106ng / ml. When the shikonin is β,β-dimethylacryl shikonin, the amount of β,β-dimethylacryl shikonin is, for example, 6ng / ml to 25ng / ml, preferably 8ng / ml to 21ng / ml, more preferably 9.9ng / ml to 19.8ng / ml. When the shikonin is isovaleryl shikonin, the amount of isovaleryl shikonin is, for example, 30ng / ml to 100ng / ml, preferably 40ng / ml to 90ng / ml, more preferably 43.8ng / ml to 87.5ng / ml. When the shikonin is α-methyl-n-butyrylshikonin, the amount of the α-methyl-n-butyrylshikonin is, for example, 30 ng / ml to 100 ng / ml, preferably 40 ng / ml to 90 ng / ml, and more preferably 43.8 ng / ml to 87.5 ng / ml.

[0038] The administration form of the agent for promoting keratinization of epidermal keratinocytes of the present disclosure can be oral or parenteral. The parenteral administration can be transdermal administration, application (contact) to the skin, etc. The application to the skin can include application to the oral mucosa, that is, application to or contact with epithelial cells in the oral cavity. The application to the skin can also include administration or injection into the skin or subcutaneously via the skin surface in addition to or instead of application to the skin surface. The administration or injection into the skin via the skin surface can be performed, for example, using a microneedle.

[0039] The dosage form of the agent for promoting keratinization of epidermal keratinocytes according to 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. When the administration form is oral administration, examples of the dosage form include tablets, pills, capsules, granules, powders, liquids, etc.

[0040] The agent for use in promoting keratinization of epidermal keratinocytes of the present disclosure may, for example, contain additives as necessary, and when used as a composition, the additives preferably contain pharma- ceutically acceptable additives or pharma-ceutically 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 keratinization-promoting effect of epidermal keratinocytes.

[0041] 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, α-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 flavors such as cocoa powder, peppermint, aromatic powder, peppermint oil, borneol, and cinnamon powder, sweeteners, and acidulants. Examples of the binder include hydroxypropyl cellulose, hydroxypropyl methyl 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, crosslinked polyvinylpyrrolidone, and sodium starch glycolate. Examples of the stabilizer include 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; sorbic acid, etc. Examples of the coating agent include macrogols such as hypromellose and macrogol 6000, talc, titanium oxide, etc.

[0042] When the composition for use in promoting keratinization of epidermal keratinocytes according to the present disclosure is an orally administered composition, specific examples of the orally administered composition include beverages, foods, pharmaceutical products (pharmaceutical products), quasi-drug products (pharmaceutical products), etc.

[0043] When the agent or composition for promoting keratinization of epidermal keratinocytes of the present disclosure is used for transdermal administration or application to the skin (hereinafter also referred to as "external skin preparation"), the form of the external 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), external skin preparations for local or whole body use, medicinal and / or cosmetic preparations to be applied to the scalp and hair, bath additives to be used by being added to bath water, 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 soap, liquid soap and hand wash; massage preparations, cleansing preparations, hair removal preparations, depilatories, shaving treatments, aftershave lotions, pre-shave lotions, shaving creams, makeup cosmetics such as foundations, lipsticks, blushers, eye shadows, eyeliners and mascaras; perfumes, nail beautifying preparations, nail beautifying enamel, nail beautifying enamel removers, poultices, plasters, tapes, sheets, patches, aerosols, toothpastes and mouthwashes such as mouthwash. 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 hair care products, poultices, plasters, tapes, sheets, aerosols, etc. Examples of the other preparations include underarm odor prevention or deodorants, antiperspirants, sanitary products, sanitary cotton, wet tissues, etc.

[0044] The topical skin preparation can be produced, as necessary, by arbitrarily selecting and / or combining the components and / or additives exemplified below within a range that does not impair the keratinization-promoting effect of epidermal keratinocytes.

[0045] (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, or hydrogenated products of these oils and fats (hardened oils, etc.).

[0046] (2) Waxes Beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0047] (3) Mineral oil Liquid paraffin, Vaseline, paraffin, ozokeride, ceresin, microcrystalline wax, etc.

[0048] (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; synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

[0049] (5) Alcohol 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.

[0050] (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.

[0051] (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, etc.

[0052] (8) Metal soaps Examples of such stearate include aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, and zinc undecylenate.

[0053] (9) Gums, sugars or water-soluble polymeric 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 their esters, trehalose or their derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan with an alkylene (C2-C4) oxide such as ethylene oxide added thereto, 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.

[0054] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkyl amine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylate type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonate 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 nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymeric surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), etc.

[0055] (11) Various vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), Vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acid, nicotinic acid, pantothenic acid, biotin, choline, inositol, 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, 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, γ-oryzanol, orotic acid, vitamin P group (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0056] (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, and amino acid derivatives thereof such as sulfates, phosphates, nitrates, citrates, or pyrrolidone carboxylic acid.

[0057] (13) Additives The skin topical preparation may further contain various additives derived from animals or plants. The additives may be added by carrying out processing that is conventionally carried out according to the type and form of the product to which they are to be added, and may be selected from various materials. The processing may be, for example, any of the following processes selected and / or combined: crushing, milling, washing, hydrolysis, fermentation, purification, squeezing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.

[0058] The solvent used for the extraction can be selected in consideration of the purpose of use, type, or subsequent processing of the product to be provided. The extraction solvent is preferably, for example, one or a mixture of two or more selected from lower alcohols such as water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, or hydrous lower alcohols, polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, glycerin, or hydrous polyhydric alcohols, and various organic solvents such as acetone and ethyl acetate. However, when the content of the organic solvent is not preferable depending on the use, for example, only water may be used, or ethanol that can be easily removed after extraction may be employed, and it may be used alone or as an arbitrary mixture with water, or the squeezed extract may be used.

[0059] When the additive derived from the plant or animal-based raw material is used for external preparations for whole body or local use, cosmetics, etc., the external skin preparation can, for example, protect the skin and hair, moisturize, improve the touch and texture, impart flexibility, relieve irritation, relieve stress by fragrance, activate cells (prevent cell aging), suppress inflammation, improve skin and hair quality, prevent and improve rough skin, promote hair growth, prevent hair loss, impart gloss, have a cleansing effect, relieve fatigue, promote blood flow, have a warm bath effect, and other cosmetic effects. In addition, effects such as fragrance addition, deodorization, thickening,防腐, and buffering can also be expected.

[0060] The external skin preparation can, for example, further expect various cosmetic and pharmaceutical effects of the known raw material materials, and by combining these, it is possible to enhance the effects aimed at in the present disclosure and make a product expecting multifunctional effects.

[0061] <Agent or composition for inducing the expression of the TGM1 gene> In another aspect, the present disclosure provides an agent or composition capable of inducing the expression of the TGM1 gene. Agents for use in suppressing the expression of the TGM1 gene of the present disclosure include sikokinin, β-hydroxyisovaleryl sikokinin, acetyl sikokinin, isobutyryl sikokinin, β,β-dimethylacrylyl sikokinin, isovaleryl sikokinin, and / or α-methyl-n-butyryl sikokinin. Compositions for use in inducing the expression of the TGM1 gene of the present disclosure include sikokinin, β-hydroxyisovaleryl sikokinin, acetyl sikokinin, isobutyryl sikokinin, β,β-dimethylacrylyl sikokinin, isovaleryl sikokinin, and / or α-methyl-n-butyryl sikokinin. According to the agent or composition for inducing the expression of the TGM1 gene of the present disclosure, an effect of promoting the keratinization of epidermal keratinocytes can be obtained.

[0062] The "induction of the expression of the TGM1 gene" means that the expression level of the TGM1 gene is promoted or increased, and may also mean a change from a state where the TGM1 gene is not expressed to a state where it is expressed. The expression of the TGM1 gene can be evaluated, for example, by measuring the expression level of the mRNA of the TGM1 gene according to Example 1 described below.

[0063] The agent for inducing the expression of the TGM1 gene of the present disclosure can promote the expression of the TGM1 gene, for example, by being used for an administration subject. Thereby, the agent for promoting the expression of the TGM1 gene of the present disclosure can obtain, for example, an effect of promoting the keratinization of epidermal keratinocytes. The usage conditions (administration conditions) of the agent for inducing the expression of the TGM1 gene of the present disclosure can refer to the description of the usage conditions of the agent for use in promoting the keratinization of the epidermal keratinocytes of the present disclosure.

[0064] <Agent or composition for use in inducing the expression of the IVL gene> In another aspect, the present disclosure provides an agent or composition capable of inducing the expression of the IVL gene. Agents for use in suppressing the expression of the IVL gene of the present disclosure include sikokinin, β-hydroxyisovaleryl sikokinin, acetyl sikokinin, isobutyryl sikokinin, β,β-dimethylacrylyl sikokinin, isovaleryl sikokinin, and / or α-methyl-n-butyryl sikokinin. Compositions for use in inducing the expression of the IVL gene of the present disclosure include sikokinin, β-hydroxyisovaleryl sikokinin, acetyl sikokinin, isobutyryl sikokinin, β,β-dimethylacrylyl sikokinin, isovaleryl sikokinin, and / or α-methyl-n-butyryl sikokinin. According to the agent or composition for inducing the expression of the IVL gene of the present disclosure, a keratinization promoting effect on epidermal keratinocytes can be obtained.

[0065] The "induction of the expression of the IVL gene" means that the expression level of the IVL gene is promoted or increased, 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 IVL gene can be evaluated, for example, by measuring the expression level of the mRNA of the IVL gene according to Example 1 described below.

[0066] The agent for inducing the expression of the IVL gene of the present disclosure can promote the expression of the IVL gene, for example, by being used for an administration subject. Thereby, the agent for promoting the expression of the IVL gene of the present disclosure can obtain, for example, a keratinization promoting effect on epidermal keratinocytes. The usage conditions (administration conditions) of the agent for inducing the expression of the IVL gene of the present disclosure can refer to the description of the usage conditions of the agent for use in promoting the keratinization of the epidermal keratinocytes of the present disclosure.

[0067] <Agent or composition for use in inducing the expression of the FLG gene> In another aspect, the present disclosure provides an agent or composition capable of inducing the expression of the FLG gene. Agents for use in suppressing the expression of the FLG gene of the present disclosure include cichonin, β-hydroxyisovaleryl cichonin, acetyl cichonin, isobutyryl cichonin, β,β-dimethylacrylyl cichonin, isovaleryl cichonin, and / or α-methyl-n-butyryl cichonin. Compositions for use in inducing the expression of the FLG gene of the present disclosure include cichonin, β-hydroxyisovaleryl cichonin, acetyl cichonin, isobutyryl cichonin, β,β-dimethylacrylyl cichonin, isovaleryl cichonin, and / or α-methyl-n-butyryl cichonin. According to the agent or composition for inducing the expression of the FLG gene of the present disclosure, an effect of promoting keratinization of epidermal keratinocytes can be obtained.

[0068] The "induction of FLG gene expression" means that the expression level of the FLG gene is promoted or increased, and may also mean a change from a state without expression of the FLG gene to a state with expression. The expression of the FLG gene can be evaluated, for example, by measuring the expression level of the mRNA of the FLG gene according to Example 1 described below.

[0069] The agent for inducing the expression of the FLG gene of the present disclosure can promote the expression of the FLG gene, for example, by being used for an administration subject. Thereby, the agent for promoting the expression of the FLG gene of the present disclosure can obtain, for example, an effect of promoting keratinization of epidermal keratinocytes. The usage conditions (administration conditions) of the agent for inducing the expression of the FLG gene of the present disclosure can refer to the description of the usage conditions of the agent for use in promoting keratinization of the epidermal keratinocytes of the present disclosure.

[0070] <Agent or composition for use in inducing the expression of the CERS1 gene> In another embodiment, the present disclosure provides an agent or composition capable of inducing the expression of the CERS1 gene. The agent for use in suppressing the expression of the CERS1 gene of the present disclosure includes shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin. The composition for use in inducing the expression of the CERS1 gene of the present disclosure includes shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin. According to the agent or composition for inducing the expression of the CERS1 gene of the present disclosure, it is possible to obtain the effect of promoting cornification of epidermal keratinocytes.

[0071] The "induction of CERS1 gene expression" means promotion or increase in the expression level of the CERS1 gene, and may mean a change from a state in which the CERS1 gene is not expressed to a state in which the CERS1 gene is expressed. The expression of the CERS1 gene can be evaluated, for example, by measuring the expression level of the mRNA of the CERS1 gene according to Example 1 described later.

[0072] The expression inducer of the CERS1 gene of the present disclosure can promote the expression of the CERS1 gene, for example, by using it on a subject to be administered. As a result, the expression inducer of the CERS1 gene of the present disclosure can obtain, for example, an effect of promoting cornification of epidermal keratinocytes. The use conditions (administration conditions) of the expression inducer of the CERS1 gene of the present disclosure can be explained by referring to the use conditions of the agent for promoting cornification of epidermal keratinocytes of the present disclosure.

[0073] <Method for promoting keratinization of epidermal keratinocytes> In another aspect, the present disclosure discloses a method capable of promoting keratinization of epidermal keratinocytes. The method for promoting keratinization of epidermal keratinocytes of the present disclosure uses the keratinization promoter and / or composition of epidermal keratinocytes of the present disclosure. According to the method for promoting keratinization of epidermal keratinocytes of the present disclosure, for example, it is expected that an effect of promoting keratinization of epidermal keratinocytes can be obtained.

[0074] The method for promoting keratinization of epidermal keratinocytes of the present disclosure includes a use step of using, for a subject, an agent and / or a composition for promoting keratinization of the epidermal keratinocytes of the present disclosure. The use may be, for example, contact with the skin or the like, or administration.

[0075] In the method for promoting keratinization of epidermal keratinocytes of the present disclosure, the use step may be, for example, in vitro or in vivo It may also be carried out. For the subject (administration subject) and administration conditions of the method for promoting keratinization of epidermal keratinocytes of the present disclosure, for example, the descriptions of the administration subject and administration conditions in the agent and / or composition for promoting keratinization of epidermal keratinocytes of the present disclosure can be incorporated by reference.

[0076] <Method for inducing expression of TGM1 gene> In another aspect, the present disclosure discloses a method capable of inducing the expression of the TGM1 gene. The method for inducing the expression of the TGM1 gene of the present disclosure uses the agent and / or composition for inducing the expression of the TGM1 gene of the present disclosure. According to the method for inducing the expression of the TGM1 gene of the present disclosure, for example, it is expected that an effect of inducing the expression of the TGM1 gene can be obtained.

[0077] The method for inducing the expression of the TGM1 gene of the present disclosure includes a use step of using, for a subject, an agent and / or a composition for inducing the expression of the TGM1 gene of the present disclosure. The use may be, for example, contact with the skin or the like, or administration.

[0078] In the method for inducing the expression of the TGM1 gene of the present disclosure, the use step may be, for example, in vitro or in vivo It may also be carried out. For the subject (administration subject) and administration conditions of the method for inducing the expression of the TGM1 gene of the present disclosure, for example, the descriptions of the administration subject and administration conditions in the agent and / or composition for inducing the expression of the TGM1 gene of the present disclosure can be incorporated by reference.

[0079] <Method for inducing expression of IVL gene> In another aspect, the present disclosure discloses a method capable of inducing the expression of the IVL gene. The method for inducing the expression of the IVL gene of the present disclosure uses the agent and / or composition for inducing the expression of the IVL gene of the present disclosure. According to the method for inducing the expression of the IVL gene of the present disclosure, for example, it is expected that an effect of inducing the expression of the IVL gene can be obtained.

[0080] The method for inducing the expression of the IVL gene of the present disclosure includes a use step of using, for a subject, the agent and / or composition for inducing the expression of the IVL gene of the present disclosure. The use may be, for example, contact with the skin or the like, or administration.

[0081] In the method for inducing the expression of the IVL gene of the present disclosure, the use step is, for example, in vitro or in vivo It may be carried out. For the subject (administration subject) and administration conditions of the method for inducing the expression of the IVL gene of the present disclosure, for example, the description of the administration subject and administration conditions in the agent and / or composition for inducing the expression of the IVL gene of the present disclosure can be incorporated by reference.

[0082] <Method for inducing expression of FLG gene> In another aspect, the present disclosure discloses a method capable of inducing the expression of the FLG gene. The method for inducing the expression of the FLG gene of the present disclosure uses the agent and / or composition for inducing the expression of the FLG gene of the present disclosure. According to the method for inducing the expression of the FLG gene of the present disclosure, for example, it is expected that an effect of inducing the expression of the FLG gene can be obtained.

[0083] The method for inducing the expression of the FLG gene of the present disclosure includes a use step of using, for a subject, the agent and / or composition for inducing the expression of the FLG gene of the present disclosure. The use may be, for example, contact with the skin or the like, or administration.

[0084] In the method for inducing the expression of the FLG gene of the present disclosure, the use step is, for example, in vitro orin vivo It may also be performed. The subject (administration subject) and administration conditions of the method for inducing the expression of the FLG gene of the present disclosure can, for example, refer to the description of the administration subject and administration conditions in the agent and / or composition used for inducing the expression of the FLG gene of the present disclosure.

[0085] <Method for inducing the expression of the CERS1 gene> In another aspect, the present disclosure discloses a method capable of inducing the expression of the CERS1 gene. The method for inducing the expression of the CERS1 gene of the present disclosure uses the agent and / or composition for inducing the expression of the CERS1 gene of the present disclosure. According to the method for inducing the expression of the CERS1 gene of the present disclosure, for example, it is expected that an effect of inducing the expression of the CERS1 gene can be obtained.

[0086] The method for inducing the expression of the CERS1 gene of the present disclosure includes a step of using, for a subject, the agent and / or composition for inducing the expression of the CERS1 gene of the present disclosure. The use may be, for example, contact with the skin or the like, or administration.

[0087] In the method for inducing the expression of the CERS1 gene of the present disclosure, the use step may be, for example, in vitro or in vivo It may also be performed. The subject (administration subject) and administration conditions of the method for inducing the expression of the CERS1 gene of the present disclosure can, for example, refer to the description of the administration subject and administration conditions in the agent and / or composition used for inducing the expression of the CERS1 gene of the present disclosure.

[0088] <Use> The present disclosure relates to the use of an agent and / or composition for use in promoting keratinization of epidermal keratinocytes. The present disclosure relates to the use of an expression inducer and / or composition of the TGM1 gene for use in inducing expression of the TGM1 gene. The present disclosure relates to the use of an expression inducer and / or composition of the IVL gene for use in inducing expression of the IVL gene. The present disclosure relates to the use of an expression inducer and / or composition of the FLG gene for use in inducing expression of the FLG gene. The present disclosure relates to the use of an expression inducer and / or composition of the CERS1 gene for use in inducing expression of the CERS1 gene.

[0089] The present disclosure relates to the use of an agent and / or composition for use in promoting keratinization of epidermal keratinocytes, for producing an agent for use in promoting keratinization of epidermal keratinocytes, or a composition for use in promoting keratinization of epidermal keratinocytes. The present disclosure relates to the use of an expression inducer and / or composition of the TGM1 gene, for producing an agent for use in inducing expression of the TGM1 gene, or a composition for use in inducing expression of the TGM1 gene. The present disclosure relates to the use of an expression inducer and / or composition of the IVL gene, for producing an agent for use in inducing expression of the IVL gene, or a composition for use in inducing expression of the IVL gene. The present disclosure relates to the use of an expression inducer and / or composition of the FLG gene, for producing an agent for use in inducing expression of the FLG gene, or a composition for use in inducing expression of the FLG gene. The present disclosure relates to the use of an expression inducer and / or composition of the CERS1 gene, 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. EXAMPLES

[0090] 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. In addition, "mol / l" may be abbreviated as "M".

[0091] [Example 1] It has been confirmed that the shikonins disclosed herein exhibit a keratinization-promoting effect on epidermal keratinocytes.

[0092] (1) Quantitative analysis of shikonin components The content of each shikonin in the shikonin extract was examined. Specifically, the examination was carried out by HPLC analysis. 0.015% or 0.03% (v / v, the same applies below) shikonin liquid (BG) (manufactured by Ichimaru Falcos) was prepared using ethanol. After the preparation, HPLC analysis was carried out under the following HPLC measurement conditions. After the analysis, the quantitative value of each shikonin was calculated by converting the molecular weight ratio of each shikonin based on the calibration curve of acetyl shikonin. The calculated content of each shikonin in 0.015% or 0.03% shikonin liquid (BG) is shown in Table 1. Note that "shikonin liquid (BG)" is a shikonin extract, and is an extract of the root of Lithospermum erythrorhizon Siebold & Zucc. (Boraginaceae) with a slightly alkaline solution of a mixture of ethanol and 1,3-butylene glycol.

[0093] (HPLC measurement conditions) HPLC system: LC-20 series (Shimadzu Corporation) Column: Mightysil RP-18 GP 5 μm 250 x 4.6 mm (Kanto Chemical Co., Ltd.) Flow rate: 1.0ml / min Column temperature: 40℃ Detector: Ultraviolet spectrophotometer (measurement wavelength: 520 nm, Shimadzu Corporation, SPD-M20A) Mobile phase: 0 min → 30 min Acetonitrile / 0.1% phosphoric acid solution mixture (50:50) → (100:0)

[0094] [Table 1]

[0095] (2) Expression level of keratinization-related genes by shikonins NHEK cells, which are human keratinocytes (KK-4009, frozen NHEK (NB), derived from a newborn, manufactured by Kurabo Industries, Ltd.), were seeded on a 12-well plate. The medium used was KGM (trademark) Gold Keratinocyte Growth Medium BulletKit (trademark) medium (manufactured by Lonza) containing 0.06 mmol / l calcium, from which transferrin, hydrocortisone, and epinephrine had been removed (KGM Tfn- / HC- / Epfn-, the same applies below in the Examples). After the seeding, the NHEK cells were cultured until they reached 100% confluence. After the culture, the medium in each well was replaced with Syconix Liquid (BG) or KGM Tfn- / HC- / Epfn- medium supplemented with various shikonins at the concentrations contained in Syconix Liquid (BG). In the positive control group, 1 mmol / l Ca 2+ The medium was replaced with KGM Tfn- / HC- / Epfn- medium supplemented with . After the replacement, the NHEK cells were cultured for 6 hours or 16 hours. After the culture, total RNA was extracted using RNeasy mini kit (manufactured by QIAGEN) according to the attached protocol. After the extraction, cDNA was synthesized using ReverTra Ace (registered trademark) qPCR RT Master Mix (manufactured by TOYOBO) based on the measured concentration value of total RNA. After the synthesis, quantitative analysis of gene expression levels by real-time PCR was performed from the synthesized cDNA using THUNDERBIRD (registered trademark) Next SYBR (registered trademark) qPCR Mix (manufactured by TOYOBO). In the real-time PCR, the following primer set for TGM1 gene, primer set for IVL gene, primer set for FLG gene, primer set for CERS1 gene, and primer set for RSP18 gene (forward primer: sequence number 1, reverse primer: sequence number 2) were used as the primer set for each gene. As the PCR device, Thermal Cycler Dice (registered trademark) Real Time System (manufactured by Takara Bio Inc.) was used. Data analysis was performed using the accompanying software. Statistical analysis was performed using JMP8 with the Dunnett test. The results are shown in Figures 1 to 4.

[0096] Figure 1 is a graph showing the gene expression level of TGM1 in NHEK cells. In Figure 1, the vertical axis shows the relative value of the gene expression level of TGM1, and the horizontal axis shows the type of sample. For the sample type, two groups of Syconix Liquid (BG) were used: 0.015% (left of the data) and 0.03% (right of the data), and two groups of various shikonins were used: Syconix Liquid (BG) 0.015% equivalent (left of the data) and Syconix Liquid (BG) 0.03% equivalent (right of the data). The values ​​on the vertical axis in FIG. 1 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The numbers on the vertical axis in Figure 1 are as follows: NC group: 1.0, Syconix Liquid (BG) 0.015% group: 1.22, Syconix Liquid (BG) 0.03% group: 1.19, Acetyl shikonin 0.015% equivalent group: 1.04, Acetyl shikonin 0.03% equivalent group: 1.20, Isobutyryl shikonin 0.015% equivalent group: 1.20, Isobutyryl shikonin 0.03% equivalent group: 1.04, β,β-Dimethyl acryl shikonin 0.015% equivalent group: 1.22, β,β-Dimethyl acryl shikonin 0.03% equivalent group: 1.19, Isovaleryl shikonin 0.015% equivalent group: 1.25, Isovaleryl shikonin 0.03% equivalent group: 1.25, α-Methyl-n-butyryl shikonin 0.015% equivalent group: 1.30, α-Methyl-n-butyryl shikonin 0.03% equivalent group: 1.28, PC group: 2.86 As shown in Figure 1, the expression level of the TGM1 gene was increased in the acetylshikonin group, isobutyrylshikonin group, β,β-dimethylacrylshikonin group, isovalerylshikonin group, and α-methyl-n-butyrylshikonin group compared to the NC (negative control) group. These results demonstrated that acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin promote TGM1 production in epidermal keratinocytes.

[0097] Figure 2 is a graph showing the gene expression level of IVL in NHEK cells. In Figure 2, the vertical axis shows the relative value of the gene expression level of IVL, and the horizontal axis shows the type of sample. For the sample type, two groups of Syconix Liquid (BG) were used: 0.015% (left of the data) and 0.03% (right of the data), and two groups of various shikonins were used: Syconix Liquid (BG) 0.015% equivalent (left of the data) and Syconix Liquid (BG) 0.03% equivalent (right of the data). The values ​​on the vertical axis in FIG. 2 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The values ​​on the vertical axis in Figure 2 are as follows: NC group: 1.0, Syconix Liquid (BG) 0.015% group: 1.39, Syconix Liquid (BG) 0.03% group: 1.39, Acetyl shikonin 0.015% equivalent group: 1.12, Acetyl shikonin 0.03% equivalent group: 1.51, Isobutyryl shikonin 0.015% equivalent group: 1.57, Isobutyryl shikonin 0.03% equivalent group: 1.12, β,β-Dimethyl acryl shikonin 0.015% equivalent group: 1.06, β,β-Dimethyl acryl shikonin 0.03% equivalent group: 1.43, Isovaleryl shikonin 0.015% equivalent group: 1.47, Isovaleryl shikonin 0.03% equivalent group: 1.52, α-Methyl-n-butyryl shikonin 0.015% equivalent group: 1.49, α-Methyl-n-butyryl shikonin 0.03% equivalent group: 1.59, PC group: 2.21 As shown in Figure 2, the expression level of the TGM1 gene was increased in the acetylshikonin group, isobutyrylshikonin group, β,β-dimethylacrylshikonin group, isovalerylshikonin group, and α-methyl-n-butyrylshikonin group compared to the NC (negative control) group. These results demonstrated that acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin promote IVL production in epidermal keratinocytes.

[0098] Figure 3 is a graph showing the expression level of FLG gene in NHEK cells. In Figure 3, the vertical axis shows the relative value of the expression level of FLG gene, and the horizontal axis shows the type of sample. For the type of sample, two groups of Syconix Liquid (BG) were used: 0.015% (left of the data) and 0.03% (right of the data), and two groups of various shikonins were used: Syconix Liquid (BG) 0.015% equivalent (left of the data) and Syconix Liquid (BG) 0.03% equivalent (right of the data). The values ​​on the vertical axis in FIG. 3 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The values ​​on the vertical axis in Figure 3 are as follows: NC group: 1.0, Syconix Liquid (BG) 0.015% group: 1.00, Syconix Liquid (BG) 0.03% group: 1.11, β-Hydroxy isovaleryl shikonin 0.015% equivalent group: 1.11, Acetyl shikonin 0.015% equivalent group: 1.18, Acetyl shikonin 0.03% equivalent group: 1.13, Isobutyryl shikonin 0.015% equivalent group: 1.18, Isobutyryl shikonin 0.03% equivalent group: 1.08, β, β-Dimethyl acryl shikonin 0.015% equivalent group: 1.21, β,β-Dimethyl acryl shikonin 0.03% equivalent group: 1.23, Isovaleryl shikonin 0.015% equivalent group: 1.13, α-Methyl-n-butyryl shikonin 0.015% equivalent group: 1.66, α-Methyl-n-butyryl shikonin 0.03% equivalent group: 1.52, PC group: 1.10 As shown in Figure 3, the expression level of FLG gene was increased in the β-hydroxyisovalerylshikonin group, acetylshikonin group, isobutyrylshikonin group, β,β-dimethylacrylshikonin group, isovalerylshikonin group, and α-methyl-n-butyrylshikonin group compared to the NC (negative control) group. These results demonstrated that β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin promote FLG production in epidermal keratinocytes.

[0099] Figure 4 is a graph showing the gene expression level of CERS1 in NHEK cells. In Figure 4, the vertical axis shows the relative value of the CERS1 gene expression level, and the horizontal axis shows the type of sample. For the sample type, two groups of Syconix Liquid (BG) were used: 0.015% (left of the data) and 0.03% (right of the data), and two groups of various shikonins were used: Syconix Liquid (BG) 0.015% equivalent (left of the data) and Syconix Liquid (BG) 0.03% equivalent (right of the data). The values ​​on the vertical axis in FIG. 4 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The values ​​on the vertical axis in Figure 4 are as follows: NC group: 1.0, Shikonix Liquid (BG) 0.015% group: 1.17, Shikonix Liquid (BG) 0.03% group: 1.56, Shikonin 0.015% equivalent group: 1.41, Isobutyryl shikonin 0.015% equivalent group: 1.25, Isobutyryl shikonin 0.03% equivalent group: 1.14, β,β-Dimethyl acryl shikonin 0.015% equivalent group: 1.02, β,β-Dimethyl acryl shikonin 0.03% equivalent group: 1.20, Isovaleryl shikonin 0.015% equivalent group: 1.53, Isovaleryl shikonin 0.03% equivalent group: 1.22, α-Methyl-n-butyryl shikonin 0.015% equivalent group: 1.67, α-Methyl-n-butyryl shikonin 0.03% equivalent group: 1.27, PC group: 1.08 As shown in Figure 4, the expression level of the CERS1 gene was increased in the shikonin group, isobutyrylshikonin group, β,β-dimethylacrylshikonin group, isovalerylshikonin group, and α-methyl-n-butyrylshikonin group compared to the NC (negative control) group. These results demonstrated that shikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin promote CERS1 production in epidermal keratinocytes.

[0100] (3) Expression level test of TGM1 gene and IVL gene by shikonin mixture Next, we investigated whether the combination of shikonins would also have the effect of promoting keratinization of epidermal keratinocytes. The investigation was carried out by measuring the expression levels of the TGM1 gene and the IVL gene. Specifically, the investigation was carried out in the same manner as in Example 1 (2), except for changing the concentration of shikonic liquid (BG) and changing various shikonins to a mixture of shikonins. The mixture of shikonins used was mixture A (Mixture of shikonin derivative A) of acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, and isovaleryl shikonin, and mixture B (Mixture of shikonin derivative B) of acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, and α-methyl-n-butyryl shikonin. These results are shown in Figures 5 to 6.

[0101] Fig. 5 is a graph showing the expression level of TGM1 gene in NHEK cells, in which the vertical axis indicates the relative value of the expression level of TGM1 gene, and the horizontal axis indicates the type of sample. The values ​​on the vertical axis in FIG. 5 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The values ​​on the vertical axis in Figure 5 are as follows: NC group: 1.0, Mixture A (Mixture of shikonin derivative A) group: 1.28, Mixture B (Mixture of shikonin derivative B) group: 1.17, Syconix Liquid (BG) 0.001% group: 1.09, Syconix Liquid (BG) 0.003% group: 1.19, Syconix Liquid (BG) 0.01% group: 1.28, Syconix Liquid (BG) 0.03% group: 1.30, PC group: 1.86 As shown in Figure 5, the expression level of the TGM1 gene was increased in the mixture A and mixture B groups compared to the NC (negative control) group. These results demonstrated that acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin promote TGM1 production in epidermal keratinocytes. Furthermore, as shown in the Syconix Liquid (BG) group in Figure 5, it was also suggested that Syconix Liquid (BG) does not contain any substances that inhibit differentiation promotion.

[0102] Fig. 6 is a graph showing the expression level of IVL gene in NHEK cells, in which the vertical axis indicates the relative value of the expression level of IVL gene, and the horizontal axis indicates the type of sample. The values ​​on the vertical axis in FIG. 6 indicate the values ​​for each group, with the value for the negative control (NC) group being set at 1.0 (1.00). The values ​​on the vertical axis in Figure 6 are as follows: NC group: 1.0, Mixture A (Mixture of shikonin derivative A) group: 1.40, Mixture B (Mixture of shikonin derivative B) group: 1.15, Syconix Liquid (BG) 0.001% group: 1.24, Syconix Liquid (BG) 0.003% group: 1.11, Syconix Liquid (BG) 0.01% group: 1.41, Syconix Liquid (BG) 0.03% group: 1.53, PC group: 0.96 As shown in Figure 6, the expression level of the IVL gene was increased in the mixture A and mixture B groups compared to the NC (negative control) group. These results demonstrated that acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and α-methyl-n-butyrylshikonin promote IVL production in epidermal keratinocytes. As shown in the Syconix Liquid (BG) group in Figure 6, it was also suggested that Syconix Liquid (BG) does not contain any substances that inhibit differentiation promotion.

[0103] The sequences and other information of the primers used in Example 1 above are shown in Table 2 below.

[0104] [Table 2]

[0105] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-mentioned 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.

[0106] <Additional Notes> Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following: <Agent for use in promoting keratinization of epidermal keratinocytes> (Appendix 1) An agent for use in promoting keratinization of epidermal keratinocytes, comprising shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 2) The agent described in Appendix 1, containing acetylshikonin. (Appendix 3) An agent described in Appendix 1 or 2, containing the β,β-dimethylacrylshikonin. (Appendix 4) An agent described in any of Appendices 1 to 3, comprising a shikon extract containing shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin. (Appendix 5) 5. An agent for application to the skin comprising an agent according to any one of claims 1 to 4. (Appendix 6) 5. An agent for oral administration comprising the agent according to any one of claims 1 to 4. <Composition for use in promoting keratinization of epidermal keratinocytes> (Appendix 7) A composition for use in promoting keratinization of epidermal keratinocytes, comprising shikonin, β-hydroxy isovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 8) The composition described in Appendix 7, comprising acetylshikonin. (Appendix 9) A composition described in Appendix 7 or 8, comprising the β,β-dimethylacrylshikonin. (Appendix 10) A composition described in any of Appendices 7 to 9, comprising a shikon extract containing shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin. (Appendix 11) A composition for application to the skin comprising a composition according to any one of claims 7 to 10. (Appendix 12) A composition for oral administration, comprising the composition according to any one of Supplementary Notes 7 to 10. (Supplementary Note 13) A composition according to any one of Supplementary Notes 7 to 12, comprising the agent according to any one of Supplementary Notes 1 to 6. <Agent for inducing the expression of the TGM1 gene> (Supplementary Note 14) An agent for inducing the expression of the Transglutaminase 1 (TGM1) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 15) The agent according to Supplementary Note 14, comprising the acetyl shikonin. (Supplementary Note 16) The agent according to Supplementary Note 14 or 15, comprising the β,β-dimethylacryl shikonin isovaleryl shikonin. (Supplementary Note 17) The agent according to any one of Supplementary Notes 14 to 16, comprising a shikon extract containing the shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 18) An agent for topical application to the skin, comprising the agent according to any one of Supplementary Notes 14 to 17. (Supplementary Note 19) An agent for oral administration, comprising the agent according to any one of Supplementary Notes 14 to 17. <Composition for inducing the expression of the TGM1 gene> (Supplementary Note 20) A composition for inducing the expression of the TGM1 gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 21) The composition according to Appendix 20, comprising the acetyl shikonin. (Appendix 22) The composition according to Appendix 20 or 21, comprising the β,β-dimethylacryl shikonin. (Appendix 23) The composition according to any one of Appendices 20 to 22, comprising a shikon extract containing the shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 24) A composition for topical application to the skin, comprising the composition according to any one of Appendices 20 to 23. (Appendix 25) A composition for oral administration, comprising the composition according to any one of Appendices 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 inducing the expression of the <IVL gene>> (Appendix 27) An agent for inducing the expression of the involucrin (IVL) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 28) The agent according to Appendix 27, comprising the acetyl shikonin. (Appendix 29) The agent according to Appendix 27 or 28, comprising the β,β-dimethylacryl shikonin. (Appendix 30) The agent according to any one of Appendices 27 to 29, comprising a shikon extract containing the shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 31) An agent for topical application to the skin, comprising the agent according to any one of Appendices 27 to 30. (Appendix 32) An agent for oral administration, comprising the agent according to any one of Appendices 27 to 30. <Composition for inducing the expression of the IVL gene> (Appendix 33) A composition for inducing the expression of the involucrin (IVL) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 34) The composition according to Appendix 33, comprising the acetyl shikonin. (Appendix 35) The composition according to Appendix 33 or 34, comprising the β,β-dimethylacryl shikonin. (Appendix 36) The composition according to any one of Appendix 33 to 35, comprising a shikon extract containing the shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 37) A composition for topical application to the skin, comprising the composition according to any one of Appendix 33 to 36. (Appendix 38) A composition for oral administration, comprising the composition according to any one of Appendix 33 to 36. (Appendix 39) The composition according to any one of Appendix 33 to 38, comprising the agent according to any one of Appendix 27 to 32. <Agent for inducing the expression of the FLG gene> (Appendix 40) An agent for inducing the expression of the filaggrin (FLG) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 41) The agent according to Appendix 40, comprising a shikonin extract containing shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 42) An agent for topical application to the skin, comprising the agent according to Appendix 40 or 41. (Appendix 43) An agent for oral administration, comprising the agent according to Appendix 40 or 41. <Composition for inducing the expression of the FLG gene> (Appendix 44) A composition for inducing the expression of the filaggrin (FLG) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Appendix 45) The composition according to Supplementary Note 44, comprising a shikonin extract containing the above-mentioned shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 46) A composition for topical application to the skin, comprising the composition according to Supplementary Note 44 or 45. (Supplementary Note 47) A composition for oral administration, comprising the composition according to Supplementary Note 44 or 45. (Supplementary Note 48) A composition according to any one of Supplementary Notes 44 to 47, comprising an agent according to any one of Supplementary Notes 40 to 43. (Agent for inducing the expression of the CERS1 gene) (Supplementary Note 49) An agent for inducing the expression of the Ceramide Synthase 1 (CERS1) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 50) The agent according to Supplementary Note 49, comprising the above-mentioned isovaleryl shikonin. (Supplementary Note 51) The agent according to Supplementary Note 49 or 50, comprising the above-mentioned α-methyl-n-butyryl shikonin. (Supplementary Note 52) The agent according to any one of Supplementary Notes 49 to 51, comprising a shikonin extract containing the above-mentioned shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 53) An agent for topical application to the skin, comprising the agent described in any one of Supplementary Notes 49 to 52. (Supplementary Note 54) An agent for oral administration, comprising the agent described in any one of Supplementary Notes 49 to 52. <Composition for inducing the expression of the CERS1 gene> (Supplementary Note 55) A composition for inducing the expression of the Ceramide Synthase 1 (CERS1) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 56) The composition according to Supplementary Note 5, comprising the isovaleryl shikonin. (Supplementary Note 57) The composition according to Supplementary Note 55 or 56, comprising the α-methyl-n-butyryl shikonin. (Supplementary Note 58) The composition according to any one of Supplementary Notes 55 to 57, comprising a shikonin extract containing the shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethylacryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin. (Supplementary Note 59) A composition for topical application to the skin, comprising the composition according to any one of Supplementary Notes 55 to 58. (Supplementary Note 60) A composition for oral administration, comprising the composition according to any one of Supplementary Notes 55 to 58. (Supplementary Note 61) The composition according to any one of Supplementary Notes 55 to 60, comprising the agent described in any one of Supplementary Notes 49 to 54.

Industrial Applicability

[0107] As described above, according to the present disclosure, it is possible to provide an agent that can induce the effect of promoting the expression of keratinization-related genes in epidermal keratinocytes and is applicable mainly 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 promoting keratinization of epidermal keratinocytes, inducing expression of the transglutaminase 1 (TGM1) gene, inducing expression of the involucrin (IVL) gene, inducing expression of the filaggrin (FLG) gene, and / or inducing expression of the ceramide synthase 1 (CERS1) gene, comprising shikonin, β-hydroxyisovaleryl shikonin, acetyl shikonin, isobutyryl shikonin, β,β-dimethyl acryl shikonin, isovaleryl shikonin, and / or α-methyl-n-butyryl shikonin.

2. The agent according to claim 1, comprising acetylshikonin.

3. The agent according to claim 1 or 2, containing the β,β-dimethylacrylshikonin.

4. The agent according to claim 1 or 2, comprising a lithospermum extract containing shikonin, β-hydroxyisovalerylshikonin, acetylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, and / or α-methyl-n-butyrylshikonin.

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