Agent for use in maintaining structure of hyaluronic acid

Abstract

Provided is an agent or the like for use in maintaining the structure of hyaluronic acid.　The agent according to the present disclosure is for maintaining the structure of hyaluronic acid, and contains a compound represented by formula (1) or a salt thereof. The hyaluronic acid has a molecular weight of 600-2000 kDa.

Description

Agents for maintaining the structure of hyaluronic acid

[0001] The present disclosure relates to an agent for use in maintaining the structure of hyaluronic acid.

[0002] The skin is the first line of defense against direct external environmental influences and plays an important role in maintaining the internal environment of the body. Therefore, although the skin's functions do not completely cease, they gradually decline with age, exposure to ultraviolet rays, and exposure to chemicals, and signs of aging such as wrinkles, age spots, dullness, and sagging skin become apparent.

[0003] Hyaluronic acid (HA) is a linear mucopolysaccharide consisting of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine. In vivo, hyaluronic acid is widely present in the skin, eyes, cartilage, synovial membrane, synovial fluid, etc. In the skin, hyaluronic acid is present in the dermis and contributes to skin moisturizing and elasticity. Conventionally, hyaluronic acid and materials that inhibit its degradation have been used in cosmetics and the like intended for skin moisturizing and anti-aging (Patent Document 1, Non-Patent Document 1).

[0004] In vivo degradation of hyaluronic acid in humans and other living organisms (including the dermis of the skin) includes, for example, KIAA1199 (HYBID)-dependent degradation of hyaluronic acid (Non-Patent Document 2).

[0005] Japanese Patent Application Laid-Open No. 2018-135272

[0006] Shintaro Inoue, Glucosamine Research 5: 4-10, 2009 Chemistry and Biology 55(2): 119-127 (2017)

[0007] Therefore, an object of the present disclosure is to provide an agent for use in maintaining the structure of hyaluronic acid.

[0008] In order to achieve the above object, the agent of the present disclosure is an agent for maintaining the structure of hyaluronic acid, and comprises a compound represented by the following formula (1) or a salt thereof, wherein the hyaluronic acid has a molecular weight of 600 to 2000 kDa: In the formula (1), R １ and R ２may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom.

[0009] The agent of the present disclosure is an agent for use in suppressing the expression of a HYBID gene, and comprises a compound represented by the following formula (1) or a salt thereof: In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom.

[0010] The method for maintaining the structure of hyaluronic acid of the present disclosure uses the agent of the present disclosure.

[0011] The method for inhibiting expression of the HYBID gene of the present disclosure uses the agent of the present disclosure.

[0012] The compound of the present disclosure or a salt thereof is represented by the following formula (2) or (3):

[0013] According to the present disclosure, for example, it is possible to provide an agent for use in maintaining the structure of hyaluronic acid.

[0014] FIG. 1 is a diagram of the structural formula of the compound. FIG. 2 is a schematic diagram of the skin monitoring test in Example 3. FIG. 3 is a photograph showing the state of each gel in Example 6. FIG. 4 is a graph showing the results of HPLC analysis in Example 9. FIG. 5 is a diagram showing the fractionation scheme of the extract of Artemisia capillaris flower heads in Example 10. FIG. 6 is a graph showing the gene expression level of HYBID upon addition of histamine in Example 10. FIG. 7 is a graph showing the gene expression level of HYBID upon addition of histamine in Example 10. FIG. 8 is a graph showing the gene expression level of HYBID upon addition of histamine in Example 10. FIG. 9 is a graph showing the gene expression level of HYBID upon addition of histamine in Example 10. FIG. 10 is a graph showing the gene expression level of HYBID in Example 10. FIG. 11 is a graph showing the expression level of miR-486-5p in Example 11. FIG. 12 is a graph showing the expression level of miR-486-5p in Example 11.

[0015] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0016] <Agent or composition for use in maintaining the structure of hyaluronic acid> As described above, the agent or composition for use in maintaining the structure of hyaluronic acid of the present disclosure comprises a compound represented by the following formula (1) or a salt thereof, and the hyaluronic acid has a molecular weight of 600 to 2000 kDa. In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom.

[0017] The agent or composition for maintaining the structure of hyaluronic acid of the present disclosure is characterized by comprising the compound represented by formula (1) or its salt, and other configurations and conditions are not particularly limited.The agent or composition for maintaining the structure of hyaluronic acid of the present disclosure comprises the compound represented by formula (1) or its salt, so it can maintain the structure of hyaluronic acid in skin.Therefore, the agent or composition for maintaining the structure of hyaluronic acid of the present disclosure is expected to contribute to, for example, the moisturizing of epidermis.

[0018] Hereinafter, examples of each substituent in the compound represented by formula (1) will be described. In the description of each substituent, unless otherwise specified, specific examples in the description of other substituents can be used. Furthermore, unless otherwise specified in the following description, the description of the compound represented by formula (1) can be used, for example, to describe the salt of the compound represented by formula (1).

[0019] R １ and R ２ are each a hydrogen atom, a hydroxy group, or a linear or branched alkoxy group having 1 to 5 carbon atoms. Specific examples of the alkoxy group include a methyloxy group (methoxy group), an ethyloxy group (ethoxy group), a propyloxy group (propoxy group), an isopropyloxy group (isopropoxy group), and a butyloxy group (butoxy group). The butyl may be any of n-butyl, sec(s)-butyl, iso(i)-butyl, and tert(t)-butyl (the same applies hereinafter). The R １ is preferably, for example, a hydrogen atom or a hydroxy group. ２ is preferably a hydrogen atom, a hydroxy group, or a methyloxy group.

[0020] R ３is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom. Specific examples of the hydroxyalkyl group include a hydroxymethyl group; hydroxyethyl groups such as a 1-hydroxyethyl group or a 2-hydroxyethyl group; hydroxypropyl groups such as a 1-hydroxypropyl group, a 2-hydroxypropyl group, or a 3-hydroxypropyl group; hydroxybutyl groups such as a 1-hydroxybutyl group, a 2-hydroxybutyl group, a 3-hydroxybutyl group, or a 4-hydroxybutyl group; hydroxypentyl groups such as a 1-hydroxypentyl group, a 2-hydroxypentyl group, a 3-hydroxypentyl group, a 4-hydroxypentyl group, or a 5-hydroxypentyl group; hydroxyhexyl groups such as a 1-hydroxyhexyl group, a 2-hydroxyhexyl group, a 3-hydroxyhexyl group, a 4-hydroxyhexyl group, a 5-hydroxyhexyl group, or a 6-hydroxyhexyl group; and hydroxyheptyl groups such as a 1-hydroxyheptyl group, a 2-hydroxyheptyl group, a 3-hydroxyheptyl group, a 4-hydroxyheptyl group, a 5-hydroxyheptyl group, a 6-hydroxyheptyl group, or a 7-hydroxyheptyl group. Examples of the alkyl group include a methyl group; an ethyl group; a propyl group such as an n-propyl group or an iso(i)-propyl group; a butyl group such as an n-butyl group, an i-butyl group, a t-butyl group or an s-butyl group; a pentyl group such as an n-pentyl group, an i-pentyl group or a t-pentyl group; a hexyl group such as an n-hexyl group, an i-hexyl group or a t-hexyl group; and a heptyl group such as an n-heptyl group, an i-heptyl group or a t-heptyl group. Examples of the cyclic alkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, and a cyclodecyl group. In the cyclic alkyl group, one or more hydrogen atoms may be substituted with a hydroxy group or a carboxyl group. ３is, for example, preferably a linear or branched hydroxyalkyl group having 1 to 5 carbon atoms, or a linear or branched alkyl group having 1 to 5 carbon atoms, and more preferably a 1-hydroxypropyl group, a 2-hydroxypropyl group, a 3-hydroxypropyl group, a 1-hydroxybutyl group, a 2-hydroxybutyl group, a 3-hydroxybutyl group, a 4-hydroxybutyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a t-butyl group, or an s-butyl group.

[0021] The compound represented by formula (1) may be, for example, caffeic acid or a derivative thereof. The caffeic acid is a compound represented by the following formula (4). The compound represented by formula (4) may also be referred to as, for example, caffeic acid, (E)-3-(3,4-dihydroxyphenyl)prop-2-enoic acid. The derivative of caffeic acid is, for example, a compound in which the caffeic acid is modified with an arbitrary substituent. The derivative of caffeic acid includes, for example, chlorogenic acids. The chlorogenic acids are compounds in which the caffeic acid or ferulic acid is ester-bonded with quinic acid.

[0022] As a specific example, the caffeic acid or its derivative may be, for example, a compound of the formula (1) in which the R １ is a hydroxy group, and the R ２ is a hydroxy group, and the R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, and is preferably a hydroxypropyl group, a hydroxybutyl group, a propyl group, a butyl group, or a cyclohexyl group. The above explanations can be applied to the substituent of the cyclic alkyl group, for example.

[0023] As a specific example, the caffeic acid derivative is preferably a compound represented by the following formula (2) or a salt thereof. The compound represented by the following formula (2) can also be referred to as, for example, 3-hydroxybutyl(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoate.

[0024] As a specific example, the caffeic acid derivative is preferably a compound represented by the following formula (3) or a salt thereof. The compound represented by the following formula (3) can also be referred to as, for example, 4-hydroxybutan-2-yl(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoate.

[0025] In the present disclosure, the caffeic acid or its derivative may be in the form of a salt, hydrate, or solvate. The caffeic acid or its derivative may be an isomer. The isomer may be, for example, a tautomer or a stereoisomer. The tautomer or the stereoisomer may be, for example, all theoretically possible tautomers or stereoisomers.

[0026] The salt of caffeic acid or its derivative is not particularly limited, and may be, for example, a pharmaceutically acceptable salt. The caffeic acid or its derivative may form, for example, an acid addition salt or a salt with a base depending on the type of substituent. The pharmaceutically acceptable salt is not particularly limited, and examples of the pharmaceutically acceptable salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt; aliphatic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, and triethanolamine salt; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, and isoquinoline salt; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt; arginine salt, lysine salt, aspartate salt, and glutamic acid salt. inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, and perchlorate; aliphatic organic acid or aromatic organic acid salts such as acetate, propionate, succinate, glycolate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate, hydroxymaleate, pyruvate, phenylacetate, benzoate, 4-aminobenzoate, anthranilate, 4-hydroxybenzoate, salicylate, 4-aminosalicylate, pamoate, gluconate, and nicotinate; sulfonate salts such as methanesulfonate, isethionate, ethanesulfonate, benzenesulfonate, halobenzenesulfonate, p-toluenesulfonate, toluenesulfonate, naphthalenesulfonate, sulfanilate, and cyclohexylsulfamate; and the like.

[0027] In the present disclosure, the agent may contain Artemisia capillaris extract and / or Honeysuckle extract as the caffeic acid or its derivative.

[0028] In this disclosure, "Artemisia capillaris" refers to a plant belonging to the species Artemisia capillaris Thunb. (Compositae) of the genus Artemisia in the family Asteraceae (Compositae). The herbal name for Artemisia capillaris is "Inchinkou," which refers to the flower head. Artemisia capillaris is used as a folk medicine for anti-inflammatory, choleretic, antipyretic, and diuretic purposes.

[0029] The Artemisia capillaris extract may be, for example, a plant extract. The plant extract can be produced, for example, by performing solvent extraction on Artemisia capillaris flowers. The Artemisia capillaris flowers include, for example, inflorescences, inflorescence spikes, and / or inflorescence-banded branches and leaves. The Artemisia capillaris extract may be, for example, homemade or a commercially available product. When the Artemisia capillaris extract is an extract of Artemisia capillaris flowers, the flowers may be harvested flowers themselves or processed products obtained by drying and / or pulverization. Examples of commercially available products include Falcorex Artemisia capillaris B (manufactured by Ichimaru Falcos Co., Ltd.) and Falcorex Artemisia capillaris E (manufactured by Ichimaru Falcos Co., Ltd.). For a method for producing the Artemisia capillaris extract, see the Examples described below.

[0030] Examples of the solvent used for extracting Artemisia capillaris 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; polyhydric alcohols or hydrous 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 (molecular weight 100 to 100,000); organic solvents such as acetone, ethyl acetate, diethyl ether, dimethyl ether, ethyl methyl ether, dioxane, 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.) whose normality has been appropriately adjusted. The solvent is preferably 1,3-butylene glycol. The solvent may be used alone or in combination of two or more. The extraction can be performed, for example, by immersing the plant in the solvent at room temperature (about 10°C to 30°C) for 5 to 10 days.

[0031] The extract of Artemisia capillaris may be a purified product after extraction. The purified product can be obtained, for example, by decomposition by adding an acid (e.g., hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or an alkali (e.g., 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 (e.g., ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity), filtration using filter paper, membrane filter, ultrafiltration membrane, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, decolorization, prolonged static storage, etc. The above treatments may be performed alone or in combination.

[0032] In the present disclosure, "hyaluronic acid" refers to a polysaccharide having a structure in which disaccharide units of glucuronic acid and N-acetylglucosamine are linked together. The salt of hyaluronic acid is not particularly limited, and may be any salt that is acceptable for food or pharmaceutically acceptable, such as sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, and ammonium salt.

[0033] Said hyaluronic acid can be, for example, extracted from cockscomb or other animal or plant tissue, and then concentrated or enzymatically treated, or can be produced by the fermentation of hyaluronic acid-producing microorganisms such as the microorganism of the genus Streptococcus.Above-mentioned hyaluronic acid can be, for example, either crude extract or purified product, but it is preferable to use the one with purity of 90% or more, because it is less likely to produce coloration or unpleasant odor when stored.

[0034] In the present disclosure, "maintaining the structure of hyaluronic acid" means maintaining the molecular weight of hyaluronic acid. The lower and upper limits of the molecular weight of hyaluronic acid can be found in the Examples described below. The lower limit of the molecular weight of hyaluronic acid is preferably 600 kDa, more preferably 1200 kDa. The upper limit of the molecular weight of hyaluronic acid is preferably 2000 kDa, more preferably 1600 kDa. The molecular weight of hyaluronic acid is, for example, 600 to 2000 kDa, preferably 1200 to 1600 kDa.

[0035] In this disclosure, "honeysuckle" refers to a plant belonging to the species Lonicera japonica in the genus Lonicera in the family Caprifoliaceae. The herbal name for honeysuckle is Nindo (honeysuckle) for its leaves and Kinginka (golden silver flower) for its flowers. Honeysuckle herbal medicine is used as a folk remedy for diuresis and fever reduction, and honeysuckle decoction is used as a gargle.

[0036] The honeysuckle extract may be, for example, a plant extract. The plant extract can be produced, for example, by subjecting the honeysuckle plant to solvent extraction. The honeysuckle plant may be, for example, a whole plant or a plant part. Examples of the plant part include leaves; flowers including inflorescences, spikes, and fringe branches and leaves; or a mixture thereof. The honeysuckle extract may be, for example, homemade or a commercially available product. Examples of commercially available products include the following: Falcorex Honeysuckle FB: an extract prepared from honeysuckle flowers using 1,3-butylene glycol solution as a solvent, manufactured by Ichimaru Falcos Co., Ltd. Falcorex Honeysuckle FE: an extract prepared from honeysuckle flowers using ethanol solution as a solvent, manufactured by Ichimaru Falcos Co., Ltd. Falcorex Honeysuckle SB: an extract prepared from honeysuckle leaves using 1,3-butylene glycol solution as a solvent, manufactured by Ichimaru Falcos Co., Ltd.

[0037] The agent for maintaining the structure of hyaluronic acid of the present disclosure may contain, for example, compounds such as cinnamic acid or its derivatives, p-coumaric acid or its derivatives, or ferulic acid or its derivatives, instead of the caffeic acid or its derivatives. These compounds are analogs of the caffeic acid or its derivatives, and are therefore expected to function as active ingredients (compounds) for maintaining the structure of hyaluronic acid. These compounds may be in the form of, for example, a salt, a hydrate, or a solvate, or may be an isomer. Specific examples of these compounds include the compounds shown in Figure 1.

[0038] The agent for maintaining the structure of hyaluronic acid of the present disclosure can be used for example by being used for administration subject, and can maintain the structure of hyaluronic acid.The use condition (administration condition) of the agent for maintaining the structure of hyaluronic acid of the present disclosure is not particularly limited, and for example, can suitably set administration form, administration time, dosage etc. according to the type etc. of administration subject.

[0039] The agent for maintaining the structure of hyaluronic acid of the present disclosure may be used, for example, in vivo or in vitro.

[0040] The subject of administration of the agent for maintaining the structure of hyaluronic acid of the present disclosure is not particularly limited.When the agent for maintaining the structure of hyaluronic acid of the present disclosure is used in vivo, the subject of administration can be, for example, human or non-human animals other than human.The non-human animals can be, for example, mammals such as mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, guinea pig, etc., birds, etc.When the agent for maintaining the structure of hyaluronic acid of the present disclosure is used in vitro, the subject of administration can be, for example, cells, tissues, organs, etc., the cells can be, for example, cells collected from living body, cultured cells, etc., and the tissue or organ can be, for example, tissues (living tissues) or organs collected from living body, etc.

[0041] In the following topical skin preparations (e.g., transdermal or skin application preparations or compositions) or the following orally administered preparations or orally administered compositions containing the agent for maintaining the structure of hyaluronic acid disclosed herein, the amount of the extract of Artemisia capillaris flower may be within the range that exerts the effect of maintaining the structure of hyaluronic acid, i.e., an effective amount.

[0042] The administration form of the agent for maintaining the structure of hyaluronic acid of the present disclosure can be oral or parenteral administration.The parenteral administration can be transdermal administration, application (contact) to the skin, etc.The application to the skin can also mean application to the oral mucosa, that is, application to or contact with epithelial cells in the oral cavity.Furthermore, the 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.The administration or injection into the skin via the skin surface can be carried out, for example, using a microneedle.

[0043] The dosage form of the agent for maintaining the structure of hyaluronic acid of the present disclosure is not particularly limited, and can be suitably determined according to the dosage form.The dosage form can be, for example, liquid or solid.When the dosage form is oral administration, the dosage form can be, for example, tablet, pill, capsule, granule, powder, liquid etc.

[0044] The agent for maintaining the structure of hyaluronic acid of the present disclosure can, for example, contain additives as necessary, and when used as composition, said additives preferably contain pharmaceutically acceptable additives or pharmaceutically acceptable carriers.Among said additives, there is no particular limitation, and for example, there can be listed base raw materials, excipients, colorants, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring agents such as perfumes, etc.In the present disclosure, the amount of said additives to be added is not particularly limited, as long as it does not hinder the effect of maintaining the structure of hyaluronic acid.

[0045] 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, macrogols such as Macrogol 6000, talc, titanium oxide, etc.

[0046] When the composition for use in maintaining the structure of hyaluronic acid of the present disclosure is an orally administered composition, specific examples of the orally administered composition include, for example, beverages, foods, pharmaceuticals (or similar), quasi-drugs (or similar), etc.

[0047] When the agent or composition for maintaining the structure of hyaluronic acid 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 can be ampule, capsule, powder, granule, liquid, gel, foam, emulsion, sheet, mist, spray, etc. according to the form of use.The form of use can be, for example, medicine (or medicines); quasi-drug (or medicines); topical or whole body external skin preparations; medicinal and / or cosmetic preparations that are applied to scalp and hair; bath additives that are used by being added 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.

[0048] The topical skin preparation can be prepared by optionally selecting and / or combining the following components and / or additives as needed, provided that the effect of maintaining the structure of hyaluronic acid is not impaired.

[0049] (1) Various oils and fats: avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange roughy 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.).

[0050] (2) Waxes: beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0051] (3) Mineral oils: liquid paraffin, petrolatum, paraffin, ozokerite, ceresin, microcrystalline wax, etc.

[0052] (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.

[0053] (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.

[0054] (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.

[0055] (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.

[0056] (8) Metallic soaps: aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0057] (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.

[0058] (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.

[0059] (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 acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: ergocalciferol Vitamin E group: vitamin E or its derivatives, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin P group (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0060] (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.

[0061] (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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] <HYBID Gene Expression Inhibitor or Composition> In another aspect, the present disclosure provides an agent or composition capable of inhibiting the expression of the HYBID gene. The HYBID gene expression inhibitor or composition of the present disclosure comprises a compound represented by the following formula (1) or a salt thereof, wherein the hyaluronic acid has a molecular weight of 600 to 2000 kDa: In the formula (1), R １ and R ２may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom.

[0066] The HYBID gene expression inhibitor or composition of the present disclosure is characterized by comprising a compound represented by formula (1) or a salt thereof, and other configurations and conditions are not particularly limited.The HYBID gene expression inhibitor or composition of the present disclosure comprises a compound represented by formula (1) or a salt thereof, and therefore can inhibit the degradation of hyaluronic acid by HYBID and maintain the structure of hyaluronic acid in the skin.Therefore, the HYBID gene expression inhibitor or composition of the present disclosure is expected to contribute to, for example, moisturizing the epidermis.The description of the compound in the HYBID gene expression inhibitor or composition of the present disclosure can be cited from the description of the agent or composition used to maintain the structure of hyaluronic acid of the present disclosure.

[0067] In the present disclosure, "HYBID" (Hyaluronan binding protein involved in hyaluronan depolymerization, KIAA1199) refers to a hyaluronan binding protein. HYBID is known to contribute to the degradation of hyaluronan. HYBID binds to high-molecular-weight (over 1000 kDa) hyaluronan and degrades it into intermediate-sized fragments with molecular weights of approximately 10 kDa.

[0068] <Method for maintaining the structure of hyaluronic acid> In another aspect, the present disclosure provides a method for maintaining the structure of hyaluronic acid.The method for maintaining the structure of hyaluronic acid of the present disclosure uses the agent or composition for maintaining the structure of hyaluronic acid of the present disclosure.The method for maintaining the structure of hyaluronic acid of the present disclosure uses the agent or composition for maintaining the structure of hyaluronic acid of the present disclosure, so it can maintain the structure of hyaluronic acid in skin.Therefore, the method for maintaining the structure of hyaluronic acid of the present disclosure is expected to contribute to, for example, moisturizing the epidermis.

[0069] The method for maintaining the structure of hyaluronic acid of the present disclosure includes an administration step of administering to a subject an agent and / or composition for use in maintaining the structure of hyaluronic acid of the present disclosure.

[0070] In the method for maintaining the structure of hyaluronic acid of the present disclosure, the administration step can be carried out, for example, in vitro or in vivo.The subject (administration subject) and administration conditions of the method for maintaining the structure of hyaluronic acid of the present disclosure can be, for example, the explanation of the administration subject and administration conditions of the agent or composition for maintaining the structure of hyaluronic acid of the present disclosure.

[0071] <Method for inhibiting expression of HYBID gene> In another aspect, the present disclosure provides a method for inhibiting the expression of the HYBID gene. The method for inhibiting expression of the HYBID gene of the present disclosure uses the expression inhibitor or composition of the HYBID gene of the present disclosure. Because the method for inhibiting expression of the HYBID gene of the present disclosure uses the expression inhibitor or composition of the HYBID gene of the present disclosure, it can inhibit the degradation of hyaluronic acid by HYBID and maintain the structure of hyaluronic acid in the skin. Therefore, the method for inhibiting expression of the HYBID gene of the present disclosure is expected to contribute to, for example, moisturizing the epidermis.

[0072] The method for inhibiting the expression of the HYBID gene of the present disclosure includes an administration step of administering to a subject an agent or composition for inhibiting the expression of the HYBID gene of the present disclosure.

[0073] In the method for inhibiting the expression of the HYBID gene disclosed herein, the administration step may be carried out, for example, in vitro or in vivo. The subject (administration subject) and administration conditions of the method for inhibiting the expression of the HYBID gene disclosed herein can be, for example, the same as the explanation of the administration subject and administration conditions of the agent or composition used to maintain the structure of hyaluronic acid disclosed herein.

[0074] <Use> In another aspect, the present disclosure relates to use of an agent and / or composition for maintaining hyaluronic acid structure. The present disclosure relates to use of an agent and / or composition for inhibiting the expression of the HYBID gene for inhibiting the expression of the HYBID gene.

[0075] The present disclosure relates to the use of an agent and / or composition for maintaining hyaluronic acid structure, for producing an agent and / or composition for maintaining hyaluronic acid structure.The present disclosure relates to the use of an agent and / or composition for inhibiting the expression of the HYBID gene, for producing an agent and / or composition for inhibiting the expression of the HYBID gene.

[0076] <Compound> In another embodiment, the compound of the present disclosure is a compound or a salt thereof represented by the following formula (2) or (3): The description of the compound of the present disclosure can be referenced to the description of the compound in the agent or composition for use in maintaining the structure of hyaluronic acid of the present disclosure.

[0077] The compound of the formula (2) or (3) can be produced, for example, by esterifying caffeic acid with 1,3-butylene glycol.

[0078] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. The percentages indicating the amount of active ingredient added are by weight unless otherwise specified.

[0079] <Materials> Normal human adult skin fibroblasts: KF-4109 (Kurabo Industries, Ltd.), cells from humans aged 40 years or older, hereinafter referred to as "AD cells". Normal human neonatal foreskin skin fibroblasts: KF-4009 (Kurabo Industries, Ltd.), hereinafter referred to as "NB cells". Artemisia capillaris flower extract: Falcorex Artemisia capillaris B (Ichimaru Pharcos, Inc.)

[0080] Reference Example 1 Maintenance of the structure of hyaluronic acid was investigated using the RT-PCR method.

[0081] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 5% FBS was used, and in this Reference Example 1, DMEM containing 0.25% FBS was used. ２ The cells were cultured at 37°C.

[0082] (2) Preparation of hyaluronic acid (HA) The following hyaluronic acids were prepared: H2: HA with a molecular weight of 1200 kDa to 1600 kDa M2: HA with a molecular weight of 600 kDa to 1120 kDa S2: HA with a molecular weight of 40 kDa to 80 kDa U2: HA with a molecular weight of 5 kDa to 10 kDa

[0083] (3) Confirmation of maintenance of hyaluronic acid structure (RT-PCR) 6 x 10 ４ NB cells were seeded into a 6-well plate and cultured until they reached 75% confluence. After the culture, the medium was replaced with DMEM containing 0.25% FBS, and after 24 hours of culture, the medium was replaced with new DMEM containing 0.25% FBS. HA was added to the NB cells after the culture replacement to a final concentration of 10 μg / mL, and the following experimental groups were prepared. (Experimental Groups) Control group: Group to which HA was not added H2-added group: Group to which H2 was added M2-added group: Group to which M2 was added S2-added group: Group to which S2 was added U2-added group: Group to which U2 was added

[0084] After preparing the experimental groups, CO ２ The cells were cultured in an incubator at 37°C for 1 to 2 hours. After 1 to 2 hours of culture, TNF-α was added to the experimental group (including the control group) to a final concentration of 1 ng / mL in order to induce inflammation in the cells. After the addition, CO２ The cells were cultured in an incubator at 37°C for 6 hours. mRNA was purified from the cultured cells. The mRNA was purified using a QIAshredder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. RT-PCR was then performed using primer pairs corresponding to each target factor, as shown in Table 1 below, to analyze changes in expression levels by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative changes in expression levels. Note that "MMP" in Table 1 below refers to matrix metalloproteinase.

[0085]

[0086] The results of mRNA quantification are shown in Table 2 below. Table 2 below shows values ​​when the value of the control group (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to one decimal place. In Table 2 below, ** indicates a significant difference (p<0.01) compared to the value of the control group in Dunnett's test, and *** indicates a significant difference (p<0.001) compared to the value of the control group in Dunnett's test.

[0087]

[0088] As shown in Table 2 above, it was found that in order to maintain the structure of hyaluronic acid, the lower limit of the molecular weight is preferably 600 kDa and the upper limit of the molecular weight is preferably 2000 kDa.

[0089] Reference Example 2 Histamine-induced expression of HYBID in NB cells was examined using RT-PCR.

[0090] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 5% FBS was used, and in Reference Example 2, DMEM containing 0.25% FBS was used. ２After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence, after which the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours, after which the medium was replaced with fresh DMEM containing 0.25% FBS.

[0091] (2) Experimental groups The following experimental groups were prepared using histamine, a substance that enhances hyaluronic acid decomposition activity, for the NB cells after the replacement. (Experimental groups) - No addition group: group to which histamine was not added - 0.1 addition group: group to which histamine was added to a final concentration of 0.1 μmol / L - 1.0 addition group: group to which histamine was added to a final concentration of 1.0 μmol / L - 10 addition group: group to which histamine was added to a final concentration of 10 μmol / L

[0092] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２ The cells were cultured at 37°C for 24 hours in an incubator. After the culture, mRNA was purified from the cells. The mRNA was purified using a QIAshredder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Subsequently, RT-PCR was performed using primer pairs corresponding to each target factor listed in Table 3 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). Furthermore, the amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative level changes.

[0093]

[0094] These results are shown in Table 4 below. Table 4 below shows values ​​when the value of the non-added group (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to two decimal places. In Table 4 below, ** indicates a significant difference (p<0.01) when compared with the value of the non-added group in Dunnett's test, and *** indicates a significant difference (p<0.001) when compared with the value of the non-added group in Dunnett's test.

[0095]

[0096] As shown in Table 4 above, it was found that the expression of HYBID in NB cells was enhanced by the addition of histamine (a substance that enhances hyaluronic acid decomposition activity).

[0097] Example 1 The expression of HYBID in NB cells induced by histamine when an extract of Artemisia capillaris flowers was added was examined using RT-PCR.

[0098] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 5% FBS was used, and in this Example 1, DMEM containing 0.25% FBS was used. ２ After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0099] (2) Experimental Groups The following experimental groups were prepared using an extract of Artemisia capillaris flower for the NB cells after replacement. (Experimental Groups) No addition group: group to which the extract and histamine were not added Control group: group to which the extract was not added and histamine was added 0.125 addition group: group to which the extract was added to a final concentration of 0.125% and histamine was added 0.25 addition group: group to which the extract was added to a final concentration of 0.25% and histamine was added 0.5 addition group: group to which the extract was added to a final concentration of 0.5% and histamine was added 1.0 addition group: group to which the extract was added to a final concentration of 1.0% and histamine was added

[0100] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 3 hours. After the culture, histamine was added to the experimental groups (except for the non-added group) to a final concentration of 10 µmol / L in order to induce inflammation in the cells. After the addition, CO ２ The cells were cultured at 37°C for 24 hours in an incubator. Histamine was added to enhance HYBID expression, as in Reference Example 2. mRNA was purified from the cultured cells. The mRNA was purified using QIAshreder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. RT-PCR was then performed using primer pairs corresponding to each target factor listed in Table 5 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0101]

[0102] These results are shown in Table 6 below. Table 6 below shows values ​​when the value of the non-added group (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to two decimal places. In Table 6 below, the mark ** indicates a significant difference (p<0.01) when compared with the value of the control group in the Dunnett test, and the mark *** indicates a significant difference (p<0.001) when compared with the value of the control group in the Dunnett test.

[0103]

[0104] As shown in Table 6 above, it was found that the addition of an extract of Artemisia capillaris flower suppressed the expression of HYBID in NB cells in an inflamed state.

[0105] Example 2: The expression of miR-600 in NB cells and AD cells was examined when Artemisia capillaris flower extract was added. Specifically, RT-PCR was used to examine whether the addition of Artemisia capillaris flower extract enhanced the expression of miR-600. MicroRNA (miRNA) is a short-stranded (20-25 base) RNA (non-coding RNA) that does not produce protein. The microRNA binds to the messenger RNA (mRNA) of a gene with the same sequence as itself, degrading the mRNA or inhibiting its translation into protein, thereby suppressing the expression of the gene. One such microRNA, miR-600, is an inhibitor of HYBID (Reference 1). Reference 1: Sun, Junfeng et al. “LncRNA TUG1 promoted KIAA1199 expression via miR-600 to accelerate cell metastasis and epithelial-mesenchymal transition in colorectal cancer.” Journal of experimental & clinical cancer research : CR vol. 37,1 106. 18 May. 2018, doi:10.1186 / s13046-018-0771-x

[0106] (1) Cells 6×10４ NB cells or AD cells were seeded into a 6-well plate and cultured until the cells reached 75% confluence. After the culture, the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours. After that, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0107] (2) Experimental Groups The following experimental groups were prepared using an extract of Artemisia capillaris flower on the NB cells or AD cells after the replacement. (Experimental Groups) - NB cell-free group: group to which the extract was not added to NB cells - AD cell-free group: group to which the extract was not added to NB cells - NB cell 1.0-added group: group to which the extract was added to a final concentration of 1.0% to NB cells - AD cell 1.0-added group: group to which the extract was added to a final concentration of 1.0% to NB cells

[0108] (3) Examination of miR-600 expression After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 6 hours. After the culture, total RNA was purified and extracted using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, Cat. No. / ID: 217684). After the purification and extraction, the expression level of miRNA (miR-600) was measured using real-time PCR. Complementary DNA (cDNA) was synthesized from the total RNA using the Mir-X™ miRNA First-Strand Synthesis Kit (Clontech Laboratories, Inc. (Takara Bio Inc.), product code: 638313, Takara code: Z8313N). After the synthesis, RT-PCR was performed using the Mir-X™ miRNA qRT-PCR SYBR Kit (manufactured by Clontech Laboratories, Inc.), the primers listed in Table 7 below, and the Thermal Cycler Dice Real Time System TP800 (manufactured by Clontech Laboratories, Inc. (Takara Bio Inc.)). Then, miRNA expression between the experimental groups was compared using the Delta-Delta-CT method.

[0109]

[0110] The thermal cycling conditions for the RT-PCR were as follows: Step 1: 40 cycles of two-step PCR consisting of 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds. Step 2: After Step 1, one cycle of dissociation steps consisting of 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds was performed.

[0111] These results are shown in Table 8 below. Table 8 below shows values ​​when the value of the group to which NB cells were not added (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to two decimal places. In Table 8 below, * indicates a significant difference (p<0.05) in comparison with the value of the group to which NB cells were not added in Student's t-test, and † indicates a significant difference (p<0.05) in comparison with the value of the group to which AD cells were not added in Student's t-test.

[0112]

[0113] As shown in Table 8, the expression level of miR-600 decreased in the group to which AD cells were not added compared to the group to which NB cells were not added. Furthermore, the expression level of miR-600 increased in the group to which AD cells or NB cells were added compared to the group to which AD cells or NB cells were not added. These results demonstrate that the addition of Artemisia capillaris flower extract enhances the expression of miR-600 in AD cells and NB cells.

[0114] Example 3 A composition containing an extract of Artemisia capillaris flowers was subjected to a skin monitoring test in humans.

[0115] (1) Preparation of Lotions First, a lotion containing the ingredients shown in Table 9 below (hereinafter also referred to as "lotion described in Table 9") and a lotion containing the ingredients shown in Table 10 below (hereinafter also referred to as "lotion described in Table 10") were prepared for use in the skin monitoring test. As shown in Table 9 below, the lotion described in Table 9 contains 1% of Artemisia capillaris flower extract.

[0116]

[0117]

[0118] (2) Human Skin Monitoring Test (External Skin Preparation) A human skin monitoring test was conducted on the lotion prepared in Example 3(1). Specifically, the test was conducted between January 31, 2022 and March 4, 2022, by applying the lotion shown in Table 9 and the lotion (placebo) shown in Table 10 to predetermined areas on the face of normal human subjects (13 men and women with an average age of 43.6 years). The lotion shown in Table 9 was applied to predetermined areas on one half of the subject's face at predetermined intervals, and the lotion shown in Table 10 was applied to predetermined areas on the other half of the subject's face at predetermined intervals. This predetermined interval was twice a day (morning and evening) for four weeks. The predetermined area was the "entire face."

[0119] FIG. 2 shows a schematic diagram of the skin monitoring test. Skin sagging was measured as shown in FIG. 2. Specifically, a staple (No. 11-1M (MAX Corporation)) was hooked onto the underside of adhesive tape with a donut-shaped hole in the center (DermaLab™ Combo (Cotex Technologies) adhesive tape for ELASTICITY probes). As shown in FIG. 2, the adhesive tape was attached at the intersection of a line drawn horizontally from under the nose and a line drawn vertically from the outer corner of the eye, where the inner lower part of the donut-shaped circle of the adhesive tape intersected.

[0120] The weight in Figure 2 is a weight (5 g to 20 g). The weight and clip were fixed with a string, and the clip was hooked onto the stapler pin. The weight was attached to each subject in advance, and the weight was heavy enough to move the cheek by 1 to 2 mm due to gravity.

[0121] To confirm the appearance of the face before and after the weight was applied, photographs of the left and right faces were taken using a VISIA™ Evolution (Canfield Scientific). The photographs were taken before the test and four weeks after the test. Images taken before and after the weight was applied were superimposed using photo editing software (Photoshop). Then, image analysis software (Digital Microscope VHX-5000 (Keyence Corporation)) was used to measure the adhesive tape movement distance and adhesive tape length before the test and after the weight was applied four weeks after the test. The adhesive tape movement distance after the weight was applied was calculated as an estimated converted value from the adhesive tape length. The measurement results are shown in Tables 11 and 12 below.

[0122]

[0123] Table 11 shows the results of measuring the distance traveled. In Table 11, the value before the test (week 0) is represented as 1. In Table 11, the mark * indicates a significant difference (p<0.05) in the Wilcoxon Rank-Sum Test compared with the value at week 0 (lotion application group in Table 9). As shown in Table 11, the measured value after 4 weeks of the test in which the lotion in Table 10 was applied was 1.13, while the measured value after 4 weeks of the test in which the lotion in Table 9 was applied was 0.71. These results demonstrate that the lotion in Table 9 containing an extract of Artemisia capillaris flower can suppress sagging of facial skin.

[0124]

[0125] Table 12 shows the results of measuring the length of the adhesive tape after 4 weeks of testing. In Table 12, * indicates a significant difference (p<0.05) in the Wilcoxon Rank-Sum Test compared with the value (100) of the lotion application group shown in Table 10. As shown in Table 12, when the value of the lotion application group shown in Table 10 was set to 100, the value of the lotion application group shown in Table 9 was 71.20. These results demonstrate that the lotion shown in Table 9, which contains an extract of Artemisia capillaris flower, can suppress sagging of facial skin.

[0126] (3) Human Skin Monitoring Test 2 (External Skin Preparation) A human skin monitoring test was conducted on the lotion prepared in Example 3(1). Specifically, the test was conducted between January 31, 2022 and March 4, 2022, by applying the lotion shown in Table 9 and the lotion (placebo) shown in Table 10 to predetermined areas on the face of normal human subjects (13 men and women with an average age of 43.6 years). The lotion shown in Table 9 was applied to predetermined areas on one half of the subject's face at predetermined intervals, and the lotion shown in Table 10 was applied to predetermined areas on the other half of the subject's face at predetermined intervals. This predetermined interval was twice a day (morning and evening) for four weeks. The predetermined area was the "entire face."

[0127] The size (total size) and depth of eyelid wrinkles were measured before application of the lotion (week 0) and four weeks after application of the lotion. The eyelids included the upper eyelid and the outer corner of the eye. The presence or absence of eyelid wrinkles was measured using an ANTERA 3D (registered trademark, Gadelius Medical Co., Ltd.).

[0128] The measurements were performed by setting the average value of the measurement results of the subjects in each group before application of the lotion as "100," and calculating the measurement results of the subjects in each group four weeks after application of the lotion as relative values. In the measurements, the value (100) before application of the lotion was determined by measuring three times at one site per person, calculating the average value of the three measurements, and collecting and aggregating the calculated average values ​​for 13 people, and the average value of these values ​​was set to 100. The measurement results four weeks after application of the lotion were calculated as the rate of change compared to the value before application of the lotion. These results are shown in Table 13 below.

[0129]

[0130] Table 13 shows the results of measuring the presence or absence of eyelid wrinkles 4 weeks after application of the lotion. In Table 13, the * mark indicates a significant difference (p<0.05) in the Wilcoxon Rank-Sum Test compared with the value (100) of the lotion application group shown in Table 10. As shown in Table 13, when the value of the lotion application group shown in Table 10 was set to 100, the value of the lotion application group shown in Table 9 was 93.51. These results demonstrate that the lotion shown in Table 9, which contains an extract of Artemisia capillaris flower, reduces the size and depth of wrinkles.

[0131] (4) Human Skin Monitoring Test 3 (External Skin Preparation) A human skin monitoring test was conducted on the lotion prepared in Example 3(1). Specifically, the test was conducted between January 31, 2022 and March 4, 2022, by applying the lotion shown in Table 9 and the lotion (placebo) shown in Table 10 to predetermined areas on the face of normal human subjects (13 men and women with an average age of 43.6 years). The lotion shown in Table 9 was applied to predetermined areas on one half of the subject's face at predetermined intervals, and the lotion shown in Table 10 was applied to predetermined areas on the other half of the subject's face at predetermined intervals. This predetermined interval was twice a day (morning and evening) for four weeks. The predetermined area was the "entire face."

[0132] The moisture distribution of the skin was measured before application of the lotion (week 0) and 4 weeks after application of the lotion. The measurement was performed using a Corneometer (CM825, manufactured by Courage+Khazaka). When the average moisture distribution measured for the 13 subjects at week 0 (before application) for the lotion application group shown in Table 9 or the lotion application group shown in Table 10 was set to 100, the moisture distribution measured for the lotion application group shown in Table 9 at week 0 (before application) was 107.73, and the moisture distribution measured for the lotion application group shown in Table 10 at week 4 after application was 98.98. These results demonstrate that the lotion shown in Table 9 containing Artemisia capillaris flower extract has the effect of maintaining a predetermined skin moisture content.

[0133] Example 4 The expression of HYBID in NB cells induced by histamine when an extract of Artemisia capillaris flower or an extract of Honeysuckle flower was added was examined using RT-PCR.

[0134] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 5% FBS was used, and in Example 4, DMEM containing 0.25% FBS was used. ２ After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0135] (2) Experimental Groups: The following experimental groups were prepared using Artemisia capillaris flower extract or Honeysuckle extract for the NB cells after replacement. The Artemisia capillaris flower extract and Honeysuckle extract used were as follows:・Artemisia capillaris flower extract: Falcorex artemisia B, manufactured by Ichimaru Falcos Co., Ltd. ・Honeysuckle flower extract: Falcorex honeysuckle FB, manufactured by Ichimaru Falcos Co., Ltd. (Experimental groups) ・No addition group: Group to which the extract and histamine were not added ・Control group: Group to which the extract was not added and histamine was added ・0.125 Honeysuckle FB addition group: Group to which Falcorex honeysuckle FB was added to a final concentration of 0.125% and histamine was added ・0.25 Honeysuckle FB addition group: Group to which Falcorex honeysuckle FB was added to a final concentration of 0.25% ・0.5 Honeysuckle FB addition group: Group to which Falcorex honeysuckle FB was added to a final concentration of 0.5% and histamine was added 1.0 Honeysuckle FB addition group: A group to which Falcorex Honeysuckle FB was added to a final concentration of 1.0% and histamine was added. 0.125 Artemisia capillaris B addition group: A group to which Falcorex Artemisia B was added to a final concentration of 0.125% and histamine was added. 0.25 Artemisia capillaris B addition group: A group to which Falcorex Artemisia B was added to a final concentration of 0.25% and histamine was added. 0.5 Artemisia capillaris B addition group: A group to which Falcorex Artemisia B was added to a final concentration of 0.5% and histamine was added. 1.0 Artemisia capillaris B addition group: A group to which Falcorex Artemisia B was added to a final concentration of 1.0% and histamine was added.

[0136] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 3 hours. After the culture, histamine was added to the experimental groups (except for the non-added group) to a final concentration of 10 µmol / L in order to induce inflammation in the cells. After the addition, CO ２The cells were cultured at 37°C for 24 hours in an incubator. Histamine was added to enhance HYBID expression. mRNA was purified from the cultured cells. The mRNA was purified using QIAshreder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. RT-PCR was then performed using primer pairs corresponding to each target factor listed in Table 14 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0137]

[0138] These results are shown in Table 15 below. Table 15 below shows values ​​when the value of the non-added group (change in expression level in the relative quantification) is set to 1. In addition, in Table 15 below, * indicates a significant difference (p<0.05) when compared with the value of the non-added group in Dunnett's test, and *** indicates a significant difference (p<0.001) when compared with the value of the non-added group in Dunnett's test.

[0139]

[0140] As shown in Table 15, it was found that the expression of HYBID was suppressed in inflammatory NB cells by the addition of Artemisia capillaris flower extract or honeysuckle flower extract.

[0141] Example 5 The expression of HYBID in NB cells induced by IL-1β when an extract of Artemisia capillaris flower or an extract of Honeysuckle flower was added was examined using RT-PCR.

[0142] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 5% FBS was used, and in Example 5, DMEM containing 0.25% FBS was used. ２After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0143] (2) Experimental Groups: The following experimental groups were prepared using Artemisia capillaris flower extract or Honeysuckle extract for the NB cells after replacement. The Artemisia capillaris flower extract and Honeysuckle extract used were as follows: Artemisia capillaris flower extract: Falcorex artemisia B, manufactured by Ichimaru Falcos Co., Ltd. Honeysuckle flower extract: Falcorex honeysuckle FB, manufactured by Ichimaru Falcos Co., Ltd. Honeysuckle leaf extract: Falcorex honeysuckle SB, manufactured by Ichimaru Falcos Co., Ltd. (Experimental groups) No addition group: Group to which the extract and IL-1β were not added Control group: Group to which the extract was not added and IL-1β was added 1.0 Honeysuckle FB addition group: Group to which Falcorex honeysuckle FB was added to a final concentration of 1.0% and IL-1β was added 1.0 Honeysuckle SB addition group: Group to which Falcorex honeysuckle SB was added to a final concentration of 1.0% and IL-1β was added 1.0 Artemisia capillaris B addition group: A group to which Falcorex Artemisia capillaris B was added to a final concentration of 1.0% and IL-1β was added.

[0144] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 3 hours. After the culture, IL-1β was added to the experimental groups (except for the non-added group) to a final concentration of 50 ng / ml in order to induce inflammation in the cells. After the addition, CO ２The cells were cultured for 24 hours at 37°C in an incubator. IL-1β was added to enhance HYBID expression. mRNA was purified from the cultured cells. The mRNA was purified using QIAshredder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Subsequently, RT-PCR was performed using primer pairs corresponding to each target factor listed in Table 16 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative changes in expression levels.

[0145]

[0146] These results are shown in Table 17 below. Table 17 below shows values ​​when the value of the non-added group (change in expression level in the relative quantification) is set to 1. In addition, in Table 17 below, ** indicates a significant difference (p<0.01) when compared with the value of the non-added group in Dunnett's test, and *** indicates a significant difference (p<0.001) when compared with the value of the non-added group in Dunnett's test.

[0147]

[0148] As shown in Table 17, it was found that the expression of HYBID was suppressed in inflammatory NB cells by the addition of Artemisia capillaris flower extract, honeysuckle flower extract, or honeysuckle leaf extract.

[0149] Example 6 Using collagen gel, it was investigated whether the maintenance of the structure of the water-soluble gel differs depending on the molecular size of hyaluronic acid.

[0150] (1) Gel A gel was prepared using a collagen gel culturing kit (Nitta Gelatin Co., Ltd., 638-00781) according to the instructions included in the kit. For experiments using collagen gel, see, for example, Reference 2 below. Reference 2: Takahashi, Yu et al. "Drug cytotoxicity screening using human intestinal organoids propagated with extensive cost-reduction strategies." Scientific Reports, Vol. 13, 1, 5407, 3 Apr. 2023, doi:10.1038 / s41598-023-32438-2

[0151] (2) Hyaluronic acid The following HAs were prepared: H2: HA with a molecular weight of 1200 kDa to 1600 kDa U2: HA with a molecular weight of 5 kDa to 10 kDa

[0152] (3) Experimental Groups The HA was added to the gel to a final concentration of 50 μg / ml to prepare the following experimental groups. (Experimental Groups) H2-Added Group: Group to which H2 was added U2-Added Group: Group to which U2 was added

[0153] (4) Measurement of gel height After preparing the experimental group, the experimental group was left to stand at 37° C. for 30 minutes. After the standing, the state of the gel in the experimental group was visually confirmed, and the height of the gel was measured. These results are shown in FIG.

[0154] Figure 3 is a photograph showing the state of each gel. If the gel height in the H2-added group is 100, the gel height in the U2-added group was 90. As shown in Figure 3, the gel height in the U2-added group was lower than that in the H2-added group, as indicated by the arrow. These results indicate that the U2-added group has a lower ability to retain water molecules than the H2-added group, and is unable to maintain the structure of the water-soluble gel. It also suggests that the molecular weight of HA is important for maintaining the gel structure.

[0155] Example 7 The expression of HYBID in NB cells when an extract of Artemisia capillaris flowers was added was examined using the RT-PCR method.

[0156] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 10% FBS was used, and in Example 7, DMEM containing 0.25% FBS was used. ２ After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0157] (2) Experimental Groups After the replacement, the following experimental groups were prepared using an extract of Artemisia capillaris flower for the NB cells. (Experimental Groups) No addition group: A group to which the extract was not added 1.0 addition group: A group to which Artemisia capillaris flower extract was added to a final concentration of 1.0 μmol / L

[0158] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 3 hours. After the culture, the cells were replaced with fresh DMEM containing 0.25% FBS. After the replacement, the cells were incubated in CO ２ The cells were cultured in an incubator at 37°C for 24 hours. mRNA was purified from the cultured cells. The mRNA was purified using QIAshredder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. RT-PCR was then performed using primer pairs corresponding to each target factor listed in Table 18 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative level changes.

[0159]

[0160] These results are shown in Table 19 below. Table 19 below shows values ​​when the value of the non-added group (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to two decimal places. In Table 19 below, the *** symbol indicates a significant difference (p<0.001) compared to the value of the non-added group in the Student's t-test.

[0161]

[0162] As shown in Table 19 above, it was found that the addition of an extract of Artemisia capillaris flower suppressed the expression of HYBID in NB cells.

[0163] Example 8 The expression of HYBID in NB cells or AD cells when an extract of Artemisia capillaris flowers was added was examined using the RT-PCR method.

[0164] (1) Cells NB cells and AD cells were prepared. For pre-culture, DMEM containing 10% FBS was used, and in Example 8, DMEM containing 0.25% FBS was used. 5% CO ２ After the incubation, 6 × 10 ４ NB cells or AD cells were seeded into a 6-well plate and cultured until the cells reached 75% confluence. After the culture, the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours. After that, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0165] (2) Experimental Groups The following experimental groups were prepared using an extract of Artemisia capillaris flower on the NB cells or AD cells after the replacement. (Experimental Groups) NB-free group: A group of NB cells to which the extract was not added AD-free group: A group of AD cells to which the extract was not added AD1.0-added group: A group to which Artemisia capillaris flower extract was added to a final concentration of 1.0 μmol / L

[0166] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, ２The cells were cultured in an incubator at 37°C for 3 hours. After the culture, the cells were replaced with fresh DMEM containing 0.25% FBS. After the replacement, the cells were incubated in CO ２ The cells were cultured in an incubator at 37°C for 24 hours. mRNA was purified from the cultured cells. The mRNA was purified using QIAshredder and RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. RT-PCR was then performed using primer pairs corresponding to each target factor listed in Table 20 below, and changes in expression levels were analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (Takara Bio Inc.). The amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0167]

[0168] These results are shown in Table 21 below. Table 21 below shows values ​​when the value of the NB-free group (change in expression level in the relative quantification) is set to 1. The values ​​are rounded to one decimal place. In Table 21 below, the *** symbol indicates a significant difference (p<0.001) compared with the value of the AD-free group in Dunnett's test.

[0169]

[0170] As shown in Table 21, it was found that the addition of Artemisia capillaris flower extract suppressed the expression of HYBID in AD cells. It was also found that the addition of Artemisia capillaris flower extract in AD cells resulted in the same level of HYBID expression as in NB cells.

[0171] Example 9 The distribution of histamine in NB cells was examined when an extract of Artemisia capillaris flower was added.

[0172] (1) NB cells NB cells were prepared. For pre-culture, DMEM containing 10% FBS was used, and in Example 9, DMEM containing 0.25% FBS was used. 5% CO ２ After the incubation, 6 × 10 ４ NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0173] (2) Experimental Groups The following experimental groups were prepared using the Artemisia capillaris flower extract for the NB cells after replacement. (Experimental Groups) No addition group: group to which the extract and histamine were not added Control group: group to which the extract was not added but histamine was added 0.5 addition group: group to which the extract was added to a final concentration of 0.5% 1.0 addition group: group to which the extract was added to a final concentration of 1.0%

[0174] (3) Examination of hyaluronic acid distribution (HPLC) After preparing the experimental groups, ２ The cells were cultured in an incubator at 37°C for 3 hours. After the culture, histamine was added to the experimental groups (except for the non-added group) to a final concentration of 10 µmol / L in order to induce inflammation in the cells. After the addition, CO ２ The cells were cultured in an incubator at 37°C for 2 hours. Histamine was added to enhance the expression of HYBID. Fluoresceinamine-labeled sodium hyaluronate (H2, average molecular weight 1.2 million to 1.6 million, FAHA-H2, Iwai Chemicals Co., Ltd.) was added to the cultured cells. After the addition, CO ２The cells were cultured in an incubator at 37°C for 48 hours. After the culture, the culture medium was collected. For experiments using FAHA, see, for example, Reference 3 (Figure 4) and Reference 4 (Figure 1, Supplemental Figure 1). The distribution of hyaluronic acid in the culture medium was examined by HPLC. The HPLC conditions were as follows. The results are shown in Figure 4. Reference 3: Tobisawa, Yuki et al. “The cell surface hyaluronidase TMEM2 is essential for systemic hyaluronan catabolism and turnover.” The Journal of biological chemistry vol. 297,5 (2021): 101281. doi:10.1016 / j.jbc.2021.101281 Reference 4: Yoshida, Hiroyuki et al. al. “KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,14 (2013): 5612-7. doi:10.1073 / pnas.1215432110

[0175] Using the culture medium, the distribution of hyaluronic acid was examined by HPLC under the following measurement conditions. The results are shown in Figure 4 and Table 22 below. (HPLC conditions) Apparatus: SHIMADZU LC-20A Prominence (Shimadzu Corporation) Column: TSKgel G5000PWXL (Tosoh Corporation) Eluent: 0.2 mol / L NaCl Flow rate: 0.5 mL / min Detector wavelength: Excitation wavelength 490 nm, Fluorescence wavelength 525 nm

[0176]

[0177] Figure 4 is a graph showing the results of HPLC analysis. In Figure 4, the vertical axis represents fluorescence intensity, and the horizontal axis represents time (minutes). Table 22 shows the peak-top fluorescence intensity values ​​for each group shown in Figure 4. Table 22 shows values ​​when the value (fluorescence intensity) of the non-added group is set to 100. The values ​​are rounded to the fourth decimal place. As shown in Figure 4 and Table 22, the peak-top fluorescence intensity was lower in the histamine-added groups (control group, 0.5-added group, 1.0-added group) compared to the non-added group. These results suggest that histamine promotes HYBID, and that HYBID decomposes a certain amount of hyaluronic acid into fragments.

[0178] [Example 10] It was confirmed that caffeic acid derivatives inhibit HYBID production.

[0179] (1) Testing of Component Fraction 1 and HYBID Gene Expression Levels Components contained in Artemisia capillaris flower extract were analyzed. Specifically, Artemisia capillaris flower extract (ACFE) was fractionated according to the scheme shown in Figure 5. The raw material used for the fractionation was purchased from Tochimoto Tenkaido Co., Ltd. Specifically, the fractionation was performed by adding water to an extract (ACFE) obtained by extracting Artemisia capillaris flower heads with 30% butylene glycol (BG). After the addition, the resulting solution was converted to a 15% BG aqueous solution, which was then subjected to crude fractionation using a column packed with Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), yielding four fractions, Fr. 1 to Fr. 4. The HYBID expression inhibitory activity of these four fractions was then examined. In this study, HYBID gene expression was confirmed in the same manner as in Example 1, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used and the four fractions were used instead of the extract. These results are shown in Figure 6.

[0180] Figure 6 is a graph showing the expression level of the HYBID gene when histamine was added. In Figure 6, the vertical axis shows the relative expression level of the HYBID gene, with the non-addition group (NT) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 6, the expression level of the HYBID gene was reduced in the Fr. 2 fraction compared to the control group. These results demonstrate that the Fr. 2 fraction contains a component that inhibits HYBID production.

[0181] (2) Testing of Component Fraction 2 and HYBID Gene Expression Levels The components contained in Fr. 2 were further fractionated using preparative HPLC under the following measurement conditions. Four fractions, Fr. 2-1, Fr. 2-2, Fr. 2-3, and Fr. 2-4, were obtained through this fractionation. The HYBID expression inhibitory activity of these four fractions was then examined. The HYBID gene expression was confirmed in the same manner as in Example 1, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used and the four fractions were used instead of the extract. These results are shown in Figures 7 and 8.

[0182] Figure 7 is a graph showing the expression level of the HYBID gene upon the addition of histamine. In Figure 7, the vertical axis shows the relative expression level of the HYBID gene, with the non-addition group (NT) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 7, the expression level of the HYBID gene was reduced in the Fr. 2-2 and Fr. 2-3 fractions compared to the control group. These results demonstrate that the Fr. 2-2 and Fr. 2-3 fractions contain components that inhibit HYBID production.

[0183] Figure 8 is a graph showing the level of HYBID gene expression upon the addition of histamine. In Figure 8, the vertical axis shows the relative value of the level of HYBID gene expression, with the non-addition group (NT) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 8, the level of HYBID gene expression decreased in a concentration-dependent manner in the Fr. 2-2 fraction. These results demonstrate that the Fr. 2-2 fraction contains a component that inhibits HYBID production, and that the HYBID production is inhibited in a concentration-dependent manner by the Fr. 2-2 fraction.

[0184] (3) Structural Analysis Structural analysis of Fr. 2-2 and Fr. 2-3, which consist of single components, was carried out by NMR analysis using the following analytical conditions. As a result of the analysis, Fr. 2-2 (Compound 1) was identified as 1-caffeoyl-3-hydroxybutane represented by chemical formula (2), and Fr. 2-3 (Compound 2) was identified as 3-caffeoyl-1-hydroxybutane represented by chemical formula (3). The NMR measurement values ​​are shown below. The 1-caffeoyl-3-hydroxybutane was obtained as a yellow solid, and the 3-caffeoyl-1-hydroxybutane was obtained as a yellow oil. (NMR analysis conditions) Optical rotations: JASCO P-1020 polarimeter (JASCO Corporation) Solvent: MeOH UV spectra: Shimadzu UV-3100 spectrometer (Shimadzu Corporation) Solvent: MeOH HRESIMS: Shimadzu LCMS-IT-TOF spectrometer (Shimadzu Corporation) Ion mode: negative ion mode NMR: JEOL JNM-ECA-500 spectrometer (JEOL Ltd.) Solvent: MeOH-d4

[0185] (NMR measurement values) 1-Caffeoyl-3-hydroxybutane (Compound 1): [α] D +4.0° (c = 0.1 g / dL, MeOH). UV λMeOH max nm (logε): 218 (4.11), 243 (3.97), 329 (4.20). calcd. for C 13 H 1５ O5 251.0925 (MH), found 251.0911. 1H NMR (500 MHz, MeOH-d4): δ = 7.53 (d, J = 15.8 Hz, 1H), 7.03 (d, J = 1.9 Hz, 1H), 6.94 (dd, J = 8.0, 1.9 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.25 (d, J = 15.8 Hz, 1H), 4.27 (br t, J = 6.5 Hz, 2H), 3.90 (dqd, J = 8.0, 6.3, 4.6 Hz, 1H), 1.83 (dtd, 14.2, 7.1, 4.6 Hz, 1H), 1.77 (ddt, 14.2, 8.0, 6.0 Hz, 1H), 1.21 (d, J = 6.3 Hz, 3H). 13 C NMR (125 MHz, MeOH-d4): δ = 169.4, 149.6, 146.9 (2C), 127.7, 122.9, 116.5, 115.10, 115.07, 65.5, 62.7, 39.0, 23.8.

[0186] 3-Caffeoyl-1-hydroxybutane (Compound 2):[α] D +3.6° (c = 0.1 g / dL, MeOH). UV λMeOH max nm (logε): 217 (3.99), 234 (3.80), 243 (3.80), 300 (3.89), 328 (3.98). calcd. for C 13 H 15 O5 251.0925 (M-H), found 251.0911. 1H NMR (500 MHz, MeOH-d4): δ = 7.52 (d, J = 16.0 Hz, 1H), 7.03 (d, J = 1.9 Hz, 1H), 6.94 (dd, J = 8.2, 1.9 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 6.24 (d, J = 16.0 Hz, 1H), 5.12 (dqd, J = 8.2, 6.3, 4.9 Hz, 1H), 3.62 (br t, J = 6.6 Hz, 2H), 1.89 (ddt, 14.0, 8.2, 6.6 Hz, 1H), 1.81 (dtd, 14.0, 6.6, 4.9 Hz, 1H), 1.30 (d, J = 6.3 Hz, 3H). 13 C NMR (125 MHz, MeOH-d4): δ = 169.0, 149.6, 146.8, 146.7, 127.7, 122.9, 116.5, 115.5, 115.1, 69.5, 59.3, 39.9, 20.6.

[0187] (4) Testing HYBID Gene Expression Levels Using Identified Compounds The isolated Compounds 1 and 2 were further examined for their inhibitory effect on HYBID expression. The HYBID gene expression was confirmed in the same manner as in Example 1, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used and Compound 1, Compound 2, and Fraction 2 were used instead of the extract. The results are shown in Figure 9.

[0188] 9 is a graph showing the expression level of HYBID gene upon the addition of histamine. In FIG. 9, the vertical axis shows the relative expression level of HYBID gene, with the non-addition group (NT) set at 100, and the horizontal axis shows the experimental group. As shown in FIG. 9, the expression level of HYBID gene was reduced in Compound 1, Compound 2, and Fr. 2 fractions compared to the control group. These results indicate that Compound 1 and Compound 2 are compounds that suppress HYBID production in inflammatory NHDF cells.

[0189] (5) HYBID Gene Expression Test The inhibitory effects of extract (ACFE), Fr. 2 fraction, and isolated Compound 1 from Artemisia capillaris flower heads were examined. Specifically, HYBID gene expression was confirmed in the same manner as in Example 7, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used and ACFE, Fr. 2 fraction, and Compound 1 were used instead of the extract. These results are shown in Figure 10.

[0190] Figure 10 is a graph showing the expression level of the HYBID gene. In Figure 10, the vertical axis shows the relative expression level of the HYBID gene, with the control group (group to which no extract was added, Control) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 10, the expression level of the HYBID gene was reduced in ACFE, Fr. 2 fraction, and Compound 1 compared to the control group. These results demonstrate that ACFE, Fr. 2 fraction, and Compound 1 suppress HYBID production.

[0191] [Example 11] It was confirmed that Artemisia annua extract inhibits HYBID production.

[0192] (1) Expression level test of miR-486-5p in NB cells using Artemisia capillaris flower extract. miR-486-5p is known as an upstream microRNA that directly targets the 3'-UTR region of HYBID (Reference 5). Therefore, we investigated whether Artemisia capillaris flower extract suppresses the expression of miR-486-5p. Specifically, 6 x 10 ４NB cells were seeded into a 6-well plate and cultured until 75% confluent. After the culture, the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours. The medium was then replaced with fresh DMEM containing 0.25% FBS. After the replacement, the cells were cultured for 6 hours in the presence or absence of AFCF. After the culture, total RNA was purified and extracted using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN) to measure the expression of miR-486-5p. After the purification and extraction, the expression level of miRNA (miR-486-5p) was measured using real-time PCR. Complementary DNA (cDNA) was synthesized from the total RNA using the Mir-X™ miRNA First-Strand Synthesis Kit (Clontech Laboratories, Inc. (Takara Bio Inc.), product code: 638313, Takara code: Z8313N). After the synthesis, RT-PCR was performed using the Mir-X (registered trademark) miRNA qRT-PCR SYBR Kit (manufactured by Clontech Laboratories, Inc.), the primers listed in Table 23 below, and the Thermal Cycler Dice Real Time System TP800 (manufactured by Clontech Laboratories, Inc. (Takara Bio Inc.)). Then, miRNA expression between the experimental groups was compared using the Delta-Delta-CT method. The results are shown in FIG. 11. Reference 5: Jiao, Xuehua et al. “KIAA1199, a Target of MicoRNA-486-5p, Promotes Papillary Thyroid Cancer Invasion by Influencing Epithelial-Mesenchymal Transition (EMT).” Medical science monitor: international medical journal of experimental and clinical research vol. 25 6788-6796. 10 Sep. 2019, doi:10.12659 / MSM.918682

[0193]

[0194] Figure 11 is a graph showing the expression level of miR-486-5p. In Figure 11, the vertical axis shows the relative expression level of miR-486-5p, with the control group (group without extract, Control) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 11, the expression level of miR-486-5p increased in the ACFE-added group compared to the control group. These results demonstrate that ACFE increases the expression level of miR-486-5p and suppresses HYBID production in NB cells.

[0195] (2) Testing the Expression Level of miR-486-5p in AD Cells by Artemisia capillaris flower extract We investigated whether Artemisia capillaris flower extract also suppresses the expression of miR-486-5p in cells other than NHDF cells. Specifically, the same method as in Example 11(1) was used, except that AD cells were used instead of NB cells. These results are shown in Figure 12.

[0196] Figure 12 is a graph showing the expression level of miR-486-5p. In Figure 12, the vertical axis shows the relative expression level of miR-486-5p, with the control group (group without extract, Control) set at 100, and the horizontal axis shows the experimental group. As shown in Figure 12, the expression level of miR-486-5p increased in the ACFE-added group compared to the control group. These results demonstrate that ACFE increases the expression level of miR-486-5p and suppresses HYBID production in AD cells.

[0197] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described 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.

[0198] This application claims priority based on Japanese Patent Application No. 2023-214475 filed on December 20, 2023, and Japanese Patent Application No. 2024-016827 filed on February 7, 2024, the disclosures of which are incorporated herein in their entireties.

[0199] <Supplementary Notes> Some or all of the above embodiments and examples can be described as the following supplementary notes, but are not limited to the following: <Agent for maintaining the structure of hyaluronic acid> (Supplementary Note 1) An agent for maintaining the structure of hyaluronic acid, comprising a compound represented by the following formula (1) or a salt thereof, wherein the hyaluronic acid has a molecular weight of 600 to 2000 kDa: In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom. １ is a hydrogen atom or a hydroxy group. ２ is a hydrogen atom, a hydroxy group, or a methyloxy group. ３ is a linear or branched hydroxyalkyl group having 1 to 5 carbon atoms, a linear or branched alkyl group having 1 to 5 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms. １ is a hydroxy group, and ２ is a hydroxy group, and ３is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted. (Appendix 6) The agent according to any of Appendices 1 to 5, wherein the compound represented by formula (1) or the salt thereof is a compound represented by the following formula (2) or (3) or a salt thereof: (Appendix 7) The agent according to any one of Appendices 1 to 6, wherein the hyaluronic acid has a molecular weight of 1200 to 1600 kDa. (Appendix 8) The agent according to any one of Appendices 1 to 7, comprising an extract of Artemisia capillaris and / or an extract of Japanese honeysuckle as the compound represented by formula (1) or a salt thereof. <An agent for suppressing expression of HYBID gene> (Appendix 9) An agent for use in suppressing expression of HYBID gene, comprising a compound represented by the following formula (1) or a salt thereof: In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted, or a hydrogen atom. １ is a hydrogen atom or a hydroxy group. ２ is a hydrogen atom, a hydroxy group, or a methyloxy group. ３ is a linear or branched hydroxyalkyl group having 1 to 5 carbon atoms, a linear or branched alkyl group having 1 to 5 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms. １ is a hydroxy group, and ２ is a hydroxy group, and ３is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cyclic alkyl group having 3 to 10 carbon atoms which may be substituted. (Appendix 14) The agent according to any of Appendices 9 to 13, wherein the compound represented by formula (1) or the salt thereof is a compound represented by the following formula (2) or (3) or a salt thereof: (Appendix 15) The agent according to any one of Appendices 9 to 14, comprising an extract of Artemisia capillaris and / or an extract of honeysuckle as the compound represented by the following formula (1) or its salt. <Method for maintaining the structure of hyaluronic acid> (Appendix 16) A method for maintaining the structure of hyaluronic acid, using the agent according to any one of Appendices 1 to 8. (Appendix 17) The maintenance method according to Appendices 16, comprising an administration step of administering the agent to a subject. (Appendix 18) The maintenance method according to Appendices 16 or 17, wherein the agent is used in vitro or in vivo. <Method for suppressing expression of HYBID gene> (Appendix 19) A method for suppressing expression of HYBID gene, using the agent according to any one of Appendices 9 to 15. (Appendix 20) The expression suppression method according to Appendices 19, comprising an administration step of administering the agent to a subject. (Appendix 21) The method for inhibiting expression according to Appendix 19 or 20, wherein the agent is used in vitro or in vivo. <Use> (Appendix 22) Use of a compound represented by formula (1) according to any one of Appendixes 1 to 8 or a salt thereof for use in maintaining the structure of hyaluronic acid. (Appendix 23) Use of a compound represented by formula (1) according to any one of Appendixes 9 to 15 or a salt thereof for use in inhibiting the expression of the HYBID gene. <Compound> (Appendix 24) A compound represented by the following formula (2) or (3) or a salt thereof:

[0200] As described above, the present disclosure provides an agent for maintaining the structure of hyaluronic acid, which is extremely useful in the fields of cosmetics, topical skin preparations, etc.

Claims

1. An agent for maintaining the structure of hyaluronic acid, comprising a compound represented by the following formula (1) or a salt thereof, wherein the hyaluronic acid has a molecular weight of 600 to 2000 kDa: In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ represents a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms, or a hydrogen atom.

2. The above R １ The agent according to claim 1 , wherein is a hydrogen atom or a hydroxyl group.

3. The above R ２ The agent according to claim 1 or 2, wherein is a hydrogen atom, a hydroxyl group, or a methyloxy group.

4. The above R ３ is a linear or branched hydroxyalkyl group having 1 to 5 carbon atoms, a linear or branched alkyl group having 1 to 5 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms.

5. The above R １ is a hydroxy group, ２ is a hydroxy group, ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms.

6. The agent according to any one of claims 1 to 5, wherein the compound represented by formula (1) or a salt thereof is a compound represented by the following formula (2) or (3) or a salt thereof:

7. The agent according to any one of claims 1 to 6, wherein the hyaluronic acid has a molecular weight of 1200 to 1600 kDa.

8. The agent according to any one of claims 1 to 7, comprising an extract of Artemisia capillaris and / or an extract of Honeysuckle as the compound represented by formula (1) or a salt thereof.

9. An agent for use in suppressing expression of a HYBID gene, comprising a compound represented by the following formula (1) or a salt thereof: In the formula (1), R １ and R ２ may be the same or different and each independently represents a hydrogen atom, a hydroxyl group, or a linear or branched alkoxy group having 1 to 5 carbon atoms; R ３ represents a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms, or a hydrogen atom.

10. The above R １ The agent according to claim 9 , wherein is a hydrogen atom or a hydroxyl group.

11. The above R ２ The agent according to claim 9 or 10, wherein is a hydrogen atom, a hydroxyl group, or a methyloxy group.

12. The above R ３ is a linear or branched hydroxyalkyl group having 1 to 5 carbon atoms, a linear or branched alkyl group having 1 to 5 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms.

13. The above R １ is a hydroxy group, ２ is a hydroxy group, ３ is a linear or branched hydroxyalkyl group having 1 to 7 carbon atoms, a linear or branched alkyl group having 1 to 7 carbon atoms, or an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms.

14. The agent according to any one of claims 9 to 13, wherein the compound represented by formula (1) or a salt thereof is a compound represented by the following formula (2) or (3) or a salt thereof:

15. The agent according to any one of claims 9 to 14, comprising an extract of Artemisia capillaris and / or an extract of Honeysuckle as the compound represented by the following formula (1) or its salt:

16. A method for maintaining the structure of hyaluronic acid, comprising using an agent according to any one of claims 1 to 8.

17. The method of claim 16, further comprising administering to a subject the agent.

18. The method of claim 16 or 17, wherein the agent is used in vitro or in vivo.

19. A method for inhibiting expression of the HYBID gene, comprising using an agent according to any one of claims 9 to 15.

20. A method for inhibiting expression as described in claim 19, comprising an administration step of administering the agent to a subject.

21. The method for inhibiting expression according to claim 19 or 20, wherein the agent is used in vitro or in vivo.

22. A compound represented by the following formula (2) or (3), or a salt thereof:

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