Agent for use in maintaining the structure of hyaluronic acid

Abstract

Provided are agents and the like for use in maintaining the structure of hyaluronic acid. The agent of the present disclosure is an agent for maintaining the structure of hyaluronic acid, and contains 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. [Chemical Formula 1] JPEG0007701773000049.jpg40170

Description

Technical Field

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

Background Art

[0002] As the forefront directly affected by the external environment, the skin undertakes an important function of maintaining the internal environment of the living body. Therefore, although the functions of the skin do not completely stop, with aging, ultraviolet exposure, skin exposure to chemical substances, etc., the functions of the skin gradually decline, and aging symptoms such as wrinkles, spots, dullness, and sagging become apparent.

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

[0004] In vivo in humans (including the dermis of the skin, etc.), as a degradation mode of hyaluronic acid, for example, a KIAA1199 (HYBID)-dependent hyaluronic acid degradation mode can be mentioned (Non-Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, the present disclosure aims to provide an agent for maintaining the structure of hyaluronic acid and the like.

[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 contains a compound represented by the following formula (1) or a salt thereof. The hyaluronic acid has a molecular weight of 600 to 2000 kDa, agent:

Chemical formula

[0009] The agent of the present disclosure is an agent for suppressing the expression of the HYBID gene, and contains a compound represented by the following formula (1) or a salt thereof, agent:

Chemical formula

[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 suppressing the 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).

Chemical formula

Chemical formula

[0013] According to the present disclosure, for example, an agent for use in maintaining the structure of hyaluronic acid can be provided.

Brief Description of Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0015] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can incorporate the description of other disclosures.

[0016] <Agent or Composition for Use in Maintaining the Structure of Hyaluronic Acid> The agent or composition for use in maintaining the structure of hyaluronic acid of the present disclosure contains, as described above, 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.

Chemical Formula

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

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

[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. Specifically, the alkoxy group is, for example, a methyloxy group (methoxy group), an ethyloxy group (ethoxy group), a propyloxy group (propoxy group), an isopropyloxy group (isopropoxy group), a butyloxy group (butoxy group), etc. The butyl may be any of n-butyl, sec(s)-butyl, iso(i)-butyl, tert(t)-butyl (the same applies hereinafter). The R 1 is preferably, for example, a hydrogen atom or a hydroxy group. The R 2 is preferably a hydrogen atom, a hydroxy group, or a methyloxy group.

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

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

Chemical formula

[0022] As a specific example, the caffeic acid or a derivative thereof is, for example, in the compound of the formula (1), where the R 1 is a hydroxy group, 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 an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms, preferably a hydroxypropyl group, hydroxybutyl group, propyl group, butyl group, or cyclohexyl group. The substituent of the cyclic alkyl group can refer to the foregoing description, for example.

[0023] As a specific example, the derivative of caffeic acid is preferably, for example, 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.

Chemical formula

[0024] As a specific example, the derivative of caffeic acid is preferably, for example, 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.

Chemical formula

[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. Examples of the isomer include tautomers or stereoisomers. Examples of the tautomer or stereoisomer include all theoretically possible tautomers or stereoisomers.

[0026] The salt of the caffeic acid or its derivative is not particularly limited, and for example, it is a pharmaceutically acceptable salt. The caffeic acid or its derivative forms an acid addition salt or a salt with a base depending on the type of substituent. The pharmaceutically acceptable salt is not particularly limited. For example, the pharmaceutically acceptable salt includes 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; amino acid salts such as arginine salt, lysine salt, aspartate salt, and glutamate salt; inorganic acid salts such as hydrochloride salt, sulfate salt, nitrate salt, phosphate salt, carbonate salt, hydrogen carbonate salt, and perchlorate salt; aliphatic organic acid or aromatic organic acid salts such as acetate salt, propionate salt, succinate salt, glycolate salt, lactate salt, maleate salt, fumarate salt, tartrate salt, malate salt, citrate salt, ascorbate salt, hydroxymaleate salt, pyruvate salt, phenylacetate salt, benzoate salt, 4-aminobenzoate salt, anthranilate salt, 4-hydroxybenzoate salt, salicylate salt, 4-aminosalicylate salt, pamoate salt, gluconate salt, and nicotinate salt; sulfonate salts such as methanesulfonate salt, isethionate salt, ethanesulfonate salt, benzenesulfonate salt, halobenzenesulfonate salt, p-toluenesulfonate salt, toluenesulfonate salt, naphthalenesulfonate salt, sulfanilate salt, and cyclohexylsulfamate salt; and the like.

[0027] In the present disclosure, the agent may contain a mugwort extract and / or a honeysuckle extract as the caffeic acid or its derivative.

[0028] In the present disclosure, "mugwort" refers to the Asteraceae family (Asteraceae (Compositae)) Artemisia genus Artemisia ) of the Artemisia princeps species Artemisia capillaris Thunb . Compositae ) means a plant belonging thereto. The crude drug name of the Artemisia princeps is also called inchinko for the capitulum. The crude drug of the Artemisia princeps is used as a folk medicine for anti-inflammatory, cholagogic, antipyretic, and diuretic purposes.

[0029] The extract of the Artemisia princeps may be, for example, a plant extract. The plant extract can be produced, for example, by subjecting the flowers of the Artemisia princeps to solvent extraction. The flowers of the Artemisia princeps include, for example, capitula, flower spikes, and / or leafy branches with flowers. The extract of the Artemisia princeps may be prepared at home or a commercially available product may be used. When the extract of the Artemisia princeps is an extract of the flowers of the Artemisia princeps, the flowers may be the collected flowers themselves or processed products that have been dried and / or pulverized, etc. Examples of the commercially available products include Falcorex Artemisia princeps B (manufactured by Ichimaru Pharcos Co., Ltd.), Falcorex Artemisia princeps E (manufactured by Ichimaru Pharcos), etc. For the method for producing the extract of the Artemisia princeps, reference can be made to the examples described later.

[0030] The solvents used for extracting the kawarayomogi include, for example, 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; acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) or alkalis (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) with appropriately regulated concentrations; and the like. The solvent is preferably 1,3-butylene glycol. The solvent may be used, for example, alone or in combination of two or more. The extraction can be carried out, 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 the kawarayomogi may be a purified product after extraction. The purified product can be obtained, for example, by decomposition by adding an acid (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, organic acid, etc.) or an alkali (sodium hydroxide, calcium hydroxide, ammonia, etc.), fermentation or metabolic conversion by microorganisms, component adsorption by ion exchange resins, activated carbon, diatomaceous earth, etc., fractionation using chromatography having various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.), filtration using filter paper, membrane filter, ultrafiltration membrane, etc., pressurization or depressurization, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-term storage by standing, etc. One of the above treatments may be carried out, or they may be carried out in combination.

[0032] In the present disclosure, "hyaluronic acid" means a polysaccharide having a structure in which disaccharide units of glucuronic acid and N-acetylglucosamine are linked. The salt of the hyaluronic acid is not particularly limited and may be any food or pharmaceutically acceptable salt, and examples thereof include sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt and the like.

[0033] The hyaluronic acid may be, for example, obtained by extraction from rooster combs or other animal or plant tissues, followed by concentration, production or enzymatic treatment, or may be produced by fermentation using hyaluronic acid-producing microorganisms such as microorganisms of the genus Streptococcus. The hyaluronic acid may be, for example, either a crude extract or a purified product, but it is preferable to use a product having a purity of 90% or more because it has less coloring and off-odor when stored.

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

[0035] In the present disclosure, "honeysuckle" means a plant belonging to the genus Lonicera ( Caprifoliaceae ) of the family Caprifoliaceae ( Lonicera ) of the genus Lonicera ( Lonicera japonica ). The crude drug name of the honeysuckle is also called leaf as "Nindou" (honeysuckle) and flower as "Kinkinka" (Japanese honeysuckle). The crude drug of the honeysuckle is used as a drug for diuresis and antipyretic as a folk medicine, and the decoction of the honeysuckle is used as a gargle solution.

[0036] The extract of the honeysuckle 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 part of a plant. Examples of the part of the plant include leaves; flowers including terminal flowers, flower spikes, and leafy branchlets with flowers; or mixtures thereof. The extract of the honeysuckle may be prepared at home or a commercially available product may be used. Examples of the commercially available products are as follows. · Falcorex Honeysuckle FB: An extract of honeysuckle flowers, prepared with a 1,3 - butylene glycol solution as a solvent, manufactured by Ichimaru Pharcos Co., Ltd. · Falcorex Honeysuckle FE: An extract of honeysuckle flowers, prepared with an ethanol solution as a solvent, manufactured by Ichimaru Pharcos Co., Ltd. · Falcorex Honeysuckle SB: An extract of honeysuckle leaves, prepared with a 1,3 - butylene glycol solution as a solvent, manufactured by Ichimaru Pharcos Co., Ltd.

[0037] The agent for maintaining the structure of hyaluronic acid of the present disclosure may contain, for example, cinnamic acid or its derivative, p - coumaric acid or its derivative, or ferulic acid or its derivative, etc., instead of the caffeic acid or its derivative. Since these compounds are analogs of the caffeic acid or its derivative, they are expected to function as active ingredients (compounds) for maintaining the structure of hyaluronic acid. These compounds may be in the form of salts, hydrates, or solvates, or may be isomers. Specific examples of these compounds include, for example, the compounds shown in Figure 1.

[0038] The agent for maintaining the structure of hyaluronic acid of the present disclosure can maintain the structure of hyaluronic acid, for example, by being used for an administration subject. The usage conditions (administration conditions) of the agent for maintaining the structure of hyaluronic acid of the present disclosure are not particularly limited, and for example, the administration form, administration timing, dosage, etc. can be appropriately set according to the type of the administration subject, etc.

[0039] The agent for maintaining the structure of hyaluronic acid of the present disclosure can be used, for example, in vivo and can also be used in in vitro and can also be used in

[0040] The administration target 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 in vivo used, the administration target includes, for example, humans or non-human animals excluding humans. Examples of the non-human animals include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, and birds. When the agent for maintaining the structure of hyaluronic acid of the present disclosure is in vitro used, the administration target includes, for example, cells, tissues, organs, etc. The cells include, for example, cells collected from a living body, cultured cells, etc. The tissues or organs include, for example, tissues (biological tissues) or organs collected from a living body.

[0041] In the following external skin preparation (for example, a transdermal administration or a skin application agent or composition) or the following oral administration agent or oral administration composition containing the agent for maintaining the structure of hyaluronic acid of the present disclosure, the blending amount of the extract of the flower of Artemisia princeps Pamp. may be within a range that exhibits an effect of maintaining the structure of hyaluronic acid, that is, an effective amount.

[0042] The administration form of the agent for maintaining the structure of hyaluronic acid of the present disclosure includes oral administration or parenteral administration. The parenteral administration includes transdermal administration, application (contact) to the skin, etc. The application to the skin may include application to the oral mucosa, that is, application or contact to the epithelial cells in the oral cavity. Also, the application to the skin may include, in addition to or instead of application to the skin surface, administration or injection into the skin or subcutaneous tissue through the skin surface. The administration or injection into the skin through 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 appropriately determined according to the administration form, for example. The dosage form includes, for example, liquid and solid forms. When the administration form is oral administration, the dosage form includes, for example, tablets, pills, capsules, granules, powders, liquids, etc.

[0044] The agent for maintaining the structure of hyaluronic acid of the present disclosure may contain additives, for example, as necessary. When used as a composition, the additives preferably include pharmaceutically acceptable additives or pharmaceutically acceptable carriers. The additives are not particularly limited and include, for example, base materials, excipients, coloring agents, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring agents such as flavoring and deodorizing agents. In the present disclosure, the blending amount of the additives is not particularly limited as long as it does not interfere with the effect of maintaining the structure of hyaluronic acid.

[0045] The excipients include, for example, sugar derivatives such as lactose, lactose hydrate, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and inorganic excipients such as calcium sulfate. Examples of the coloring agent include yellow ferric oxide. Examples of the lubricant include metal stearates such as stearic acid, calcium stearate, magnesium stearate; talc; polyethylene glycol; silica; hydrogenated vegetable oil. Examples of the flavoring and odor-masking agent include spices such as cocoa powder, peppermint oil, aromatic powder, peppermint oil, borneol, cinnamon powder, sweeteners, acidulants. Examples of the binder include hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, macrogol. Examples of the disintegrant include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, sodium carboxymethyl starch, crosslinked polyvinyl pyrrolidone, sodium starch glycolate. Examples of the stabilizer include paraoxybenzoic acid esters such as methyl paraben, propyl paraben; alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalkonium chloride; phenols such as phenol, cresol; thimerosal; dehydroacetic acid; sorbic acid. Examples of the coating agent include hypromellose, macrogol such as macrogol 6000, talc, titanium oxide.

[0046] When the composition for maintaining the structure of hyaluronic acid of the present disclosure is an oral administration composition, specific examples of the oral administration composition include, for example, beverages, foods, pharmaceuticals, quasi-drugs, etc.

[0047] When an agent or composition for maintaining the structure of hyaluronic acid according to the present disclosure is used for transdermal administration or application to the skin (hereinafter also referred to as a "topical skin preparation"), the form of the topical skin preparation may be, depending on the form of use, an ampoule, a capsule, a powder, a granule, a liquid, a gel, a foam, an emulsion, a sheet, a mist, a spray agent, etc. The forms of use include, for example, pharmaceuticals; quasi-drugs; topical or systemic topical skin preparations; pharmaceutical and / or cosmetic preparations applied to the scalp and hair; bath agents added to bath water; other preparations; etc. The topical or systemic topical skin preparations include, for example, basic cosmetics such as lotion, emulsion, cream, ointment, lotion, oil, pack, etc., facial cleansers or skin cleansers such as solid soap, liquid soap, hand wash, etc., massage agents, cleansing agents, hair removal agents, depilatory agents, beard shaving agents, aftershave lotion, pre-shave lotion, shaving cream, foundation, lipstick, blush, eyeshadow, eyeliner, mascara, etc., makeup cosmetics, perfumes, nail cosmetics, nail enamel, nail enamel remover, poultices, plasters, tape agents, sheet agents, patches, aerosol agents, gargles (such as dentifrice and mouthwash), etc. The pharmaceutical and / or cosmetic preparations applied to the scalp and hair include, for example, shampoo agents, rinse agents, hair treatment agents, pre-hair treatment agents, permanent solutions, hair dyes, hair styling agents, hair tonics, hair growth and nourishing agents, poultices, plasters, tape agents, sheet agents, aerosol agents, etc. The other preparations include, for example, axillary odor preventives or deodorants, antiperspirants, sanitary products, sanitary napkins, wet tissues, etc.

[0048] The topical skin preparation can be optionally selected and / or used in combination with the components and / or additives exemplified below, as long as it does not interfere with the effect of maintaining the structure of hyaluronic acid, as necessary.

[0049] (1) Various oils and fats Avocado oil, almond oil, perilla oil, sesame oil, olive oil, orange oil, orange raffia oil, sesame oil, cocoa 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, peppermint oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, candelilla wax, coconut oil, beef tallow, lard, squalene, squalane, pristane or hydrogenated products (such as hardened oils) of these oils and fats, etc.

[0050] (2) Waxes Beeswax, carnauba wax, whale wax, 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 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, lanolin fatty acid and other natural fatty acids, isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, isopentanoic acid and other synthetic fatty acids.

[0053] (5) Alcohols Ethanol, isopropanol, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol and other natural alcohols, 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol and other synthetic alcohols.

[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, arabinitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[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, etc.

[0056] (8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0057] (9) Gum substances, saccharides or water-soluble polymer compounds Arabic gum, benzoin gum, dammar gum, guaiac resin, Irish moss, karaya gum, tragacanth gum, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide are added, low molecular weight chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salts, hyaluronic acid or its salts, chondroitin sulfate or its salts, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate salts, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their cross-linked polymers, carboxyvinyl polymer, polyethyleneimine, etc.

[0058] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfate esters, alkyl phosphate esters), cationic surfactants (alkyl amine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonate type amphoteric surfactants, phosphate ester type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), etc.

[0059] (11) Various vitamins Group A vitamins: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A); Group B vitamins: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin, choline, inositols; Group C vitamins: ascorbic acid or its derivatives; Group D vitamins: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol; Group E vitamins: vitamin E or its derivatives, ubiquinones; Group K vitamins: phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4); others, essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, Group P vitamins (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, etc., and their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidonecarboxylic acid.

[0061] (13) Additives The topical skin preparation may further contain various additives derived from animals or plants. The additives can be arbitrarily selected from various materials and added after performing processing commonly carried out according to the type and form of the product to be added. The processing can be, for example, treatment arbitrarily selected and / or combined from among pulverization, flour production, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.

[0062] The solvent used for the extraction can be selected taking into consideration the purpose and type of the product to be used, or the subsequent processing. The extraction solvent is preferably one or a mixture of two or more selected from various organic solvents such as water, lower alcohols or water-containing lower alcohols such as water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol, polyhydric alcohols or water-containing polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, and glycerin, acetone, and ethyl acetate. However, when the inclusion of an organic solvent is not preferable depending on the application, the extraction solvent may be water alone or ethanol, which is easy to remove after extraction, may be used alone or in any mixture with water, or may be extracted by squeezing.

[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, for example, cosmetic effects such as protection of the skin and hair, moisturizing, improving feel and texture, imparting softness, easing irritation, relieving stress through fragrance, cell activation (preventing cell aging), suppressing inflammation, improving skin and hair quality, preventing and improving rough skin, hair growth, hair care, preventing hair loss, imparting shine, a cleansing effect, relieving fatigue, promoting blood flow, and a hot bath effect, as well as fragrance, deodorizing, thickening, preserving, buffering, and other effects.

[0064] In addition to the above, the topical skin preparation can be made into a product that is expected to have various cosmetic and medicinal effects of each of the raw material materials that are known to date, and by combining these, it is possible to enhance the effect intended by this disclosure and create a product that is expected to have multifunctional effects.

[0065] <HYBID遺伝子の発現抑制剤または組成物> In another embodiment, the present disclosure provides an agent or composition capable of suppressing the expression of the HYBID gene. The agent or composition for suppressing the expression of the HYBID gene of the present disclosure comprises a compound or a salt thereof represented by the following formula (1), and the hyaluronic acid has a molecular weight of 600 to 2000 kDa: [Chemical] In the above formula (1), R 1 and R 2 may be the same or different, and each independently is a hydrogen atom, a hydroxy 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, an optionally substituted cyclic alkyl group having 3 to 10 carbon atoms, or a hydrogen atom.

[0066] The HYBID gene expression inhibitor or composition thereof of the present disclosure is characterized by containing the compound represented by the above formula (1) or a salt thereof, and other configurations and conditions are not particularly limited. Since the HYBID gene expression inhibitor or composition of the present disclosure contains the compound represented by the above formula (1) or a salt thereof, it can inhibit hyaluronic acid degradation by HYBID and maintain the structure of hyaluronic acid in the skin. Therefore, according to the HYBID gene expression inhibitor or composition of the present disclosure, for example, it is expected to contribute to epidermal moisturization. The description of the compound in the HYBID gene expression inhibitor or composition thereof of the present disclosure can incorporate the description of the agent or composition for maintaining the structure of hyaluronic acid of the present disclosure.

[0067] In the present disclosure, "HYBID" (Hyaluronan binding protein involved in hyaluronan depolymerization, KIAA1199) means a hyaluronic acid-binding protein. The above HYBID is known to contribute to the degradation of hyaluronic acid. The above HYBID binds to hyaluronic acid of a high molecular size (more than 1000 kDa) and degrades the hyaluronic acid into intermediate-sized fragments having a molecular weight of about 10 kDa.

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

[0069] The method for maintaining the structure of hyaluronic acid according to 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 according to the present disclosure.

[0070] In the method for maintaining the structure of hyaluronic acid according to the present disclosure, the administration step may be performed, for example, in vitro or in vivo The subject (administration target) and administration conditions of the method for maintaining the structure of hyaluronic acid according to the present disclosure can refer to, for example, the description of the administration target and administration conditions in the agent or composition for use in maintaining the structure of hyaluronic acid according to the present disclosure.

[0071] <Method for suppressing expression of HYBID gene> In another aspect, the present disclosure provides a method capable of suppressing the expression of the HYBID gene. The method for suppressing the expression of the HYBID gene according to the present disclosure uses an inhibitor or composition for suppressing the expression of the HYBID gene according to the present disclosure. Since the method for suppressing the expression of the HYBID gene according to the present disclosure uses an inhibitor or composition for suppressing the expression of the HYBID gene according to the present disclosure, the degradation of hyaluronic acid by HYBID can be inhibited, and the structure of hyaluronic acid in the skin can be maintained. Therefore, according to the method for suppressing the expression of the HYBID gene of the present disclosure, it is expected to contribute to moisturizing the epidermis, for example.

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

[0073] In the method for suppressing the expression of the HYBID gene of the present disclosure, the administration step may be, for example, in vitro or in vivo performed in this manner. The subject (administration subject) and administration conditions of the method for suppressing the expression of the HYBID gene of the present disclosure can, for example, make use of the description of the administration subject and administration conditions in the agent or composition for use in maintaining the structure of hyaluronic acid of the present disclosure.

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

[0075] The present disclosure is the use of an agent and / or composition for use in maintaining the hyaluronic acid structure for manufacturing an agent and / or composition for use in maintaining the hyaluronic acid structure. The present disclosure is the use of an agent and / or composition for suppressing the expression of the HYBID gene for manufacturing an agent and / or composition for suppressing the expression of the HYBID gene.

[0076] <Compound> In another aspect, 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 make use of the description of the compound in the agent or composition for use in maintaining the hyaluronic acid structure of the present disclosure.

Chemical formula

Chemical formula

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

Example

[0078] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercial reagents were used based on their protocols unless otherwise indicated. Note that percentages indicating the addition amounts of active ingredients, etc. mean weight % unless otherwise specified.

[0079] <Material> · Normal human adult dermal fibroblasts: KF-4109 (manufactured by Kuraray Co., Ltd.), cells from humans aged 40 or older, hereinafter referred to as "AD cells". · Normal human neonatal foreskin dermal fibroblasts: KF-4009 (manufactured by Kuraray Co., Ltd.), hereinafter referred to as "NB cells". · Extract of the flowers of Artemisia keiskeana: Falcorex Artemisia keiskeana B (manufactured by Ichimaru Pharcos Co., Ltd.)

[0080] [Reference Example 1] Regarding the structural maintenance of hyaluronic acid, it was examined 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. Culturing was performed under conditions of 5% CO2 and 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 the structural maintenance of hyaluronic acid (RT-PCR) 6×10 4Individual NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After the above culture, the medium was replaced with DMEM containing 0.25% FBS, and after culturing for 24 hours, it was replaced with fresh DMEM containing 0.25% FBS. For the NB cells after the replacement, the HA was added to a final concentration of 10 μg / mL to prepare the following experimental groups. (Experimental group) · Control group: The group without HA addition · H2 addition group: The group with H2 addition · M2 addition group: The group with M2 addition · S2 addition group: The group with S2 addition · U2 addition group: The group with U2 addition

[0084] After preparing the above experimental groups, they were further cultured in a CO2 incubator at 37°C for 1 - 2 hours. 1 - 2 hours after the culture, to make the cells in an inflammatory state, TNF-α was added to the above experimental groups (including the control group) to a final concentration of 1 ng / mL. After the addition, they were cultured in a CO2 incubator at 37°C for 6 hours. mRNA was purified from the cultured cells. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Then, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 1 below, and the change in the expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, the amplification result of RPS18 (ribosomal protein S18) was used as a reference for the analysis of the relative amount change. Note that "MMP" in Table 1 below is matrix metalloprotease.

[0085]

Table 1

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

[0087] [Table 2]

[0088] As shown in Table 2 above, it was found that for the maintenance of 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] Regarding the expression of HYBID induced by histamine in NB cells, it was examined using the RT-PCR method.

[0090] (1) NB cells NB cells were prepared. For the pre-culture, DMEM containing 5% FBS was used, and in this Reference Example 2, DMEM containing 0.25% FBS was used. Culturing was performed under the conditions of 5% CO2 and 37 °C. After the said culturing, 6 × 10 4 individual NB cells were seeded in a 6-well plate and cultured until they reached a 75% confluent state. Then, they were replaced with DMEM containing 0.25% FBS, cultured for 24 hours, and then replaced with new DMEM containing 0.25% FBS.

[0091] (2) Experimental groups For the NB cells after the said replacement, the following experimental groups were prepared using histamine, which is a substance that enhances hyaluronic acid degrading activity. (Experimental groups) · Non-added group: The group to which histamine was not added · 0.1 added group: The group to which histamine was added to a final concentration of 0.1 μmol / l · Group 1.0 addition: The group to which histamine was added to a final concentration of 1.0 μmol / l · Group 10 addition: The 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, they were further cultured at 37 °C for 24 hours in a CO2 incubator. After the culture, mRNA was purified from the cells. The purification of mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Thereafter, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 3 below, and the change in expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, as a reference for the analysis of the relative amount change, the amplification result of RPS18 (ribosomal protein S18) was used.

[0093]

Table 3

[0094] These results are shown in Table 4 below. Table 4 below shows the values when the value of the non-addition group (change in expression level by the relative quantification) is set to 1. The values are the values rounded off to the third decimal place. Also, in Table 4 below, the ** mark indicates a significant difference (p < 0.01) by comparison with the value of the non-addition group in the Dunnett test, and the *** mark indicates a significant difference (p < 0.001) by comparison with the value of the non-addition group in the Dunnett test.

[0095]

Table 4

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

[0097] [Example 1] Regarding the expression of HYBID induced by histamine in NB cells when an extract of the flowers of Artemisia princeps Pamp. was added, it was examined using the RT-PCR method.

[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. The cells were cultured under the conditions of 5% CO2 and 37 °C. After the above culture, 6×10 4 cells were seeded in a 6-well plate and cultured until they reached a 75% confluent state. Then, they were replaced with DMEM containing 0.25% FBS, cultured for 24 hours, and then replaced with fresh DMEM containing 0.25% FBS.

[0099] (2) Experimental groups For the NB cells after the above replacement, the following experimental groups were prepared using the extract of the flowers of Artemisia princeps Pamp. (Experimental groups) · Non-addition group: A group without addition of the extract and histamine · Control group: A group without addition of the extract and with addition of histamine · 0.125 addition group: A group with addition of the extract at a final concentration of 0.125% and addition of histamine · 0.25 addition group: A group with addition of the extract at a final concentration of 0.25% and addition of histamine · 0.5 addition group: A group with addition of the extract at a final concentration of 0.5% and addition of histamine · 1.0 addition group: A group with addition of the extract at a final concentration of 1.0% and addition of histamine

[0100] (3) Confirmation of gene expression of HYBID (RT-PCR) After preparing the experimental groups, they were further cultured at 37°C for 3 hours in a CO2 incubator. After the culture, to induce a state of inflammation in the cells, histamine was added to the experimental groups (excluding the non-addition group) to a final concentration of 10 μmol / l. After the addition, they were cultured at 37°C for 24 hours in a CO2 incubator. The addition of histamine was performed to enhance the expression of HYBID, similar to Reference Example 2. mRNA was purified from the cells after the culture. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Then, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 5 below, and the change in the expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, for the reference of the analysis of the relative amount change, the amplification result of RPS18 (ribosomal protein S18) was used.

[0101]

Table 5

[0102] These results are shown in Table 6 below. Table 6 below shows the values when the value of the non-addition group (the change in the expression level by the relative quantification) is set to 1. The values are those with the third decimal place rounded off. Also, in Table 6 below, the ** mark indicates a significant difference (p < 0.01) by comparison with the value of the control group in the Dunnett test, and the *** mark indicates a significant difference (p < 0.001) by comparison with the value of the control group in the Dunnett test.

[0103]

Table 6

[0104] As shown in Table 6 above, it was found that in NB cells with a flame symptom state, the addition of the extract of the flowers of Artemisia princeps Pamp. suppressed the expression of HYBID.

[0105] [Example 2] The expression of miR-600 in NB cells and AD cells when the extract of the flowers of Artemisia princeps Pamp. was added was examined. Specifically, regarding whether the addition of the extract of the flowers of Artemisia princeps Pamp. enhanced the expression of miR-600, it was examined using the RT-PCR method. MicroRNA (miRNA) is short-chain (20-25 bases) RNA (non-coding RNA) that does not produce proteins. The microRNA binds to the messenger RNA (mRNA) of a gene having the same sequence as itself, and suppresses the expression of the gene by degrading the mRNA or inhibiting translation into a protein. miR-600, which is one of the microRNAs, 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 6×10 4 Individual NB cells or AD cells were seeded in a 6-well plate and cultured until they reached a 75% confluent state. After the culture, they were replaced with DMEM containing 0.25% FBS and cultured for 24 hours. Then, they were replaced with new DMEM containing 0.25% FBS.

[0107] (2) Experimental groups For the NB cells or AD cells after the replacement, the following experimental groups were prepared using the extract of the flowers of *Artemisia keiskeana*. (Experimental group) · NB cell non-added group: The group in which the extract was not added to NB cells · AD cell non-added group: The group in which the extract was not added to NB cells · NB cell 1.0 added group: The group in which the extract was added to NB cells at a final concentration of 1.0% · AD cell 1.0 added group: The group in which the extract was added to NB cells at a final concentration of 1.0%

[0108] (3) Examination of the expression of miR-600 After the preparation of the above experimental groups, the cells were further cultured in a CO₂ incubator at 37 °C for 6 hours. After the culture, total RNA was purified and extracted using the miRNeasy Tissue / Cells Advanced Mini Kit (manufactured by 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 (trademark) miRNA First-Strand Synthesis Kit (manufactured by Clontech Laboratories, Inc. (Takara Bio Inc.), product code: 638313, Takara code: Z8313N). After the synthesis, RT-PCR was performed using the Mir-X (trademark) miRNA qRT-PCR SYBR Kit (manufactured by Clontech Laboratories, Inc.), the primers described in Table 7 below, and the Thermal Cycler Dice Real Time System TP800 (manufactured by Clontech Laboratories, Inc. (Takara Bio Inc.)). Then, the miRNA expression among the experimental groups was compared by the Delta-delta-CT method.

[0109]

Table 7

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

[0111] These results are shown in Table 8 below. Table 8 below shows the values when the NB cell-free group (change in expression level in the relative quantification) is set to 1. The values are those with the third decimal place rounded off. Also, in Table 8 below, the * mark indicates a significant difference (p < 0.05) compared to the value of the NB cell-free group in the Student's t-test, and the † mark indicates a significant difference (p < 0.05) compared to the value of the AD cell-free group in the Student's t-test.

[0112]

Table 8

[0113] As shown in Table 8 above, the expression level of miR-600 decreased in the AD cell-free group compared to the NB cell-free group. Also, the expression level of miR-600 increased in the AD cell-added group and the NB cell-added group, respectively, compared to the AD cell-free group or the NB cell-free group. From these results, it was found that the addition of the extract of the flowers of Artemisia princeps Pamp. enhances the expression of miR-600 in AD cells and NB cells.

[0114] [Example 3] A human skin monitoring test and the like were conducted on a composition containing the extract of the flowers of Artemisia princeps Pamp.

[0115] (1) Preparation of lotion First, for use in the skin monitor test, a lotion containing the components shown in Table 9 below (hereinafter also referred to as the "lotion described in Table 9") and a lotion containing the components shown in Table 10 below (hereinafter also referred to as the "lotion described in Table 10") were prepared. As shown in Table 9 below, the lotion described in Table 9 contains 1% of an extract of the flowers of Artemisia princeps Pamp.

[0116]

Table 9

[0117]

Table 10

[0118] (2) Skin monitor test in humans (topical skin preparation) A skin monitor test in humans was conducted on the lotion prepared in Example 3(1) above. Specifically, the lotion described in Table 9 and the lotion described in Table 10 (placebo) were applied to predetermined sites on the faces of normal human subjects (13 men and women with an average age of 43.6 years) between January 31, 2022, and March 4, 2022. The lotion described in Table 9 was applied at predetermined intervals to a predetermined site on half of the subjects' faces, and the lotion described in Table 10 was applied at predetermined intervals to a predetermined site on the other half of the subjects' faces. This predetermined interval was twice a day (morning and evening) for 4 weeks. The predetermined site was "the entire face".

[0119] Figure 2 shows a schematic diagram of the skin monitor test. As described in Figure 2, the sagging of the skin was measured. Specifically, on the cheeks of the subjects, a hotchkiss needle (No. 11-1M (Max Co., Ltd.)) was hooked under the lower side of an adhesive tape with a donut-shaped hole in the center (adhesive tape for the ELASTICITY probe of DermaLabTM Combo (manufactured by Cotex Technologies)). As shown in Figure 2, the application position of the adhesive tape was set as the intersection where a line drawn horizontally from under the nose on the face and a line drawn vertically from the outer corner of the eye overlapped with the intersection where the lower part inside the donut circle of the adhesive tape overlapped.

[0120] The plumb bob in Fig. 2 is a weight (5 g to 20 g). The plumb bob and the clip were fixed with a string, and the clip side was hooked onto the staple of the stapler. In addition, the weight was added to each of the subjects in advance, and a weight that caused the cheek to move 1-2 mm by gravity was used.

[0121] In order to check the state of the face before and after the load of the weight, left and right face photos were taken using VISIA™ Evolution (manufactured by Canfield Scientific). The photographing was performed before the test and 4 weeks after the test. Using photo processing software (Photoshop), the photographed images before and after the load of the weight were superimposed. Then, using image analysis software (Digital Microscope VHX-5000 (manufactured by KEYENCE CORPORATION)), the moving distance of the adhesive tape and the length of the adhesive tape after the load of the weight before the test and 4 weeks after the test were measured. The moving distance of the adhesive tape after the load of the weight was calculated by estimating a conversion value from the length of the adhesive tape. The measured results are shown in Table 11 and Table 12 below.

[0122]

Table 11

[0123] Table 11 shows the results of measuring the moving distance. In Table 11, the value before the test (0 week) is shown as 1. In Table 11, the * mark indicates a significant difference (p < 0.05) by comparison with the value at 0 week (lotion application group in Table 9) in the Wilcoxon Rank-Sum Test. As shown in Table 11, the measured value 4 weeks after the test in which the lotion described in Table 10 was applied was 1.13, whereas the measured value 4 weeks after the test in which the lotion described in Table 9 was applied was 0.71. From these results, it was found that the lotion described in Table 9 containing the extract of the flowers of Artemisia keiskeana can suppress sagging of the facial skin.

[0124]

Table 12

[0125] Table 12 shows the results of measuring the length of the adhesive tape after 4 weeks of the test. In Table 12, the * mark indicates a significant difference (p < 0.05) compared with the value (100) of the lotion application group described in Table 10 in the Wilcoxon Rank-Sum Test. As shown in Table 12, when the value of the lotion application group described in Table 10 was set to 100, the value of the lotion application group described in Table 9 was 71.20. From these results, it was found that the lotion described in Table 9 containing the extract of the flowers of Artemisia princeps Pamp. can suppress sagging of facial skin.

[0126] (3) Skin monitor test 2 (external preparation for skin) in humans A skin monitor test in humans was conducted on the lotion prepared in Example 3(1). Specifically, the lotion described in Table 9 and the lotion described in Table 10 (placebo) were applied to predetermined parts of the faces of normal human subjects (13 men and women with an average age of 43.6 years), and the test was conducted between January 31, 2022 and March 4, 2022. The lotion described in Table 9 was applied to predetermined parts of one half of the subjects' faces at predetermined intervals, and the lotion described in Table 10 was applied to predetermined parts of the other half of the subjects' faces at predetermined intervals. This predetermined interval was twice a day (morning and evening) for 4 weeks. The predetermined part was "the whole face".

[0127] Before the application of the lotion (week 0) and 4 weeks after the application of the lotion, the size (overall size) and depth of the wrinkles of the eyelids were measured. The eyelids include the upper eyelids and the outer corners of the eyes. The presence or absence of wrinkles on the eyelids was measured using ANTERA 3D (registered trademark, Gadelious Medical Co., Ltd.).

[0128] The measurement was performed by calculating the relative values of the measurement results of the subjects in each group 4 weeks after the application of the lotion, with the average value of the measurement results of the subjects in each group before the application of the lotion set as "100". In the measurement, the value (100) before the application of the lotion was measured 3 times at 1 site per person, the average value of the 3 measurements was calculated, and the average value of the calculated values was aggregated from 13 people and the average value of the values was set as 100. The measurement result 4 weeks after the application of the lotion is a value obtained by calculating the rate of change compared to the value before the application of the lotion. These results are shown in Table 13 below.

[0129]

Table 13

[0130] Table 13 shows the results of the measurement of the presence or absence of crow's feet after 4 weeks of application of the lotion. In Table 13, the * mark indicates a significant difference (p < 0.05) by comparison with the value (100) of the lotion application group described in Table 10 in the Wilcoxon Rank-Sum Test. As shown in Table 13, when the value of the lotion application group described in Table 10 was set as 100, the value of the lotion application group described in Table 9 was 93.51. From these results, it was found that the lotion described in Table 9 containing the extract of the flowers of Artemisia princeps Pamp. reduced the size and depth of crow's feet.

[0131] (4) Human Skin Monitor Test 3 (Topical Skin Preparation) A skin monitor test was conducted on humans for the lotion prepared in Example 3(1) above. Specifically, the lotion described in Table 9 above and the lotion described in Table 10 above (placebo) were applied to predetermined parts of the faces of normal human subjects (13 men and women with an average age of 43.6 years), and the test was conducted between January 31, 2022 and March 4, 2022. The lotion described in Table 9 was applied to a predetermined part of half of the subject's face at predetermined intervals, and the lotion described in Table 10 was applied to a predetermined part of the other half of the subject's face at predetermined intervals. This predetermined interval was twice a day (morning and evening) for 4 weeks. The predetermined part was "the entire face".

[0132] Before the application of the lotion (week 0) and 4 weeks after the application of the lotion, the measurement of the skin moisture distribution was carried out. The measurement was performed using a Corneometer (CM825, manufactured by Courage+Khazaka). When the average value of the measurement of the moisture distribution of the 13 subjects at week 0 (before the application) in the lotion application group described in Table 9 or the lotion application group described in Table 10 was set to 100 respectively, after 4 weeks of application, the lotion application group described in Table 9 was 107.73, and the lotion application group described in Table 10 was 98.98. From these results, it was found that the lotion described in Table 9 containing the extract of the flowers of Kawarayomogi has the effect of maintaining a predetermined skin moisture amount.

[0133] [Example 4] Regarding the expression of HYBID induced by histamine in NB cells when an extract of the flowers of Kawarayomogi or an extract of the flowers of Honeysuckle was added, it was examined using the RT-PCR method.

[0134] (1) NB cells NB cells were prepared. DMEM containing 5% FBS was used for pre-culture, and DMEM containing 0.25% FBS was used in Example 4. The cells were cultured under the conditions of 5% CO2 and 37°C. After the culture, 6×10 4NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. Then, they were replaced with DMEM containing 0.25% FBS and cultured for 24 hours, after which they were replaced with fresh DMEM containing 0.25% FBS.

[0135] (2) Experimental groups For the NB cells after the replacement, the following experimental groups were prepared using the extract of the flowers of *Artemisia keiskeana* or the extract of *Wisteria floribunda*. The extracts of the flowers of *Artemisia keiskeana* and *Wisteria floribunda* used were as follows. · Extract of the flowers of *Artemisia keiskeana*: Falcorex Artemisia keiskeana B, manufactured by Ichimaru Pharcos Co., Ltd. · Extract of the flowers of *Wisteria floribunda*: Falcorex Wisteria floribunda FB, manufactured by Ichimaru Pharcos Co., Ltd. (Experimental groups) · Non-added group: The group without the addition of the above extract and histamine · Control group: The group without the addition of the above extract and with the addition of histamine · 0.125% Wisteria floribunda FB-added group: The group with the addition of Falcorex Wisteria floribunda FB at a final concentration of 0.125% and the addition of histamine · 0.25% Wisteria floribunda FB-added group: The group with the addition of Falcorex Wisteria floribunda FB at a final concentration of 0.25% · 0.5% Wisteria floribunda FB-added group: The group with the addition of Falcorex Wisteria floribunda FB at a final concentration of 0.5% and the addition of histamine · 1.0% Wisteria floribunda FB-added group: The group with the addition of Falcorex Wisteria floribunda FB at a final concentration of 1.0% and the addition of histamine · 0.125% Artemisia keiskeana B-added group: The group with the addition of Falcorex Artemisia keiskeana B at a final concentration of 0.125% and the addition of histamine · 0.25% Artemisia keiskeana B-added group: The group with the addition of Falcorex Artemisia keiskeana B at a final concentration of 0.25% and the addition of histamine · 0.5% Artemisia keiskeana B-added group: The group with the addition of Falcorex Artemisia keiskeana B at a final concentration of 0.5% and the addition of histamine · Group with 1.0% Artemisia princeps Pamp. var. orientalis added: Group with falcolekks Artemisia princeps Pamp. var. orientalis added at a final concentration of 1.0% and histamine added

[0136] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, they were further cultured at 37°C for 3 hours in a CO2 incubator. After the culture, in order to make the cells in an inflammatory state, histamine was added to the experimental groups (excluding the non-added group) at a final concentration of 10 μmol / l. After the addition, they were cultured at 37°C for 24 hours in a CO2 incubator. The addition of histamine was performed to enhance the expression of HYBID. mRNA was purified from the cells after the culture. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Then, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 14 below, and the change in the expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, for the reference of the relative amount change analysis, the amplification result of RPS18 (ribosomal protein S18) was used.

[0137]

Table 14

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

[0139]

Table 15

[0140] As shown in Table 15 above, it was found that in NB cells with a flammatory state, the addition of the extract of the flowers of Artemisia keiskeana or the addition of the extract of the flowers of Lonicera japonica suppressed the expression of HYBID.

[0141] [Example 5] Regarding the expression of HYBID induced by IL-1β in NB cells when the extract of the flowers of Artemisia keiskeana or the extract of the flowers of Lonicera japonica was added, it was examined using the RT-PCR method.

[0142] (1) NB cells NB cells were prepared. DMEM containing 5% FBS was used for pre-culture, and DMEM containing 0.25% FBS was used in Example 5. They were cultured under the conditions of 5% CO2 and 37 °C. After the above culture, 6×10 4 NB cells were seeded in a 6-well plate and cultured until they reached a 75% confluent state. Then, they were replaced with DMEM containing 0.25% FBS, cultured for 24 hours, and then replaced with new DMEM containing 0.25% FBS.

[0143] (2) Experimental groups For the NB cells after the above replacement, the following experimental groups were prepared using the extract of the flowers of Artemisia keiskeana or the extract of Lonicera japonica. The extract of the flowers of Artemisia keiskeana and the extract of Lonicera japonica used the following extracts. · Extract of the flowers of Artemisia keiskeana: Falcorex Artemisia keiskeana B, manufactured by Ichimaru Pharcos Co., Ltd. · Extract of the flowers of Lonicera japonica: Falcorex Lonicera japonica FB, manufactured by Ichimaru Pharcos Co., Ltd. · Extract of the leaves of Lonicera japonica: Falcorex Lonicera japonica SB, manufactured by Ichimaru Pharcos Co., Ltd. (Experimental groups) · Unadded group: The group without the addition of the above extract and IL-1β · Control group: The group without the addition of the above extract and with the addition of IL-1β · Group with 1.0% addition of Passiflora edulis Sims f. flavicarpa Deg: Passiflora edulis Sims f. flavicarpa Deg was added to a final concentration of 1.0%, and the group to which IL-1β was added · Group with 1.0% addition of Passiflora suberosa L.: Passiflora suberosa L. was added to a final concentration of 1.0%, and the group to which IL-1β was added · Group with 1.0% addition of Artemisia princeps Pamp.: Artemisia princeps Pamp. was added to a final concentration of 1.0%, and the group to which IL-1β was added

[0144] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, they were further cultured at 37°C for 3 hours in a CO2 incubator. After the culture, in order to make the cells in an inflammatory state, IL-1β was added to the experimental groups (excluding the non-added group) to a final concentration of 50 ng / ml. After the addition, they were cultured at 37°C for 24 hours in a CO2 incubator. The addition of IL-1β was performed to enhance the expression of HYBID. mRNA was purified from the cells after the culture. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Then, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 16 below, and the change in the expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, for the reference of the relative amount change analysis, the amplification result of RPS18 (ribosomal protein S18) was used.

[0145]

Table 16

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

[0147] [Table 17]

[0148] As shown in Table 17 above, it was found that the expression of HYBID was suppressed by the addition of the extract of the flowers of Artemisia princeps Pamp., the addition of the extract of the flowers of Vinca major L., or the addition of the extract of the leaves of Vinca major L. in NB cells in the inflammatory state.

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

[0150] (1) Gel Using a Collagen Gel Culturing Kit (manufactured by Nitta Gelatin Inc., 638-00781), a gel was prepared according to the procedure manual included in the kit. For experiments using a collagen gel, for example, Reference 2 below can be referred to. 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 HA was 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 To the gel, the HA was added to a final concentration of 50 μg / ml, and the following experimental groups were prepared. (Experimental groups) · H2 addition group: The group to which H2 was added · U2 addition group: The group to which U2 was added

[0153] (4) Measurement of gel height After the preparation of the experimental groups, they were left standing at 37°C for 30 minutes. After the standing, the state of the gels in the experimental groups was visually confirmed, and the height of the gels was measured. These results are shown in Figure 3.

[0154] Figure 3 is a photograph showing the state of each gel. Assuming the value of the gel height in the H2 addition group is 100, the value of the gel height in the U2 addition group was 90. As shown in Figure 3, compared with the H2 addition group, in the U2 addition group, the gel height decreased to the extent of the arrow. From these results, it was found that the U2 addition group had a lower ability to retain water molecules compared with the H2 addition group and could not maintain the structure of the water-soluble gel. Also, it was suggested that the molecular weight of HA is important for maintaining the gel structure.

[0155] [Example 7] Regarding the expression of HYBID in NB cells when an extract of the flowers of Artemisia scoparia was added, it 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 this Example 7, DMEM containing 0.25% FBS was used. They were cultured under the conditions of 5% CO2 and 37°C. After the culture, 6×10 4Individual NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. Subsequently, they were replaced with DMEM containing 0.25% FBS and cultured for 24 hours, and then replaced with fresh DMEM containing 0.25% FBS.

[0157] (2) Experimental groups For the NB cells after the replacement, the following experimental groups were prepared using the extract of the flowers of *Artemisia keiskeana* Miq.. (Experimental groups) · Unadded group: The group without the addition of the extract · 1.0 addition group: The group with the extract of the flowers of *Artemisia keiskeana* Miq. added to a final concentration of 1.0 μmol / l

[0158] (3) Confirmation of HYBID gene expression (RT-PCR) After the preparation of the experimental groups, they were further cultured at 37 °C in a CO₂ incubator for 3 hours. After the culture, they were replaced with fresh DMEM containing 0.25% FBS. After the replacement, they were cultured at 37 °C in a CO₂ incubator for 24 hours. mRNA was purified from the cells after the culture. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Subsequently, RT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 18 below, and the change in the expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, the amplification result of RPS18 (ribosomal protein S18) was used as a reference for the analysis of the relative amount change.

[0159]

Table 18

[0160] These results are shown in Table 19 below. Table 19 below shows the values when the values of the non-added group (change in expression level in the relative quantification) are set to 1. The values are those obtained by rounding to the third decimal place. In Table 19 below, the *** mark indicates a significant difference (p < 0.001) compared to the values of the non-added group in the Student's t-test.

[0161] [Table 19]

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

[0163] [Example 8] Regarding the expression of HYBID in NB cells or AD cells when the extract of the flowers of *Artemisia princeps* was added, it 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. The cells were cultured under the conditions of 5% CO2 and 37 °C. After the above culture, 6 × 10 4 individual NB cells or AD cells were seeded in a 6-well plate and cultured until they reached a 75% confluent state. After the above culture, the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours. Then, the medium was replaced with fresh DMEM containing 0.25% FBS.

[0165] (2) Experimental groups For the NB cells or AD cells after the above replacement, the following experimental groups were prepared using the extract of the flowers of *Artemisia princeps*. (Experimental groups) · NB non-added group: The group without the addition of the extract to NB cells · AD non-added group: The group without the addition of the extract to AD cells ·AD1.0 addition group: A group in which an extract of Artemisia keiskeana flowers was added to AD cells at a final concentration of 1.0 μmol / l

[0166] (3) Confirmation of HYBID gene expression (RT-PCR) After preparing the experimental groups, they were further cultured in a CO2 incubator at 37°C for 3 hours. After the culture, they were replaced with fresh DMEM containing 0.25% FBS. After the replacement, they were cultured in a CO2 incubator at 37°C for 24 hours. mRNA was purified from the cells after the culture. The purification of the mRNA was performed using QIAshreder and RNeasy Mini Kit (manufactured by QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (manufactured by Takara Bio Inc.) to synthesize cDNA. Then, RT-PCR was performed using primer pairs corresponding to each target factor shown in Table 20 below, and the change in expression level was analyzed by relative quantification. The RT-PCR was performed using TB Green Premix Ex Taq (manufactured by Takara Bio Inc.). Also, for the reference of the relative amount change analysis, the amplification result of RPS18 (ribosomal protein S18) was used.

[0167]

Table 20

[0168] These results are shown in Table 21 below. Table 21 below shows the values when the value of the NB-unadded group (the change in expression level by the relative quantification) is set to 1. The values are those rounded to the second decimal place. Also, in Table 21 below, the *** mark indicates a significant difference (p < 0.001) by comparison with the value of the AD-unadded group in the Dunnett test.

[0169]

Table 21

[0170] As shown in Table 21 above, it was found that in AD cells, the addition of the extract of the flowers of *Artemisia princeps* Pamp. suppressed the expression of HYBID. Also, in AD cells, it was found that the addition of the extract of the flowers of *Artemisia princeps* Pamp. resulted in an expression level of HYBID comparable to that of NB cells.

[0171] [Example 9] The distribution of histamine in NB cells when the extract of the flowers of *Artemisia princeps* Pamp. was added was examined.

[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. The cells were cultured under the conditions of 5% CO2 and 37 °C. After the above culture, 6×10 4 cells of NB were seeded in a 6-well plate and cultured until they reached 75% confluence. Then, they were replaced with DMEM containing 0.25% FBS, cultured for 24 hours, and then replaced with fresh DMEM containing 0.25% FBS.

[0173] (2) Experimental groups For the NB cells after the above replacement, the following experimental groups were prepared using the extract of the flowers of *Artemisia princeps* Pamp. (Experimental groups) · Non-added group: The group without the addition of the extract and histamine · Control group: The group without the addition of the extract and with the addition of histamine · 0.5-added group: The group with the extract added to a final concentration of 0.5% · 1.0-added group: The group with the extract added to a final concentration of 1.0%

[0174] (3) Examination of the distribution of hyaluronic acid (HPLC) After preparing the experimental groups, they were further cultured in a CO2 incubator at 37°C for 3 hours. After the culturing, to induce a pro-inflammatory state in the cells, histamine was added to the experimental groups (excluding the non-addition group) to a final concentration of 10 μmol / l. After this addition, the cells were cultured in a CO2 incubator at 37°C for 2 hours. The addition of histamine was performed to enhance the expression of HYBID. Fluorescein amine-labeled sodium hyaluronate (H2, average molecular weight 1.2 million to 1.6 million, FAHA-H2, Iwai Chemical Co., Ltd.) was added to the cells after the culturing. After the addition, the cells were cultured in a CO2 incubator at 37°C for 48 hours. After the culturing, the culture medium was collected. For the experiment using the FAHA, for example, the following References 3 (Figure 4) and References 4 (Figure 1, Supplemental Figure 1) can be referred to. The distribution of hyaluronic acid was examined using HPLC with the culture medium. The conditions for HPLC are as follows. These 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. “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 above culture solution, the distribution of hyaluronic acid was examined by HPLC under the following measurement conditions. These results are shown in FIG. 4 and Table 22 below. (HPLC Conditions) Apparatus: SHIMADZU LC-20A Prominence (manufactured by Shimadzu Corporation) Column: TSKgel G5000PWXL (manufactured by 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] [Table 22]

[0177] FIG. 4 is a graph showing the results of HPLC analysis. In FIG. 4, the vertical axis represents fluorescence intensity, and the horizontal axis represents time (minutes). Table 22 above shows the fluorescence intensity values at the peak tops of each group shown in FIG. 4. Table 22 shows the values when the value of the non-added group (fluorescence intensity) is set to 100. The above values are the values obtained by rounding off the fourth decimal place. As shown in FIG. 4 and Table 22 above, compared with the non-added group, in the groups to which histamine was added (control group, 0.5 addition group, 1.0 addition group), the fluorescence intensity at the peak top was low. From these results, it was suggested that histamine enhanced HYBID, and HYBID decomposed a certain number of hyaluronic acids into fragments.

[0178] [Example 10] It was confirmed that the caffeic acid derivative suppresses HYBID production.

[0179] (1) Component fractionation 1 and gene expression level test of HYBID The components contained in the extract of the flowers of Artemisia capillaris Thunb. were analyzed. Specifically, the extract of the capitulum of Artemisia capillaris Thunb. (Artemisia capillaris flower extract: ACFE) was fractionated into components according to the scheme shown in Fig. 5. The raw material used for the component fractionation was purchased from Shibamoto Tenkaido Co., Ltd. Specifically, for the component fractionation, water was added to the extract (ACFE) obtained by extracting the capitulum of Artemisia capillaris Thunb. with 30% butylene glycol (BG). After making it a 15% BG aqueous solution by the addition, it was roughly fractionated with a column packed with Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) to obtain four fractions, Fr.1 to Fr.4. Next, regarding the four fractions, the HYBID expression inhibitory effect was examined. The examination was carried out in the same manner as in Example 1 except that human NHDF (Normal Human Dermal Fibroblasts) cells were used for the cells and the four fractions were used instead of the extract, and the gene expression of HYBID was confirmed. These results are shown in Fig. 6.

[0180] Fig. 6 is a graph showing the gene expression level of HYBID when histamine is added. In Fig. 6, the vertical axis indicates the relative value of the expression level of the HYBID gene when the non-added group (NT) is set to 100, and the horizontal axis indicates the experimental group. As shown in Fig. 6, compared with the control group, the expression level of the HYBID gene decreased in the Fr.2 fraction. From these results, it was found that the Fr.2 fraction contains a component that suppresses the production of HYBID.

[0181] (2) Component fractionation 2 and test of HYBID gene expression level Regarding the components contained in Fr.2, further fractionation was performed using preparative HPLC under the following measurement conditions. By this fractionation, four fractions, Fr.2-1, Fr.2-2, Fr.2-3, and Fr.2-4, were obtained. Next, the HYBID expression inhibitory effect was examined for the four fractions. The examination confirmed the gene expression of HYBID in the same manner as in Example 1, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used for the cells and the four fractions were used instead of the extract. These results are shown in FIGS. 7 and 8.

[0182] FIG. 7 is a graph showing the gene expression level of HYBID when histamine is added. In FIG. 7, the vertical axis indicates the relative value of the expression level of the HYBID gene when the non-added group (NT) is set to 100, and the horizontal axis indicates the experimental groups. As shown in FIG. 7, compared with the control group, the expression levels of the HYBID gene decreased in the Fr.2-2 and Fr.2-3 fractions. From these results, it was found that the Fr.2-2 and Fr.2-3 fractions contain components that suppress HYBID production.

[0183] FIG. 8 is a graph showing the gene expression level of HYBID when histamine is added. In FIG. 8, the vertical axis indicates the relative value of the expression level of the HYBID gene when the non-added group (NT) is set to 100, and the horizontal axis indicates the experimental groups. As shown in FIG. 8, in the Fr.2-2 fraction, the expression level of the HYBID gene decreased in a concentration-dependent manner. From these results, it was found that the Fr.2-2 fraction contains components that suppress HYBID production, and the HYBID production is suppressed in a concentration-dependent manner by the Fr.2-2 fraction.

[0184] (3) Structural analysis The structural analysis of Fr.2-2 and Fr.2-3 consisting of a single component was carried out by NMR analysis using the following analysis conditions. As a result of the above 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 measured values of NMR are shown below. Note that the 1-Caffeoyl-3-hydroxybutane was obtained as a yellow solid, and the 3-Caffeoyl-1-hydroxybutane was obtained as a yellow oil. (Conditions for NMR analysis) ·Optical rotaions Apparatus: JASCO P-1020 polarimeter (manufactured by JASCO Corporation) Solvent: MeOH ·UV spectra Apparatus: Shimadzu UV-3100 spectrometer (manufactured by Shimadzu Corporation) Solvent: MeOH ·HRESIMS Apparatus: Shimadzu LCMS-IT-TOF spectrometer (manufactured by Shimadzu Corporation) Ion mode: negative ion mode ·NMR Apparatus: JEOL JNM-ECA-500 spectrometer (manufactured by JEOL Ltd.) Solvent: MeOH-d4

[0185] (Measured values of NMR) 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 O5251.0925 (M-H), found 251.0911.1 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 13C 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 O5251.0925 (M-H), found 251.0911. 11H 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 13C 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) Gene expression level test of HYBID by the identified compound Furthermore, for the isolated Compound 1 and Compound 2, the inhibitory effect on HYBID expression was examined. The examination was carried out in the same manner as in Example 1 except that human NHDF (Normal Human Dermal Fibroblasts) cells were used for the cells and Compound 1, Compound 2, and Fr.2 fraction were used instead of the extract, and the gene expression of HYBID was confirmed. These results are shown in Figure 9.

[0188] Figure 9 is a graph showing the gene expression level of HYBID when histamine is added. In Figure 9, the vertical axis represents the relative value of the expression level of the HYBID gene when the non-added group (NT) is set to 100, and the horizontal axis represents the experimental groups. As shown in Figure 9, compared with the control group, the expression levels of the HYBID gene decreased in Compound 1, Compound 2, and Fr.2 fraction. From these results, it was found that Compound 1 and Compound 2 are compounds that suppress HYBID production in NHDF cells in the inflammatory state.

[0189] (5) Test for gene expression level of HYBID The inhibitory effect of the extract of the capitulum of Artemisia princeps Pamp. (ACFE), Fr.2 fraction, and isolated Compound 1 on HYBID expression was examined. Specifically, the gene expression of HYBID was confirmed in the same manner as in Example 7, except that human NHDF (Normal Human Dermal Fibroblasts) cells were used for the cells, 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 gene expression level of HYBID. In Figure 10, the vertical axis represents the relative value of the expression level of the HYBID gene when the control group (extract non-added group, Control) is set to 100, and the horizontal axis represents the experimental groups. As shown in Figure 10, compared with the control group, the expression levels of the HYBID gene decreased in ACFE, Fr.2 fraction, and Compound 1. From these results, it was found that ACFE, Fr.2 fraction, and Compound 1 suppress HYBID production.

[0191] [Example 11] It was confirmed that the Artemisia princeps Pamp. extract suppresses HYBID production.

[0192] (1) Test for the expression level of miR-486-5p in NB cells by the extract of the flower of Artemisia princeps Pamp. As an upstream microRNA directly targeting the 3'-UTR region of HYBID, miR-486-5p is known (Reference 5). Therefore, it was examined whether the extract of the flowers of Artemisia vulgaris suppresses the expression of miR-486-5p. Specifically, 6×10 4 NB cells were seeded in a 6-well plate and cultured until they reached 75% confluence. After the above culture, the medium was replaced with DMEM containing 0.25% FBS and cultured for 24 hours. Then, the medium was 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, in order to measure the expression of miR-486-5p, total RNA was purified and extracted using the miRNeasy Tissue / Cells Advanced Mini Kit (manufactured by QIAGEN). 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 (trademark) miRNA First-Strand Synthesis Kit (manufactured by 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 described in Table 23 below, and the Thermal Cycler Dice Real Time System TP800 (manufactured by Clontech Laboratories, Inc. (Takara Bio Inc.)). Then, the miRNA expression between the experimental groups was compared by the Delta-delta-CT method. These 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]

Table 23

[0194] Figure 11 is a graph showing the expression level of miR-486-5p. In Figure 11, the vertical axis indicates the relative value of the expression level of miR-486-5p when the control group (the group without extract added, Control) is set to 100, and the horizontal axis indicates the experimental groups. As shown in Figure 11, compared with the control group, the expression level of miR-486-5p increased in the ACFE-added group. From these results, it was found that ACFE increased the expression level of miR-486-5p and suppressed HYBID production in NB cells.

[0195] (2) Test on the expression level of miR-486-5p in AD cells by the extract of the flowers of Artemisia vulgaris It was examined whether the extract of the flowers of Artemisia vulgaris suppresses the expression of miR-486-5p in cells other than NHDF cells. Specifically, it was performed in the same manner as in Example 11(1) 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 represents the relative value of the expression level of miR-486-5p when the control group (the group without extract addition, Control) is set to 100, and the horizontal axis represents the experimental groups. As shown in Figure 12, the expression level of miR-486-5p increased in the ACFE addition group compared with the control group. From these results, it was found that ACFE increases the expression level of miR-486-5p and suppresses HYBID production in AD cells.

[0197] As described above, the present disclosure has been described with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those 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, and incorporates the entire disclosure thereof herein.

[0199] <Supplementary Note> Some or all of the above embodiments and examples can be described as follows, but are not limited thereto. <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, agent:

Chemical Formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Industrial Applicability

[0200] As described above, according to the present disclosure, agents for maintaining the structure of hyaluronic acid and the like can be provided. Therefore, the present disclosure can be said to be extremely useful, for example, in the fields of cosmetics, external skin preparations, and the like.

Claims

1. An agent for maintaining the structure of hyaluronic acid, The present invention includes a compound represented by the following formula (2) or (3) or a salt thereof: The hyaluronic acid has a molecular weight of 600 kDa to 2000 kDa. 【Chemistry 2】 【Chemistry 3】

2. The agent according to claim 1 , wherein the hyaluronic acid has a molecular weight of 1200 kDa to 1600 kDa.

3. The agent according to claim 1 or 2, comprising an extract of Artemisia capillaris and / or an extract of Honeysuckle as the compound represented by the formula (2) or (3) or a salt thereof.

4. An agent for use in suppressing expression of a HYBID gene, comprising: An agent comprising a compound represented by the following formula (2) or (3) or a salt thereof: 【Chemistry 2】 【Chemistry 3】

5. The agent according to claim 4, comprising an extract of Artemisia capillaris and / or an extract of honeysuckle as the compound or its salt represented by the following formula (2) or (3):

6. A compound represented by the following formula (2) or (3), or a salt thereof: 【Chemistry 2】 【Chemistry 3】

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