JP2024046365A - Transglutaminase production promoter and applications thereof - Google Patents

Abstract

To provide a transglutaminase production promoter that can improve the production of transglutaminase, which influences the formation processes of skin's epidermal and keratin cells.SOLUTION: A transglutaminase production promoter that includes 3-hydroxybutyric acid and/or a salt thereof is prepared. The 3-hydroxybutyric acid and/or a salt thereof may include the R-isomer. The transglutaminase production promoter may be a promoter that promotes the production of transglutaminase 1. The transglutaminase production promoter may be a promoter that promotes the production of transglutaminase during the keratin cell formation process. The transglutaminase production promoter may be a promoter that promotes the production of transglutaminase on dry skin.SELECTED DRAWING: None

Description

The present invention relates to a transglutaminase production promoter containing 3-hydroxybutyric acid and/or its salt, and its use.

Human skin has a three-layer structure consisting of the epidermis, dermis, and subcutaneous tissue, stacked in this order from the surface side. Furthermore, on the surface side of the epidermis, the stratum corneum (horny layer) is formed as the outermost layer of the skin. This keratin is formed of multiple keratinocytes (keratinocytes) stacked like tiles or bricks, and intercellular lipids such as sphingolipids (ceramides) that exist between these keratinocytes. In addition, the keratin stores moisture to moisturize the skin. More specifically, NMF (natural moisturizing factor) that stores moisture is held within the keratinocytes, and moisture is also held between the keratinocytes together with the intercellular lipids. In particular, the outer wall structure of the keratinocyte is called the cornified envelope (CE), and is formed of various constituent proteins, and is responsible for the barrier function of the skin.

The keratinocytes are formed when keratinocytes (basal cells) formed in the basal layer at the deepest part of the epidermis are pushed up by newly developed keratinocytes and change into spinous cells and granular cells while migrating to the skin surface. During this process, proteins that make up CE, such as involucrin (IVN) and filaggrin (FLG), are crosslinked by the action of transglutaminase 1 (TGM-1) to form isopeptide bonds (glutamyl-lysine bonds), forming strong, water-insoluble CE. In addition, TGM-1 also has the function of binding proteins that make up CE to intercellular lipids, and in the above process, it can bind intercellular lipids to the surface of CE to form a lamellar structure. Therefore, on the surface of the outer wall of keratinocytes that have mature CE, the intercellular lipids bound to CE are arranged in layers to form a membrane on the surface of the CE, forming a lipid envelope.

In healthy skin, keratinocytes are formed into a uniform shape by strong CE, and are regularly arranged with the CE surrounded by a lipid envelope, allowing NMF and moisture to be sufficiently retained in the keratin. However, when the skin becomes rough due to aging or allergies, the regularity of the keratinocytes is lost, and moisture cannot be sufficiently retained in the keratin, causing dullness and roughness. Therefore, the production ability of transglutaminase 1 in epidermal cells has a significant impact on the health of the skin.

On the other hand, 3-hydroxybutyric acid (3HB or BHB), a type of ketone body, and its salts are endogenous substances produced in the liver and contained in breast milk, and have been reported to have various functions on the skin.

JP 2017-200883 A (Patent Document 1) discloses a cosmetic composition containing 3HB. This document describes the effects of the cosmetic, including a whitening effect (effect as a whitening cosmetic) evaluated using tyrosinase activity (amount of melanin produced per unit protein amount) using human pigment cells, a cell activation effect (effect as a cell activation cosmetic) evaluated using MTT reducing activity using fibroblasts and epidermal cells in a nutritionally deficient or aged state, and a moisturizing effect (effect as a moisturizing cosmetic) measured by the moisture content of the stratum corneum of the forearm.

Japanese Patent No. 6804352 (Patent Document 2) discloses a collagenase MMP1 and MMP3 production inhibitor containing R-3-hydroxybutyric acid as an active ingredient. In this document, the effect of suppressing the production of collagenase MMP1 and 3 is evaluated using normal human skin fibroblast cells and an aging model (senescence cells) in which normal cells are treated with hydrogen peroxide.

Japanese Patent No. 6979918 (Patent Document 3) discloses a human senescent cell activator containing 3-hydroxybutyric acid alkyl ester as an active ingredient. In this document, the collagen content of senescent cells obtained by treating human normal fibroblasts with hydrogen peroxide solution was measured for the human senescent cell activation effect.

JP 2017-200883 A Japanese Patent No. 6804352 Patent No. 6979918

However, Patent Documents 1 to 3 do not describe transglutaminase.

Therefore, an object of the present invention is to provide a transglutaminase production promoter capable of improving the production of transglutaminase, which acts on the formation process of skin epidermal cells and corneocytes, and uses thereof.

As a result of intensive research to solve the above problems, the inventors discovered that 3-hydroxybutyric acid and/or its salts can promote the production of transglutaminase, which acts on processes such as the formation of epidermal cells and keratinocytes in the skin, and thus completed the present invention.

That is, the transglutaminase production promoter as aspect [1] of the present invention contains 3-hydroxybutyric acid and/or a salt thereof.

Aspect [2] of the present invention is an embodiment in which the 3-hydroxybutyric acid and/or its salt contains the R form in the above aspect [1].

Aspect [3] of the present invention is an aspect in which the production of transglutaminase 1 is promoted in the above aspect [1] or [2].

Aspect [4] of the present invention is an aspect of any one of aspects [1] to [3] above, in which the production of transglutaminase is promoted during the formation of keratinocytes.

Aspect [5] of the present invention is any of aspects [1] to [4] above, in which the production of transglutaminase is promoted in dry skin.

The present invention also includes, as aspect [6], a composition that contains 3-hydroxybutyric acid and/or a salt thereof and is for promoting the production of transglutaminase.

Aspect [7] of the present invention is an embodiment in which the composition of aspect [6] is a transdermal composition (or external composition).

Aspect [8] of the present invention is an embodiment in which the composition of Aspect [6] or [7] is a liquid or semi-solid composition.

Aspect [9] of the present invention is an embodiment in which the composition of any one of the above aspects [6] to [8] is a skin care product.

The present invention also includes, as aspect [10], a protein production promoter for promoting the production of proteins constituting the cornified envelope, the protein production promoter containing 3-hydroxybutyric acid and/or a salt thereof. It will be done.

Aspect [11] of the present invention is the aspect of aspect [10], in which the protein is involucrin and/or filaggrin.

The present invention also includes, as aspect [12], a cornified envelope forming ability enhancer containing 3-hydroxybutyric acid and/or a salt thereof.

The present invention also includes, as aspect [13], a lipid envelope forming ability enhancer containing 3-hydroxybutyric acid and/or a salt thereof.

The present invention also includes, as embodiment [14], a skin barrier function improver that contains 3-hydroxybutyric acid and/or a salt thereof.

The present invention also includes, as aspect [15], a method for promoting the production of transglutaminase during the formation of keratinocytes by permeating the skin with a transglutaminase production promoter according to any one of aspects [1] to [5] above.

The present invention also includes, as aspect [16], a method for normalizing keratin by infiltrating the transglutaminase production promoter according to any one of aspects [1] to [5] into the skin.

The present invention also includes, as aspect [17], a method for improving the barrier function of the skin by permeating the transglutaminase production promoter according to any one of aspects [1] to [5] into the skin. It will be done.

Aspect [18] of the present invention is any of aspects [15] to [17] above, in which the transglutaminase production promoter is repeatedly and intermittently infiltrated into the skin for 24 hours or more.

In the present invention, since the transglutaminase production promoter contains 3-hydroxybutyric acid (also referred to as "3HB" or "BHB") and/or a salt thereof, it is possible to promote the production of transglutaminase in vivo. Therefore, it can promote the production of transglutaminase 1 during the formation process of corneocytes, forming a strong cornified envelope and lipid envelope, and improving the skin's barrier function (i.e., increasing the physical strength of the skin surface and moisturizing function). function) can be improved. Furthermore, not only the production of transglutaminase 1 but also the production of involucrin (IVN) and filaggrin (FLG), which are proteins that constitute CE, can be improved, so that a stronger CE can be formed. In particular, not only healthy skin but also dry skin due to skin diseases such as aging and allergies (especially dry skin due to aging) can be normalized by penetrating the skin with a transglutaminase production promoter. , it has high moisturizing properties and can regenerate moisturized skin. Furthermore, the barrier function of the skin is improved, and the corneocytes are regularly arranged in a tile pattern to regenerate normal corneum, making it more resistant to external stimuli such as ultraviolet rays, physical forces, and pathogenic bacteria. I can handle it.

FIG. 1 is a graph comparing the amount of 3HB added and the change in the amount of TGM-1 mRNA expression in Example 1. FIG. 2 is a processed photograph showing the difference in TGM-1 mRNA expression level depending on the presence or absence of 3HB in Example 1. FIG. 3 is a graph comparing the amount of 3HB added and the change in IVN mRNA expression level in Example 1. FIG. 4 is a graph comparing the amount of 3HB added and the change in the amount of FLG mRNA expression in Example 1. FIG. 5 is a graph comparing the amount of transepidermal water transpiration between Example 2 and Comparative Example 1. FIG. 6 is a graph comparing the moisture content of the stratum corneum between Example 2 and Comparative Example 1. FIG. 7 is a graph comparing the moisturizing ability of Example 2 and Comparative Example 1. FIG. 8 is a set of photographs comparing the stratum corneum stained with brilliant green and gentian violet before and after use of the liquid composition in Example 2 and Comparative Example 1. FIG. 9 is a graph comparing the average values of the stratum corneum cell areas in Example 2 and Comparative Example 1. FIG. 10 is a graph comparing the multiple peeling rates of Example 2 and Comparative Example 1. FIG. 11 is a set of processed photographs comparing the stratum corneum stained with dansylcadaverine before and after use of the liquid composition in Example 2 and Comparative Example 1. FIG. 12 is a graph comparing TGM-1 activity between Example 2 and Comparative Example 1. FIG. 13 is a processed photograph comparing the stratum corneum dyed with Nile red in Example 2 and Comparative Example 1 before and after using the liquid composition. FIG. 14 is a graph comparing the degree of Nile Red staining between Example 2 and Comparative Example 1.

[Transglutaminase production promoter]
The transglutaminase production promoter of the present invention contains 3-hydroxybutyric acid and/or a salt thereof [hereinafter may be collectively referred to as "3HB (salt)"], and 3HB (salt) acts as an active ingredient.

The transglutaminase production promoter of the present invention can promote transglutaminase production in a wide variety of living organisms, from microorganisms to mammals. Transglutaminase is not particularly limited as long as it can cross-link proteins (such as cross-linking between glutamine and lysine), but among the eight isozymes, transglutaminase 1 (TGM-1), TGM-3 and TGM-5 are preferred, and TGM-1 is particularly preferred. In particular, TGM-1 (CE protein cross-linking enzyme) promotes the binding of proteins that make up CE during the process in which basal cells in the basal layer of the epidermis form corneocytes via spiny cells and granule cells. , can also promote the binding of CE to intercellular lipids. Therefore, the present invention also includes a cornified envelope forming ability improver containing 3HB (salt) as an active ingredient and a lipid envelope forming ability improving agent containing 3HB (salt) as an active ingredient.

3HB (or BHB) may be an optical isomer (R-form or S-form) or a racemate, but from the viewpoint of biocompatibility etc., R-form (R-3-hydroxybutyric acid) It is preferable to include at least.

The proportion of R isomers in 3HB, particularly the optical purity (enantiomer or optical isomer excess), is, for example, 50% e.e. or more (e.g., 80% e.e. or more), preferably 90% e.e. or more (e.g., 95 to 100% e.e.), and more preferably 98 to 100% e.e. (e.g., 99 to 100% e.e., particularly substantially 100% e.e.). If the optical purity is too low, there is a risk of reduced biocompatibility.

Furthermore, the R form [(R)3HB] and the S form [(S)3HB] and/or the racemic form may be used in combination, but the mass proportion of the R form in 3HB is 10% by mass or more. The content is preferably 50% by mass or more, more preferably 90% by mass or more, and most preferably 100% by mass. When the proportion of R-isomer is high, when used as cosmetics or medicines, the biocompatibility is high, so that the function of 3HB can be efficiently expressed in vivo.

As 3HB, commercially available products may be used. Examples of commercially available products include chemically synthesized 3HB and 3HB produced by fermentation using microorganisms. Among these, fermentation-produced 3HB (fermentation-derived 3HB) is preferred because of its high purity of the R-isomer, and fermentation-produced 3HB using microorganisms from biomass raw materials (resources derived from living organisms) is particularly preferred.

3HB may be 3HB alone in the form of an acid, 3HB salt alone in the form of a salt, or a combination (mixture) of 3HB and 3HB salt. Among these, 3HB salt alone is preferred from the viewpoint of ease of handling.

Examples of the types of salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as magnesium salts and calcium salts; ammonium salts; amine salts; and salts with basic amino acids. It will be done. These 3HB salts (3HB salts) can be used alone or in combination of two or more. Among these, sodium salts, magnesium salts, and calcium salts are preferred because they have relatively low deliquescent properties and are easy to handle, and sodium salts are particularly preferred because the balance of various properties can be easily adjusted.

The proportion of 3HB (salt) in the transglutaminase production promoter may be 10% by mass or more, preferably 50% by mass or more, more preferably 80% by mass or more, more preferably 90% by mass or more, most preferably It is 100% by mass.

The transglutaminase production promoter of the present invention may further contain a 3HB oligomer. The average degree of polymerization of the 3HB oligomer may be 2 or more, for example, 2 to 10, preferably 2 to 5, further preferably 2 to 4, more preferably 2 to 3, and most preferably 2. The 3HB oligomer may be an oligomer that is inevitably mixed in during the manufacturing process of 3HB, etc.

The proportion of 3HB oligomers may be 10 parts by mass or less, preferably 5 parts by mass or less, more preferably 3 parts by mass or less, and most preferably 1 part by mass or less, per 100 parts by mass of 3HB (salt) (active ingredient). The transglutaminase production promoter of the present invention may not substantially contain 3HB oligomers, and it is particularly preferable that it does not contain 3HB oligomers.

The transglutaminase production promoter of the present invention can be used to promote the production of transglutaminase in the body of a mammal such as a human, and is preferably used to promote the production of transglutaminase in the epidermis of a medium-sized or large mammal (particularly a human).

The method of application (or administration) of the transglutaminase production promoter of the present invention may be an oral application method or a parenteral application method. Of these, the parenteral application method is preferred. Examples of parenteral application methods include an inhalation application method, an injection application method, a transdermal application method, and a nasal application method. Of these, the transdermal application method is preferred because it can effectively promote the production of transglutaminase in the epidermis.

The transdermal application method is not particularly limited as long as it allows the transglutaminase production promoter applied to the skin surface to penetrate into the outer skin. Specific methods include, for example, applying the transglutaminase production promoter directly to the skin by dropping or spraying, or using a carrier impregnated with the transglutaminase production promoter (a mask made of non-woven fabric, cotton, sponge, film, etc.). ) may be brought into contact with the skin. The application site on human skin is not particularly limited, and can be applied to the skin of the entire body including the scalp and lips.

The application amount (or dosage) of the transglutaminase production promoter of the present invention is determined as appropriate depending on the application target, age and body weight of the application target, application time, form of the transglutaminase production promoter, application route, application method, etc. You can choose. For example, when applied transdermally, the transglutaminase production promoter of the present invention may be applied to the necessary site (skin, etc.) about 1 to 10 times, preferably 1 to 5 times per day. times, more preferably 1 to 4 times, more preferably 1 to 3 times, and most preferably 1 to 2 times.

When the transglutaminase production promoter of the present invention is applied to the skin, it can not only promote the production of transglutaminase 1 in the epidermis during the formation of keratinocytes, but also promote the production of involucrin and filaggrin, which are proteins that constitute CE, and therefore normal keratinocytes with strong CE and lipid envelope can be formed. The cycle in which keratinocytes change from basal cells to keratinocytes and are exfoliated as dirt on the skin surface is called "turnover", and this cycle is generally estimated to be about 4 to 6 weeks. Therefore, the transglutaminase production promoter of the present invention is preferably applied intermittently (or periodically) repeatedly for a predetermined number of days so as to affect turnover, and may be applied intermittently repeatedly for, for example, 24 hours or more, preferably for 1 week or more (e.g., 1 to 20 weeks), more preferably for 4 weeks or more, more preferably for 6 weeks or more, and most preferably for 8 weeks or more. For example, if applied 1 to 2 times a day for 4 weeks or more, keratinocytes can be replaced with normal keratinocytes by the transglutaminase production promoter of the present invention.

The transglutaminase production promoter of the present invention can generate (regenerate) normal corneocytes, so even if the skin is dry due to aging or allergies, it can be applied repeatedly to the skin to restore normal dry skin. can be converted into In particular, the effect of promoting transglutaminase production is more effective in dry skin than in healthy skin. Therefore, the transglutaminase production promoter of the present invention is preferably applied to dry skin, and particularly preferably applied to dry skin due to aging for the purpose of anti-aging.

In this specification and claims, "dry skin" includes not only skin with low moisture content, but also skin with low moisture content and rough skin (dull skin, roughness, hangnails, wrinkles, etc.).

[Composition for promoting the production of transglutaminase]
The composition of the present invention is a composition for promoting the production of transglutaminase, and contains 3HB (salt), preferably (R)3HB (salt), as an active ingredient.

The composition of the present invention may be a liquid composition, a semi-solid composition, or a solid composition. That is, in the composition of the present invention, (R)3HB (salt) is blended as the aforementioned transglutaminase production promoter into the liquid composition, semisolid composition, or solid composition.

Examples of liquid compositions include solutions, drinks, suspensions, emulsions, syrups, and injections.

Examples of semi-solid compositions (semi-solid agents) include gels, creams, slurries, and pastes.

Examples of solid compositions (solid preparations) include powders, fine granules, granules, pills, tablets, flakes, cakes, gummies, nougat, films, and capsules.

Among these, external compositions (or external preparations), such as liquid compositions and semi-solid compositions, are preferable because the composition of the present invention is particularly effective for the skin and can be easily applied transdermally. , liquid compositions are particularly preferred.

The active ingredients in the composition, including preferred embodiments, are the same as those described above as transglutaminase production promoters.

The proportion of the active ingredient [3HB (salt)] in the composition can be selected from a range of about 0.01 to 99% by mass, preferably 0.1 to 95% by mass, more preferably 0.5 to 90% by mass. be. The proportion of active ingredients may be selected depending on the form of the composition.

The method of application of the composition of the present invention, including preferred embodiments, is the same as the method of application described for the transglutaminase production promoter. Therefore, the composition of the present invention is preferably a transdermal composition.

The transdermal composition of the present invention may be a transdermal liquid composition or a transdermal semi-solid composition.

(Transdermal liquid composition)
In the transdermal liquid composition (or external liquid composition), the proportion of the active ingredient is, for example, 0.01% by mass or more, preferably 0.1% by mass or more, more preferably 0.2% by mass or more, more preferably 0.3% by mass or more, particularly preferably 0.5% by mass or more, and most preferably 0.8% by mass or more in the transdermal liquid composition. If the proportion of the active ingredient is too small, there is a risk that the production of transglutaminase cannot be promoted. In addition, in the transdermal liquid composition, the proportion of the active ingredient is, for example, 30% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less, even more preferably 3% by mass or less, particularly preferably 2% by mass or less, and most preferably 1.5% by mass or less in the transdermal liquid composition. The proportion of the active ingredient may be 0.1 to 5% by mass (particularly 0.3 to 3% by mass) in the transdermal liquid composition. If the proportion of the active ingredient is too high, there is a risk that the handling properties may be reduced.

The transdermal liquid composition of the present invention may further contain a liquid base. The liquid base includes a solvent, a liquid oil, etc.

Although the solvent may be a lipophilic solvent, a hydrophilic solvent is preferable from the viewpoint of safety. Examples of the hydrophilic solvent include water, lower aliphatic alcohols (eg, C _1-4 alkyl alcohols such as ethanol and isopropanol), and the like. These solvents can be used alone or in combination of two or more. Examples of liquid oils include animal and vegetable oils (e.g., jojoba oil, olive oil, coconut oil, camellia oil, macadamian nut oil, avocado oil, corn oil, sesame oil, wheat germ oil, linseed oil, castor oil, etc.), and mineral oils. Examples include oils based on oils (eg, liquid paraffin, polybutene, silicone oil, etc.), synthetic oils (eg, synthetic ester oils, synthetic polyether oils, etc.). These liquid oils can be used alone or in combination of two or more.

As a liquid base, a hydrophilic solvent such as water or ethanol may be used in combination with a liquid oil as an additive (oil). Of these liquid bases, water, lower alcohols or mixtures thereof are preferred, with water and/or ethanol (especially water) being particularly preferred.

The proportion of the liquid base in the transdermal liquid composition is 50 to 99.9% by mass, preferably 60 to 99% by mass, more preferably 70 to 95% by mass, even more preferably 80 to 93% by mass, and most preferably 85 to 90% by mass.

The transdermal liquid composition of the present invention may further contain a humectant. Examples of humectants include alkylene glycols (e.g., ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, pentanediol, polyethylene glycol, polyoxyethylene-polyoxypropylene block copolymer, polyalkylene glycol or its monoalkyl ester such as diethylene glycol monoethyl ether, polyoxyethylene polyoxypropylene dimethyl ether), water-soluble vinyl polymers (e.g. polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyacrylic acid, polyacrylamide) ), polyhydric alcohols (e.g., glycerin such as concentrated glycerin, pentaerythritol, diglycerin, diglycerin propylene oxide adduct, etc.), organic acids (e.g., lactic acid, sodium lactate, sodium pyrrolidone carboxylate, etc.), amino acids (e.g., serine, glycine, threonine, alanine, etc.), sugars (e.g., sugar alcohols such as xylitol, sorbitol, maltitol; polysaccharides such as hyaluronic acid, sodium hyaluronate, sodium chondroitin sulfate, chondroitin heparin, etc.), water-soluble Cellulose ethers (eg, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), proteins (eg, vitronectin, fibronectin, keratin, elastin, royal jelly, sericin, etc.), and the like.

These moisturizers can be used alone or in combination. Of these moisturizers, alkylene glycols such as dipropylene glycol, 1,3-butylene glycol, pentanediol (pentylene glycol), and polyethylene glycol, polyhydric alcohols such as glycerin, and sugars such as sodium hyaluronate are preferred.

The proportion of the humectant is, for example, 1 to 100 parts by weight, preferably 3 to 80 parts by weight, more preferably 5 to 50 parts by weight, more preferably 8 to 30 parts by weight, and most preferably 1 to 100 parts by weight, based on 100 parts by weight of the liquid base. Preferably it is 10 to 20 parts by mass.

The transdermal liquid composition of the present invention may further contain an emollient. Examples of emollients include higher fatty acids or oils (e.g., oleic acid, isostearic acid, oxystearic acid, oleyl alcohol, cetearyl alcohol, isopropyl stearate, octyl oxystearate, glycerin oxystearate, decyl oleate, octyldodecyl 12-stearoyl stearate, polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, polyoxyethylene hydrogenated castor oil, triethylhexanoin, dimethicone, etc.), waxes (e.g., lanolin, lanolin alcohol, lanolin oil, polyoxyethylene lanolin, polyoxyethylene propylene lanolin, honey, etc.), and the like. wax, polyoxyethylene beeswax, etc.), sugars (e.g., polyoxyethylene methyl glycoside, methyl sesquistearate glycoside, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene sorbitan oleate, polyoxyethylene sucrose oleate, polyoxyethylene methyl dioleate glycoside, polyoxyethylene methyl glycoside sesquistearate, etc.), ceramides, squalane, squalene, honey, emulsions of oily components (e.g., emulsions of oily components such as triglyceride oil, squalane, and ester oil emulsified with a nonionic emulsifier such as monoglyceride, etc.), etc.

These emollients can be used alone or in combination of two or more. Of these emollients, preferred are higher alcohols such as cetearyl alcohol, oils and fats such as triethylhexanoin and dimethicone, polyoxyethylene group-containing sugars such as polyoxyethylene methyl glycoside, oils and fats such as glycerin oxystearate and polyoxyethylene hydrogenated castor oil, and waxes such as beeswax and lanolin.

The proportion of the emollient may be, for example, 50 parts by mass or less, preferably 0.01 to 30 parts by mass, more preferably 0.1 to 20 parts by mass, even more preferably 0.5 to 20 parts by mass, and most preferably 1 to 10 parts by mass, per 100 parts by mass of the liquid base.

The transdermal liquid composition of the present invention may further contain a surfactant. Surfactants may be used as solubilizers and emulsifiers. The surfactant may be any of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants.

In addition, in this specification and the claims, "POE" means polyoxyethylene (polyethylene oxide), and "POP" means polyoxypropylene (polypropylene oxide).

Examples of anionic surfactants include glycerin fatty acid esters such as glyceryl monostearate (glyceryl stearate); higher fatty acid soaps such as sodium laurate; higher alkyl sulfate ester salts such as sodium lauryl sulfate; alkyl ether sulfate ester salts such as sodium POE-lauryl sulfate and triethanolamine POE-lauryl sulfate; N-acyl sarcosine salts such as sodium N-lauroyl sarcosine; sodium N-stearoyl-N-methyl taurate, sodium N-myristoyl-N-methyl taurate, sodium N- Examples include higher fatty acid amide sulfonates such as sodium lauryl-N-methyl taurate; phosphate ester salts such as sodium POE-oleyl ether phosphate; sulfosuccinates such as sodium di-2-ethylhexyl sulfosuccinate; alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate and triethanolamine dodecylbenzene sulfonate; N-acyl glutamates such as sodium N-lauroyl-L-glutamate and disodium N-stearoyl-L-glutamate; and sulfated oils (e.g., turmeric oil).

Examples of nonionic surfactants include POE-sorbitan fatty acid esters such as POE-sorbitan monooleate, POE-sorbitan monostearate, POE-sorbitan monooleate, and POE-sorbitan tetraoleate; POE-sorbit fatty acid esters such as POE-sorbitol, POE-sorbitol monooleate, POE-sorbitolpentaoleate, and POE-sorbitol monostearate; sucrose fatty acid esters such as sucrose laurate; POE-glycerol monostearate; POE-glycerin fatty acid esters such as POE-glycerin monoisostearate and POE-glycerin triisostearate; POE-fatty acid esters (for example, POE-distearate, POE-monodioleate, ethylene glycol distearate, etc.); POE-alkyl Ethers (e.g., POE-lauryl ether, POE-oleyl ether, POE-stearyl ether, POE-behenyl ether, POE-2-octyldodecyl ether, POE-cholestanol ether, etc.); Pluronic types (e.g., Pluronic® ); POE/POP-alkyl ethers (e.g., POE/POP-cetyl ether, POE/POP-2-decyltetradecyl ether, POE/POP-monobutyl ether, POE/POP-hydrogenated lanolin, POE/ POP-glycerin ether, etc.); Tetra-POE/tetra-POP-ethylenediamine condensates (e.g. Tetronic, etc.); POE-castor oil hydrogenated castor oil derivatives (e.g. POE-castor oil, POE-hydrogenated castor oil, POE-hardened castor oil monoisostearate, POE-hydrogenated castor oil triisostearate, POE-hydrogenated castor oil monopyroglutamic acid monoisostearate diester, POE-hydrogenated castor oil maleic acid, etc.); POE-beeswax lanolin derivatives (e.g. POE-sorbitol); beeswax, etc.); alkanolamides (e.g., coconut oil fatty acid diethanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide, etc.); POE-propylene glycol fatty acid ester; POE-alkylamine; POE-fatty acid amide; alkyl ethoxydimethylamine oxide; Examples include trioleyl phosphate.

Examples of the cationic surfactant include stearyltrimethylammonium chloride; cetylpyridinium chloride; benzalkonium chloride; and benzethonium chloride.

Examples of the amphoteric surfactant include phospholipid-based amphoteric surfactants such as hydrogenated lecithin; imidazoline-based amphoteric surfactants; and betaine-based surfactants.

Silicone-based surfactants can also be used as surfactants. Examples of silicone-based surfactants include polyether-modified silicones (adducts in which at least ethylene oxide is added to polydimethylsiloxane).

These surfactants can be used alone or in combination of two or more. Among these surfactants, anionic surfactants such as glyceryl stearate, sodium lauryl sulfate, triethanolamine lauryl ether sulfate, disodium lauryl polyoxyethylene sulfosuccinate, and nonionic surfactants such as POE-hydrogenated castor oil. Amphoteric surfactants such as hydrogenated lecithin and hydrogenated lecithin are preferred.

The proportion of the surfactant is, for example, 0.01 to 5 parts by weight, preferably 0.03 to 3 parts by weight, more preferably 0.05 to 1 part by weight, and more preferably 0.1 to 0.5 parts by weight, most preferably 0.2 to 0.3 parts by weight.

The transdermal liquid composition of the present invention may further contain a preservative. Preservatives include antiseptics, bactericides or antibacterial agents, antioxidants, ultraviolet absorbing agents or ultraviolet scattering agents, etc.

Examples of preservatives include phenoxyethanol, chlorphenesin, benzoic acid, salicylic acid, sorbic acid, and paraoxybenzoic acid alkyl esters (parabens).

Examples of the bactericidal or antibacterial agent include sodium benzoate.

Examples of antioxidants include inorganic acids such as phosphoric acid and sodium pyrosulfite; organic acids such as malonic acid, succinic acid, ascorbic acid, maleic acid, fumaric acid, and edetic acid; phosphorus compounds such as cephalin and phytic acid; and polyphenols such as anthocyanins.

Examples of ultraviolet absorbing or scattering agents include tocopherol acetate, ethylhexyl methoxycinnamate, ethylhexyl 4-(N,N-dimethylamino)benzoate, t-butyl methoxydibenzoylmethane, oxybenzone, zinc oxide, and titanium oxide.

These preservatives can be used alone or in combination of two or more. Among these preservatives, preservatives such as phenoxyethanol are preferred.

The proportion of the preservative is, for example, 0.01 to 10 parts by weight, preferably 0.05 to 5 parts by weight, more preferably 0.1 to 3 parts by weight, and more preferably 0. .2 to 1 part by weight, most preferably 0.3 to 0.5 part by weight.

The transdermal liquid composition of the present invention may further contain a fragrance. The fragrance may be a natural fragrance or a synthetic fragrance.

Examples of natural fragrances include fruit essences or oils (vanillin, lemon oil, etc.) such as strawberry, blueberry, apple, plum, orange, lemon, lime, vanilla, and pepper; fruit peels such as orange, white grape, grapefruit, and lemon; Bark essence or oil such as Nikki (cinnamon); Bark powder such as cinnamon powder; Root vegetable essence or oil such as ginger; Root vegetable powder such as ginger powder; Seeds such as vanilla beans and cacao powder. Examples include foliage-based powders; foliage-based essences or oils such as peppermint, spearmint, rosemary, and perilla; foliage-based powders such as peppermint powder; and flower-based essences or oils such as jasmine, lavender, rose, rosemary, and hyacinth.

Examples of synthetic fragrances include benzyl acetate, linalyl acetate, citral, citronellal, citronellol, cis-jasmine, cis-3-hexenol, and menthol.

These fragrances can be used alone or in combination.

The proportion of the fragrance is, for example, 0.001 to 5 parts by mass, preferably 0.003 to 1 part by mass, more preferably 0.005 to 0.5 parts by mass, and more preferably 0.01 to 0.1 parts by weight, most preferably 0.015 to 0.05 parts by weight.

The transdermal liquid composition of the present invention may further contain a pH adjuster. Examples of the pH adjuster include bases such as sodium hydrogen carbonate; acids such as citric acid, sodium citrate, and sodium monohydrogen phosphate; and borax. These pH adjusters can be used alone or in combination of two or more. Among these, organic acids such as citric acid and sodium citrate are preferred. The proportion of the pH adjuster can be appropriately selected depending on the desired pH.

The transdermal liquid composition of the present invention may further contain a physiologically active or pharmacologically active ingredient.

Physiologically active ingredients (or pharmacologically active ingredients) include, for example, cell activators (e.g. riboflavin, pyridoxine, nicotinic acid, pantothenic acid, α-tocopherol or derivatives thereof; plant extracts such as saxifrage extract), skin roughness prevention agents, etc. whitening agents (e.g., ascorbic acid or its derivatives, cysteine, placenta extract, arbutin, kojic acid, rucinol) , ellagic acid, chamomile extract, etc.), stain and freckle inhibitors (e.g., tyrosinase activity inhibitors, melanin reducing agents, etc.), acne suppressants (e.g., keratin softeners such as sulfur, anti-inflammatory agents, adrenal corticosteroids, sebum secretion) emollients (e.g., salicylic acid or its derivatives, urea, etc.), anti-inflammatory agents (e.g., allantoin, guaiazulene, glycyrrhizic acid or its salts, glycyrrhetinic acid or its salts, ε-aminocaproic acid, tranexamic acid, Examples include ibuprofen, indomethacin, zinc oxide, derivatives thereof; plant extracts such as arnica extract, etc.), skin itch suppressants, and blood circulation promoters. These physiologically active ingredients can be used alone or in combination of two or more.

The proportion of the physiologically active ingredient is, for example, 0.001 to 10 parts by mass, preferably 0.01 to 5 parts by mass, more preferably 0.03 to 3 parts by mass, even more preferably 0.05 to 2 parts by mass, and most preferably 0.1 to 1 part by mass, per 100 parts by mass of the liquid base.

The liquid composition of the present invention may further contain other components.

Other ingredients include, for example, astringents or antiperspirants (e.g., oxyacids such as lactic acid, tartaric acid, or their salts; aluminum compounds such as aluminum chloride; zinc compounds such as zinc sulfate and zinc sulfonate; proanthocyanidins; extracts of tannin-containing plants such as witch hazel and white birch; chinese laurel extract, rhubarb extract, horsetail extract, etc.), coenzymes (e.g., coenzyme Q10, etc.), amino acids (e.g., tryptophan, etc.), cooling agents (e.g., menthol or its derivatives, camphor, thymol, etc.), inorganic salts (e.g., sodium sulfate, potassium chloride, etc.), fibers (e.g., synthetic fibers such as nylon fibers, natural natural fibers, etc.), binders (e.g., carboxymethylcellulose, sodium carboxymethylcellulose, carrageenan, etc.), chelating agents or sequestering agents (e.g., gluconic acid, citric acid, succinic acid, ascorbic acid, sodium edetate, sodium 1-hydroxyethane-1,1-diphosphonate, phosphoric acid, sodium hexametaphosphate, etc.), reducing agents (e.g., thioglycolic acid or its salts, etc.), enzymes (e.g., lipase, protease, etc.), thickeners (e.g., gelatin, gluten, fish protein, etc.), basic agents, oxidizing agents, cleansing agents, cooling agents, colorants, opacifying agents, solidifying agents, plasticizers, etc.

These other components can be used alone or in combination of two or more.

The proportion of other ingredients is, for example, 0.001 to 30 parts by mass, preferably 0.01 to 20 parts by mass, more preferably 0.03 to 10 parts by mass, even more preferably 0.05 to 5 parts by mass, and most preferably 0.1 to 3 parts by mass, relative to 100 parts by mass of the liquid base.

The pH of the transdermal liquid composition of the present invention is, for example, 3 to 9, preferably 4 to 8, more preferably 5 to 7, more preferably 5.5 to 6.5, and most preferably 5.8 to 6. .2.

When the transdermal liquid composition of the present invention contains a liquid base, it can be produced by mixing the active ingredient, the liquid base, and, if necessary, components other than the active ingredient and the liquid base. A method of manufacturing by dissolving or dispersing the components in a liquid base is preferred.

(Semi-solid composition for transdermal use)
In the semisolid composition for transdermal use (or semisolid composition for external use), the proportion of the active ingredient is 0.01% by mass or more in the semisolid composition for transdermal use, and 0.1% by mass or more is It is preferably 0.2% by mass or more, more preferably 0.3% by mass or more, particularly preferably 0.5% by mass or more, and most preferably 0.8% by mass or more. If the proportion of the active ingredient is too low, there is a risk that transglutaminase production may not be promoted. Further, in the transdermal semisolid composition, the proportion of the active ingredient is, for example, 30% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less. , more preferably 3% by mass or less, particularly preferably 2% by mass or less, and most preferably 1.5% by mass or less. The proportion of the active ingredient may be 0.1 to 5% by weight (in particular 0.3 to 3% by weight) in the transdermal semisolid composition. Furthermore, if the proportion of the active ingredient is too high, there is a risk that the handling properties will be reduced.

The transdermal semisolid composition of the present invention may further contain a liquid base. Examples of the liquid base include the liquid bases exemplified in the section of the liquid composition for transdermal use. The above liquid bases can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition.

The proportion of the liquid base in the transdermal semisolid composition is 10 to 95% by weight, preferably 30 to 90% by weight, more preferably 50 to 85% by weight, more preferably 60 to 80% by weight, and most preferably is 65 to 75% by mass.

The semi-solid composition for transdermal use of the present invention may further contain an emollient. Examples of the emollient include the emollients exemplified in the section on the liquid composition for transdermal use. The emollients may be used alone or in combination of two or more. The preferred embodiments are the same as those of the liquid composition. In particular, in the semi-solid composition for transdermal use, multiple emollients may be combined, for example, oils and fats, higher fatty acids or derivatives thereof, and waxes may be combined.

The proportion of the emollient agent may be, for example, 1 part by mass or more, for example 1 to 1000 parts by mass, preferably 5 to 100 parts by mass, and more preferably 10 to 80 parts by mass, based on 100 parts by mass of the liquid base. , more preferably 15 to 50 parts by weight, most preferably 20 to 30 parts by weight. If the proportion of the emollient agent is too small, there is a possibility that the semi-solid state cannot be maintained, and if the proportion of the emollient is too large, there is a possibility that the handleability will be deteriorated.

The transdermal semisolid composition of the present invention may further contain a gelling agent. Examples of gelling agents include dextran, pullulan, agar, pectin, alginic acid, sodium alginate, carrageenan, galactomannan, glucomannan, curdlan, gum arabic, gum tragacanth, gellan gum, gum karaya, guar gum, xanthan gum, locust bean gum, and tara gum. Water-soluble or water-swellable polysaccharides such as , tamarind seed gum, and psyllium seed gum; proteins such as collagen, casein, albumin, gelatin, and royal jelly; and inorganic water-soluble polysaccharides such as bentonite, organically modified bentonite, aluminum magnesium silicate, and silicic anhydride. Examples include polymers. These gelling agents can be used alone or in combination of two or more. Among these gelling agents, water-swellable polysaccharides such as xanthan gum are preferred.

The proportion of the gelling agent is, for example, 0.01 to 100 parts by mass, preferably 0.03 to 10 parts by mass, more preferably 0.05 to 1 part by mass, even more preferably 0.08 to 0.5 parts by mass, and most preferably 0.1 to 0.3 parts by mass, relative to 100 parts by mass of the liquid base.

The transdermal semisolid composition of the present invention may further contain a humectant. Examples of the humectant include the humectants exemplified in the section of the liquid composition for transdermal use. The humectants can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition.

The proportion of the moisturizer is, for example, 1 to 100 parts by weight, preferably 3 to 80 parts by weight, more preferably 5 to 50 parts by weight, even more preferably 8 to 30 parts by weight, and most preferably 10 to 20 parts by weight, per 100 parts by weight of the liquid base.

The transdermal semisolid composition of the present invention may further contain a surfactant. Examples of the surfactant include the surfactants exemplified in the section of the liquid composition for transdermal use. The surfactants can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition.

The proportion of the surfactant is, for example, 0.01 to 30 parts by weight, preferably 0.1 to 20 parts by weight, more preferably 0.5 to 15 parts by weight, and more preferably 1 to 10 parts by weight, most preferably 3 to 5 parts by weight.

The transdermal semisolid composition of the present invention may further contain a preservative. Examples of the preservative include the preservatives exemplified in the section of the liquid composition for transdermal use. The above preservatives can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition.

The proportion of the preservative is, for example, 0.01 to 10 parts by mass, preferably 0.05 to 5 parts by mass, even more preferably 0.1 to 3 parts by mass, even more preferably 0.2 to 1 part by mass, and most preferably 0.3 to 0.5 parts by mass, per 100 parts by mass of the liquid base.

The transdermal semisolid composition of the present invention may further contain a fragrance. Examples of perfumes include the perfumes exemplified in the section of the liquid composition for transdermal use. The above fragrances can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition.

The proportion of the flavoring is, for example, 0.001 to 5 parts by mass, preferably 0.003 to 1 part by mass, even more preferably 0.005 to 0.5 parts by mass, even more preferably 0.01 to 0.1 parts by mass, and most preferably 0.015 to 0.05 parts by mass, relative to 100 parts by mass of the liquid base.

The transdermal semisolid composition of the present invention may further contain a pH adjuster. Examples of the pH adjuster include the pH adjusters exemplified in the section of the liquid composition for transdermal use. The pH adjusters can be used alone or in combination of two or more. Preferred embodiments are also the same as those for the transdermal liquid composition. The proportion of the pH adjuster can be appropriately selected depending on the desired pH.

The semi-solid composition for transdermal use of the present invention may further contain a physiologically active ingredient. Examples of the physiologically active ingredient include the physiologically active ingredients exemplified in the section on the liquid composition for transdermal use. The physiologically active ingredients may be used alone or in combination of two or more kinds. The preferred embodiments are also the same as those of the liquid composition for transdermal use.

The proportion of the physiologically active ingredient is, for example, 0.001 to 10 parts by mass, preferably 0.01 to 5 parts by mass, more preferably 0.03 to 3 parts by mass, even more preferably 0.05 to 2 parts by mass, and most preferably 0.1 to 1 part by mass, per 100 parts by mass of the liquid base.

The semi-solid composition for transdermal use of the present invention may further contain other components. Examples of the other components include the other components exemplified in the section on the liquid composition for transdermal use. The components can be used alone or in combination of two or more. The preferred embodiments are the same as those of the liquid composition for transdermal use.

The proportion of other components is, for example, 0.001 to 30 parts by weight, preferably 0.01 to 20 parts by weight, more preferably 0.03 to 10 parts by weight, and more preferably 0.05 to 5 parts by weight, most preferably 0.1 to 3 parts by weight.

The pH of the semi-solid transdermal composition of the present invention is, for example, 3 to 9, preferably 4 to 8, more preferably 5 to 7, even more preferably 5.5 to 6.5, and most preferably 5.8 to 6.2.

When the transdermal semisolid composition of the present invention contains an emollient, it can be produced by mixing the active ingredient, the emollient, and, if necessary, components other than the active ingredient and the emollient.

(Applications of transdermal composition)
The transdermal composition (external preparation) of the present invention can be used as various cosmetics, cosmetics, pharmaceuticals, or quasi-drugs.

Examples of cosmetics include moisturizing cosmetics, whitening cosmetics, UV care cosmetics, cleansing agents, and scrubbing agents.

Cosmetics include basic cosmetics, skin care cosmetics, makeup cosmetics, UV care cosmetics, body cosmetics, hair care cosmetics, etc.

Examples of basic cosmetics include skin lotions, beauty serums, milky lotions, creams, facial cleansers, and cleansing cosmetics (oil, gel, cream, lotion, milk cosmetics, etc.).

Examples of skin care cosmetics include lotions, milky lotions, gel creams, skin care creams, and massage lotions.

Examples of makeup cosmetics include makeup bases, BB creams, foundations (powder foundations, liquid foundations, cream foundations, emulsion foundations, etc.), lipsticks/lip glosses, eye shadows, eyeliners, eyebrow pencils, mascaras, etc.

Examples of UV care cosmetics include emulsion-like, lotion-like, gel-like, cream-like, and spray-like sunscreen cosmetics.

Examples of body cosmetics include body cleansers, body lotions, body milks, body creams, body powders, and body massage products.

Examples of hair care cosmetics include shampoos, conditioners, treatments, after-bath treatments, tonics, hair styling products, hair growth products, and hair creams.

Examples of pharmaceuticals or quasi-drugs include medicated cosmetics, liquids, aerosols, creams, gels (jelly), ointments, poultices, and liniments.

Among these uses, since the effects of the transdermal composition of the present invention are easily expressed, cosmetics, cosmetics, pharmaceuticals, or quasi-drugs that are used in a manner that they are allowed to penetrate into the skin without being rinsed off immediately after use. is preferred.

Furthermore, the transdermal composition of the present invention is particularly preferably a skin care product for improving the barrier function of the skin. Skin care products may be transdermal liquid compositions such as lotion, serum, lotion, milk, emulsion, mist, etc., or transdermal semi-transdermal compositions such as creams, gels, balms, foams (bubbles), etc. It may be a solid composition. The transdermal liquid composition and transdermal semisolid composition may be beauty compositions or cosmetic compositions (cosmetics), or may be aging care products for anti-aging purposes.

The present invention will be explained in more detail below based on Examples, but the present invention is not limited by these Examples. The raw materials used in the examples and the evaluation methods for the liquid compositions prepared in the examples are as follows.

[material]
3HB: Crystalline (R) 3HB sodium, "OHALOS (registered trademark)" manufactured by Osaka Gas Chemical Co., Ltd.
BG: 1,3-butylene glycol, wetting agent PD: Pentylene glycol, “Diol PD” manufactured by Kyukyu Alcohol Kogyo Co., Ltd., wetting agent Citric acid: pH adjuster Sodium citrate: pH adjuster Ethanol: Has a wetting function Liquid base phenoxyethanol: Preservative PEG-60 hydrogenated castor oil: Polyoxyethylene hydrogenated castor oil, solubilizer.

Example 1
It is known that the expression of glucose transporter 1 (GLUT1) decreases with aging, and the amount of intracellular glucose uptake decreases. Therefore, as a senescent cell model, GLUT1 knockdown epidermal keratinocytes (hereinafter referred to as "GLUT1 KD cells") in which GLUT1 was knocked down were used to evaluate characteristics related to skin barrier function.

[Preparation of GLUT1 KD cells]
Normal human epidermal keratinocytes (manufactured by Kurabo Industries, hereinafter referred to as "NHEK") were grown in a 96-well plate using a growth medium (manufactured by Kurabo Industries, Ltd., "HuMedia-KG2", hereinafter referred to as "KG2") for 1.5 minutes. The cells were seeded at a density of ×10 ⁴ cells/well and cultured for 24 hours.

Next, the growth medium in which NHEK was grown was replaced with a basal medium (“HuMedia-KB2” manufactured by Kurabo Industries, hereinafter referred to as “KB2”) containing siGLUT1 (manufactured by Thermo) at a concentration of 100 nM, and incubated at 37°C. GLUT1 KD cells were generated by incubation for 24 hours. As a negative control, NHEK treated with KB2 containing non-target siRNA (siControl; manufactured by Thermo) was prepared.

[Evaluation of epidermal differentiation and moisturizing-related gene mRNA expression by 3HB treatment using GLUT1 KD cells]
The medium in which GLUT1 KD cells were cultured or the negative control medium (medium in which stationary cells were cultured) was mixed with three types of KB2 (basal medium without 3HB, basal medium containing 3HB at a concentration of 25.0 mM, and basal medium containing 3HB at a concentration of 50.0 mM). The medium was replaced with a basal medium containing 3HB, and incubated at 37°C for 24 hours.

After incubation, total RNA was extracted from the cells, and cDNA was synthesized using reverse transcriptase. Using the cDNA as a template, various mRNAs were subjected to relative quantification (ΔΔCt method) by real-time PCR. As various mRNAs, mRNAs encoding the production of transglutaminase 1 (TGM-1), involucrin (IVN), or filaggrin (FLG) were used. The expression levels of various mRNAs were expressed as relative values to the expression level of mRNA encoding the production of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). It is believed that the higher the expression level of mRNA, the more active the production of the target protein is, and therefore the higher the production level.

[Evaluation results of TGM-1 mRNA expression level]
The evaluation results of TGM-1 mRNA expression level are shown in Table 1 and FIG. 1.

In Tables 1 to 3, "S.D." means standard deviation, "p vs. 0 mM (siControl)" means the p-value for stationary cells not containing 3HB, and "p vs. 0 mM (siGLUT1)" means the p-value for GLUT1 KD cells not containing 3HB. In Figure 1, "**" means that the p-value for stationary cells not containing 3HB is less than 0.01 (p<0.01), and "♯♯" means that the p-value for GLUT1 KD cells not containing 3HB is less than 0.01 (p<0.01).

As is clear from the results in Table 1 and Figure 1, the expression level of TGM-1 mRNA in GLUT1 KD cells without 3HB is about 1/2 that of normal cells; It can be confirmed that the amount increases as a result of this. In particular, the fact that the expression level of GLUT1 KD cells, which was lower than that of stationary cells when 3HB was not included, is conversely expressed more than stationary cells when 3HB is included. It has been shown that it works effectively in

GLUT1 KD cells cultured in a basal medium containing no 3HB and 50.0 mM 3HB were stained with 50 mM dansylcadaverine (a fluorescent dye that binds to TGM-1) and photographed under ultraviolet light using a fluorescent microscope. The stronger the blue fluorescence, the higher the TGM-1 activity. The photographs were processed to binarize the fluorescence intensity (threshold: 125), and the processed photographs are shown in Figure 2. In Figure 2, the white areas represent cells stained with dansylcadaverine (i.e., cells producing TGM-1), and it can be seen that a large amount of TGM-1 is produced in GLUT1 KD cells cultured in a basal medium containing 3HB at a concentration of 50.0 mM (with 3HB in the figure) compared to GLUT1 KD cells cultured in a basal medium not containing 3HB (without 3HB in the figure).

[Evaluation results of IVN mRNA expression level]
The evaluation results of IVN mRNA expression level are shown in Table 2 and FIG. 3.

In addition, "#" in FIG. 3 means that the p value for GLUT1 KD cells that do not contain 3HB is less than 0.05 (p<0.05), and "**" and "##" indicate that It is similar to

As is clear from the results in Table 2 and Figure 3, the IVN mRNA expression level in GLUT1 KD cells is lower than that in stationary cells when 3HB is not included, but it can be confirmed that it increases when 3HB is included. In particular, when 3HB is included, the expression level in GLUT1 KD cells is significantly increased compared to stationary cells.

[Evaluation results of FLG mRNA expression level]
The evaluation results of FLG mRNA expression level are shown in Table 3 and FIG. 4.

In Figure 4, "*" means that the p-value for stationary cells not containing 3HB is less than 0.05 (p<0.05), and "#" is the same as in Figure 3.

As is clear from the results in Table 3 and Figure 4, the expression level of FLG mRNA in GLUT1 KD cells is low compared to stationary cells when 3HB is not included, but it can be confirmed that it increases when 3HB is included. In particular, when 3HB is included, the expression level in GLUT1 KD cells is significantly increased compared to stationary cells.

Example 2 and Comparative Example 1
[Preparation of liquid composition]
Purified water, wetting agent, 3HB, and pH adjuster were sequentially added to a glass beaker and stirred uniformly with a stirring rod, and then ethanol, preservative, solubilizer, fragrance, and purified water were sequentially added and stirred in the same manner. . After measuring the pH and adjusting the pH to 6 with a pH adjuster, the mixture was filtered through a mesh (200 to 400 mesh) to obtain a liquid composition. Note that in Comparative Example 1, 3HB was not added. The composition of the liquid composition is shown in Table 4.

[Measurement method in human clinical trials]
The liquid composition was administered twice a day to 9 subjects [8 men, 1 woman, average age 43 years (35 to 56 years)] whose skin was found to have a tendency to dry according to skin measurements conducted in advance. It was applied to the face twice, and the transepidermal water loss (TEWL), stratum corneum water content, and stratum corneum analysis shown below were evaluated in a double-blind test. The liquid composition was applied by applying a liquid composition containing 3HB (Example 2) and a liquid composition not containing 3HB (Comparative Example 1, Placebo) to the right half of the face and the left half of the face. Subjects waited for 15 minutes after entering the room under conditions of room temperature: 20°C ± 1°C and humidity: 50% ± 5%, and after acclimatization, the subjects were examined for transepidermal water loss (TEWL), stratum corneum water content, and stratum corneum analysis. It was measured by the following method. The study was approved by an external ethics committee, and the study period is 8 weeks from January to March 2022.

(Transepidermal water loss (TEWL))
Before the start of the test and after 8 weeks, a Tewameter (Courage+Khazaka Electronic Co., Ltd. "Tewameter") connected as a probe to a multi-dermatometer (Courage+Khazaka Electronic Co., Ltd. "Multi Probe Adapter MPA6") was used. er TM Hex") on both cheekbones of the subject. Transepidermal water loss (TEWL) was measured at the lower site.

The measurement results are shown in FIG. In addition, "++" in FIG. 5 means that the p value of Example 2 is less than 0.01 (p<0.01). As is clear from FIG. 5, TEWL decreased after 8 weeks in Example 2 using a liquid composition containing 3HB compared to Comparative Example 1 using a liquid composition not containing 3HB.

(Moisture content of stratum corneum)
Before the start of the study and after 8 weeks, the moisture content of the stratum corneum was measured under both cheekbones of the subjects using a Corneometer (Corneometer CM825, manufactured by Courage+Khazaka Electronic Co.) connected as a probe to a multi-skin measuring device (Multi Probe Adapter MPA6, manufactured by Courage+Khazaka Electronic Co.).

The measurement results are shown in FIG. Note that "*" in FIG. 6 means that the p value of Comparative Example 1 is less than 0.05 (p<0.05). As is clear from FIG. 6, Example 2, which used a liquid composition containing 3HB, had a lower stratum corneum water content than Comparative Example 1, which used a liquid composition that did not contain 3HB, before the start of the test. Despite this, the surprising result was that the moisture content of the stratum corneum increased after 8 weeks.

(moisturizing ability)
As the moisturizing ability of the skin, the ratio of the stratum corneum water content to TEWL measured by the above method (stratum water content/TEWL or W/T) was calculated, and the calculation results are shown in FIG. 7. As is clear from FIG. 7, compared to Comparative Example 1 using a liquid composition that does not contain 3HB, in Example 2 using a liquid composition containing 3HB, the moisturizing state of the skin (moisturizing ability) has been improved.

(Stratum corneum analysis)
Before the start of the study and after 8 weeks, the examiner used Scotch tape (registered trademark) to strip the stratum corneum (keratin) from the areas below both cheekbones of the subjects, and evaluated the following items for the collected stratum corneum.

(1) Cell area and degree of multiple peeling by BG staining The first layer of the stratum corneum sample collected with Sellotape was cut and pasted onto a slide glass, and transferred onto the slide glass by immersing it in xylene overnight. Hereinafter, this operation will be referred to as transcription.

Next, the transferred stratum corneum sample was immersed in an aqueous solution containing brilliant green and gentian violet (BG staining solution) and stained.

Furthermore, the cells were washed with running water, air-dried, sealed using Marinol (manufactured by Muto Kagaku Co., Ltd.), and then images were taken using a microscope (n=3).

The photograph obtained is shown in Figure 8. In the photograph in Figure 8, the dark colored areas indicate that the keratin layers overlap, causing multiple peeling. As is clear from Figure 8, it can be confirmed that multiple peeling is suppressed after 8 weeks in Example 2, which used a liquid composition containing 3HB, compared to Comparative Example 1, which used a liquid composition not containing 3HB.

Meanwhile, the average value of the stratum corneum cell area (μm ² ) and the multiple peeling rate (%) were calculated for each sample by image analysis. The calculated average value of the stratum corneum cell area is shown in FIG. 9, and the multiple peeling rate is shown in FIG. 10. The multiple peeling rate was the ratio of the area of the multiple peeling portion to the total stratum corneum cell area in the image. In addition, in FIG. 9, "++" means that the p value of Example 2 is less than 0.01 (p<0.01). As is clear from FIG. 9, the increase in the average value of the stratum corneum cell area after the 8-week test in Example 2 was 65.72 μm ² , which was a large increase due to the use of the liquid composition, whereas the increase in Comparative Example 1 was 16.64 μm ² , which was a smaller increase than that in Example 2. Furthermore, as is clear from FIG. 10, the change in the multiple peeling rate before and after the 8-week test in Example 2 decreased to -0.22%, whereas the change in Comparative Example 1 increased to 1.19%. From these results, it was confirmed that the state of the stratum corneum was improved in Example 2 compared to Comparative Example 1.

(2) TGM-1 Activity The collected third stratum corneum samples were immersed in phosphate buffered saline [PBS(-)] containing dansylcadaverine at a concentration of 50 µM and incubated at 37°C overnight.

Next, the surface to which the stratum corneum was attached after being washed and air-dried was attached to a glass slide, and transferred onto the slide glass by immersing it in xylene overnight.

To evaluate the cross-linking ability of transglutaminase, blue fluorescence derived from dansylcadaverine, which was added as a fluorescent substrate, was photographed using a fluorescence microscope (n=3).

FIG. 11 shows a processed photograph (image-processed photograph) in which the photographed photograph was binarized (threshold value: 125) by the density of fluorescence through image processing. As is clear from FIG. 11, a large amount of TGM-1 was produced after 8 weeks in Example 2, which used a liquid composition containing 3HB, compared to Comparative Example 1, which used a liquid composition that did not contain 3HB. It can be confirmed that

Meanwhile, the average fluorescence intensity (A.U.) of the blue color excluding the multiple peeled areas of the stratum corneum was calculated by image analysis and used as TGM-1 activity. The results of the calculated average fluorescence intensity (TGM-1 activity) are shown in Figure 12. As is clear from Figure 12, the increase in average fluorescence intensity after the 8-week test in Example 2 was 0.20 A.U., while the increase in Comparative Example 1 was 0.05 A.U., which was smaller than that of Example 2.

(3) Nile red staining The second layer of the stratum corneum sample was cut and then transferred onto a slide glass. Next, 75% glycerin containing Nile Red (lipophilic fluorescent dye) at a concentration of 3 μg/mL was dropped into the transferred stratum corneum and sealed, and images were taken using a fluorescence microscope (n=3). The stronger the red fluorescence, the greater the amount of intercellular lipids present.

FIG. 13 shows a processed photograph (image-processed photograph) in which the photographed photograph was binarized (threshold value: 125) by the density of fluorescence through image processing. As is clear from FIG. 13, compared to Comparative Example 1 using a liquid composition that does not contain 3HB, in Example 2 using a liquid composition containing 3HB, the amount of intercellular lipids present is greater after 8 weeks. This can be confirmed. Therefore, it can be seen that in Example 2, the lipid envelope was formed more uniformly over a wider range than in Comparative Example 1.

On the other hand, by image analysis, the average red fluorescence intensity (A.U.) excluding the multiple peeled portions of the stratum corneum was calculated, and was defined as the Nile Red staining degree. The results of the calculated average fluorescence intensity (Nile red staining intensity) are shown in FIG. In addition, "+" in FIG. 14 means that the p value of Example 2 is less than 0.05 (p<0.05). As is clear from FIG. 14, the amount of increase in the average fluorescence intensity of Example 2 after the 8-week test was 6.80A. U. On the other hand, the average fluorescence intensity of Comparative Example 1 was 2.53A. U. , which was smaller than that of Example 2.

Since the transglutaminase production promoter of the present invention can promote the production of transglutaminase in vivo, it can be used for various applications such as beauty products, cosmetics, foods, pharmaceuticals, and quasi-drugs. Even in the skin, normal CE and lipid envelope can be formed, and skin with normalized keratin can be regenerated, so it can be suitably used in skin care products such as aging care products.

Claims (18)

A transglutaminase production promoter containing 3-hydroxybutyric acid and/or its salt. The transglutaminase production promoter according to claim 1, wherein the 3-hydroxybutyric acid and/or its salt contains the R form. The transglutaminase production promoter according to claim 1 or 2, which promotes the production of transglutaminase 1. The transglutaminase production promoter according to claim 1 or 2, which promotes the production of transglutaminase during the formation process of corneocytes. The transglutaminase production promoter according to claim 1 or 2, which promotes the production of transglutaminase in dry skin. A composition containing 3-hydroxybutyric acid and/or a salt thereof and for promoting the production of transglutaminase. The composition according to claim 6, which is a transdermal composition. The composition according to claim 6 or 7, which is a liquid or semi-solid composition. The composition according to claim 6 or 7, which is a skin care product. A protein production promoter for promoting the production of proteins that constitute the cornified envelope, the protein production promoter containing 3-hydroxybutyric acid and/or a salt thereof. The protein production promoter according to claim 10, wherein the protein is involucrin and/or filaggrin. A cornified envelope forming ability enhancer containing 3-hydroxybutyric acid and/or its salt. A lipid envelope forming ability enhancer containing 3-hydroxybutyric acid and/or a salt thereof. A skin barrier function improver containing 3-hydroxybutyric acid and/or its salt. A method for promoting the production of transglutaminase during the formation process of corneocytes by permeating the transglutaminase production promoter according to claim 1 or 2 into the skin. A method for normalizing keratin by penetrating the transglutaminase production promoter according to claim 1 or 2 into the skin. A method for improving the barrier function of the skin by permeating the transglutaminase production promoter according to claim 1 or 2 into the skin. The method according to claim 15, in which the transglutaminase production promoter is permeated into the skin repeatedly and intermittently for 24 hours or more.

Images