JP7382181B2 - Composition for suppressing skin barrier function decline - Google Patents

Description

The present invention relates to a composition for suppressing decline in skin barrier function.

The epidermis is located at the outermost layer of the skin, and has a barrier function that prevents the invasion of foreign substances from the outside and evaporation of water within the body, and plays the role of protecting the inside of the living body. Environmental factors such as poor lifestyle habits, sunlight, and changes in temperature and humidity impair the role played by the epidermis through the formation of wrinkles, irregular texture, and inflammation.

Among the epidermis, the stratum corneum plays a central role in skin barrier function. The stratum corneum barrier is constructed by constantly repeating the normal turnover cycle of the epidermis. The stratum corneum barrier mainly consists of corneocytes differentiated from cornified cells and intercellular lipids that fill the space between the corneocytes. The main component of intercellular lipids is ceramide, and the intercellular lipids exhibit a regularly arranged lamellar structure. Ceramide is synthesized through several types of enzymatic reactions such as serine palmitoyltransferase (SPT) during the differentiation process of epidermal cells.

The cell membrane of a corneocyte is thick, and on the inside thereof there is a lining structure called a cornified cell envelope (CE), also called a marginal zone. The proteins that make up CE are extremely stable against physical and chemical stimuli, and play a role in reinforcing cell membranes. Further, outside the CE there is a lipid envelope (corneocyte-bound lipid envelope; CLE). CLE functions as a scaffold that builds a lamellar structure of intercellular lipids on its outside. In this way, corneocytes exhibit a membranous structure with each other through CE on the inside of the cell membrane, CLE on the outside of the cell membrane, and intercellular lipids, and a corneum barrier is constructed.

By the way, it is known that transglutaminase-1 (TGM1) is deeply involved in the formation of CE. As keratinocytes differentiate into keratinocytes, TGM1 promotes the formation of CE by crosslinking proteins that constitute CE, such as involucrin (INV). Furthermore, TGM1 promotes the formation of CLE by catalyzing ester bonds between glutamic acid residues of scaffold proteins deposited on the cell membrane side and very long chain fatty acids (ω-hydroxyceramide). Therefore, TGM1 is involved in both the formation of CE and CLE, and is responsible for constructing the stratum corneum barrier as the stratum corneum matures.

Carbonylated protein (CP) is a type of oxidized protein. CP is a reaction product between amino groups in proteins and aldehyde compounds produced during lipid peroxidation processes. Carbonylation of proteins impairs their original functions. In epidemiological studies to date, it has been observed in skin exposed to sunlight, in dry environments, and in the skin of atopic dermatitis patients. There is also a report that the amount of CP increases more significantly in autumn and winter, which are in a low temperature and low humidity environment, than in spring and summer. Furthermore, Patent Document 1 describes that the water content of the stratum corneum is reduced by treating pigskin with acrolein, which oxidizes proteins and induces the production of CP.

Apple fruit extract is a complex product rich in catechin monomers, phenolic carboxylic acids such as chlorogenic acid, flavonoids such as phlorizin, and catechin multimeric structures such as procyanidins, and has excellent antioxidant power. For this reason, it is attracting attention as an aging care material (anti-aging material). Patent Document 2 describes that apple fruit extract suppresses the production of ultraviolet light-induced CP in human skin fibroblasts. Further, Patent Document 3 describes that an apple fruit extract suppresses the production of acrolein derivative-induced CP in human epidermal cell lines.

Japanese Patent Application Publication No. 2005-249672 Japanese Patent Application Publication No. 2012-031133 JP2017-095432A

Patent Document 1 describes that the moisture content of the stratum corneum decreases due to carbonylated protein (CP) internally generated in the stratum corneum of pigskin due to acrolein. Further, Patent Documents 2 and 3 describe that apple fruit extract suppresses internal production of CP due to ultraviolet irradiation or acrolein treatment.

However, Patent Documents 1 to 3 do not describe the influence of CP already generated outside cells on skin tissue or keratinocytes. That is, no changes in the state or function of skin tissues and keratinocytes that have come into contact with extracellular CP have been known.

Therefore, the present invention aims to elucidate the effects of carbonylated proteins already produced extracellularly on skin tissues and keratinocytes, and to clarify the effects of extracellular carbonylated proteins and intracellular carbonylated proteins produced thereby on skin tissues and keratinocytes. An object of the present invention is to provide a composition for improving the effect on cells.

The present inventors have carried out intensive studies to solve the above problems, and have discovered that when epidermal keratinocytes are treated with extracellular CP, new CP is generated in the keratinocytes. We discovered the possibility that external CP may be deposited. And, surprisingly, the present inventors found that extracellular CP reduces transglutaminase-1 (TGM1) and involucrin (INV) gene expression and TGM1 activity in keratinocytes.

Furthermore, the present inventors have found that by treating an epidermal tissue model with extracellular CP, transepidermal water transpiration (TEWL) increases and skin barrier function decreases. This decrease in skin barrier function due to extracellular CP may be caused by inhibition of CE formation through decreased expression levels of TGM1 and INV and decreased TGM1 activity.

Furthermore, surprisingly, the present inventors found that the decrease in skin barrier function associated with the increase in TEWL due to extracellular CP was suppressed by apple fruit extract. As a result, apple fruit extract exhibits the effect of suppressing the decline in skin barrier function, thereby suppressing the inhibition of CE formation, suppressing the decline in the expression levels of TGM1 and INV, and suppressing the decline in TGM1 activity. there's a possibility that.

Based on these findings, the present inventors finally solved the problems of the present invention by developing a method for reducing skin barrier function that contains a Rosaceae plant fruit extract such as apple fruit extract as an active ingredient. We have succeeded in creating a composition for suppressing cornified envelope formation, a composition for suppressing a decrease in transglutaminase expression, a composition for suppressing a decrease in transglutaminase activity, and a composition for suppressing a decrease in involucrin expression. The present invention was completed based on these findings and successful examples.

Therefore, according to one aspect of the present invention, the following composition is provided.
[1] A composition for suppressing deterioration of skin barrier function, containing a Rosaceae plant fruit extract as an active ingredient.
[2] A composition for inhibiting cornified envelope formation, which contains a Rosaceae plant fruit extract as an active ingredient.
[3] A composition for suppressing a decrease in transglutaminase expression or a composition for suppressing a decrease in transglutaminase activity, which contains a Rosaceae plant fruit extract as an active ingredient.
[4] The composition according to [3], wherein the transglutaminase is intrakeratinocyte transglutaminase.
[5] The composition according to any one of [3] to [4], wherein the transglutaminase is transglutaminase-1.
[6] A composition for suppressing a decrease in involucrin expression, which contains a Rosaceae plant fruit extract as an active ingredient.
[7] The composition according to [6], wherein the involucrin is intrakeratinocyte involucrin.
[8] The composition according to any one of [1] to [7], wherein the Rosaceae plant fruit extract is an apple fruit extract.

According to the composition of one aspect of the present invention, a decrease in skin barrier function, inhibition of cornified envelope formation, decrease in transglutaminase expression, decrease in transglutaminase activity, and/or decrease in involucrin expression brought about by extracellular carbonylated proteins is suppressed. can do.

In addition, the composition of one embodiment of the present invention has a rich track record of use on the human body as an active ingredient, such as a fruit extract of a Rosaceae plant, as a cosmetic ingredient. I can do it.

The composition that is one embodiment of the present invention is expected to be widely used for general purposes depending on the usage mode. For example, the composition that is one embodiment of the present invention is expected to be used as a skin care cosmetic by taking the form of a lotion, serum, milky lotion, cream, or the like. In addition, for example, the composition that is an embodiment of the present invention has a cell protection effect, an anti-aging effect (anti-aging), an antioxidant effect, an anti-wrinkle effect, a radical scavenging effect, a skin quality maintenance effect, a skin quality improvement effect, and a whitening effect. Physiological beauty effects, such as ``moisturizing the skin,'' ``replenishing and maintaining skin moisture,'' ``protecting the skin,'' and ``preventing skin dryness.'' It is expected to be used as a cosmetic.

FIG. 1 is a diagram showing the results of observing the locations of carbonylated proteins (CP) in human skin tissue, as described in the Examples. FIG. 2A is a diagram showing the results of measuring the amount of CP in NHEK cells treated with CP-BSA as described in the Examples. FIG. 2B is a diagram showing the results of measuring the amount of CP in NHEK cells treated with CP-BSA as described in the Examples. FIG. 3A is a diagram evaluating the effect of CP-BSA treatment on the expression level of skin moisturizing-related genes in NHEK cells, as described in Examples. FIG. 3B is a diagram showing the results of measuring the amount of TGM1 mRNA expression in NHEK cells treated with CP-BSA as described in the Examples. FIG. 4A is a diagram evaluating the effect of CP-BSA treatment on reducing the expression level of TGM1 protein in the cell membrane of keratinocytes, as described in the Examples. FIG. 4B is a diagram evaluating the effect of CP-BSA treatment on reducing TGM1 activity in the cell membrane of keratinocytes, as described in the Examples. FIG. 5 is a diagram evaluating the reduction in transepidermal water transpiration (TEWL) in an epidermal model by CP-BSA treatment, as described in the Examples. FIG. 6 is a diagram evaluating the increase in CP amount in an epidermal model by CP-BSA treatment, as described in the Examples.

The details of the composition of one embodiment of the present invention will be described below, but the technical scope of the present invention is not limited only by the matters in this item, and the present invention can be applied to various forms as long as the object is achieved. It can take various forms.

Unless otherwise specified, each term herein is used in the sense commonly used by those skilled in the art and should not be construed as having an unduly limiting meaning. Furthermore, since the theories and speculations made in this specification were made based on the knowledge and experience of the present inventors, the technical scope of the present invention is not limited solely to such theories and speculations. It is not something that will be done.

A "composition" is not particularly limited in its commonly used meaning, but is, for example, a combination of two or more substances, specifically a combination of an active ingredient and another substance. Examples include those consisting of a combination of two or more active ingredients, and more specifically, those consisting of a combination of one or more active ingredients and one or more solid components or solvent components. Examples include solid compositions and liquid compositions.
The term "and/or" means any one of the listed related items, or any or all combinations of two or more.
"Content" is synonymous with concentration, and means the ratio of the amount of the component to the total amount of the composition. However, the total content of the components does not exceed 100%. In this specification, unless otherwise specified, the unit of content means "mass% (wt%)".
In the present specification, "~" in the numerical range is a range that includes the numbers before and after that, for example, "0% by mass to 100% by mass" is 0% by mass or more and 100% by mass or less. means a certain range.

"Skin barrier function deterioration suppressing effect" means suppressing the increase in transepidermal water transpiration. Deterioration of skin barrier function occurs, for example, when extracellular carbonylated proteins come into contact with skin tissue and/or keratinocytes.
"Conified envelope formation inhibiting action" means an action that inhibits the formation of a cornified envelope and/or the maintenance of the formed cornified envelope. Inhibition of cornified envelope formation occurs, for example, when extracellular carbonylated proteins come into contact with skin tissue and/or keratinocytes.
"Transglutaminase" is not particularly limited as long as it is an enzyme that catalyzes a protein crosslinking reaction by forming an isopeptide bond between a glutamine residue and a lysine residue in a protein; for example, transglutaminase-1, Examples include transglutaminase-3 and transglutaminase-5, but transglutaminase-1, which is deeply involved in the formation of cornified envelopes and lipid envelopes, is preferred.
"Transglutaminase expression reduction suppression effect" and "involucrin expression reduction suppression effect" refer to the effect of suppressing the reduction in the expression level of the respective genes, the effect of suppressing the reduction in the translation amount of the gene product (protein), and the effect of suppressing a reduction in the production of a gene product.
"Transglutaminase activity reduction suppressing effect" refers to the effect of suppressing transglutaminase activity reduction.
The inhibitory effect on the decrease in skin barrier function, the inhibitory effect on the inhibition of cornified envelope formation, the inhibitory effect on the decrease in transglutaminase expression, the inhibitory effect on the decrease in transglutaminase activity, and the inhibitory effect on the decrease in involucrin expression are collectively referred to as "effective effects." The effective action refers to any one, two, three, four, or all five of these actions.

The matters that can be derived from the facts shown in the examples described later are as follows.
When we confirmed the localization of carbonylated proteins (CP) in the skin, we found that CP is present throughout the epidermis in the skin of elderly people. From this, it was inferred that CP in the epidermis, including the living cell layer, reduces the skin's moisturizing function. Extracellular CP led to the deposition and production of CP within epidermal cells and suppressed transglutaminase-1 (TGM1) expression and activity. This fact suggests that the activity of TGM1 is reduced in the epidermis where CP is present, and the formation of cornified envelope (CE) and lipid envelope (CLE) may be inhibited. CLE, which is present in the outermost layer of CE, is said to contribute to stabilizing the intercellular lipid lamellar structure. From this, it is considered that CP affects the differentiation of keratinocytes and induces the maturation failure of the stratum corneum, thereby becoming a factor in the decline in skin moisturizing function.

Previous studies have shown that extracellular CP increases intracellular reactive oxygen species (ROS) levels and increases intracellular oxidation status. Therefore, one possible reason for the decrease in TGM1 expression is that ROS production by CP is triggered, CP is generated within cells, and skin moisturizing-related proteins decrease.

When we verified the effect of apple fruit extract, which has antioxidant properties, on CP production, we found that the presence of CP increases TEWL, and the results of measuring transepidermal water loss (TEWL) using a reconstituted cultured epidermis model show that the presence of CP increases TEWL, and apple It was confirmed that pretreatment of fruit extracts tends to suppress the increase in TEWL. Therefore, it was revealed that CP reduces the stratum corneum barrier function, but apple fruit extract suppresses the reduction. It is thought that TGM1 is involved in the decrease in the stratum corneum barrier function caused by CP. Furthermore, it is presumed that the pretreatment of the apple fruit extract suppressed the deposition and/or production of CP within cells, thereby suppressing the decline in skin barrier function. From the above, apple fruit extract is expected to suppress the deposition and/or production of CP in the epidermis, thereby suppressing the decrease in TGM1 expression, and normalizing the skin moisturizing function.

The composition of one aspect of the present invention has an effect of suppressing the decline in skin barrier function, an effect of suppressing the inhibition of cornified envelope formation, an effect of suppressing the decrease of transglutaminase expression, an effect of suppressing the decrease of transglutaminase activity, and an effect of suppressing the decrease in transglutaminase activity, and an effect of suppressing the decrease in transglutaminase activity, and an effect of suppressing the decrease in transglutaminase activity, due to the fruit extract of the Rosaceae plant contained therein. It has at least one effective effect selected from the group consisting of an effect of suppressing expression decline. The composition of one aspect of the present invention has an effective effect, and is typically used as a composition for suppressing a decline in skin barrier function, a composition for suppressing inhibition of cornified envelope formation, a composition for suppressing a decline in transglutaminase expression, and a composition for suppressing a decline in transglutaminase expression. It takes the form of a composition for suppressing a decrease in glutaminase activity and a composition for suppressing a decrease in involucrin expression, and furthermore, a composition for cell protection, an anti-aging composition (anti-aging composition), an antioxidant composition, and an anti-wrinkle composition. The composition may take the form of a composition, a composition for scavenging radicals, a composition for maintaining skin quality, a composition for improving skin quality, a composition for whitening, and the like. Another aspect of the present invention is a method for treating, preventing, suppressing, or improving skin abnormalities such as wrinkles and dry skin, and aging by applying a Rosaceae plant fruit extract to an individual.

A composition according to one embodiment of the present invention contains a Rosaceae plant fruit extract as an active ingredient. Rosaceae plants include, for example, apples, strawberries, pears, loquats, quince, etc., but apples are preferred because they contain many polyphenols having antioxidant power.

Apples are not particularly limited as long as they are plants of the genus Malus , and examples include apples ( Malus domestica , Malus pumila ), and specifically, varieties such as Fuji, Orin, Kogyoku, Mutsu, Tsugaru, and Asahi. , Delicious, Jonagold, and Sunshine apples. Hereinafter, fruits of Rosaceae plants will be explained using apple fruits as an example.

The part of the apple fruit to be extracted is not particularly limited, and includes, for example, the whole fruit, pulp, pericarp, and seeds, but it is preferable to extract the part containing the pulp, which is rich in polyphenols. The apple fruit may be a mature fruit or an immature fruit (young fruit), but since the pulp contains a lot of polyphenols, it is preferable to be an immature fruit (young fruit), and each apple fruit contains a large amount of polyphenols. It is more preferable that the fruit is an immature fruit (young fruit) that is thinned when the weight is about 5 g to 20 g.

The apple fruit extract may be an apple fruit extract obtained by extracting apple fruits, or may be a commercially available apple fruit extract, and is not particularly limited.

The method for obtaining an apple fruit extract is not particularly limited as long as it extracts a large amount of polyphenols from apple fruits, and any conventionally known method can be used. Examples of the method for obtaining apple fruit extract include the method described in JP-A No. 2005-179373. Specifically, apple fruits are mixed with alcohol such as ethanol or methanol, crushed, soaked and squeezed as they are, or extracted while heating and refluxing to obtain a crude extract, and then the obtained crude extract is concentrated under reduced pressure. After removing alcohol, centrifugation and filtration, or distribution and filtration with an organic solvent such as hexane or chloroform is performed to obtain a clear extract, and then the resulting clear extract is used to selectively adsorb and elute polyphenols. The polyphenol fraction is adsorbed by passing it through a column filled with an adsorbent, and then washed by passing distilled water through the adsorbed column, and then the polyphenol fraction is removed by passing an aqueous alcohol solution such as aqueous ethanol through the column. After elution and recovery, the resulting polyphenol fraction is concentrated under reduced pressure to distill off the alcohol to obtain an apple fruit extract. The apple fruit extract may be an apple fruit extract powder that has been subjected to a drying process such as spray drying or freeze drying.

Commercially available apple fruit extracts include, for example, those sold as "ApplePhenon (registered trademark)" by BGG Japan, "ApplePhenon (registered trademark) Apple Phenon C-100", "ApplePhenon (registered trademark) Apple Phenon SH", and Examples include "ApplePhenon (registered trademark) apple procyanidin", but when the composition of one embodiment of the present invention is used as an external skin composition such as cosmetics, "ApplePhenon (registered trademark) apple phenon C-100" is preferable.

The apple fruit extract preferably contains a large amount of polyphenols such as catechin, phenolic carboxylic acids such as chlorogenic acid, flavonoids such as phlorizin, and catechin multimer structures such as procyanidin. Although the content of polyphenols in the apple fruit extract is not particularly limited, it is preferably 95% by mass or more based on the total amount of the apple fruit extract, for example.

The content of the apple fruit extract is not particularly limited as long as it has an effective effect, and can be set as appropriate depending on the form and usage of the composition, the expected degree of effect, etc. When the composition of one embodiment is used as a skin external composition, the dry mass is preferably 0.00005% by mass to 1% by mass, more preferably 0.0005% by mass to 0.1% by mass. .

The composition of one embodiment of the present invention is not particularly limited in terms of the degree of effective action; This effect is to the extent that it is possible.

The effective action of the composition of one embodiment of the present invention is evaluated by the method described in the Examples below. As a summary, the inhibitory effect on skin barrier function decline is evaluated by measuring transepidermal water loss (TEWL) using an epidermal model. The inhibitory effect on cornified envelope formation is evaluated by measuring the mRNA levels of transglutaminase and involucrin in TEWL and epidermal keratinocytes. The inhibitory effect on the decrease in transglutaminase expression and the inhibitory effect on the decrease in involucrin expression are evaluated by measuring the amounts of transglutaminase and involucrin mRNA in epidermal keratinocytes. The inhibitory effect on reducing transglutaminase activity is evaluated by measuring the amount of transglutaminase protein in epidermal keratinocytes. Note that "measuring" includes both quantitatively confirming and qualitatively observing.

The animal to which the composition of one embodiment of the present invention is used is not particularly limited. For example, the animal to be used includes animals, especially mammals. Mammals include humans, dogs, cats, cows, horses, etc. Among these, humans are preferred.

The usage and dosage of the composition of one embodiment of the present invention, such as the amount used per time, the amount used per day, the period of use, and the interval between uses, are not particularly limited, and are appropriately set according to the mode of use, the condition of the individual using it, etc. can be done. The usage interval is, for example, once, twice, three times or several times a day for a certain period of time, that is, one or more days, preferably one week or more, more preferably two weeks or more, and even more preferably one month or more. Examples include continuous application. Although it is preferred that the composition of one aspect of the present invention be applied daily, it is not necessary to apply the composition of one aspect of the present invention every day as long as it is applied continuously over a period of time.

When the composition of one embodiment of the present invention is used as a composition for external use on the skin, the apple fruit extract is dissolved in a water-soluble solvent in advance, and then added to a desired content to form a liquid composition. It can be done.

The composition of one embodiment of the present invention can be used as a composition for parenteral or oral cosmetics, quasi-drugs, food and drink products, pharmaceuticals, animal feed, etc., by itself or by containing it together with other ingredients. Although the composition can take the form of a product, it is preferably a parenteral composition because it can be applied directly to the target area where it exerts an effective effect, and more preferably a composition for cosmetics and quasi-drugs. preferable. Therefore, one specific embodiment of the present invention is a cosmetic composition or a quasi-drug composition containing at least a Rosaceae plant fruit extract as an active ingredient.

The cosmetic composition is not particularly limited in terms of usage as long as it is applied to the skin, and examples thereof include skin care cosmetics, makeup cosmetics, fragrance cosmetics, body care cosmetics, all-in-one cosmetics, etc. Specifically, lotions, serums, milky lotions, creams, foundations, sunscreens, packs, BB creams, face powders, hand creams, cleansers, face washes, makeup bases, concealers, blushers, eyeshadows, eyeliners, eyebrows. , lipstick, mascara, lipstick, etc., and can take the form of solid, powder, liquid, gel, spray, etc. Cosmetic compositions exhibit effective effects, such as cell protection effects, anti-aging effects, antioxidant effects, anti-wrinkle effects, radical scavenging effects, skin quality maintenance effects, skin quality improvement effects, and whitening effects. Physiological beauty effects and/or ``moisturizing the skin'' effect, ``replenishing and maintaining skin moisture'' effect, ``protecting the skin'' effect, and ``preventing skin dryness'' that are recognized as cosmetics. Preferably, it is a lotion, serum, milky lotion, or cream that has the effect of "making fine wrinkles caused by dryness less noticeable."

In addition to the Rosaceae plant fruit extract, the cosmetic composition may contain other ingredients that can be commonly used in cosmetics, as long as they can solve the problems of the present invention.

Other ingredients commonly used in cosmetics are not particularly limited, and include, for example, bases such as water and organic solvents, oily ingredients, humectants, cooling agents, preservatives, chelating agents, pH adjusters, antioxidants, and ultraviolet rays. Examples include absorbers, ultraviolet scattering agents, thickeners, emulsifiers, solubilizers, whitening agents, vitamins, and various other medicinal ingredients, powders, fragrances, and coloring materials. The contents of other ingredients that can be commonly used in cosmetics can be appropriately determined by those skilled in the art, as long as they do not interfere with solving the problems of the present invention. Some of the other components are listed below, but these are just examples and are not limiting.

Examples of oily components include neopentyl glycol diethylhexanoate, neopentyl glycol dicaprate, triethylhexanoin, ethylhexyl palmitate, isopropyl palmitate, pentaerythrityl tetraisostearate, cetyl 2-ethylhexanoate, isononyl isononanoate, Ester oils such as isotridecyl isononanoate, diisopropyl sebacate, pentaerythrityl tetra-2-ethylhexanoate, diethylhexyl succinate, alkyl benzoates having 12 to 15 carbon atoms; octyldodecanol, oleyl alcohol, cetyl alcohol, stearyl alcohol, iso Higher alcohols such as stearyl alcohol, hexyldecanol, 2-decyltetradecinol; silicone oils such as dimethicone, methylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, higher alcohol-modified silicone oil; hydrogenated polyisobutene, fluid Straight and branched chain hydrocarbon oils such as paraffin, light isoparaffin, petrolatum, squalane, squalene, isohexadecane, isododecane; undecylenic acid, oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, myristic acid, arachidonic acid , higher fatty acids such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and isostearic acid; animal oils such as liquid lanolin; fluorinated oils such as fluoropolyether and perfluoroalkyl ether silicone; olive oil, jojoba oil, lavender oil, and evening primrose. oil, avocado oil, camellia oil, macadamia nut oil, corn oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, sasanquat oil, castor oil, linseed oil, safflower oil, cottonseed oil, perilla oil, soybean oil, Natural oil components such as peanut oil, tea seed oil, kaya seed oil, rice bran oil, and rice germ oil can be used, and one type of these can be used alone or two or more types can be used in combination. The content of the oily component is preferably about 0% by mass to 40% by mass.

Examples of humectants include glycerin, diglycerin, trehalose, butylene glycol (BG), dipropylene glycol (DPG), xylitol, maltitol, maltose, sorbitol, glucose, fructose, hydrolyzed elastin, sodium lactate, cyclodextrin, Examples include pyrrolidone carboxylic acid, and one type thereof can be used alone or two or more types can be used in combination. The content of the humectant is preferably about 0% by mass to 15% by mass.

Examples of the refreshing agent include lower alcohols such as ethanol, L-menthol, camphor, menthoxypropyl diol, and the like, and one type of these can be used alone or two or more types can be used in combination. The content of the refreshing agent is preferably about 0% by mass to 10% by mass.

Examples of preservatives include propylparaben, methylparaben, benzoic acid, salicylic acid, carbolic acid, sorbic acid, parachlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichlorocarbanilide, photosensitizer, phenoxyethanol, ethylhexylglycerin. , alkanediols, etc., and one type of these can be used alone or two or more types can be used in combination. The content of the preservative is preferably about 0% by mass to 5% by mass.

Examples of the chelating agent include alanine, disodium edetate, sodium polyphosphate, sodium metaphosphate, sodium phytate, trisodium phosphate, etc., and one of these may be used alone or in combination of two or more. Can be used. The content of the chelating agent is preferably about 0% by mass to 1% by mass.

Examples of the pH adjuster include lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, malic acid, sodium malate, etidronic acid, potassium carbonate, sodium hydrogen carbonate, ammonium hydrogen carbonate, sodium hydroxide, potassium hydroxide, Examples include arginine, triethanolamine, monoethanolamine, etc., and one type of these can be used alone or two or more types can be used in combination. The content of the pH adjuster is preferably about 0% by mass to 1% by mass.

Examples of antioxidants include vitamin E and its derivatives such as dibutylhydroxytoluene, butylhydroxyanisole, and d-δ-tocopherol; thiotaurine, evening primrose extract, β-carotene, catechin compounds, flavonoid compounds, and polyphenol compounds. , these can be used alone or in combination of two or more. The content of the antioxidant is preferably about 0% by mass to 1% by mass.

Examples of thickeners include water-soluble polymers, specifically hyaluronic acid, sodium hyaluronate, acetylated hyaluronic acid, acetylated sodium hyaluronate, chondroitin sulfate, sodium chondroitin sulfate, fucoidan, and tuberose polysaccharide. Water-soluble polysaccharides such as, Examples include synthetic polymers such as carbomer, polyacrylic acid, sodium polyacrylate, and acrylic acid polymers such as (meth)acrylic acid/alkyl (meth)acrylate copolymers. These thickeners can be used alone or in combination of two or more. The content of the thickener is preferably 0% by mass to 1% by mass.

Examples of emulsifiers include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, and more specifically, sorbitan stearate, polyglyceryl-10 diisostearate, and triglyceride. Polyoxyethylene isostearate hydrogenated castor oil, polyoxyethylene cetyl ether, self-emulsifying glyceryl monostearate, decaglyceryl monomyristate, polyoxyethylene sorbitol tetraoleate, hydrogenated soy phospholipid, sodium stearoyl glutamate, monostearic acid Examples include polyethylene glycol, and one type thereof can be used alone or two or more types can be used in combination. The content of the emulsifier is preferably about 0% by mass to 5% by mass.

Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polyoxyethylene phytosterol, and polyoxyethylene cholesterol, and one of these can be used alone or two or more can be used in combination. The content of the solubilizer is preferably about 0% by mass to 5% by mass.

The cosmetic composition may contain skin-beautifying ingredients other than the Rosaceae plant fruit extract. Ingredients for beautiful skin include, for example, placenta extract, glutathione, saxifrage extract, oil-soluble licorice extract, ascorbic acid, ascorbic acid derivatives, arbutin, kojic acid, tranexamic acid, cysteine, chamomile extract, linoleic acid, rusinol, α- Whitening agents such as hydroxy acids; cell activators such as royal jelly, photosensors, cholesterol derivatives, and young calf blood extract; rough skin improving agents; nonylic acid valenylamide, nicotinic acid benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, Blood circulation promoters such as zingerone, cantharis tincture, ictamol, caffeine, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclanderate, cinnarizine, tolazoline, acetylcholine, verapamil, cephalanthine, γ-oryzanol; zinc oxide, etc. skin astringents; antiseborrheic agents such as sulfur and thianthrol; one type of these can be used alone or two or more types can be used in combination.

Quasi-drugs are not particularly limited as long as they are commonly known, but for example, they must have mild effects on the human body, are not medical devices, and are designated by the Minister of Health, Labor and Welfare. Specific examples include hair growth agents, hair dyes, and vitamin preparations.

The composition for quasi-drugs may contain, in addition to the Rosaceae plant fruit extract, other components that can be commonly used in quasi-drugs, as long as the problems of the present invention can be solved. In addition to the above-mentioned cosmetic ingredients, other ingredients include, for example, excipients, binders, disintegrants, stabilizers, flow agents, surfactants, colorants, sweeteners, lubricants, etc. Examples include additives used in the processing of quasi-drugs. The amount of the additive to be used is not particularly limited as long as it does not interfere with solving the problems of the present invention, and can be set as appropriate.

Another specific embodiment of the composition of one embodiment of the present invention is a medicated cosmetic composition. Medicinal cosmetics refer to so-called medicated cosmetics that belong to quasi-drugs. The manner of use of the medicinal cosmetic composition, the types and contents of other components, etc. can be appropriately determined by those skilled in the art, similarly to the cosmetic composition and the quasi-drug composition.

The composition of one aspect of the present invention is not particularly limited in its production method, and for example, the composition may be prepared by combining a Rosaceae plant fruit extract with a method known to those skilled in the art, depending on the usage such as cosmetics or quasi-drugs. It can be manufactured by optionally mixing other components.

The method of using the composition of one embodiment of the present invention is not particularly limited, and for example, a commonly used method can be adopted depending on the usage mode of cosmetics, quasi-drugs, etc.

The composition of one embodiment of the present invention can be a packaged composition packed in a container and sealed. The container is not particularly limited, but for example, a bag made of metal such as aluminum, paper, plastic such as PE, PP, PET, PBT, PVC, and/or glass in a single layer or a laminated state. , pouches, packs, boxes, bottle containers, tube containers, bottles, cans, and other packaging containers. Preferably, the packaged composition can be distributed and marketed independently by dispensing, filling and/or individually packaging the composition obtained by manufacturing the composition.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various embodiments as long as the problems of the present invention can be solved. can.

[1. Evaluation method]
(1-1) Confirmation of localized location of carbonylated protein (CP) in human skin tissue Frozen thin sections of skin tissue excised from the abdomen of humans (63 and 68 year old Caucasian women) were prepared using a conventional method. was created. The prepared skin tissue sections were labeled with fluorescein-5-thiosemic carbazide (FTSC) to visualize CP.

(1-2) Preparation of CP-BSA 100 mg/mL bovine serum albumin (BSA) and 300 mM acrolein were mixed at a ratio of 1:1 and reacted at 37° C. for 48 hours to carbonylate BSA. Thereafter, dialysis was performed using a regenerated cellulose dialysis tube ("T1 MWCO3500"; Thermofiscer) to remove untreated acrolein, thereby obtaining a dialysate containing CP-BSA. The production of CP-BSA was confirmed by measuring the fluorescence intensity (Ex: 360 nm, Em: 465 nm) of the obtained dialysate.

(1-3) Cell culture Normal human epidermal keratinocytes (``NHEK''; Kurabo) were cultured in a 96-well plate (culture container) at 37°C with 5% CO using Humedia-KG2 medium (Kurabo). The cells were cultured in _two incubators.

(1-4) Intracellular oxidation state of epidermal cells co-cultured with CP NHEK cells were cultured in the presence of 50 μg/mL, 75 μg/mL or 100 μg/mL BSA; The cells were cultured for 24 hours in the presence of either of them; or in the absence of any addition (control). Next, the medium in the culture plate was removed by decantation, and the cells were fixed with methanol at -20°C. After removing methanol and washing the cells three times with PBS, a solution diluted with PBS to give a 2 μmol/L Hoechst 33342 solution was added, and the cells were treated at 37°C for 15 minutes to remove the inside of the nucleus. Stained. After removing the Hoechst 33342 solution and washing three times with PBS, a FTSC solution diluted with Mes-Na buffer to 20 μmol/L was added, and the cells were treated at 37°C for 1 hour. CP was stained.

After removing the FTSC solution and washing three times with PBS, 100 μL/well of PBS was added and the fluorescence intensity (Hoechst 33342: Ex: 340 nm, Em: 465 nm; FTSC: 485 nm, Em: 535 nm) was measured.

(1-5) Gene expression measurement (real-time PCR)
Similarly to 1-4 above, NHEK cells were subjected to conditions in which 150 μg/ml CP-BSA, 150 μg/ml BSA, or neither was added (control). Total RNA was extracted from cells using "RNeasy Mini Kit" (Qiagen). The reverse transcription reaction was performed using "ReverTraAce qPCR RT Master Mix" (Toyobo) according to the manufacturer's instructions. Real-time PCR was performed on the cDNA obtained by the reverse transcription reaction using "StepOne real-time PCR system" and "PowerUp SYBR Green Master Mix" (Thermo Fisher Scientific) according to the manufacturer's instructions.

(1-6) Transglutaminase-1 (TGM1) cell-based ELISA
A medium containing 150 μg/ml CP-BSA or 150 μg/ml BSA was added to NHEK cells cultured in plates, and the cells were treated according to cell culture conditions. As a control, CP-BSA and a medium containing no BSA were used. 24 hours after treatment, cells were fixed using 4% paraformaldehyde-PBS solution. A solution prepared by diluting "anti-TGM1 antibody Ab167657" (Abcam) with PBS as a primary antibody was added to each well and allowed to react overnight at 4°C.

Next, after removing the primary antibody and washing the cells with PBS, a reaction was performed using the secondary antibody "VECTA STAIN Elite Mouse IgG kit PK-6101" (VECTOR). After the reaction, the cells were subjected to a coloring reaction with 3,3',5,5'-tetramethylbenzidine (TMB) (Thermo Fisher Scientific), and the coloring reaction was stopped with 2M H ₂ SO ₄ . After stopping the reaction, the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

(1-7) TGM1 activity measurement using monodansylcadaverine (MDC) NHEK cells were treated with CP-BSA (50 μg/mL, 75 μg/mL and 100 μg/mL) or BSA (50 μg/mL) in the same manner as in 1-6 above. mL, 75 μg/mL, and 100 μg/mL) or conditions without any addition (control). After 24 hours of treatment, the medium was removed by decantation, and 100 μL/well of Humedia-KG2 containing MDC, which was adjusted to a final MDC concentration of 75 μM, was added. Plates were incubated for 24 hours at 37°C in a 5% _CO2 incubator. After incubation, the medium was removed by decantation. Each well was washed three times with 200 μL of HBSS (GIBCO), and then the fluorescence intensity was measured with 100 μL of HBSS added (Ex.: 335 nm, Em.: 512 nm).

(1-8) Intracellular oxidation state of CP co-cultured epidermal cells after cell reseeding NHEK cells were subjected to CP-BSA treatment in the same manner as in 1-6 above. After 24 hours of treatment, the cells were peeled off the plate and collected in order to wash the outside of the cells. The cells were collected by treating NHEK cells with a trypsin solution ("HK-3120"; Kurabo) and then acting on a trypsin neutralization solution ("HK-3220"; Kurabo). The collected cells were centrifuged (1,000 rpm, 3 minutes) and resuspended using Humedia-KG2 medium, and then the cells were seeded on another culture plate and cultured for 24 hours. The cultured cells were then subjected to CP staining by FTSC in the same manner as in 1-4 above, and the fluorescence intensity was measured.

(1-9) Measurement of transepidermal water loss (TEWL) using an epidermal model As an epidermal model, “LabCyte EPI-MODEL 6D” (Japan Tissue Engineering Co., Ltd.), a 6-day culture product of LabCyte EPI-MODEL, was used. there was. After acclimatizing the epidermal model for 1 hour in assay medium (Japan Tissue Engineering Co., Ltd.), apple fruit extract (AP) (“ApplePhenon® C-100”; BGG Japan Co., Ltd.) was added to the final concentration. It was added to the medium at a concentration of 200 μg/mL and cultured for 24 hours (hereinafter referred to as "AP pretreatment"). Thereafter, the medium was removed, and an assay medium containing BSA or CP-BSA at a final concentration of 150 μg/mL was added (however, only the assay medium was used as a control), and cultured for 3 days. During the culture period, TEWL was measured every day using a water transpiration measuring device (“VAPO SCAN”; Nippon Ash Co., Ltd.). At the time of TEWL measurement, the TEWL measurement was performed after allowing the sample to stand for 20 minutes with the lid open on a heat block set at 37°C.

(1-10) CP staining in epidermal model For the epidermal model cultured for 3 days in 1-9 above, frozen thin sections were prepared according to a conventional method. The prepared epidermal model sections were labeled with FTSC to visualize CP.

(1-11) Statistical processing As the statistical analysis software, the Excel add-in software "Statcel4" included with "4Step Excel Statistics 4th Edition" (OMS Publishing) was used. As a test, a Tukey-Kramer multiple comparison test was performed.

[2. Evaluation of the influence of extracellular carbonylated proteins on epidermal keratinocytes]
(2-1) Confirmation of CP localization in human skin In order to confirm the localization of CP in skin tissue, frozen sections taken from abdominal skin of elderly people were stained with FTSC. The results are shown in Figure 1. As shown in FIG. 1, CP-derived fluorescence was confirmed in the entire epidermal layer of the skin tissue.

(2-2) Changes in skin moisture-related proteins due to extracellular CP In order to verify the effects of extracellular CP on epidermal keratinocytes, NHEK cells were subjected to CP-BSA treatment. In this verification, CP-BSA mimics various proteins in a dysfunctional state that exist outside, rather than inside, cells. The results are shown in FIGS. 2A and 2B. As shown in FIG. 2A, the amount of CP was increased in NHEK cells treated with CP-BSA. Further, as shown in FIG. 2B, even when the cells were washed and the medium was replaced after the CP-BSA treatment, the cells were still stained by CP staining. From this, it was found that epidermal keratinocytes may take CP-BSA into the cells through phagocytosis or the like, or may deposit or pass through the cell membrane.

Next, in order to investigate the effect of CP on genes related to skin moisturization, gene expression analysis was performed using real-time PCR. The results are shown in FIGS. 3A and 3B. As shown in FIGS. 3A and 3B, CP was found to reduce the gene expression of TGM1 and involucrin (INV), which play a central role in the formation of the cornified envelope (CE) in the cell membrane of keratinocytes.

TGM1 is an enzyme that binds to the cell membrane of keratinocytes, whose expression increases with keratinization, and which bridges scaffold proteins and mediates ester bonds between glutamine residues of scaffold proteins and very long chain fatty acids. (For example, see the literature by Ishizuka et al. (Jpn. J. Clin. Immunol., 40 (6) 416-427 (2017)). As a result, TGM1 not only forms a CE but also forms a lipid envelope outside the CE. (CLE). Therefore, reduced expression or dysfunction of TGM1 results in inhibition of the formation of CE and CLE, which may be a cause of phyllodes ichthyosis.

INV plays a role as a scaffold protein that is deposited on the cell membrane side of keratinocytes in the first step of CE formation. INV is a substrate of TGM1, forms CE, and can serve as a scaffold for CLE. However, it is known that the CE of INV-deficient mice has no significant morphological changes compared to the wild type, suggesting a compensatory role of the CE scaffold protein.

Based on the above results and the knowledge known so far, we believe that decreased expression of TGM1 due to CP may lead to a decrease in skin barrier function through inhibition of CE formation and/or inhibition of CLE formation. The expression level of TGM1 protein in the cell membrane of cells and the effect of reducing TGM1 activity were evaluated. The results are shown in FIGS. 4A and 4B. As shown in FIGS. 4A and 4B, it was confirmed that the expression level of TGM1 protein decreased and TGM1 activity decreased in a CP-BSA concentration-dependent manner.

[3. Suppressing effect of apple fruit extract on deterioration of skin barrier function of extracellular carbonylated proteins]
As mentioned above, TGM1 is an important factor for CE formation, and a decrease in TGM1 protein and TGM1 activity causes weakening of CE and inhibits the formation of the stratum corneum barrier, which plays a central role in skin barrier function. it is conceivable that. Therefore, the influence of extracellular CP on stratum corneum barrier formation was evaluated.

Changes in transepidermal water loss (TEWL) in a cultured epidermal model reconstituted by extracellular CP were evaluated. Furthermore, in this evaluation system, the influence of apple fruit extract (AP) on changes in TEWL in an epidermal model due to extracellular CP was evaluated. The results are shown in Table 1. Further, a graph of the Day 1 results in Table 1 is shown in FIG.

As shown in Table 1 and FIG. 5, TEWL showed a high value one day after the epidermal model was treated with CP-BSA. This revealed that extracellular CP causes a decrease in skin barrier function in epidermal tissue.

However, AP pretreatment decreased TEWL 1 day after CP-BSA treatment. This revealed that apple fruit extract suppresses the decline in skin barrier function caused by extracellular CP in the epidermal tissue. From the results of Day 1, it is thought that CP causes a delay in the maturation of the stratum corneum. In actual human skin, the stratum corneum usually peels off due to turnover. However, in the epidermal model, the stratum corneum does not peel off and is layered. Therefore, in the epidermal model, it is considered that after Day 2, the TEWL value reached a limit at which it would not decrease any further. Therefore, it is presumed that the Day 1 results serve as a model for the phenomenon that occurs in human skin.

After culturing for 3 days, the epidermal model was stained for CP using FTSC. The results are shown in FIG. As shown in FIG. 6, the CP staining images also revealed that the CP amount in the epidermal model was reduced by AP pretreatment.

From the above results, the following findings were obtained.
The skin barrier function of the epidermal tissue that comes into contact with extracellular CP decreases. However, if the epidermal tissue is exposed to apple fruit extract in advance, the deterioration of the skin barrier function caused by subsequent contact with CP is suppressed. Furthermore, in keratinocytes that have come into contact with extracellular CP, the expression levels of TGM1 and INV involved in CE formation decrease, and further, TGM1 activity decreases. This suggests that the decrease in skin barrier function due to extracellular CP may be brought about by inhibition of CE formation through decreased TGM1 expression, decreased TGM1 activity, and/or decreased INV expression. Therefore, since apple fruit extract suppresses the decline in skin barrier function, apple fruit extract has the effect of suppressing the inhibition of CE formation, the effect of suppressing the decrease of TGM1 expression, the effect of suppressing the decrease of TGM1 activity, and the effect of suppressing the decrease of INV expression. is suggested.

The composition according to one aspect of the present invention has an effect of suppressing a decline in skin barrier function, an effect of suppressing an inhibition of cornified envelope formation, an effect of suppressing a decrease in transglutaminase-1 expression, an effect of suppressing a decrease in transglutaminase-1 activity, and/or an effect of suppressing a decrease in involucrin expression. For the beauty and/or use of individuals who expect cell protection effects, anti-aging effects, anti-oxidation effects, anti-wrinkle effects, radical scavenging effects, skin quality maintenance effects, skin quality improvement effects, whitening effects, etc. It can contribute to health.

Claims (7)

A composition for suppressing deterioration of skin barrier function, containing apple pulp extract as an active ingredient. A composition for inhibiting cornified envelope formation, which contains apple pulp extract as an active ingredient. A composition for suppressing a decrease in transglutaminase expression or a composition for suppressing a decrease in transglutaminase activity, which contains an apple pulp extract as an active ingredient. 4. The composition of claim 3, wherein the transglutaminase is intrakeratinocyte transglutaminase. The composition according to any one of claims 3 to 4, wherein the transglutaminase is transglutaminase-1. A composition for suppressing a decrease in involucrin expression, which contains apple pulp extract as an active ingredient. 7. The composition of claim 6, wherein the involucrin is intrakeratinocyte involucrin.