Preventing and / or ameliorating agent, screening method, and beauty method

Abstract

The present invention addresses the problem of providing: an agent that is based on a new idea, is for preventing and / or ameliorating skin sagging and / or pore conspicuousness caused by the skin sagging, and is capable of easily obtaining a cosmetic effect similar to that of HIFU irradiation; and a new cosmetic method using the agent.　A preventing and / or ameliorating agent according to the present invention contains a high-intensity focused ultrasound (HIFU) irradiation-like effect agent, and is for preventing and / or ameliorating skin sagging and / or pore conspicuousness caused by the skin sagging.

Description

Prevention and / or improvement agent, screening method, and beauty method

[0001] The present invention relates to a prevention and / or improvement agent, a screening method, a beauty method, an agent having an action similar to high-intensity focused ultrasound (HIFU) irradiation, and an agent for promoting the expression of the Wnt2 gene.

[0002] In recent years, in the skin care industry, high-intensity focused ultrasound (HIFU) irradiation has attracted attention as a non-invasive treatment for improving skin laxity and the like. HIFU irradiation is a technique that focuses ultrasonic waves on a single point under the skin and instantaneously raises the temperature of the irradiation site. By performing HIFU irradiation, local thermal damage can be caused to the subcutaneous tissue (mainly the fascia), and a lifting effect of tightening the fascia can be obtained without performing an incision operation. Regarding HIFU irradiation, cosmetic effects such as prevention and / or improvement of skin laxity and enlarged pores have been reported.

[0003] In addition to thermal damage, HIFU irradiation is also known to increase the production of extracellular matrix in skin cells. For example, in Non-Patent Document 1, it has been reported that by performing HIFU irradiation on aged skin cells, dermal collagen and elastic fibers can be increased through the regulation of caveolin-1 and the reduction of p53 activity. The increase in the production of extracellular matrix that occurs in skin cells by such HIFU irradiation is considered to be an important factor for realizing cosmetic effects, but the detailed mechanism of action has not been fully elucidated.

[0004] Since HIFU irradiation requires special equipment and specialized knowledge, the number of medical institutions performing HIFU irradiation is limited at present. Therefore, HIFU irradiation is not easily accessible to everyone. In addition, people's demands for beauty are increasing day by day, and there is also a need for proposals of options tailored to individual lifestyles, skin conditions, or skin characteristics.

[0005] Seyon Oh et al., Cells 2023, 12(18), 2275

[0006] This invention has been made in view of the above circumstances, and aims to clarify the mechanism of action of HIFU irradiation and to provide a novel concept of a preventive and / or corrective agent for skin sagging and / or enlarged pores caused by said skin sagging, which can easily obtain the same cosmetic effects as HIFU irradiation, as well as a new cosmetic method using the same.

[0007] The inventors believed that if they could fully elucidate the mechanism of action by which HIFU irradiation increases extracellular matrix and reproduce it using a simple method, it might be possible to easily achieve cosmetic effects equivalent to HIFU irradiation without causing thermal damage. As a result of diligent research to solve the above problem, the inventors found that when HIFU irradiation was performed on three-dimensional cultured skin, the expression of several genes, including the Wnt2 (wingless-type MMTV integration site family member 2) gene and the SELE (Selectin E) gene, increased. Furthermore, the inventors confirmed that adding Wnt2 protein or SELE protein to fibroblasts increased the production of extracellular matrix (particularly elastin and fibronectin, which can serve as a foundation for other extracellular matrix). From these findings, the inventors concluded that the induction of extracellular matrix formation-related genes, such as the Wnt2 gene and SELE gene, is important for the preventive and / or ameliorative effects of HIFU irradiation on skin sagging and enlarged pores. Furthermore, the inventors realized that by utilizing this knowledge, they could develop a cosmetic product that could easily achieve the same effects as HIFU irradiation. The inventors then screened cosmetic ingredients using the presence or absence of induction of the Wnt2 gene as an indicator, and discovered that certain plant extracts exhibited HIFU irradiation-like effects. The gist of the present invention is as follows.

[0008] [1] A preventive and / or corrective agent for skin sagging and / or enlarged pores caused by skin sagging, comprising a high-intensity focused ultrasound (HIFU) irradiation-like agent. [2] The preventive and / or corrective agent of [1], wherein the HIFU irradiation-like agent is a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae, and Araliaceae. [3] The preventive and / or corrective agent of [2], wherein the plant is at least one selected from the group consisting of loquat, grape, and ginseng, and the plant extract is extracted from at least one part selected from the group consisting of root, stem, leaf, and fruit of the plant. [4] The preventive and / or corrective agent of [3], wherein the plant extract comprises a mixed extract of loquat leaf extract and grape fruit extract. [5] The preventive and / or corrective agent of [1] or [2], wherein the HIFU irradiation-like agent has the effect of increasing the expression level of the Wnt2 gene in skin cells. [6] The HIFU irradiation-like agent is the preventive and / or corrective agent of [5], having a fibronectin expression-promoting effect due to an increase in the expression level of the Wnt2 gene. [7] A screening method characterized by screening for cosmetic ingredients effective in preventing and / or improving skin sagging and / or enlarged pores caused by skin sagging, using as an indicator a gene whose expression changes when a cultured skin model is irradiated with high-intensity focused ultrasound (HIFU). [8] The screening method of [7], comprising at least the steps of applying a test substance to cultured skin cells and measuring the gene expression in the cultured skin cells. [9] The screening method of [7] or [8], wherein the gene is at least one selected from the group consisting of the Wnt2 gene, ZFP36 gene, mir-21 gene, CCN3 gene, ICAM1 gene, SELE gene, CYR61 gene, and CSF2 gene.

[10] A cosmetic method for improving and / or enhancing skin condition, characterized by using a high-intensity focused ultrasound (HIFU) irradiation-like agent.

[11] A high-intensity focused ultrasound (HIFU) irradiation-like agent containing as an active ingredient a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae and Araliaceae.

[12] An expression enhancer for the Wnt2 gene, containing a plant extract extracted from at least one plant selected from the group consisting of loquat, grape, and ginseng.

[13] An expression enhancer for the Wnt2 gene according to

[12] , used to reproduce the genetic changes that occur in skin cells due to high-intensity focused ultrasound (HIFU) irradiation.

[14] Use of a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae, and Araliaceae as an HIFU irradiation-like agent.

[15] Use of a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae, and Araliaceae as an agent to prevent and / or improve skin laxity and / or enlarged pores caused by said skin laxity.

[16] Use of a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae, and Araliaceae in the manufacture of an HIFU irradiation-like agent.

[17] Use of a plant extract extracted from at least one plant selected from the group consisting of Rosaceae, Vitaceae and Araliaceae in the manufacture of an agent for preventing and / or improving skin laxity and / or enlarged pores resulting from said skin laxity.

[0009] According to the present invention, it is possible to provide a novel agent for preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging, which can easily obtain the same cosmetic effects as HIFU irradiation, as well as a novel cosmetic method using the same.

[0010] This is a photograph showing the results of immunohistochemistry performed in Test 2. This is a graph showing the fluorescence intensity of the immunohistochemistry performed in Test 2. This is a photograph showing the results of immunohistochemistry performed in Test 3. This is a graph showing the results of qPCR performed in Test 4. This is a graph showing the results of qPCR performed in Test 4. This is a graph showing the results of qPCR performed in Test 4. This is a graph showing the results of qPCR performed in Test 4. This is a graph showing the results of qPCR performed in Test 5. This is a graph showing the number of hair follicles measured in Test 6. This is a graph showing the results of qPCR performed in Test 7. This is a graph showing the results of qPCR performed in Test 8.

[0011] The present invention will be described in detail below. Unless otherwise specified, terms used herein shall be interpreted in the sense commonly used in the art.

[0012] <First Embodiment: Preventive and / or Remedial Agent> The preventive and / or remedial agent according to the first embodiment of the present invention contains a HIFU irradiation-like agent. Here, a HIFU irradiation-like agent refers to a component capable of giving the same effect as when HIFU is irradiated onto a subject. Although there are multiple effects that can be obtained by irradiating a subject with HIFU, the HIFU irradiation-like agent only needs to be able to give at least some of these effects. The HIFU irradiation-like agent is a substance that, for example, by the method described in the Examples or a method similar thereto, is confirmed to have at least one effect selected from the group consisting of an effect of increasing the expression of the Wnt2 gene, an effect of increasing the expression of the CSF2 gene, an effect of increasing the expression of the ZFP36 gene, an effect of suppressing the expression of the mir-21 gene, an effect of suppressing the expression of the CCN3 gene, an effect of increasing the expression of the ICAM1 gene, an effect of increasing the expression of the SELE gene, and an effect of increasing the expression of the CYR61 gene (particularly, an effect of increasing the expression of the Wnt2 gene). The above effect is preferably concentration-dependent. The preventive and / or corrective agent of the present invention is a component that can serve as an alternative to HIFU irradiation. Furthermore, unlike HIFU irradiation, the preventive and / or corrective agent of the present invention does not cause thermal damage, thus avoiding the inflammatory reaction caused by thermal damage. Therefore, in some cases, the preventive and / or corrective agent of the present invention may provide superior cosmetic effects compared to HIFU irradiation.

[0013] The main effects obtained by irradiating a subject with HIFU include the prevention and / or improvement of skin sagging and / or enlarged pores caused by said skin sagging. Therefore, the use of the preventive and / or improving agent of the present invention provides the effect of preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging. In particular, the use of the preventive and / or improving agent of the present invention shows the effect of reducing the number of pores by improving the appearance of pores. However, the use of the preventive and / or improving agent of the present invention may also provide other effects. For example, the use of the preventive and / or improving agent of the present invention may provide the effect of preventing and / or improving wrinkles caused by rough skin, or an anti-aging effect.

[0014] The preventive and / or corrective agent of the present invention may contain, in addition to the HIFU irradiation-like agent, other optional components, as long as they do not impair the effects of the present invention. The HIFU irradiation-like agent and other optional components are described below.

[0015] In this specification, "HIFU irradiation" refers to high-intensity focused ultrasound irradiation, and is not particularly limited as long as it is irradiated using high-intensity focused ultrasound equipment and conditions commonly used for cosmetic and anti-aging purposes. For example, it can be performed using an irradiation device (e.g., "UltraCell" manufactured by Jeisys Medical Japan) in ultrasonic mode with an output of 0.3 to 0.8 mJ (preferably 0.5 mJ) and a depth of 1.0 to 2.0 mm (preferably 1.5 mm).

[0016] In this specification, "increased expression level" or "upregulation" of a specific gene means a significant increase in the expression level of that specific gene. The increase in the expression level of the specific gene is preferably 5% or more, more preferably 15% or more, even more preferably 30% or more, and particularly preferably 50% or more.

[0017] In this specification, "decreased expression" or "downregulation" of a specific gene means a significant decrease in the expression level of that specific gene. The decrease in the expression level of a specific gene is preferably 5% or more, more preferably 15% or more, even more preferably 30% or more, and particularly preferably 50% or more. "Change in expression" of a specific gene means that the aforementioned "increased expression" or "decreased expression" occurs.

[0018] [HIFU Irradiation-like Agents] HIFU irradiation-like agents are substances capable of producing the same effects as when HIFU irradiation is performed on a subject. Preferably, HIFU irradiation-like agents produce the same gene changes as when HIFU is irradiated onto skin cells as the HIFU irradiation-like effect. Specifically, HIFU irradiation-like agents preferably have the effect of increasing the expression level of the Wnt2 gene in skin cells, and more preferably have at least one selected from the group consisting of an effect of increasing the expression level of the CSF2 gene, an effect of increasing the expression level of the ZFP36 gene, an effect of suppressing the expression level of the mir-21 gene, an effect of suppressing the expression level of the CCN3 gene, an effect of increasing the expression level of the ICAM1 gene, an effect of increasing the expression level of the SELE gene, and an effect of increasing the expression level of the CYR61 gene, and even more preferably have at least one selected from the group consisting of an effect of increasing the expression level of the Wnt2 gene, an effect of increasing the expression level of the ZFP36 gene, and an effect of suppressing the expression level of the CCN3 gene. Furthermore, it is even more preferable that, in addition to the effect of increasing the expression level of the Wnt2 gene, it also has a fibronectin expression-promoting effect resulting from this gene.

[0019] Fibronectin is a large glycoprotein and one of the main components of the extracellular matrix (ECM) in the skin, and is involved in skin elasticity and strength. Increasing the amount of fibronectin in the skin can increase skin elasticity and strength, and consequently prevent and / or improve skin sagging and / or the enlargement of pores caused by said skin sagging.

[0020] It is known that the expression level of the fibronectin gene is regulated by genes such as the Wnt2 gene, ZFP36 gene, mir-21 gene, CCN3 gene, and CSF2 gene. In particular, increasing the expression level of the Wnt2 gene can increase the expression level of the fibronectin gene. Therefore, in skin cells, inducing the expression level of the Wnt2 gene can promote fibronectin expression. It is also thought that in skin cells, increased expression of the Wnt2 gene, or caused by increased expression of the Wnt2 gene, leads to increased expression of the CSF2 gene, increased expression of the ZFP36 gene, suppression of the mir-21 gene, and suppression of the CCN3 gene, resulting in a fibronectin expression-promoting effect.

[0021] The HIFU irradiation-like agent preferably has the effect of increasing the expression levels of the Wnt2 gene and / or SELE gene in skin cells, and consequently has an elastin expression-promoting effect.

[0022] Elastin is a protein that is a component of elastic fibers that support collagen fibers, and plays an important role in skin firmness and elasticity. Increasing the amount of elastin in the skin increases skin elasticity and strength, which in turn can prevent and / or improve skin sagging and / or the enlargement of pores caused by such skin sagging.

[0023] The expression level of the elastin gene is thought to be regulated by the Wnt2 gene, SELE gene, etc. Therefore, increasing the expression levels of the Wnt2 gene and / or SELE gene in skin cells can promote elastin expression.

[0024] Examples of HIFU irradiation-like agents include plant extracts and seaweed extracts. An extract refers to a solution or dried product obtained from a raw material such as a plant or seaweed. In addition to components derived from the raw material, the extract may also contain solvents such as water, organic solvents, or inorganic solvents, as well as other components. Plant extracts are preferred as HIFU irradiation-like agents.

[0025] Examples of plants from which plant extracts are extracted include monocots and dicots. Examples of monocots include grasses, orchids, lilies, amaryllidaceae, sedges, palms, araceae, arrowheads, asparagaceae, yamaceae, ginger, banana, iris, and pineapples. Examples of dicots include chrysanthemums, legumes, roses, nightshades, brassicas, mint, cucurbitaceae, carrots, mallows, laurels, russulaceae, sprouts, amaranthaceae, water lilies, lotus, rutaceae, Araliaceae, morning glory, violets, sesame, plantaginaceae, hemp, caryophyllaceae, meliaceae, violets, primroses, and buttercups.

[0026] Examples of plant parts from which extracts can be obtained include roots, leaves, flowers, buds, stems, shoots, bulbs, endosperm, pollen, rhizomes, fruits, fruit peels, bark, sap, resin, seeds, and pith.

[0027] Examples of plant extracts include artichoke leaf extract, Rehmannia glutinosa root extract, Angelica keiskei leaf extract, Angelica keiskei stem extract, asparagus stem extract, acerola fruit extract, acerola seed extract, Hydrangea macrophylla extract, Althaea officinalis root extract, Artemia extract, Arnica montana flower extract, Aloe vera leaf extract, Apricot kernel extract, Polygonum cuspidatum root extract, Ginkgo biloba leaf extract, Turmeric rhizome extract, Asarum sieboldii rhizome, Asarum sieboldii root extract, Prunus mume fruit extract, Edelweiss flower extract, Edelweiss leaf extract, Citrus citrus peel extract, Scutellaria baicalensis root extract, Coptis japonica root extract, and more. Sophora japonica fruit extract, Panax ginseng root extract, Nasturtium officinale leaf extract, Nasturtium officinale stem extract, Chamomile flower extract, Quince extract, Artemisia capillaris flower extract, Licorice root extract, Licorice leaf extract, Glycyrrhiza uralensis flavonoids, Rubus idaeus extract, Rubus idaeus fruit extract, Kiwi extract, Kiwi seed extract, Phellodendron amurense bark extract, Cucumis fruit extract, Lycium barbarum fruit extract, Pueraria lobata root extract, Passiflora edulis fruit extract, Gardenia fruit extract, Sasa veitchii leaf extract, Sophora flavescens root extract, Grapefruit fruit extract, Alpinia speciosa leaf extract, Geranium thunbergii flower extract, Geranium thunbergii Leaf extract, Geranium thunbergii stem extract, coffee seed extract, burdock root extract, rice bran extract, star fruit leaf extract, pomegranate peel extract, pomegranate flower extract, hawthorn extract, sansho pepper peel extract, perilla leaf extract, peony root extract, ginger rhizome extract, calamus rhizome extract, birch bark extract, honeysuckle flower extract, horsetail extract, purslane extract, linden flower extract, yarrow extract, peppermint leaf extract, sage leaf extract, mallow flower extract, Cnidium officinale rhizome extract, Swertia japonica extract, Somei Yoshino cherry leaf extract Soybean seed extract, Damask rose flower extract, Camellia sinensis leaf extract, Camellia sinensis flower extract, Clove extract, Camellia japonica seed extract, Camellia japonica leaf extract, Camellia japonica flower extract, Centella asiatica extract, Centella asiatica leaf extract, Centella asiatica stem extract, Tencha extract, Angelica acutiloba root extract, Calendula officinalis flower extract, Houttuynia cordata extract, Tomato fruit extract, Ruscus aculeatus root extract, Tilia cordata flower extract, Ziziphus jujuba fruit extract, Carrot root extract, Garlic root extract, Rosa multiflora fruit extract, Pineapple fruit extract, Coix lacryma-jobi seed extract, Papaya fruit extract, Rosa rugosa flower extract, Hamamelis virginiana leaf extract,Examples include Belamcanda chinensis extract, Isodon japonicus leaf extract, Isodon japonicus stem extract, Vaccinium bilberry leaf extract, Loquat leaf extract, Vitis vinifera seed extract, Tilia cordata flower extract, Safflower extract, Paeonia suffruticosa extract, Morus alba root bark extract, Citrus reticulata peel extract, Sapindus mukorossi extract, Sapindus mukorossi peel extract, Lithospermum erythrorhizon root extract, Melissa officinalis leaf extract, Prunus persica seed extract, Cornflower extract, Saxifraga stolonifera extract, Citrus yuzu fruit extract, Citrus yuzu seed extract, Raspberry fruit extract, Artemisia princeps extract, Artemisia princeps leaf extract, Lavandula angustifolia flower extract, Apple fruit extract, Lemon fruit extract, Astragalus sinicus extract, Rosemary leaf extract, Roman chamomile flower extract, Sanguisorba officinalis extract, etc.

[0028] The HIFU irradiation-like agent is preferably a plant extract extracted from at least one plant selected from the group consisting of the Rosaceae, Vitaceae, and Araliaceae families. Examples of Rosaceae plants include roses, apples, pears, cherries, peaches, plums, apricots, Japanese apricots, almonds, cherries, strawberries, raspberries, blackberries, quince, hawthorn, loquats, and rowan, with loquats being preferred. Examples of Vitaceae plants include grapes, wild grapes, muscadine, Virginia creeper, American ivy, wild grapes, wild grapes, cissus, benghalensis, trefoil, and triangular vine, with grapes being preferred. Examples of Araliaceae plants include Japanese angelica tree, Japanese angelica tree, Fatsia japonica, Acanthopanax sciadophylloides, Kalopanax septemlobus, Korean ginseng, Panax ginseng, Siberian ginseng, Schefflera, ivy, and Japanese ginseng, with Panax ginseng being preferred.

[0029] The plant used as the raw material for the plant extract is preferably at least one selected from the group consisting of loquat, grape, and ginseng. The plant extract is preferably extracted from at least one part of the plant selected from the group consisting of the root, stem, leaves, and fruit.

[0030] Considering the effects of the present invention comprehensively, the plant extract used as a HIFU irradiation-like agent is preferably at least one selected from the group consisting of loquat leaf extract, ginseng root extract, and grape fruit extract, and a mixed extract of loquat leaf extract and grape fruit extract is more preferable.

[0031] The content ratio of the HIFU irradiation-like agent in the preventive and / or corrective agent of the present invention is preferably 0.0001% to 5% by mass, more preferably 0.001% to 1% by mass, and even more preferably 0.003% to 0.5% by mass. If the preventive and / or corrective agent of the present invention contains two or more HIFU irradiation-like agents, the sum of the content ratios of these two or more HIFU irradiation-like agents shall be the aforementioned content ratio.

[0032] When the preventive and / or corrective agent of the present invention contains a mixed extract of loquat leaf extract and grape fruit extract as a HIFU irradiation-like agent, the amount of grape fruit extract blended with 1 part by mass of loquat leaf extract is preferably 0.1 to 10 parts by mass, more preferably 0.2 to 8 parts by mass, even more preferably 0.5 to 6 parts by mass, and particularly preferably 0.9 to 5 parts by mass.

[0033] [Optional Components] Examples of optional components included in the preventive and / or corrective agent of the present invention include whitening active ingredients, turnover promoters, anti-glycation ingredients, antioxidant ingredients, anti-aging ingredients, anti-inflammatory agents, cooling agents, bactericides, vitamins, organic acids, moisturizing ingredients, polyhydric alcohols, scrubbing agents, UV absorbing ingredients, UV scattering ingredients, astringent ingredients, peptides or their derivatives, amino acids or their derivatives, cleansing ingredients, keratin softening ingredients, cell activating ingredients, blood circulation promoting ingredients, etc. In the preventive and / or corrective agent of the present invention, these ingredients may be used individually or in combination of two or more.

[0034] Examples of the aforementioned whitening active ingredients include tocopherol, vitamin C and its derivatives, arbutin, kojic acid, placenta, ellagic acid, nicotinamide, tranexamic acid and its derivatives, hydroquinone, lucinol (registered trademark), potassium 4-methoxysalicylate, linoleic acid and its derivatives, and the like.

[0035] Examples of the aforementioned turnover-promoting agents include vitamins, keratin-softening components, and cell-activating components, which will be described later.

[0036] Examples of the anti-glycation components include plant extracts such as Budreja axillaris leaf extract, evening primrose oil, Amla fruit, fruit juice or extracts thereof, L-arginine, L-lysine, hydrolyzed casein, hydrolyzable tannins, and carnosine.

[0037] Examples of the aforementioned antioxidant components include those derived from plants (e.g., grapes, ginseng, and comfrey); proanthocyanidins, tocopherols and their derivatives, ascorbic acid and its derivatives, hesperidin and its derivatives, ergothioneine, sodium bisulfite, erythorbic acid and its salts, flavonoids, glutathione, and the like.

[0038] Examples of the aforementioned anti-aging ingredients include hydrolyzed soy protein, retinoids (retinol and its derivatives, retinoic acid, and retinal, etc.), pangamic acid, kinetin, ursolic acid, turmeric extract, sphingosine derivatives, silicon, silicic acid, N-methyl-L-serine, and mevalonolactone.

[0039] Examples of the anti-inflammatory agents include allantoin and its derivatives, glycyrrhetinic acid and its derivatives, glycyrrhizic acid and its derivatives, salicylic acid derivatives, aminocaproic acid, azulene and its derivatives, zinc oxide, tocopherol acetate, and the like.

[0040] Examples of the cooling agents include terpenes such as menthol, camphor, and geraniol (these may be d-isomers, l-isomers, or dl-isomers); and essential oils such as eucalyptus oil, bergamot oil, peppermint oil, cool mint oil, spearmint oil, and peppermint oil.

[0041] Examples of the aforementioned disinfectants include isopropylmethylphenol, salicylic acid, benzalkonium chloride, ethanol, benzethonium chloride, gluconic acid and its derivatives, photosensitizer 101, photosensitizer 201, parabens, phenoxyethanol, piroctoolamine, miconazole, and the like.

[0042] The vitamins may be either water-soluble vitamins or oil-soluble vitamins. For example, vitamin B6, pantothenic acid, nicotinic acid, vitamin B1, vitamin B2, biotin, folic acid, vitamin B12, water-soluble vitamin C, oil-soluble vitamin C, vitamin K; vitamin-like active factors such as ferulic acid, etc. may be mentioned.

[0043] As the organic acid, for example, gluconic acid, aspartic acid, aminoethylsulfonic acid, citric acid, glutamic acid, succinic acid, oxalic acid, fumaric acid, propionic acid, malic acid, salicylic acid, glycolic acid, phytic acid, tartaric acid, acetic acid, lactic acid, and salts thereof may be mentioned. As the salts, for example, salts of mineral acids such as sulfuric acid, hydrochloric acid or phosphoric acid, salts of organic acids such as maleic acid or methanesulfonic acid, alkali metal salts such as sodium or potassium, alkaline earth metal salts, ammonium salts, etc. may be mentioned.

[0044] As the moisturizing component, for example, high molecular compounds such as sodium hyaluronate, heparin-like substances, sodium chondroitin sulfate, collagen, elastin, keratin, chitin, chitosan, proteoglycan; amino acids such as glycine, aspartic acid, arginine; natural moisturizing factors such as sodium lactate, urea, sodium pyrrolidonecarboxylate; lipids such as ceramide, cholesterol, phospholipid; plant extractives such as chamomile extract, witch hazel extract, tea extract, perilla extract; polyhydric alcohols such as glycerin, etc. may be mentioned.

[0045] The polyhydric alcohol preferably has 2 to 10 carbon atoms. For example, glycerin, diglycerin, triglycerin, propylene glycol, dipropylene glycol, 1,3-butanediol, ethylene glycol, diethylene glycol, isoprene glycol, 1,3-butylene glycol, sorbitol, xylitol, erythritol, mannitol, pentanediol, hexanediol, octanediol, decanediol, neopentyl glycol, etc. may be mentioned.

[0046] Examples of the scrubbing agent include apricot kernel powder, almond shell powder, apricot kernel powder, sodium chloride granules, olive kernel powder, dried seawater granules, candelilla wax, walnut shell powder, cherry kernel powder, coral powder, charcoal powder, sawdust shell powder, polyethylene powder, anhydrous silicic acid, and the like.

[0047] Examples of the ultraviolet absorbing component include ethylhexyl methoxycinnamate, hexyl diethylaminohydroxybenzoyl benzoate, bis-ethylhexyloxyphenol methoxyphenyl triazine, ethylhexyl triazone, t-butyl methoxydibenzoylmethane, octocrylene, polysilicone-15, methylene bisbenzotriazolyl tetramethylbutylphenol, octyl dimethoxybenzylidene dioxoimidazolidine propionate, phenylbenzimidazole sulfonic acid, and the like.

[0048] Examples of the ultraviolet scattering component include inorganic compounds such as hydrous silicic acid, zinc silicate, cerium silicate, titanium silicate, zirconium oxide, cerium oxide, titanium oxide, iron oxide, zinc oxide, anhydrous silicic acid, those obtained by coating these inorganic compounds with inorganic powders such as hydrous silicic acid, aluminum hydroxide, mica, and talc, those obtained by compounding with resin powders such as polyamide, polyethylene, polyester, polystyrene, and nylon, and those treated with silicone oil, aluminum fatty acid salts, and the like.

[0049] Examples of the astringent component include alum, zinc sulfate, aluminum chloride, zinc sulfocarbolic acid, tannic acid, and the like.

[0050] Examples of the peptides or their derivatives include keratin-degrading peptides, hydrolyzed keratin, collagen, fish-derived collagen, atelocollagen, gelatin, elastin, elastin-degrading peptides, collagen-degrading peptides, hydrolyzed collagen, hydroxypropylammonium chloride hydrolyzed collagen, elastin-degrading peptides, conchiolin-degrading peptides, hydrolyzed conchiolin, silk protein-degrading peptides, hydrolyzed silk, sodium lauroyl hydrolyzed silk, soy protein-degrading peptides, hydrolyzed soy protein, wheat protein, wheat protein-degrading peptides, hydrolyzed wheat protein, casein-degrading peptides, acylated peptides (palmitoyl oligopeptides, palmitoyl pentapeptides, palmitoyl tetrapeptides, etc.).

[0051] Examples of the aforementioned amino acids or their derivatives include betaine (trimethylglycine), proline, hydroxyproline, arginine, lysine, serine, glycine, alanine, phenylalanine, β-alanine, threonine, glutamic acid, glutamine, asparagine, aspartic acid, cysteine, cystine, methionine, leucine, isoleucine, valine, histidine, taurine, γ-aminobutyric acid, γ-amino-β-hydroxybutyric acid, carnitine, carnosine, creatine, and the like.

[0052] Examples of the cleansing components include soaps selected from alkali metal salts such as potassium laurate, potassium myristate, potassium palmitate, or potassium stearate, alkanolamide salts, or amino acid salts; amino acid-based surfactants such as sodium cocoyl glutamate and sodium cocoyl methyl taurate; ether sulfate salts such as sodium laureth sulfate; ether carboxylate salts such as sodium lauryl ether acetate; sulfosuccinate salts such as sodium alkyl sulfosuccinate; fatty acid alkanolamides such as coconut oil fatty acid monoethanolamide and coconut oil fatty acid diethanolamide; monoalkyl phosphate salts such as sodium lauryl phosphate and sodium polyoxyethylene lauryl ether phosphate; betaine-type amphoteric surfactants such as coconut oil fatty acid amidopropyl dimethylaminoacetic acid betaine, lauryl dimethylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryl hydroxysulfobetaine, and lauroylamide ethyl hydroxyethyl carboxymethyl betaine hydroxypropyl sodium phosphate; and amino acid-type amphoteric surfactants such as sodium laurylaminopropionate.

[0053] Examples of the aforementioned keratin-softening ingredients include lactic acid, salicylic acid, glycolic acid, citric acid, malic acid, fruit acid, phytic acid, urea, sulfur, and the like.

[0054] Examples of the cell-activating components include amino acids such as γ-aminobutyric acid; vitamins such as retinol, thiamine, riboflavin, pyridoxine hydrochloride, and pantothenic acid; α-hydroxy acids such as glycolic acid and lactic acid; tannins, flavonoids, saponins, and photosensitizer 301.

[0055] Examples of blood circulation-promoting components include those derived from plants (for example, Angelica keiskei, Arnica, Ginkgo biloba, Fennel, Ophiopogon japonicus, Watercress, Chamomile, Roman chamomile, Carrot, Gentian, Burdock, Rice, Hawthorn, Shiitake mushroom, Ginger, Hawthorn, Juniper, Cnidium officinale, Swertia japonica, Thyme, Clove, Citrus unshiu peel, Chili pepper, Angelica acutiloba, Prunus persica, Prunus serrulata, Carrot, Garlic, Butcher's broom, Peony, Horse chestnut, Melissa, Yuzu, Coix seed, Mugwort, Rosemary, Rosehip, Citrus unshiu peel, Angelica acutiloba, Prunus persica, Peach, Apricot, Walnut, Corn, etc.); acetylcholine, ichthammol, Cantharis tincture, gamma-oryzanol, cepharanthine, trazoline, tocopherol nicotinate, glucosyl hesperidin, etc.

[0056] [Method for Manufacturing the Preventive and / or Remedial Agent] The method for manufacturing the preventive and / or remedial agent of the present invention is not particularly limited, and it can be manufactured by a conventional method by appropriately selecting and blending the essential component, the HIFU irradiation-like agent, and other components that are added as needed (the optional components mentioned above, the base or carrier described later, additives, etc.).

[0057] [Uses of the Preventive and / or Improving Agent] The preventive and / or improving agent of the present invention is used in an approach inspired by HIFU irradiation performed in cosmetic medicine, with the expectation of similar effects on the skin, such as the face, as HIFU irradiation. By using the preventive and / or improving agent of the present invention, a preventive and / or improving effect on sagging skin of the face, etc., and the resulting enlarged pores can be obtained. Thus, the preventive and / or improving agent of the present invention can be used as an alternative to HIFU irradiation, or in combination with HIFU irradiation. The preventive and / or improving agent of the present invention used for such purposes can be suitably used as an external composition such as a serum, lotion, emulsion, cream, gel cream, beauty mask (face mask), makeup base, foundation, sunscreen, cleanser, or washing agent, and more preferably as a skincare product such as a serum, lotion, emulsion, cream, or gel cream.

[0058] [Formulation (Topical Composition)] When the preventive and / or corrective agent of the present invention is a topical composition, its essential components and other optional components described above can be mixed with a base or carrier commonly used in cosmetics, pharmaceuticals, and quasi-drugs, and optionally with additives described later, in accordance with conventional methods, and emulsified or solubilized as necessary to produce topical compositions in various formulation forms.

[0059] Examples of the aforementioned base or carrier include hydrocarbons such as liquid paraffin, squalane, petrolatum, gelling hydrocarbons (such as Plastibase), ozokerite, α-olefin oligomers, and light liquid paraffin; silicone oils such as methylpolysiloxane, cyclic silicone, modified silicone, and silicone resin; oils and fats such as coconut oil, olive oil, rice bran oil, and shea butter; waxes such as jojoba oil, miura wax, candelilla wax, and lanolin; cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, octyldodecanol, isostearyl alcohol, phytosterol, and cholesterol. Examples include higher alcohols such as sterols; esters such as methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, carrageenan, polyethylene glycol, isopropyl myristate, octyldodecyl myristate, isopropyl palmitate, cetyl palmitate, isononyl isononanoate, and pentaerythritol tetra-2-ethylhexanoate; polysaccharides such as dextrin and maltodextrin; lower alcohols such as ethanol; glycol ethers such as diethylene glycol monoethyl ether; and water.

[0060] The bases or carriers described above may be used individually or in combination of two or more. Furthermore, the amounts used can be appropriately selected from a range known to those skilled in the art.

[0061] [Additives (Topical Composition)] When the preventive and / or corrective agent of the present invention is a topical composition, known additives used in cosmetics, pharmaceuticals, and quasi-drugs, such as surfactants, antioxidants, colorants, pearlescent agents, chelating agents, pH adjusters, preservatives, and thickeners, may be added, to the extent that they do not impair the effects of the present invention. These additives may be used individually or in combination of two or more.

[0062] The surfactant may be any of the following: nonionic surfactants, cationic surfactants, anionic surfactants, amphoteric surfactants, etc. For example, sorbitan fatty acid esters such as sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan diglycerol penta-2-ethylhexyl sorbitan, sorbitan diglycerol tetra-2-ethylhexyl sorbitan; propylene glycol fatty acid esters such as propylene glycol monostearate; hydrogenated castor oil derivatives such as polyoxyethylene hydrogenated castor oil 40 (HCO-40), polyoxyethylene hydrogenated castor oil 50 (HCO-50), polyoxyethylene hydrogenated castor oil 60 (HCO-60), polyoxyethylene hydrogenated castor oil 80; polyoxyethylene monolauryl Examples include polyoxyethylene sorbitan fatty acid esters such as poly(20) sorbitan (polysorbate 20), polyoxyethylene(20) sorbitan monostearate (polysorbate 60), polyoxyethylene(20) sorbitan monooleate (polysorbate 80), and polyoxyethylene(20) sorbitan isostearate; polyoxyethylene monococonut oil fatty acid glyceryl; glycerin alkyl ethers; alkyl glucosides; polyoxyalkylene alkyl ethers such as polyoxyethylene cetyl ether; amines such as stearylamine and oleylamine; and silicone-based surfactants such as polyoxyethylene methylpolysiloxane copolymers, lauryl PEG-9 polydimethylsiloxyethyl dimethicone, and PEG-9 polydimethylsiloxyethyl dimethicone.

[0063] Examples of the aforementioned antioxidants include dibutylhydroxytoluene, butylhydroxyanisole, sorbic acid, sodium sulfite, ascorbic acid, erythorbic acid, and L-cysteine ​​hydrochloride.

[0064] Examples of the coloring agents include inorganic pigments and natural pigments.

[0065] Examples of the chelating agent include EDTA disodium salt and hydroxyethanediphosphonic acid calcium disodium salt.

[0066] Examples of the pH adjusting agents include inorganic acids (hydrochloric acid, sulfuric acid, etc.), organic acids (lactic acid, sodium lactate, citric acid, sodium citrate, succinic acid, sodium succinate, etc.), inorganic bases (potassium hydroxide, sodium hydroxide, etc.), and organic bases (triethanolamine, diisopropanolamine, triisopropanolamine, etc.).

[0067] Examples of the aforementioned preservatives include benzoic acid, sodium benzoate, dehydroacetic acid, sodium dehydroacetate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, phenoxyethanol, caprylhydroxamic acid, pentanediol, hexanediol, octanediol, and other alkanediols.

[0068] Examples of the aforementioned thickening agents include vinyl-based thickening agents such as polyvinyl alcohol, polyvinylpyrrolidone, and carboxyvinyl polymer; cellulose-based thickening agents such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and carboxyethylcellulose; guar gum, pectin, pullulan, gelatin, locust bean gum, carrageenan, agar, xanthan gum, alkyl acrylate methacrylate copolymer, polyethylene glycol, bentonite, alginic acid, propylene glycol alginate, macrogol, sodium chondroitin sulfate, hyaluronic acid, sodium hyaluronate, (hydroxyethyl acrylate / sodium acryloyldimethyltaurate) copolymer, and (ammonium acryloyldimethyltaurate / vinylpyrrolidone) copolymer.

[0069] [Formulation Form (Topical Composition)] When the preventive and / or corrective agent of the present invention is a topical composition, the formulation form is not particularly limited. Examples include liquid formulations (lotions, serums, etc.), cream formulations (emulsions, creams, gel creams, balms, etc.), gel formulations, jelly formulations, sherbet formulations, aerosol formulations, foam formulations, spray formulations, patches, stick formulations, and other ointments, solid formulations, etc. These formulations can be manufactured by conventional methods, for example, the method described in the General Provisions for Formulations of the 17th Revised Japanese Pharmacopoeia. Furthermore, depending on the desired stability, feel, etc., dosage forms such as W / O, O / W, W / O / W, and O / W / O emulsions can be selected as appropriate. For example, a patch may be made by impregnating a nonwoven fabric sheet, gel sheet, etc., with a serum composition, lotion composition, emulsion composition, etc.

[0070] <Second Embodiment: Screening Method> The screening method according to the second embodiment of the present invention is characterized by screening for cosmetic ingredients that are effective in preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging, using genes whose expression changes when a cultured skin model is irradiated with HIFU as indicators. According to the screening method of the present invention, cosmetic ingredients that are effective in preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging, similar to those obtained when HIFU irradiation is performed, can be efficiently obtained.

[0071] A cultured skin model is a skin cell culture that has a multilayered structure that replicates living skin, and for example, it has a structure corresponding to at least two of the following layers: the stratum corneum, stratum granulosum, stratum spinosum, and stratum basalis. A cultured skin model contains at least keratinocytes and may also contain other cells such as melanocytes and fibroblasts.

[0072] The screening method of the present invention preferably includes at least the steps of (1) applying a test substance to skin culture cells and (2) measuring the gene expression in the skin culture cells.

[0073] Step (1): The skin culture cells are not particularly limited, but for example, epidermal cells, dermal cells, cultured skin models, etc., can be used. Examples of the cultured skin models include MEL-300A (MatTek) and EFT-400 (MatTek). In step (1), the test substance is added to the skin culture cells and cultured for, for example, 2 to 72 hours. As a negative control, skin culture cells without the test substance are prepared.

[0074] Step (2): After step (1), cells are collected, and RNA is extracted according to conventional methods such as spin column method, AGPC method, magnetic bead method, and mRNA purification method using oligo(dT) beads. Gene expression is then analyzed using DNA microarray method, RT-qPCR method, RNA-seq method, reporter assay method, Northern blot method, RNA FISH method, etc. The gene expression of the group to which the test substance was added in step (1) and the group to which it was not added (negative control) are correlated. At this time, the genes to be correlated are those that have been confirmed to change when cultured skin models are irradiated with HIFU. Examples of such genes include at least one selected from the group consisting of the Wnt2 gene, ZFP36 (Zinc Finger Protein 36 Homology) gene, mir-21 (microRNA 21) gene, CCN3 (Cellular Communication Network Factor 3) gene, ICAM1 (InterCellular Adhesion Molecule 1) gene, SELE gene, CYR61 (Cysteine-Rich Angiogenic Inducer 61) gene, and CSF2 (Colony Stimulating Factor 2) gene, with the Wnt2 gene being preferred. The Wnt2 gene, ZFP36 gene, mir-21 gene, CCN3 gene, and CSF2 gene are genes involved in the induction or suppression of fibronectin. The inventors have confirmed that in HIFU-irradiated skin models, the expression of the Wnt2 gene is promoted, the expression of the ZFP36 gene is promoted, the expression of the mir-21 gene is suppressed, the expression of the CCN3 gene is suppressed, and the expression of the CSF2 gene is promoted, the expression of the ICAM1 gene is promoted, the expression of the SELE gene is promoted, and the expression of the CYR61 gene is promoted. They have also confirmed that fibronectin expression is promoted in skin culture cells to which the Wnt2 protein is added, and that elastin expression is promoted in skin culture cells to which the Wnt2 protein or SELE protein is added. In the screening method of the present invention, it is confirmed whether the expression of each gene changes in the group to which the test substance is added in the same way as when HIFU irradiation occurs.Furthermore, the test substance that alters the expression of these genes, similar to the case of HIFU irradiation, can be determined to be a cosmetic ingredient effective in preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging.

[0075] <Third Embodiment: Beauty Method> The beauty method according to the third embodiment of the present invention is a beauty method for improving and / or enhancing the condition of the skin, characterized by the use of a HIFU irradiation-like agent. According to the beauty method of the present invention, by using a HIFU irradiation-like agent, the same effects as HIFU irradiation can be obtained, for example, the effect of preventing and / or improving sagging of the facial skin and / or enlarged pores caused by said sagging can be obtained. The specific description of the HIFU irradiation-like agent can be directly applied as described in the first embodiment above.

[0076] The cosmetic method of the present invention can be implemented, for example, as an alternative to HIFU irradiation. By implementing the cosmetic method of the present invention as an alternative to HIFU irradiation, the same effects as HIFU irradiation can be obtained more easily than with HIFU irradiation. Furthermore, while thermal damage (inflammation) of the skin is unavoidable with HIFU irradiation, the cosmetic method of the present invention does not cause such thermal damage to the skin. Therefore, it can be implemented even for individuals who cannot undergo HIFU irradiation due to their constitution or other reasons, and in some cases, a higher cosmetic effect than HIFU irradiation can be expected.

[0077] <Fourth Embodiment: HIFU Irradiation-like Agent> The HIFU irradiation-like agent according to the fourth embodiment of the present invention contains a plant extract as an active ingredient, which is extracted from at least one plant selected from the group consisting of the Rosaceae, Vitaceae, and Araliaceae families. The plant extract has already been described in the first embodiment, so a redundant explanation will be omitted.

[0078] The HIFU irradiation-like agent of the present invention provides effects similar to those of HIFU irradiation. Therefore, using a cosmetic composition containing this agent can, for example, prevent and / or improve sagging of the facial skin and / or enlarged pores caused by such sagging. The specific description of the HIFU irradiation-like agent can be directly applied to the description in the section on the preventive and / or improving agent according to the first embodiment.

[0079] <Fifth Embodiment: Wnt2 Gene Expression Promoter> The Wnt2 gene expression promoter according to the fifth embodiment of the present invention contains a plant extract extracted from at least one plant selected from the group consisting of loquat, grape, and ginseng. The plant extract has already been described in the first embodiment, so a redundant explanation will be omitted.

[0080] The Wnt2 gene is known to be involved in wound healing and tissue regeneration, such as in the liver, in addition to the production of the extracellular matrix in the skin as described above. The Wnt2 gene expression promoter of the present invention is thought to exert wound healing and tissue regeneration effects in addition to cosmetic effects by promoting Wnt2 gene expression. Furthermore, the Wnt2 gene is also known to be involved in cell reprogramming. The Wnt2 gene expression promoter of the present invention may also be used as a reagent to induce such reprogramming.

[0081] Furthermore, the present invention also includes a CCN3 gene expression inhibitor and / or ZFP36 gene expression promoter containing a plant extract obtained from at least one plant selected from the group consisting of loquat, grape, and ginseng.

[0082] The CCN3 gene is thought to act as an anti-fibrotic factor, suppressing collagen production and fibroblast activation. The CCN3 gene expression inhibitor of the present invention is expected to exert cosmetic effects, wound healing effects, and tissue regeneration effects by suppressing the expression of the CCN3 gene.

[0083] The ZFP36 gene is thought to act as an anti-inflammatory regulator. The ZFP36 gene expression promoter of the present invention is thought to suppress inflammatory responses by promoting ZFP36 gene expression, thereby enabling cosmetic effects and improvement of inflammatory diseases.

[0084] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0085] [Test 1] Analysis of changes in gene expression in a skin model induced by HIFU irradiation. The Kurabo EFT-400 skin model was used. This skin model was constructed by culturing normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) in serum-free medium. This skin model has the basic epidermal structure from the basal layer to the granular layer, spinous layer, and stratum corneum, and is characterized by having a multilayered, highly differentiated three-dimensional structure similar to the structure of human skin (dermis and epidermis). The following tests were performed using this skin model.

[0086] After the skin models arrived from the manufacturer, HIFU irradiation was performed on the skin models under conditions similar to actual HIFU irradiation on humans. The irradiation device used was the "UltraCel" manufactured by Jeisys Medical Japan, set to ultrasound mode with an output of 0.5 mJ and a depth of 1.5 mm. After HIFU irradiation, the skin models were heated to 37°C and CO2. ２ Cells were incubated at a 5% concentration and harvested from the dermis layer of the skin model after 6 and 24 hours. RNA was extracted from the harvested cells according to standard procedures, and gene expression was analyzed using DNA microarray. The DNA microarray used was "Affymetrix GeneChip® Human Gene 2.0 ST Array". In the DNA microarray analysis, the skin model 6 hours after HIFU irradiation was compared with a control incubated for 6 hours without treatment. Genes whose expression increased or decreased by 1.5 times or more due to HIFU irradiation were counted as upregulated genes and downregulated genes, respectively. In the skin model after 6 hours, changes were mainly observed in inflammation-related genes. The skin model 24 hours after HIFU irradiation was similarly compared with a control incubated for 24 hours without treatment. In the skin model after 24 hours, changes were mainly observed in extracellular matrix formation-related genes. Table 1 below shows the number of upregulated and downregulated genes 6 hours and 24 hours after HIFU irradiation, respectively.

[0087]

[0088] The following genes were selected as upregulated in the skin model by HIFU irradiation: Wnt2 gene (significantly upregulated after 24 hours), CSF2 gene (significantly upregulated after 24 hours), ZFP36 gene (significantly upregulated after 6 and 24 hours), ICAM1 gene (significantly upregulated after 6 and 24 hours), SELE gene (significantly upregulated after 6 and 24 hours), and CYR61 gene (significantly upregulated after 6 and 24 hours). The following genes were selected as downregulated in the skin model by HIFU irradiation: mir-21 gene (significantly downregulated after 24 hours) and CCN3 gene (significantly downregulated after 24 hours). These genes were related to extracellular matrix reformation and / or fibroblast proliferation-promoting pathways. Among these, we focused on the Wnt2 gene, which is a beneficial wound healing-related gene. The Wnt2 gene is involved in the formation and development of connective tissue and is essential for extracellular matrix production in the dermal tissue of the skin. We also focused on the SELE gene, a cell adhesion gene that plays an important role in the transition from inflammation to tissue repair.

[0089] [Test 2] Effects of Wnt2 protein addition to normal human dermal fibroblasts. Wnt2 protein (Abnova "H00007472-P01") was added to normal human dermal fibroblasts (Kurabo Corporation: neonatal origin) to confirm its fibrous formation promoting effect. Normal human dermal fibroblasts were cultured in Dulbecco's modified Eagle medium (Thermo Fisher Scientific "Gibco DMEM") supplemented with 10% fetal bovine serum and 1% antibiotic (anti-mycotin) at a temperature of 37°C and CO2. ２ The experiment was conducted in an environment with a concentration of 5%.

[0090] Normal human dermal fibroblasts were cultured for two weeks in a culture medium containing 0.25 μg / mL of Wnt2 protein. Normal human dermal fibroblasts were also cultured for two weeks in a medium without Wnt2 protein to serve as a control. Subsequently, both the cells with Wnt2 protein and the control cells were immunostained using fibronectin (FBN) antibody or elastin (ELN) antibody according to standard procedures.

[0091] For immunohistochemical staining, a polyclonal antibody against tropoelastin (EPC "PR398", rabbit-derived, dilution 1 / 200) and a monoclonal antibody against fibronectin (Sigma Aldrich "F7387-.2ML", mouse-derived, dilution 1 / 200) were used as antibodies.

[0092] A representative example of the results of immunohistochemistry is shown in Figure 1. Figure 2 shows a graph quantifying the fluorescence intensity derived from fibronectin or elastin as shown by immunohistochemistry. As shown in Figures 1 and 2, normal human dermal fibroblasts treated with Wnt2 protein showed increased expression of fibronectin and elastin fibers compared to the control. This indicates that Wnt2 protein has a promoting effect on the formation of fibronectin and elastin fibers in normal human dermal fibroblasts.

[0093] [Test 3] Effects of SELE protein addition to normal human dermal fibroblasts. SELE protein addition and immunostaining were performed on normal human dermal fibroblasts using the same procedure as in Test 2, except for the following changes. In Test 3, high passage (passage number: 25) normal human dermal fibroblasts (NHDF) (Kurabo Corporation: neonatal origin) were used as cultured cells. In Test 3, SELE protein (Abnova Corporation "H00006401-G01") was added instead of Wnt2 protein (addition concentration: 0 μg / mL (control), 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, or 0.4 μg / mL). Furthermore, in Experiment 3, for immunohistochemistry, a monoclonal antibody against elastin (Merck "Ms x Elastin", catalog number: MAb2503, mouse-derived, dilution ratio 1 / 250) was used as the primary antibody, and a polyclonal antibody against mouse antibody (Thermo Fisher Scientific "Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546, A11003", goat-derived, dilution ratio 1 / 250) was used as the secondary antibody.

[0094] A typical example of immunohistochemical staining results is shown in Figure 3. As shown in Figure 3, normal human dermal fibroblasts treated with SELE protein showed a concentration-dependent increase in elastin fiber expression compared to the control. This indicates that SELE protein has a concentration-dependent effect in promoting elastin fiber formation in normal human dermal fibroblasts.

[0095] [Test 4] Screening of substances affecting Wnt2 gene expression. Normal human dermal fibroblasts (Kurabo Industries Ltd.: neonatal-derived) were prepared. For the culture of normal human dermal fibroblasts, Dulbecco's modified Eagle medium (Thermo Fisher Scientific, "Gibco DMEM") supplemented with 10% fetal bovine serum and 1% antibiotic (anti-mycotin) was used as the culture medium. ２An incubator with a concentration of 5% and a temperature of 37°C was used. Normal human dermal fibroblasts were treated with candidate substances (21 types) at predetermined concentrations and cultured for 6 hours or 24 hours (6-hour induction or 24-hour induction).

[0096] After culturing, RNA was extracted from the cells using an RNA extraction kit (Promega's "Maxwell® RSC simplyRNA Cells / Tissue Kit"). Next, the concentration of the RNA was measured using a micro-spectrophotometer (Thermo Fisher Scientific's "NanoDrop® UV-Vis spectrophotometer"). Then, the RNA was reverse transcribed using a reverse transcription master mix (Toyobo's "ReverTrapAce qPCR RT Master Mix") to obtain the first-strand cDNA for qPCR. Next, the first-strand cDNA was mixed with Wnt2 gene primers and a qPCR master mix (SYBR® Green PCR Master Mix) to obtain a sample for qPCR. The obtained qPCR samples were subjected to qPCR using a real-time PCR instrument (Thermo Fisher Scientific) according to the manufacturer's protocol. This allowed for the measurement of Wnt2 gene expression levels in normal human dermal fibroblasts treated with the candidate substance. The expression level of the GAPDH gene was measured using the same procedure as for Wnt2 gene expression, except that the primers were changed to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. The expression level of the GAPDH gene was then used as an internal standard to normalize the expression level of the Wnt2 gene. Separately, the normalized Wnt2 gene expression level was measured in normal human dermal fibroblasts without the candidate substance using the same procedure and used as a control.

[0097] Using the method described above, components that increase the expression level of the Wnt2 gene in normal human dermal fibroblasts in a concentration-dependent manner were screened. As a result, the following components were selected: loquat leaf extract-containing solution (Loquat Leaf Extract CA, manufactured by Maruzen Pharmaceutical Co., Ltd.), grape fruit extract-containing solution (Chronochardi, manufactured by Ichimaru Falcos Co., Ltd.), and ginseng root extract-containing solution (Ginseng Extract LA-20, manufactured by Maruzen Pharmaceutical Co., Ltd.). Loquat leaf extract and grape fruit extract increased the expression level of the Wnt2 gene after 6 hours of induction. Ginseng root extract increased the expression level of the Wnt2 gene after both 6 hours and 24 hours of induction.

[0098] Next, the Wnt2 gene expression-promoting effect of each extract was quantitatively measured. The loquat leaf extract solution, grape fruit extract solution, and ginseng root extract solution mentioned above were prepared. In the loquat leaf extract solution, the concentration of loquat leaf extract (pure extract) was 1% of the total volume of the extract solution. In the grape fruit extract solution, the concentration of grape fruit extract (pure extract) was 4% of the total volume of the extract solution. In the ginseng root extract solution, the concentration of ginseng root extract (pure extract) was 2.4% of the total volume of the extract solution. Table 2 shows the increase in Wnt2 gene expression when the loquat leaf extract solution, grape fruit extract solution, and ginseng root extract solution were added to normal human dermal fibroblasts for 6 hours. Table 3 shows the increase in Wnt2 gene expression when the ginseng root extract solution was added to normal human dermal fibroblasts for 24 hours. In Tables 2 and 3, the "amount added (%)" for each extract indicates the ratio of the amount of extract-containing solution added to the total amount of culture medium. For example, "0.05%" means that 0.05% of the extract-containing solution was added to 99.95% of the culture medium. Tables 2 and 3 show the average expression level and SD (standard deviation) of the Wnt2 gene when each extract-containing solution was added, with the Wnt2 gene expression level in the control set to 1.00 (n=3). The results from Table 2 are graphed in Figures 4 to 6, and the results from Table 3 are graphed in Figure 7.

[0099]

[0100]

[0101] As is clear from Tables 2-3 and Figures 4-7, loquat leaf extract, grape fruit extract, and ginseng root extract increased the expression level of the Wnt2 gene in normal human dermal fibroblasts in a concentration-dependent manner. This indicates that loquat leaf extract, grape fruit extract, and ginseng root extract function as HIFU irradiation-like agents.

[0102] [Test 5] Among the extracts screened in Test 4 for promoting Wnt2 gene expression with mixed extracts, the Wnt2 gene expression-promoting effect of loquat leaf extract and grape fruit extract was measured individually or in combination. Normal human dermal fibroblasts (Kurabo Industries Ltd.: neonatal-derived) were prepared as the test subjects. For culturing normal human dermal fibroblasts, Dulbecco's modified Eagle medium (Thermo Fisher Scientific, "Gibco DMEM") supplemented with 0.1% fetal bovine serum and 1% antibiotic (anti-mycotin) was used as the culture medium, and CO2 was used. ２ An incubator with a concentration of 5% and a temperature of 37°C was used. Normal human dermal fibroblasts were added in predetermined amounts to loquat leaf extract-containing solution, grape fruit extract-containing solution, or a mixed extract-containing solution of loquat leaf extract and grape fruit extract (loquat leaf extract:grape fruit extract = 1:1), and cultured for 6 hours (6-hour induction). The amount of each extract-containing solution added was 0.10% (0.10% extract-containing solution added to 99.90% culture medium). Next, RNA extraction and qPCR were performed using the same procedure as in Experiment 4, and the expression level of the Wnt2 gene (internal standard: GAPDH gene) in normal human dermal fibroblasts to which each extract-containing solution had been added was measured. Separately, the expression level of the Wnt2 gene in normal human dermal fibroblasts without added extracts was measured using the same procedure and used as a control. The experiment was performed three times, and the mean and standard deviation (SD) were calculated. The results are shown in Table 4 and Figure 8, and Dunnett's test was used for statistical analysis. The results of the analysis show that cases where p < 0.01 are represented as ** and cases where p < 0.001 are represented as ***. In Figure 8, the Wnt2 gene expression level of the control group, which serves as the baseline, is set to 100%.

[0103]

[0104] As shown in Table 4 and Figure 8, the expression level of the Wnt2 gene increased when a mixed extract of loquat leaf extract and grape fruit extract was added, compared to the expression levels of the Wnt2 gene when loquat leaf extract or grape fruit extract was added alone. This confirms that while loquat leaf extract and grape fruit extract each have a certain effect in promoting Wnt2 gene expression, combining them exhibits a synergistic effect that further effectively promotes Wnt2 gene expression.

[0105] [Test 6] In vivo test of preventive and / or corrective agent containing HIFU irradiation-like activator The effects of the aforementioned HIFU irradiation-like activator on humans were tested on volunteers. First, a beauty serum (corresponding to a preventive and / or corrective agent) containing loquat leaf extract and grape fruit extract as HIFU irradiation-like activators was prepared.

[0106] The subjects, number of subjects, and measuring equipment used in the study are as follows: Subjects: Healthy women aged 35-59 who were concerned about their pores. Number of subjects: 32. Measuring equipment: Skin optical 3D measurement system (Canfield "Primos-CR").

[0107] In the study, participants first washed their faces with a general facial cleanser, and then rested for 20 minutes in a room maintained at a constant temperature and humidity (approximately 21°C and 50% humidity) (acclimatization). Next, the facial skin of the participants was photographed using a 3D optical skin measurement system. Based on the measured images, the number of pores on the facial skin of each participant (initial pore count) was analyzed. The mean and standard deviation of the initial pore count for all participants were then calculated.

[0108] Next, the subjects were asked to use the aforementioned serum for four weeks. The method of use was to apply the serum thoroughly to the entire face after washing, twice a day (immediately after waking up and immediately before going to bed), allowing it to penetrate.

[0109] Four weeks after starting to use the serum, the number of pores on the subjects' facial skin (number of pores after 4 weeks) was analyzed using the same method as for measuring the initial number of pores. The mean and standard deviation of the number of pores after 4 weeks were then calculated for all subjects. The initial number of pores and the number of pores after 4 weeks were then compared, and a significance test was performed using a t-test. The results are shown in Table 5 and Figure 9 below. An asterisk (*) in Table 5 indicates that p < 0.05 in the t-test.

[0110]

[0111] From the above, it has been confirmed that the preventive and / or corrective agent of the present invention is effective in preventing and / or improving the appearance of enlarged pores. Furthermore, although not measured in the examples, the preventive and / or corrective agent of the present invention contains a HIFU irradiation-like agent and increases the expression level of the Wnt2 gene in the skin in the same way as HIFU irradiation, and is therefore expected to be effective in preventing and / or improving skin sagging, similar to HIFU.

[0112] [Test 7] Effects of each extract on CCN3 gene expression: The effects of the loquat leaf extract and grape fruit extract mentioned above on the expression level of the CCN3 gene, which is downregulated in a skin model by HIFU irradiation, were confirmed.

[0113] Normal human dermal fibroblasts were prepared in the same manner as in Experiment 5. A predetermined amount of loquat leaf extract-containing solution, grape fruit extract-containing solution, or a mixed extract-containing solution of loquat leaf extract and grape fruit extract (loquat leaf extract:grape fruit extract = 1:1) was added to the normal human dermal fibroblasts, and the cells were cultured for 6 hours (6-hour induction). The amounts of each extract-containing solution added are shown in Table 6 and Figure 10. Next, RNA extraction and qPCR were performed in the same manner as in Experiment 5, except that the primer was changed to one for the CCN3 gene, and the expression level of the CCN3 gene (internal standard: GAPDH gene) in normal human dermal fibroblasts to which each extract-containing solution had been added was measured. Separately, the expression level of the CCN3 gene in normal human dermal fibroblasts without added extract was measured in the same manner and used as a control. The experiment was performed three times, and the mean and standard deviation (SD) were calculated. The results are shown in Table 6 and Figure 10, and Dunnett's test was used for statistical analysis. The results of the analysis are shown as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

[0114]

[0115] As shown in Table 6 and Figure 10, loquat leaf extract and grape fruit extract were confirmed to reduce the expression level of the CCN3 gene, similar to HIFU irradiation. Therefore, it was confirmed that loquat leaf extract and grape fruit extract function as HIFU irradiation-like agents, also in terms of suppressing CCN3 gene expression.

[0116] Furthermore, when a mixed extract of loquat leaf extract and grape fruit extract was added, the expression level of the CCN3 gene decreased compared to when loquat leaf extract or grape fruit extract was added alone. This confirms that while loquat leaf extract and grape fruit extract individually have a certain inhibitory effect on CCN3 gene expression, combining them exhibits a synergistic effect that further effectively suppresses CCN3 gene expression.

[0117] [Test 8] Effects of each extract on ZFP36 gene expression The effects of the loquat leaf extract and grape fruit extract described above on the expression level of the ZFP36 gene, which is upregulated in a skin model by HIFU irradiation, were confirmed.

[0118] The amount of each extract-containing solution added was as shown in Table 7 and Figure 11, the induction time was set to 24 hours, and the primer was changed to one for the ZFP36 gene. Except for these changes, the effect of each extract on the expression of the ZFP36 gene was confirmed in the same manner as in Experiment 7. The results are shown in Table 7 and Figure 11.

[0119]

[0120] As shown in Table 7 and Figure 11, loquat leaf extract and grape fruit extract were confirmed to increase the expression level of the ZFP36 gene, similar to HIFU irradiation. Therefore, it was confirmed that loquat leaf extract and grape fruit extract function as HIFU irradiation-like agents, also in terms of promoting ZFP36 gene expression.

[0121] Furthermore, when a mixed extract of loquat leaf extract and grape fruit extract was added, the expression level of the ZFP36 gene increased compared to when loquat leaf extract or grape fruit extract was added alone. This confirms that while loquat leaf extract and grape fruit extract individually have a certain effect in promoting the expression level of the ZFP36 gene, combining them exhibits a synergistic effect that further effectively promotes the expression level of the ZFP36 gene.

Claims

1. An agent for preventing and / or improving skin laxity and / or enlarged pores caused by said skin laxity, comprising an agent that mimics the action of high-intensity focused ultrasound (HIFU) irradiation.

2. The preventive and / or corrective agent according to claim 1, wherein the HIFU irradiation-like agent is a plant extract extracted from at least one plant selected from the group consisting of the Rosaceae, Vitaceae, and Araliaceae families.

3. The preventive and / or corrective agent according to claim 2, wherein the plant is at least one selected from the group consisting of loquat, grape and ginseng, and the plant extract is extracted from at least one part of the plant selected from the group consisting of root, stem, leaf and fruit.

4. The preventive and / or corrective agent according to claim 3, wherein the plant extract comprises a mixed extract of loquat leaf extract and grape fruit extract.

5. The HIFU irradiation-like agent has the effect of increasing the expression level of the Wnt2 gene in skin cells, as described in claim 1 or 2, for prevention and / or improvement.

6. The HIFU irradiation-like agent has a fibronectin expression-promoting effect due to its effect of increasing the expression level of the Wnt2 gene, as described in claim 5, for the prevention and / or improvement agent.

7. A screening method characterized by screening for cosmetic ingredients effective in preventing and / or improving skin sagging and / or enlarged pores caused by said skin sagging, using as an indicator genes whose expression changes when a cultured skin model is irradiated with high-intensity focused ultrasound (HIFU).

8. The screening method according to claim 7, comprising at least the steps of applying a test substance to skin culture cells and measuring the gene expression in the skin culture cells.

9. The screening method according to claim 7 or 8, wherein the gene is at least one selected from the group consisting of the Wnt2 gene, ZFP36 gene, mir-21 gene, CCN3 gene, ICAM1 gene, SELE gene, CYR61 gene, and CSF2 gene.

10. A cosmetic method for improving and / or enhancing skin condition, characterized by the use of an agent that mimics high-intensity focused ultrasound (HIFU) irradiation.

11. A high-intensity focused ultrasound (HIFU) irradiation-like agent containing a plant extract as an active ingredient, which is extracted from at least one plant selected from the group consisting of the Rosaceae, Vitaceae, and Araliaceae families.

12. A WNT2 gene expression promoter containing a plant extract obtained from at least one plant selected from the group consisting of loquat, grape, and ginseng.

13. An expression promoter for the WNT2 gene according to claim 12, used to reproduce gene changes that occur in skin cells due to high-intensity focused ultrasound (HIFU) irradiation.

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