Screening methods for anti-aging ingredients

Abstract

This invention provides a screening method for occludin expression-promoting components, using the expression level of GLUT1 in skin tissue as an indicator. [Solution] A screening method for occludin expression-promoting components using the expression level of GLUT1 in skin tissue as an indicator.

Description

Technical Field

[0001] The present invention relates to a method for screening anti-aging components.

Background Art

[0002] As a substance related to skin aging, Advanced Glycation End-products (hereinafter sometimes abbreviated as AGEs) are known. This is a substance produced by the glycation in which proteins and lipids existing in skin tissues bind to sugars. When AGEs increase in skin tissues, it is known that skin dullness and skin hardening occur (for example, Non-Patent Documents 1 and 2).

[0003] On the other hand, collagen existing in skin tissues is produced in dermal layer cells, and it is also known that its production amount decreases with aging (for example, Non-Patent Document 3).

[0004] Incidentally, involucrin is a protein existing in keratinocytes that form the stratum corneum in the epidermis and is one of the proteins that form the cornified envelope. Since the cornified envelope contributes to the physical or chemical toughness of the stratum corneum, it is important to maintain the expression level of involucrin in the body (particularly in skin tissues) (for example, Non-Patent Document 4).

[0005] Moreover, occludin is one of the proteins that form tight junctions. Since tight junctions contribute to the binding of epithelial cells and control the movement of molecules between cells, it is important to maintain the expression level of occludin in the body (for example, Non-Patent Document 5).

Prior Art Documents

Non-Patent Documents

[0006] [Non-Patent Document 1] H Ohshima et al., Skin Res. and Tech., (2009), 15: 496-502 [Non-Patent Document 2] Gomi et al., IFSCC2010, Poster [Non-Patent Document 3] James Varani et al., American Journal of Pathology, (2006), 168(6): 1861-1868 [Non-Patent Document 4] Yasuo Kitajima, Skin Barrier Function and its Control, Drug Delivery System 22-4, pp. 424-432, 2007. [Non-Patent Document 5] Relationship Between Expression of Tight Junction-Related Molecules and Perturbed Epidermal Barrier Function in UVB-irradiated Hairless Mice Takuya Yamamoto 1 , Masumi Kurasawa, Takao Hattori, Tetsuo Maeda, Hiroyuki Nakano, Hiroyuki Sasaki, Arch Dermatol Res . 2008; Feb300(2):61-8. doi: 10.1007 / s00403-007-0817-y. Epub 2007 Dec 7. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, the object of the present invention is to provide a screening method for anti-aging components. Furthermore, the object of the present invention is to provide an anti-aging agent and an anti-aging method. [Means for solving the problem]

[0008] As a result of diligent research, the inventors have found that the expression level of glucose transporter 1 (GLUT1), which is present in skin tissue, particularly in the epidermal and dermal layers, decreases with age.

[0009] Therefore, the screening method of the present invention, which solves the above problems, is a screening method that screens for anti-aging components using the expression level of GLUT1 in skin tissue as an indicator. This invention makes it possible to screen for anti-aging components.

[0010] Preferably, a component that increases the expression level of GLUT1 is selected as the anti-aging component. This invention makes it possible to select anti-aging components.

[0011] Preferably, the skin tissue is the epidermis and / or dermis. The present invention makes it possible to screen for anti-aging components that are effective in the epidermal layer and / or dermal layer.

[0012] Preferably, the anti-aging component is a component that suppresses the production of advanced glycation end products in the skin tissue. As mentioned above, AGEs are known to be one of the causes of skin aging. According to the present invention, it is possible to screen for components that can suppress the production of advanced glycation end products in skin tissue.

[0013] Preferably, the anti-aging component is a component that promotes collagen production in the skin tissue. As mentioned above, it is known that skin aging progresses as collagen production decreases with age. According to the present invention, it is possible to screen for components that can promote collagen production in skin tissue.

[0014] Moreover, the anti-aging agent of the present invention that solves the above problems contains, as an active ingredient, an anti-aging component screened by a screening method using the expression level of GLUT1 in skin tissue as an index. According to the present invention, an anti-aging agent can be provided.

[0015] Preferably, the above anti-aging agent is for suppressing the production of advanced glycation end products. As described above, AGEs are closely related to aging. Therefore, the anti-aging agent of the present invention is preferably used for the purpose of suppressing the production of AGEs.

[0016] Preferably, the above anti-aging agent is for promoting the production of collagen. As described above, it is known that when collagen production is suppressed, skin aging progresses. Therefore, the anti-aging agent of the present invention is preferably used for the purpose of promoting the production of collagen.

[0017] Moreover, the anti-aging method of the present invention that solves the above problems is an anti-aging method in which an anti-aging component screened by screening using the expression level of GLUT1 in skin tissue as an index is applied to the skin tissue. According to the present invention, it is possible to increase the expression level of GLUT1 in skin tissue decreased by aging and anti-aging.

[0018] Preferably, the anti-aging method of the present invention applies the above anti-aging agent to the skin. Since the above anti-aging agent has the effect of increasing the GLUT1 expression level in skin tissue, a high anti-aging effect can be obtained by applying it to the skin.

[0019] Furthermore, the inventors found that suppressing GLUT1 expression in skin tissue reduces the expression of involucrin and occludin, proteins produced by epidermal keratinocytes. In other words, they revealed that GLUT1 is involved in the expression of involucrin and occludin in epidermal keratinocytes. As mentioned above, involucrin contributes to the skin's barrier function, as well as to preventing rough skin and improving skin texture. Furthermore, as mentioned above, occludins form tight junctions with other proteins, contributing to the skin barrier function in the epidermis and to the control of ion permeability and other functions inside the body.

[0020] Therefore, a further objective of the present invention is to provide a screening method for components that promote the expression of involucrin or occludin. Furthermore, a further objective of the present invention is to provide an agent for promoting the expression of involucrin or occludin, and a method for promoting the expression of involucrin or occludin.

[0021] The present invention provides a screening method for solving the above problems, which screens for involucrin expression-promoting components using the expression level of GLUT1 in skin tissue as an indicator. According to the present invention, it is possible to screen for components that promote the expression of involucrin.

[0022] Preferably, the involucrin expression-promoting component is a skin barrier function-improving component. The skin barrier function is known to be impaired by dryness and irritation. Furthermore, as mentioned above, involucrin is known to contribute to the skin barrier function. Therefore, according to the present invention, it is possible to screen for components that promote the expression of involucrin and improve skin barrier function.

[0023] Furthermore, the involucrin expression promoter of the present invention, which solves the above problems, incorporates a component screened by the above-mentioned involucrin expression promoting component as an active ingredient. According to the present invention, it is possible to provide an agent containing an involucrin expression-promoting component as an active ingredient.

[0024] Furthermore, the present invention, which solves the above problems, involves applying the components screened by the above-mentioned involucrin expression promoting component screening method to the skin. Preferably, the method for promoting involucrin expression of the present invention involves applying the above-mentioned involucrin expression promoting agent to the skin. According to the present invention, the expression of involucrin in the epidermal layer (particularly in stratum corneum cells) can be promoted.

[0025] Furthermore, the screening method of the present invention that solves the above problems is a screening method that screens for occludin expression-promoting components using the expression level of GLUT1 in skin tissue as an indicator. According to the present invention, it is possible to screen for components that promote occludin expression.

[0026] Preferably, the occludin expression-promoting component is a skin barrier function-improving component. As mentioned above, occludins are known to contribute to the skin barrier function. Therefore, according to the present invention, it is possible to screen for components that promote occludin expression and improve skin barrier function.

[0027] Furthermore, the occludin expression promoter of the present invention, which solves the above problems, incorporates a component screened by the above-mentioned occludin expression promoting component as an active ingredient. According to the present invention, it is possible to provide an agent containing an occludin expression-promoting component as an active ingredient.

[0028] Furthermore, the present invention, which solves the above problems, involves applying the components screened by the above-mentioned occludin expression promoting component to the skin. Preferably, the method for promoting occludin expression of the present invention involves applying the above-mentioned occludin expression promoting agent to the skin. According to the present invention, the expression of occludin can be promoted. [Effects of the Invention]

[0029] According to the present invention, anti-aging components can be screened. According to the present invention, an anti-aging agent can be obtained. According to the present invention, an anti-aging method can be provided.

[0030] According to the present invention, it is possible to screen for components that promote the expression of involucrin or occludin. According to the present invention, an agent that promotes the expression of involucrin or occludin can be obtained. According to the present invention, a method for promoting the expression of involucrin or occludin can be provided.

[0031] According to the present invention, it is possible to screen for components that improve skin barrier function. According to the present invention, an agent that improves the skin barrier function can be obtained. According to the present invention, a method for improving skin barrier function can be provided. [Brief explanation of the drawing]

[0032] [Figure 1] This graph shows that GLUT1 expression levels in skin tissue decrease with age. (A) shows the results in normal human epidermal keratinocytes, and (B) shows the results in normal human dermal fibroblasts. [Figure 2]This graph shows the results of measuring GLUT1 expression levels in normal human epidermal keratinocytes in Test Example 2. (A) shows the experimental results using Saxifraga stolonifera extract, (B) shows the results using Thyme extract, and (C) shows the results using Hypericum perforatum extract. [Figure 3] This graph shows the results of measuring the expression level of GLUT1 in normal human dermal fibroblasts in Test Example 3. [Figure 4] This graph compares the mRNA expression levels of GLUT1 in normal human epidermal keratinocytes, measured by siRNA, in cells without GLUT1(SLC2A1) gene knockdown and in cells with GLUT1(SLC2A1) gene knockdown. [Figure 5] This graph compares the mRNA expression levels of involucrin in cells with and without GLUT1(SLC2A1) gene knockdown, as measured by the siRNA method, with those in cells with GLUT1(SLC2A1) gene knockdown. [Figure 6] This graph compares the mRNA expression levels of occludin in cells with and without GLUT1(SLC2A1) gene knockdown, as measured by the siRNA method, with those in cells with GLUT1(SLC2A1) gene knockdown. [Modes for carrying out the invention]

[0033] Preferred embodiments of the present invention will be described below, but the technical scope of the present invention is not limited to the following embodiments.

[0034] In this invention, "skin tissue" refers to the epidermis, dermis, and subcutaneous tissue that constitute the skin. In this invention, "anti-aging" refers to bringing the skin condition closer to its pre-aging state or suppressing undesirable changes that occur with aging (for example, dullness, sagging, deterioration of skin texture, hardening of the skin, etc.). "Dullness" refers to a condition where the skin loses its natural transparency, brightness, and radiance, making the entire face appear darker than its original skin tone. "Sagging" refers to a deformation of the skin caused by a decrease in the elasticity of the skin tissue. "Skin texture" refers to the fine irregularities on the surface of the skin, and "deterioration of skin texture" refers to a decrease in these irregularities on the skin's surface. In this invention, "anti-aging component" refers to a component that is effective in anti-aging as described above.

[0035] 1. Screening Method The present invention provides a screening method for anti-aging components using GLUT1 expression levels as an indicator. Here, screening includes searching for anti-aging components or candidates thereof.

[0036] In a preferred embodiment of the present invention, a component that increases the expression level of GLUT1 in skin tissue is selected as the anti-aging component. The inventors have found that the expression level of GLUT1 in skin tissue decreases with age. Furthermore, as will be discussed later, a decrease in GLUT1 expression is thought to cause undesirable changes in the skin, such as increased production of AGEs and decreased collagen production, thereby accelerating skin aging. Therefore, increasing the expression level of GLUT1 in skin tissue, which decreases with age, is effective for anti-aging according to the present invention.

[0037] In a preferred embodiment of the present invention, the skin tissue is the epidermis and / or dermis. The epidermis is the outermost layer of skin tissue, and when its condition deteriorates, causing dullness and hardening, it is the most noticeable deterioration in the appearance of the skin. Furthermore, fibroblasts in the dermis produce collagen, and when the condition of the dermis deteriorates, collagen production is suppressed, leading to sagging skin. Therefore, by screening for components that increase GLUT1 expression in the epidermis and / or dermis, it is possible to suppress aging phenomena in each layer and achieve anti-aging effects.

[0038] In the present invention, the anti-aging component may be a component that suppresses the production of AGEs. When GLUT1 expression decreases in skin tissue, it becomes more difficult for cells to take up glucose. As a result, the amount of glucose in the extracellular environment of skin tissue increases, which is expected to accelerate glycation reactions in the epidermis and promote the production of AGEs. As mentioned above, an increase in AGEs in the epidermal layer can lead to deterioration of skin texture, dullness, and hardening of the skin. Therefore, components that increase GLUT1 expression in skin tissue can suppress the production of AGEs and are effective in anti-aging.

[0039] In the present invention, the anti-aging component may be a component that promotes collagen production. When GLUT1 expression decreases in the dermis, it becomes more difficult for collagen-producing fibroblasts to take up glucose (e.g., N Nakashima et al., Metabolism, (1995) 44(4): 543-548). This is expected to reduce the collagen-producing capacity in the dermis, leading to skin sagging (e.g., M Cechowska-Pasko et al., Mol Cell Biochem, (2007) 305: 79-85, Y Ogura et al., Biomedical Engineering, (2017), 55(2): 97-102). Therefore, components that increase GLUT1 expression in the dermis can promote collagen production and are effective in anti-aging.

[0040] The anti-aging component in this invention may have both the effect of suppressing the production of AGEs and the effect of promoting the production of collagen.

[0041] The substance to be tested in the screening method of the present invention may be a pure substance, an extract of biological origin, or a mixture thereof. The term "biologically derived extract" refers not only to the extract itself derived from animals or plants, but also to fractions of the extract, purified fractions, and solvent-removed products of the extract, fraction, or purified product. Plant-derived extracts include extracts made from plants that grow naturally or are cultivated, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts. The extraction process can use the entire plant, as well as parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flower spikes, and flower buds. However, it is preferable to crush or finely chop these parts beforehand to improve extraction efficiency. Suitable extraction solvents include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol; polyhydric alcohols such as 1,3-butanediol and polypropylene glycol; ketones such as acetone and methyl ethyl ketone; and ethers such as diethyl ether and tetrahydrofuran. Specific extraction methods include, for example, adding 1 to 30 parts by mass of solvent to 1 mass of the plant tissue or its dried material to be extracted, immersing it for several days at room temperature or for several hours at a temperature near its boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography or the like.

[0042] The screening method of the present invention includes adding a test substance to a cell culture system and measuring the expression level of GLUT1 in the cells. Here, normal human keratinocytes can be used in the epidermal layer test, and normal human fibroblasts can be used in the dermal layer test. Specifically, if the expression level of GLUT1 in cells cultured with the test substance is statistically significantly higher than the expression level of GLUT1 in cells cultured without the test substance, the test substance can be determined to be a candidate for an anti-aging component. More specifically, if the expression level of GLUT1 is greater than one-fold in cells cultured without the test substance, it can be selected as a candidate for an anti-aging component.

[0043] GLUT1 expression levels can be measured using conventional methods such as mRNA measurement and immunohistochemical analysis. For example, the expression level of the gene encoding GLUT1 can be quantitatively detected by PCR using a DNA fragment containing a sequence that specifically binds to the gene's sequence as a primer. Furthermore, since the gene sequence encoding GLUT1 is publicly available, those skilled in the art can design appropriate primers. Furthermore, since commercially available antibodies exist, their expression levels in cells can be measured using these.

[0044] The present invention also relates to a method for screening involucrin expression-promoting components in skin tissue using GLUT1 expression levels as an indicator. The above-mentioned skin tissue is preferably the epidermis, and more preferably the stratum corneum. The inventors have revealed that a decrease in GLUT1 expression in skin tissue leads to a decrease in involucrin expression in skin tissue. Therefore, by using GLUT1 expression levels as an indicator, it is possible to screen for involucrin expression-promoting components.

[0045] As described above, involucrin is one of the proteins that form the cornified envelope, which contributes to the physical and chemical toughness of the stratum corneum. Therefore, when involucrin expression is promoted, the formation of the cornified envelope is promoted, the toughness of the stratum corneum is improved, and the skin's protective function is enhanced. When the skin's protective function is enhanced, the skin condition improves. The skin's protective function is, for example, the skin barrier function. In other words, when involucrin expression is promoted, skin condition improves. Therefore, the screening method for involucrin expression-promoting components according to the present invention is preferably a screening method for skin condition-improving components. Specifically, the skin condition-improving components can be skin barrier function-enhancing components.

[0046] In a preferred embodiment of the present invention, the above-mentioned skin condition improving component is a skin barrier function improving component. According to the present invention, components that improve skin barrier function can be screened using the expression level of GLUT1 as an indicator.

[0047] The present invention also relates to a method for screening occludin expression-promoting components in skin tissue using GLUT1 expression levels as an indicator. The skin tissue mentioned above is preferably the epidermis. The inventors have revealed that a decrease in GLUT1 expression in skin tissue leads to a decrease in occludin expression in skin tissue. Therefore, by using GLUT1 expression levels as an indicator, it is possible to screen for occludin expression-promoting components.

[0048] As mentioned above, occludins are one of the proteins that form tight junctions, which contribute to the binding of epithelial cells. Therefore, when occludin expression is promoted, the formation of tight junctions is promoted, the connections between epithelial cells become stronger, and the skin's protective function improves. When the skin's protective function improves, the skin condition improves. The skin's protective function is, for example, the skin barrier function. In other words, when occludin expression is promoted, skin condition improves. Therefore, the screening method for occludin expression-promoting components according to the present invention is preferably a screening method for skin condition-improving components. Specifically, the skin condition-improving components can be skin barrier function-enhancing components.

[0049] In a preferred embodiment of the present invention, the above-mentioned skin condition improving component is a skin barrier function improving component. According to the present invention, components that improve skin barrier function can be screened using the expression level of GLUT1 as an indicator.

[0050] In the methods for screening involucrin expression-promoting components and occludin expression-promoting components, the above-mentioned matters can be applied to the substance to be tested and how to obtain it, the specific screening method, and the method for measuring the expression levels of involucrin and occludin.

[0051] 2. Anti-aging agents The anti-aging agent of the present invention contains an anti-aging component as an active ingredient, which has been screened by any of the screening methods described in "1. Screening Method" above. As described above, the anti-aging components screened in this invention increase the expression level of GLUT1 and therefore have an anti-aging effect.

[0052] Preferably, the anti-aging agent is intended to suppress the production of advanced glycation end products. As mentioned above, suppressing the production of AGEs in skin tissue is effective for anti-aging.

[0053] Preferably, the above-mentioned anti-aging agent is intended to promote collagen production. As mentioned above, promoting collagen production in skin tissue is effective for anti-aging.

[0054] Preferably, the anti-aging agent is intended to suppress the production of advanced glycation end products and promote collagen production. Anti-aging agents that are effective in both inhibiting the production of advanced glycation end products and promoting collagen production are more effective in anti-aging.

[0055] The anti-aging agent of the present invention can be appropriately combined with any component used in formulation to take the form of an oral preparation or a topical skin preparation. When prepared as an oral preparation, it is preferable that the anti-aging component be in the form of a food composition containing the above-mentioned anti-aging component as an active ingredient. Specifically, it is preferable that it be in the form of a supplement having dosage forms such as general food, tablets, granules, or drinks.

[0056] In oral preparations, the content of the above-mentioned anti-aging component is typically 0.1 mg or more, preferably 1 mg or more, and more preferably 10 mg or more, per single dose, based on the dry mass of the extract, depending on the dosage form. Furthermore, the amount is usually 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less. By setting the expression level within the above range, it is possible to effectively increase the expression level of GLUT1.

[0057] When used as a topical skin preparation, examples include cosmetics, quasi-drugs, and topical medicinal products. Furthermore, these dosage forms are not particularly limited. For example, when used as a cosmetic product, it is preferable to use it in the form of a lotion, serum, emulsion, cream, gel, sun care product, etc.

[0058] The content of the above-mentioned anti-aging component in the topical skin preparation (dry weight in the case of an extract) is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, and more preferably 0.001% by mass or more. Furthermore, the amount is usually 80% by mass or less, preferably 30% by mass or less, and more preferably 10% by mass or less. By setting the expression level within the above range, it is possible to effectively increase the expression level of GLUT1.

[0059] When incorporating the above anti-aging ingredients into cosmetics, other ingredients commonly used in cosmetics, such as whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, plant and animal extracts, and active ingredients, may be included as optional ingredients, provided that they do not impair the anti-aging effect. These optional components may be obtained from commercially available sources and incorporated into the formula, or they may be synthesized using known methods and incorporated into the formula.

[0060] As for whitening ingredients, any ingredients commonly used in cosmetics can be used without particular restrictions. However, it is preferable to use ingredients that have a whitening effect through mechanisms other than promoting GLUT1 expression, in order to fully utilize the effects of each ingredient. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethyl ascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2-(triphenylmethyloxy)ethanol, 2-(triphenylmethylamino)ethanol, 2-(triphenylmethyloxy)ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N-(o-toluoyl)cysteic acid, N-(m-toluoyl) Examples include N-(p-toluyl)cysteic acid, N-(p-methoxybenzoyl)cysteic acid, N-benzoyl-serine, N-(p-methylbenzoyl)serine, N-(p-ethylbenzoyl)serine, N-(p-methoxybenzoyl)serine, N-(p-fluorobenzoyl)serine, N-(p-trifluoromethylbenzoyl)serine, N-(2-naphthoyl)serine, N-(4-phenylbenzoyl)serine, N-(p-methylbenzoyl)serine, methyl ester, N-(p-methylbenzoyl)serine ethyl ester, N-(2-naphthoyl)serine methyl ester, N-benzoyl-O-methylserine, N-(p-methylbenzoyl)-O-methylserine, N-(p-methylbenzoyl)-O-acetylserine, N-(2-naphthoyl)-O-methylserine, etc.

[0061] The amount of whitening ingredients in cosmetics is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (dry weight in the case of extracts).

[0062] As wrinkle-improving ingredients, any ingredients commonly used in cosmetics can be used without particular restrictions. However, it is preferable to use ingredients that have wrinkle-improving effects through mechanisms other than promoting GLUT1 expression, from the standpoint of fully utilizing the effects of each ingredient. Examples of vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, retinoic acid tocopherol, retinyl palmitate, and retinyl acetate. Other examples include benzyl ursolate, ursolic acid phosphate, benzyl betulinate, and benzyl acid phosphate.

[0063] The amount of wrinkle-improving ingredients in cosmetics is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (dry weight in the case of extracts).

[0064] Examples of anti-inflammatory components include clarinon, glabridin, glycyrrhizic acid, glycyrrhetinic acid, and pantothenyl alcohol, with glycyrrhizic acid and its salts, alkyl glycyrrhetinate and its salts, and glycyrrhetinic acid and its salts being preferred. The content of anti-inflammatory ingredients in cosmetics is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry weight in the case of extracts).

[0065] Examples of plant and animal-derived extracts include akebia extract, thunbergii extract, asparagus extract, avocado extract, hydrangea extract, almond extract, arnica extract, aronia extract, apricot extract, ginkgo extract, fennel extract, Japanese angelica extract, Eleutherococcus senticosus extract, Enmeisou extract, Phellodendron bark extract, Panax ginseng extract, Lamium album extract, Arctium moniliforme extract, Pueraria lobata extract, chamomile extract, carrot extract, Artemisia capillaris extract, licorice extract, kiwi extract, cucumber extract, and guava extract. Gardenia extract, bamboo grass extract, walnut extract, black rice extract, chlorella extract, mulberry extract, keiketto extract, ginger extract, gentian extract, rice extract, rice ferment extract, rice bran ferment extract, rice germ oil, salvia extract, soapwort extract, bamboo extract, sunflower extract, sansho extract, shiitake mushroom extract, rehmannia extract, lithospermum extract, perilla extract, linden extract, meadowsweet extract, ginger extract, calamus root extract, horsetail extract, stevia extract, stevia ferment, yarrow extract Gypsum extract, peppermint extract, sage extract, mallow extract, Cnidium officinale extract, Swertia japonica extract, mulberry bark extract, rhubarb extract, soybean extract, jujube extract, dandelion extract, clove extract, chili pepper extract, angelica extract, calendula extract, peach kernel extract, tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, Ophiopogon extract, lotus extract, parsley extract, birch extract, witch hazel extract, Isodon japonicus extract, cypress extract, loquat extract, f Preferred extracts include dandelion extract, butterbur extract, poria extract, loofah extract, peppermint extract, linden extract, pine extract, skunk cabbage extract, lemon balm extract, seaweed extract, peach extract, cornflower extract, eucalyptus extract, lily extract, coix seed extract, mugwort extract, lavender extract, apple extract, rooibos tea extract, reishi mushroom extract, lettuce extract, forsythia extract, astragalus extract, rosemary extract, Roman chamomile extract, and burnet extract.

[0066] The content (dry mass) of the aforementioned arbitrary animal or plant-derived extracts in the cosmetic composition is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 0.3 to 3% by mass.

[0067] In addition to the active ingredient, ingredients commonly used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, polyols such as 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, and 1,2-octanediol, fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl sulfate triethanolamine A. Anionic surfactants such as tel, cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide, amphoteric surfactants such as imidazoline (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyltaurine, sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (monostearate) Glycerin phosphate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (e.g., POE2-octyldodecyl ether), POE alkylphenyl ethers (e.g., POE nonylphenyl ether), Pluronic® type, POE·POP alkyl ethers (e.g., POE·POP2-decyltetradecyl ether), Tetronic type, POE castor oil / hydrogenated castor oil derivatives (e.g., POE castor oil, POE hydrogenated castor oil), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, and sodium lactate, which may be surface-treated.Powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, barium sulfate, etc., which may be surface-treated; inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, zinc oxide, which may be surface-treated; composite pigments such as iron oxide titanium dioxide sintered bodies, which may be surface-treated; pearlescent agents such as titanium mica, fish scale foil, bismuth oxychloride, which may be lake-formed; Red 202, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue 1, Green 201, Violet 20 Examples include organic dyes such as No. 1 and Red No. 204, organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomers, lower alcohols such as ethanol and isopropanol, vitamin A or its derivatives, vitamin B compounds such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or its derivatives, vitamin B12, vitamin B15 or its derivatives, vitamin E compounds such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate, vitamin D compounds, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0068] The present invention also relates to involucrin expression enhancers and occludin expression enhancers.

[0069] The involucrin expression promoter of the present invention contains an involucrin expression promoter as an active ingredient, which has been screened by the screening method for involucrin expression promoters described in "1. Screening Method" above. The components screened using the above screening method increase the expression level of involucrin.

[0070] Preferably, the involucrin expression promoter is a skin barrier function enhancer. According to the present invention, a skin barrier function improving agent can be provided.

[0071] The occludin expression promoter of the present invention contains, as an active ingredient, an occludin expression promoter that has been screened by the screening method for occludin expression promoters described in "1. Screening Method" above. The components screened using the above screening method increase the expression level of occludin.

[0072] Preferably, the occludin expression promoter is a skin barrier function enhancer. According to the present invention, a skin barrier function improving agent can be provided.

[0073] In involucrin expression promoters and occludin expression promoters, the above-mentioned matters may be applied to the dosage form, the content of the involucrin expression promoter and the occludin expression promoter, and optional components and their content.

[0074] 3.Anti-aging method The anti-aging method of the present invention involves applying an anti-aging component, screened by any of the screening methods described in "1. Screening Method" above, to skin tissue. Here, "applying anti-aging components to skin tissue" means enabling the anti-aging components screened by the method of the present invention to exert a GLUT1 expression-promoting effect in the user's skin tissue. Examples include topical application to the skin or application of a patch. Furthermore, the anti-aging method in this invention is a non-therapeutic method, preferably a cosmetic method, and more preferably a skin cosmetic method.

[0075] Preferably, one of the anti-aging agents described in "2. Anti-aging agents" above is applied to the skin. The anti-aging agents described in "2. Anti-aging agents" above contain components that increase the expression level of GLUT1 in skin tissue and have a high anti-aging effect. Furthermore, by applying it to the skin, it can fully exert its anti-aging effects.

[0076] The present invention also relates to methods for promoting involucrin expression and methods for promoting occludin expression.

[0077] The present invention's method for promoting involucrin expression involves applying an involucrin expression-promoting component, screened by any of the screening methods described in "1. Screening Method" above, to skin tissue.

[0078] Preferably, the involucrin expression promoter described in "2. Anti-aging agents" above is applied to the skin. The involucrin expression promoter described in "2. Anti-aging agents" above contains components that increase the expression level of GLUT1 in skin tissue and has a high involucrin expression promoting effect. Furthermore, by applying it to the skin, it can fully exert its effect of promoting involucrin expression.

[0079] Furthermore, the present invention's method for promoting occludin expression involves applying an occludin expression-promoting component, screened by any of the screening methods described in "1. Screening Method" above, to skin tissue.

[0080] Preferably, the occludin expression promoter described in "2. Anti-aging agents" above is applied to the skin. The occludin expression promoters described in "2. Anti-aging agents" above contain components that increase the expression level of GLUT1 in skin tissue and have a high occludin expression promoting effect. Furthermore, by applying it to the skin, it can fully exert its effect of promoting occludin expression.

[0081] Furthermore, the above-mentioned methods for promoting involucrin expression and occludin expression are non-therapeutic methods, preferably cosmetic methods, and more preferably cosmetic methods for the skin. [Examples]

[0082] The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to the following embodiments.

[0083] <Test Example 1> Measurement of GLUT1 expression level in skin tissue (I) Measurement of GLUT1 expression levels in the epidermal layer GLUT1 expression levels were measured in normal human epidermal keratinocytes from donors of different ages. (1) Using HuMedia-KG2 medium (manufactured by Kurabo Industries Ltd.), normal human epidermal keratinocytes collected from donors in their 30s and 50s were placed in a 48-well plate in a 3.0 × 10⁶ format. 4 Seeds were sown to a density of 1 per well and cultured for 24 hours at 37°C in a 5% CO2 environment. (2) After incubation, the culture medium was removed, HuMedia-KB2 medium (manufactured by Kurabo Industries Ltd.) was added, and the culture was further incubated at 37°C in a 5% CO2 environment for 24 hours. (3) After culturing, cultured normal human epidermal keratinocytes were harvested and mRNA was extracted according to the protocol of the RNeasy Mini Kit (QIAGEN). (4) The mRNA expression level of GLUT1 was measured using real-time qPCR. The mRNA expression level of β-actin, an endogenous control, was also measured simultaneously. Fast SYBR Green Master Mix (Thermo Fisher) was used for the measurements. The primer used was Hs_SLC2A1_1_SG QuantiTect Primer Assay (QIAGEN). (5) The mRNA expression levels of GLUT1 in donor cells of each age group were corrected for the mRNA expression levels of β-actin. The corrected mRNA expression levels of GLUT1 in donor cells of age 50 were calculated, with the corrected mRNA expression level of GLUT1 in donor cells of age 30 set to 1. The results are shown in Figure 1.

[0084] (II) Measurement of GLUT1 expression levels in the dermis We measured the expression levels of GLUT1 in normal human dermal fibroblasts from donors of different ages. In this study, the donors were in their teens and 50s. Normal human dermal fibroblasts collected from each donor were cultured in DMEM medium (SIGMA) containing 10% FBS, in 1.5 × 10⁶ well plates. 5 Seeds were seeded at a density of 1 / well and cultured for 24 hours at 37°C in a 5% CO2 environment. After culturing, the culture medium was removed, and DMEM medium containing 1% FBS was added, and cultured for 24 hours at 37°C in a 5% CO2 environment. After culturing, the expression level of GLUT1 was calculated according to the procedure from (I)(3) onward. The results are shown in Figure 1.

[0085] As shown in Figure 1, it was revealed that GLUT1 expression levels decrease with age in normal human epidermal keratinocytes and normal human dermal fibroblasts.

[0086] <Example Test 2> Screening for components that increase GLUT1 expression in the epidermal layer We screened for components that increase GLUT1 expression levels in normal human epidermal keratinocytes. (1) Using HuMedia-KG2 medium (manufactured by Kurabo Industries Ltd.), normal human epidermal keratinocytes were placed in a 24-well plate in a quantity of 5.0 × 10⁶ 4 Seeds were sown to a density of 1 per well and incubated for 24 hours at 37°C in a 5% CO2 environment. (2) After culturing, the culture medium was removed, and HuMedia-KB2 medium (manufactured by Kurabo Industries Ltd.) containing 0.5% of the test plant extract or solvent control was added, and the cells were cultured at 37°C in a 5% CO2 environment for 24 hours. Hereafter, cells treated with the culture medium containing the test plant extract will be referred to as the plant extract group, and cells treated with the culture medium containing the solvent control will be referred to as the solvent control group. (3) After culturing, cultured normal human epidermal keratinocytes were harvested and mRNA was extracted according to the protocol of the RNeasy Mini Kit (QIAGEN). (4) The mRNA expression level of GLUT1 was measured using real-time qPCR. The mRNA expression level of β-actin, an endogenous control, was also measured simultaneously. Fast SYBR Green Master Mix (Thermo Fisher) was used for the measurements. The primer used was Hs_SLC2A1_1_SG QuantiTect Primer Assay (QIAGEN). (5) The mRNA expression levels of GLUT1 in the plant extract-added group and the solvent control group were corrected using the mRNA expression levels of β-actin. The corrected mRNA expression level of GLUT1 in the plant extract-added group was calculated, with the corrected mRNA expression level of GLUT1 in the solvent control group set to 1. The results are shown in Figure 2.

[0087] As shown in Figure 2, it was found that Saxifraga stolonifera extract, Thyme extract, and Hypericum perforatum extract increased the expression level of GLUT1 in normal human epidermal keratinocytes. From the above, it has been shown that the screening method of the present invention can screen for components that increase the expression level of GLUT1 in the epidermal layer.

[0088] <Test Example 3> Screening of components that increase GLUT1 expression in the dermis We screened for components that increase GLUT1 expression in normal human dermal fibroblasts. (1) Using DMEM medium containing 10% FBS (manufactured by SIGMA), normal human dermal fibroblasts were placed in 7.0 × 10⁶ well plates in a 24-well plate. 4 Seeds were sown to a density of 1 per well and incubated for 24 hours at 37°C in a 5% CO2 environment. (2) After culturing, the culture medium was removed, and DMEM medium containing 0.5% and 1% FBS of the test plant extract or solvent control was added, and the cells were cultured at 37°C in a 5% CO2 environment for 24 hours. Hereafter, cells treated with the culture medium containing the test plant extract will be referred to as the plant extract group, and cells treated with the culture medium containing the solvent control will be referred to as the solvent control group. Subsequently, the expression level of GLUT1 in normal human dermal fibroblasts was calculated in the same manner as in (3) and onward of <Test Example 2> above. The results are shown in Figure 3.

[0089] As shown in Figure 3, royal jelly extract was found to increase the expression level of GLUT1 in normal human dermal fibroblasts. From the above, it has been shown that the screening method of the present invention can screen for components that increase the expression level of GLUT1 in the dermis.

[0090] The anti-aging agent of the present invention can be obtained by incorporating the components screened in Test Examples 2 and 3 as active ingredients. Furthermore, the anti-aging method of the present invention involves applying an anti-aging agent containing the components screened in Test Examples 2 and 3 as active ingredients to the skin.

[0091] <Test Example 4> Relationship between GLUT1 expression level and involucrin and occludin expression levels The relationship between GLUT1 expression levels and involucrin and occludin expression levels was investigated.

[0092] (I) Cell culture Human fibroblasts (NHDF) and human keratinocytes (NHEK) were used as cells. For each cell type, cells from young donors (18 and 28 years old) and older donors (54 years old) were used in the study. Each cell was cultured according to the manufacturer's recommended method. Cells were seeded in 250ml cell culture flasks (Sumitomo Bakelite # MS-21250), and when they reached 60-90% confluence, they were detached using trypsin neutralization solution (KURABO # HK-3220) and subcultured. The passage number was set to P+2-8 for human fibroblasts and P+2-5 for human keratinocytes.

[0093] (II) Knockdown of GLUT1 expression gene by introduction of siRNA (1) Cells were seeded on plates and cultured overnight at 37°C and 5% CO2 in the complete medium shown in Table 1 below until 70-90% confluence (N=3-4). For 24-well plates, 6.0-7.0 × 10⁶ cells were used per well.4 Seed cells so that each cell is 500 μL, and in a 48-well plate, 3.0-3.5 × 10¹ cells were seeded per well. 4 Cells were seeded at a density of 250 μL per cell. Table 1 shows the cell types used and the culture media used during cultivation.

[0094] [Table 1]

[0095] (2) Lipofectamin Reagent (Invitrogen) was mixed with the dedicated culture medium corresponding to each cell type (listed in Table 1 above) and diluted in a ratio of 1 to 2:50. (3) The siRNA to be introduced was diluted with dedicated medium and PBS using the ON-TARGET plus siRNA (Dharmacon) reagent (final cell addition concentration 25 nM), and mixed with diluted Lipofectamin Reagent in a 1:1 ratio. Then, it was incubated at room temperature for 15 minutes to prepare the siRNA complex introduction reagent. The siRNAs used were those listed in Table 2 below.

[0096] [Table 2]

[0097] (4) Remove the culture medium from the cells and wash them once with an equal volume of PBS. (5) After adding 200-400 μL of the special culture medium to each well, 50-100 μL of the siRNA complex introduction reagent prepared in (3) above was added, the plate was gently shaken, and after confirming under a microscope that there was no cytotoxicity, the cells were incubated at 37°C in a 5% CO2 environment for 8-48 hours.

[0098] (III) RNA extraction from cells, cDNA synthesis, and qPCR (1) After removing the culture medium from the cultured cells prepared in (II)(5) above, the cells were washed once with an equal volume of PBS to the removed medium. The cells were then suspended in 350 μL of Buffer RLT per well on ice, and the cell suspension was collected. (2) RNA was extracted from the cell suspension obtained in (1) above using QIAcube (QIAGEN #9001293). The elution volume was 30 μL per sample. (3) cDNA was synthesized using the SuperScript cDNA Synthesis kit (Life Technologies #11754250) on a thermal cycler (BIO RAD #T100) according to the manufacturer's recommended protocol. Sample RNA (involucrin and occludin RNA) was used at concentrations of 20-50 ng / 20 μL, and standard RNA was used at concentrations of 50-100 ng / 20 μL. (4) qPCR was performed using the standard method with a real-time PCR system (Applied Biosystems, QuantStudio5) to measure the mRNA expression levels of β-actin, involucrin, and occludin. Each sample was prepared with 2.5 μL of deionized water, 0.5 μL of primer, 5 μL of SYBR Green, and 2 μL of either a 10-30 fold dilution of cDNA sample or a 5-30 fold dilution of standard cDNA sample. The cycle count was set to 45-50. The primer used was the QuantiTect Primer Assay (QIAGEN #249900) shown in Table 3 below.

[0099] [Table 3]

[0100] (5) The expression level of involucrin or occludin mRNA was divided by the expression level of β-actin mRNA. The above measurement was performed three times, and the average value of the three measurements was calculated. The results are shown in Table 4 and Figures 4-6.

[0101] [Table 4]

[0102] As shown in Table 4 and Figure 4, the expression level of GLUT1 mRNA was reduced in the group in which the GLUT1 (SLC2A1) gene was knocked down (the group in which si GLUT1 was used as siRNA) compared with the group in which no genes were knocked down (the group in which si non-targeting was used as siRNA). Furthermore, in the group in which the GLUT1(SLC2A1) gene was knocked down, the expression levels of involucrin mRNA and occludin mRNA were reduced compared to the group in which the gene was not knocked down (Table 4, Figures 5 and 6). From the above findings, it became clear that a decrease in GLUT1 expression in skin tissue leads to a decrease in involucrin and occludin expression.

[0103] The results above clearly show that components that promote the expression of involucrin or occludin can be screened using GLUT1 expression levels as an indicator. Specifically, when tested using the methods described in Test Example 2 and Test Example 3 above, the component that promotes GLUT1 expression can be determined as the component that promotes involucrin expression (involucrin expression promoting component). Furthermore, the present invention's method for promoting involucrin expression involves applying an involucrin expression promoter, for example, containing the components screened in Test Examples 2 and 3 as active ingredients, to the skin.

[0104] Furthermore, when tested using the methods described in Test Example 2 and Test Example 3 above, the component that promotes GLUT1 expression can be determined as the component that promotes occludin expression (occludin expression promoting component). Furthermore, the present invention's method for promoting occludin expression involves applying an occludin expression promoter, for example, containing the components screened in Test Examples 2 and 3 as active ingredients, to the skin. [Industrial applicability]

[0105] This invention allows for the screening of components that increase the expression level of GLUT1 in skin tissue. Furthermore, we can provide an anti-aging agent containing an active ingredient that increases the expression level of GLUT1 in skin tissue, and an anti-aging method using the anti-aging agent.

[0106] Furthermore, the present invention allows for the screening of components that increase the expression levels of involucrin or occludin in skin tissue. Furthermore, we can provide an involucrin expression promoter containing an active ingredient that increases the expression level of involucrin in skin tissue, and a method for promoting involucrin expression using the involucrin expression promoter. Furthermore, we can provide an occludin expression promoter containing an active ingredient that increases the expression level of occludins in skin tissue, and a method for promoting occludin expression using the occludin expression promoter.

Claims

1. A screening method for occludin expression-promoting components, using the expression level of GLUT1 in skin tissue as an indicator.

2. The screening method according to claim 1, wherein the occludin expression promoting component is a skin condition improving component.

3. The screening method according to claim 2, wherein the skin condition improving ingredient is a skin barrier function improving ingredient.

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