Screening method for anti-aging ingredients

Abstract

To provide a method for screening an anti-ageing component using an expression amount of GLUT 1 in skin tissue as an indicator.SOLUTION: A screening method for screening an anti-aging component using an expression amount of GLUT 1 in skin tissue as an indicator.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Advanced glycation end-products (hereinafter sometimes abbreviated as AGEs) are known to be substances related to skin aging. These substances are produced by glycation, in which proteins and lipids present in skin tissue bind with sugar, and it is known that an increase in AGEs in skin tissue can cause dull skin and hardening of the skin (e.g., Non-Patent Documents 1 and 2).

[0003] On the other hand, it is known that collagen present in skin tissue is produced in dermal layer cells, but the amount of collagen produced decreases with age (eg, Non-Patent Document 3).

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

[0005] Occludin is one of the proteins that form tight junctions. Tight junctions contribute to the binding of epithelial cells and control the movement of molecules between cells, so it is important to maintain the expression level of occludin in the body (e.g., Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[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, p424-432, 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. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, an object of the present invention is to provide a method for screening an anti-aging component. A further 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 extensive research, the present inventors have found that the expression level of glucose transporter 1 (hereinafter sometimes abbreviated as GLUT1), which is present in skin tissue, particularly in the epidermis and dermis layers, decreases with age.

[0009] Therefore, the screening method of the present invention, which solves the above-mentioned problems, is a screening method for screening anti-aging components using the expression level of GLUT1 in skin tissue as an indicator. According to the present invention, anti-aging ingredients can be screened.

[0010] Preferably, a component that increases the expression level of GLUT1 is selected as the anti-aging component. According to the present invention, an anti-aging component can be selected.

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

[0012] Preferably, the anti-aging component is a component that inhibits 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 ingredients capable of suppressing the production of advanced glycation endproducts in skin tissue.

[0013] Preferably, the anti-aging ingredient is an ingredient 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 capable of promoting collagen production in skin tissue.

[0014] Furthermore, the anti-aging agent of the present invention, which solves the above-mentioned problems, contains, as an active ingredient, an anti-aging ingredient 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 anti-aging agent is for inhibiting 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 inhibiting the production of AGEs.

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

[0017] Furthermore, the anti-aging method of the present invention, which solves the above-mentioned 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 skin tissue. According to the present invention, the expression level of GLUT1 in skin tissue, which decreases with age, can be increased, thereby achieving anti-aging.

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

[0019] Furthermore, the present inventors found that suppressing GLUT1 expression in skin tissues reduced the expression of involucrin and occludin, two proteins produced by epidermal keratinocytes, indicating that GLUT1 is involved in the expression of involucrin and occludin in epidermal keratinocytes. Here, as described above, involucrin contributes to the skin's barrier function, prevention of rough skin, and fine skin texture. As described above, occludin forms tight junctions together with other proteins, contributes to the skin barrier function in the epidermis, and contributes to the control of ion permeation, etc. inside the body.

[0020] Thus, a further objective of the present invention is to provide a method for screening a component that promotes the expression of involucrin or occludin.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 screening method of the present invention which solves the above-mentioned problems is a screening method which screens for components which promote involucrin expression 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. It is known that the skin barrier function is reduced by dryness and irritation, and 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-mentioned problems, contains as an active ingredient a component screened by the above-mentioned method for screening involucrin expression promoting components. 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 method for promoting involucrin expression of the present invention, which solves the above-mentioned problems, involves applying to the skin a component screened by the above-mentioned method for screening an involucrin expression-promoting component. Preferably, the method for promoting involucrin expression of the present invention involves applying the involucrin expression promoter to the skin. According to the present invention, it is possible to promote the expression of involucrin in the epidermis layer (particularly, keratinocytes).

[0025] Furthermore, the screening method of the present invention that solves the above-mentioned problems is a screening method that screens for a component that promotes occludin expression 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 occludin.

[0026] Preferably, the occludin expression promoting component is a skin barrier function improving component. As described above, occludin 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 occludin and improve the skin barrier function.

[0027] Furthermore, the occludin expression promoter of the present invention, which solves the above-mentioned problems, contains as an active ingredient a component screened by the above-mentioned method for screening an occludin expression promoter component. 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 method for promoting occludin expression of the present invention, which solves the above-mentioned problems, comprises applying to the skin a component screened by the above-mentioned method for screening components for promoting occludin expression. Preferably, in the method for promoting occludin expression of the present invention, the occludin expression promoter is applied to the skin. According to the present invention, the expression of occludin can be promoted. Effect of the Invention

[0029] According to the present invention, anti-aging ingredients 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 the skin barrier function. According to the present invention, an agent for improving the skin barrier function can be obtained. According to the present invention, a method for improving skin barrier function can be provided. [Brief description of the drawings]

[0032] [Figure 1] 1 is a graph showing that the expression level of GLUT1 in skin tissue decreases with aging, (A) shows the results in normal human epidermal keratinocytes, and (B) shows the results in normal human dermal fibroblasts. [Diagram 2]1 is a graph showing the results of measuring the expression level of GLUT1 in normal human epidermal keratinocytes in Test Example 2. (A) shows the experimental results using Saxifrage extract, (B) shows the experimental results using Thyme extract, and (C) shows the experimental results using Hypericum perforatum extract. [Diagram 3] 1 is a graph showing the results of measuring the expression level of GLUT1 in normal human dermal fibroblasts in Test Example 3. [Figure 4] 1 is a graph comparing the expression levels of GLUT1 mRNA in cells in which the GLUT1 (SLC2A1) gene is not knocked down and in cells in which the GLUT1 (SLC2A1) gene is knocked down, as measured by the siRNA method in normal human epidermal keratinocytes. [Diagram 5] 1 is a graph comparing the level of involucrin mRNA expression in cells in which the GLUT1 (SLC2A1) gene is not knocked down and the level of involucrin mRNA expression in cells in which the GLUT1 (SLC2A1) gene is knocked down, measured by the siRNA method. [Figure 6] FIG. 13 is a graph comparing the level of occludin mRNA expression in cells in which the GLUT1 (SLC2A1) gene was not knocked down with the level of occludin mRNA expression in cells in which the GLUT1 (SLC2A1) gene was knocked down, as measured by the siRNA method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0034] In the present invention, the term "skin tissue" refers to the epidermis layer, dermis layer, and subcutaneous tissue that constitute the skin. In the present invention, "anti-aging" refers to restoring undesirable changes in skin condition caused by aging (e.g., dullness, sagging, deterioration of skin texture, hardening of the skin, etc.) to a state before aging or inhibiting such changes. "Dullness" refers to the loss of skin's natural transparency, brightness, and luster, making the entire face appear darker than its natural skin color. "Sagging" refers to deformation of the skin caused by a decrease in the elasticity of skin tissue. "Texture" refers to the fine irregularities on the surface of the skin, and "deterioration of texture" refers to a decrease in the irregularities on the skin surface. In the present invention, the term "anti-aging component" refers to the above-mentioned component having an anti-aging effect.

[0035] 1. Screening Method The screening method of the present invention is a method for screening for anti-aging components using the expression level of GLUT1 as an index. 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 present inventors have found that the expression level of GLUT1 in skin tissue decreases with aging. In addition, as described below, a decrease in the expression level of GLUT1 is thought to cause unfavorable changes in the skin, such as promoting the production of AGEs and decreasing collagen production, accelerating skin aging. Therefore, by using the present invention to increase the expression level of GLUT1 in skin tissue, which decreases with age, it is effective in anti-aging.

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

[0038] In the present invention, the anti-aging component may be a component that inhibits the production of AGEs. When the expression level of GLUT1 in skin tissue decreases, it becomes difficult for sugar to be taken up into cells. As a result, the amount of sugar in the extracellular skin tissue increases, promoting glycation reactions in the epidermis layer and promoting the production of AGEs. As mentioned above, when AGEs increase in the epidermis layer, it leads to deterioration of skin texture, dullness, and hardening of the skin. Therefore, ingredients that increase the expression level of GLUT1 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 the expression level of GLUT1 in the dermis decreases, sugar is less easily taken up by collagen-producing fibroblasts (e.g., N Nakashima et al., Metabolism, (1995) 44(4): 543-548). This is expected to result in a decrease in collagen production in the dermis, causing sagging skin (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, ingredients that increase the expression level of GLUT1 in the dermis layer can promote collagen production and are effective in anti-aging.

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

[0041] The substance to be tested in the screening method of the present invention may be any of a pure substance, an extract derived from a living organism, or a mixture thereof. The term "extracts of biological origin" refers not only to the extracts themselves derived from animals or plants, but also to a collective term including fractions of extracts, purified fractions, and products obtained by removing the solvent from the extracts, fractions, or purified products. Examples of extracts of plant origin include extracts made from wild or cultivated plants, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts. In the extraction procedure, in addition to using the whole plant, parts of the plant, such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, inflorescences, and flower buds, can be used. However, it is preferable to crush or chop these in advance to improve the extraction efficiency. Suitable examples of the extraction solvent include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, ketones such as acetone, methyl ethyl ketone, ethers such as diethyl ether, tetrahydrofuran, etc. A specific example of the extraction method is to add 1 to 30 parts by mass of a solvent to 1 mass of the part of the plant body or the dried part used for extraction, immerse the part at room temperature for several days or at a temperature near the boiling point for several hours, cool the part to room temperature, remove insoluble matter and / or the solvent as desired, and then fractionate and purify the part 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 test in the epidermis layer, and normal human fibroblasts can be used in the test in the dermis layer. Specifically, when the expression level of GLUT1 in cells cultured with the addition of a test substance is statistically significantly higher than the expression level of GLUT1 in cells cultured without the addition of the test substance, the test substance can be determined to be a candidate anti-aging ingredient. More specifically, when the expression level of GLUT1 is more than 1-fold higher than that in cells cultured without the addition of the test substance, it can be selected as a candidate for an anti-aging component.

[0043] The expression level of GLUT1 can be measured by standard methods such as mRNA measurement and immunohistochemical analysis. For example, the expression level of a gene encoding GLUT1 is quantitatively detected by carrying out PCR using, as a primer, a DNA fragment having a sequence that specifically binds to the sequence of the gene. In addition, since the gene sequence encoding GLUT1 has been made public, those skilled in the art can design appropriate primers. Furthermore, since there are commercially available antibodies, these can be used to measure the expression level in cells.

[0044] The present invention also relates to a method for screening for components that promote involucrin expression in skin tissue, using the expression level of GLUT1 as an indicator. The skin tissue is preferably the epidermis layer, more preferably the stratum corneum. The present inventors have revealed that when the expression level of GLUT1 in skin tissue decreases, the expression level of involucrin in skin tissue also decreases. Therefore, by using the expression level of GLUT1 as an indicator, it is possible to screen for components that promote involucrin expression.

[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 the expression of involucrin is promoted, the formation of the cornified envelope is promoted, the toughness of the stratum corneum is improved, and the defense function of the skin is improved. When the defense function of the skin is improved, the skin condition is improved. An example of the defense function of the skin is the skin barrier function. In other words, when the expression of involucrin is promoted, the skin condition improves. Therefore, the method for screening for an involucrin expression-promoting component according to the present invention is preferably a method for screening for a skin condition-improving component. Specifically, the skin condition-improving component can be a skin barrier function improving component.

[0046] In a preferred embodiment of the present invention, the skin condition improving component is a skin barrier function improving component. According to the present invention, it is possible to screen for ingredients that improve skin barrier function using the expression level of GLUT1 as an indicator.

[0047] The present invention also relates to a method for screening for a component that promotes occludin expression in skin tissue, using the expression level of GLUT1 as an indicator. The skin tissue is preferably the epidermis. The present inventors have revealed that when the expression level of GLUT1 in skin tissue decreases, the expression level of occludin in skin tissue also decreases. Therefore, by using the expression level of GLUT1 as an indicator, it is possible to screen for components that promote occludin expression.

[0048] As described above, occludin is one of the proteins that form tight junctions that contribute to the binding of epithelial cells. Therefore, when the expression of occludin is promoted, the formation of tight junctions is promoted, the binding of epithelial cells becomes stronger, and the defense function of the skin is improved. When the defense function of the skin is improved, the skin condition is improved. An example of the defense function of the skin is the skin barrier function. In other words, when the expression of occludin is promoted, the skin condition improves. Therefore, the method for screening for an occludin expression-promoting component according to the present invention is preferably a method for screening for a skin condition-improving component. Specifically, the skin condition-improving component can be a skin barrier function improving component.

[0049] In a preferred embodiment of the present invention, the skin condition improving component is a skin barrier function improving component. According to the present invention, it is possible to screen for ingredients that improve skin barrier function using the expression level of GLUT1 as an indicator.

[0050] In the methods for screening components that promote involucrin expression and the methods for screening components that promote occludin expression, the above-mentioned items can be applied to the substances to be tested and the methods for obtaining them, the specific screening techniques, and the methods for measuring the expression levels of involucrin and occludin.

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

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

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

[0054] Also preferably, the anti-aging agent is for suppressing the production of advanced glycation end-products and promoting the production of collagen. An anti-aging agent that is effective in both inhibiting the production of advanced glycation endoproducts and promoting collagen production would be more effective in anti-aging.

[0055] The anti-aging agent of the present invention can be appropriately combined with any ingredient used in formulation to take the form of an oral agent or an external agent for skin application. When the anti-aging ingredient is administered orally, it is preferable that the ingredient is in the form of a food composition containing the anti-aging ingredient as an active ingredient. Specifically, the ingredient is preferably in the form of a supplement having a dosage form such as a general food, tablet, granule, or drink.

[0056] The content of the anti-aging ingredient in the oral preparation is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, in terms of the dry mass of the extract per dose, depending on the dosage form. Also, the amount is usually 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less. By setting the amount within the above range, the expression level of GLUT1 can be increased effectively.

[0057] In the case of a skin external preparation, examples of the form include cosmetics, quasi-drugs, skin external medicines, etc. Furthermore, the dosage form of these is not particularly limited. For example, when used as a cosmetic product, the form of a lotion, beauty essence, milky lotion, cream, gel, sun care product, etc. is preferred.

[0058] The content of the anti-aging ingredient in the external skin preparation (dry weight in the case of an extract) is usually 0.00001 mass % or more, preferably 0.0001 mass % or more, and more preferably 0.001 mass % or more. Also, it is usually 80% by mass or less, preferably 30% by mass or less, and more preferably 10% by mass or less. By setting the amount within the above range, the expression level of GLUT1 can be increased effectively.

[0059] When the above-mentioned anti-aging component is blended in a cosmetic, whitening components, anti-wrinkle components, anti-inflammatory components, extracts derived from animals and plants, and components normally used in cosmetics other than the active ingredient may be blended as optional components within a range that does not impair the anti-aging effect. These optional components may be commercially available and used, or may be synthesized by a known method and used.

[0060] As the whitening ingredient, ingredients generally used in cosmetics can be used without any particular restrictions. However, it is preferable to use an ingredient that has a whitening effect through a mechanism other than promoting GLUT1 expression, from the viewpoint of making the most of the action and effect 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)cis Theic acid, N-(m-toluoyl)cysteic acid, 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 Examples of the serine methyl ester include 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, and N-(2-naphthoyl)-O-methylserine.

[0061] The content of the whitening ingredient in the cosmetic 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 mass in the case of an extract).

[0062] As the wrinkle-improving ingredient, ingredients generally used in cosmetics can be used without any particular restrictions. However, from the viewpoint of making the most of the action and effect of each ingredient, it is preferable to use an ingredient that has a wrinkle-improving effect by a mechanism other than promoting the expression of GLUT1. Examples of vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, and retinol acetate. In addition, examples of vitamin A or its derivatives include ursolic acid benzyl ester, ursolic acid phosphate, benzylic acid ester, and benzilic acid phosphate.

[0063] The content of the wrinkle-improving ingredient in the cosmetic 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 mass in the case of an extract).

[0064] Examples of anti-inflammatory components include clarinone, glabridin, glycyrrhizinic acid, glycyrrhetinic acid, pantothenyl alcohol, and the like, and preferred examples include glycyrrhizinic acid and its salts, alkyl glycyrrhetinates and their salts, and glycyrrhetinic acid and its salts. The content of the anti-inflammatory component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry mass in the case of an extract).

[0065] Examples of extracts derived from plants and animals include Akebia extract, Asarum extract, Asparagus extract, Avocado extract, Amacha extract, Almond extract, Arnica extract, Aronia extract, Apricot extract, Ginkgo extract, Fennel extract, Aralia extract, Eleuthero extract, Enmeiso extract, Phellodendron bark extract, Panax ginseng extract, Lamium extract, Kakyoku extract, Pueraria lobata extract, Chamomilla recutita extract, Carrot extract, Artemisia capillaris extract, Licorice extract, Kiwi extract, Cucumber extract, and Guava extract. , Gardenia extract, Kumazasa extract, Walnut extract, Black rice extract, Chlorella extract, Mulberry extract, Kaneko extract, Alpinia alba extract, Gentian extract, Rice extract, Fermented rice extract, Fermented rice bran extract, Rice germ oil, Salvia extract, Soapwort extract, Sasa extract, Sandalwood extract, Zanthoxylum extract, Shiitake mushroom extract, Rehmannia root extract, Lithospermum root extract, Perilla extract, Tilia extract, Spiraea extract, Ginger extract, Calamus root extract, Horsetail extract, Stevia extract, Fermented stevia, Ardisia maritima Geranium extract, peppermint extract, sage extract, mallow extract, cnidium extract, swertia britannica extract, soybean extract, dandelion extract, clove extract, chili pepper extract, angelica extract, calendula extract, tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, burdock extract, lotus extract, parsley extract, birch extract, witch hazel extract, sedge extract, Japanese cypress extract, loquat extract, fennel extract, Preferred extracts include dandelion extract, butterbur extract, poria extract, loofah extract, peppermint extract, linden extract, pine extract, skunk cabbage extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, lily extract, coix seed extract, mugwort extract, lavender extract, apple extract, rooibos tea extract, lychee extract, lettuce extract, forsythia extract, astragalus extract, rosemary extract, Roman chamomile extract, and burnet extract.

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

[0067] In addition to the active ingredients, other ingredients commonly used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol and other polyols, fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and triethanolamine alkyl sulfate. anionic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, laurylamine oxide, cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, laurylamine oxide, imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaine, amido betaine, sulfo betaine, etc.), acyl methyl taurine, and other amphoteric surfactants, 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 ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbitan monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic (registered trademark) types, POE·POP alkyl ethers (POE·POP 2-decyltetradecyl ether, etc.), Tetronics, POE castor oil / hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, sodium lactate, which may be surface-treated,Powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc., inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, zinc oxide, etc. which may be surface-treated, composite pigments such as sintered iron oxide titanium dioxide, etc. which may be surface-treated, pearling agents such as titanium mica, fish phosphorus foil, bismuth oxychloride, etc. which may be surface-treated, Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 20 Examples of suitable organic pigments include organic dyes such as Red No. 1 and Red No. 204; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomer; lower alcohols such as ethanol and isopropanol; vitamin A or a derivative thereof; vitamin B group such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof; vitamin E group such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D group, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0068] The present invention also relates to an agent for promoting involucrin expression and an agent for promoting occludin expression.

[0069] The involucrin expression promoter of the present invention contains as an active ingredient an involucrin expression promoting ingredient screened by the screening method for involucrin expression promoting ingredients described above in "1. Screening method." The components screened by the above screening method increase the expression level of involucrin.

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

[0071] The occludin expression promoting agent of the present invention contains, as an active ingredient, an occludin expression promoting ingredient screened by the screening method for occludin expression promoting ingredients described above in "1. Screening method." The component screened by the above screening method increases the expression level of occludin.

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

[0073] For the involucrin expression promoter and occludin expression promoter, the above-mentioned items can be applied to the dosage form, the content of the involucrin expression promoting component and the occludin expression promoting component, and the optional components and their contents.

[0074] 3.Anti-aging method The anti-aging method of the present invention involves applying to skin tissue an anti-aging ingredient screened by any of the screening methods described above in "1. Screening method." Here, "applying an anti-aging ingredient to skin tissue" refers to making the anti-aging ingredient screened by the method of the present invention capable of promoting GLUT1 expression in the skin tissue of a user, for example by applying the ingredient to the skin or by applying a patch. The anti-aging method of the present invention is a non-therapeutic method, preferably a cosmetic method, more preferably a cosmetic method for the skin.

[0075] Preferably, any of the anti-aging agents described above under "2. Anti-aging Agents" is applied to the skin. The anti-aging agents described above in "2. Anti-aging agents" contain a component that increases 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 effect.

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

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

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

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

[0080] Preferably, the occludin expression promoter described above in "2. Anti-aging agents" is applied to the skin. The occludin expression promoter described above in "2. Anti-aging agents" contains a component that increases the expression level of GLUT1 in skin tissue, and has a high effect of promoting occludin expression. Furthermore, by applying it to the skin, the effect of promoting occludin expression can be fully exerted.

[0081] The above-mentioned method for promoting involucrin expression and method for promoting 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 in the epidermis The expression level of GLUT1 was measured in normal human epidermal keratinocytes from donors of different ages. (1) Using HuMedia-KG2 medium (manufactured by Kuraray Industries, Inc.), normal human epidermal keratinocytes collected from donors in their 30s and 50s were plated on a 48-well plate at 3.0 × 10 4 The cells were seeded at 10 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. (2) After the incubation, the medium was removed, and HuMedia-KB2 medium (Kuraray Industries, Inc.) was added, followed by incubation at 37°C and 5% CO for an additional 24 hours. (3) After the culture, the cultured normal human epidermal keratinocytes were recovered and mRNA was extracted according to the protocol of the RNeasy Mini Kit (QIAGEN). (4) The expression level of GLUT1 mRNA was measured by real-time qPCR, along with the expression level of β-actin mRNA, an endogenous control. For the measurement, Fast SYBR Green Master Mix (Thermo Fisher) was used, and the primer used was Hs_SLC2A1_1_SG QuantiTect Primer Assay (QIAGEN). (5) The expression level of GLUT1 mRNA in donor cells of each age group was corrected by the expression level of β-actin mRNA. The expression level of GLUT1 mRNA after correction in donor cells in their 30s was set to 1, and the expression level of GLUT1 mRNA after correction in donor cells in their 50s was calculated. The results are shown in Figure 1.

[0084] (II) Measurement of GLUT1 expression in the dermis The expression level of GLUT1 was measured in normal human dermal fibroblasts from donors of different ages. The donors were in their teens and fifties. Normal human dermal fibroblasts collected from each donor were cultured in DMEM medium (manufactured by SIGMA) containing 10% FBS at 1.5 × 10 5 The cells were seeded at 100 cells / well and cultured for 24 hours at 37°C in a 5% CO2 environment. After culture, the medium was removed and DMEM medium containing 1% FBS was added, followed by culture for 24 hours at 37°C in a 5% CO2 environment. After culture, the expression level of GLUT1 was calculated according to the procedure from (I)(3) onwards. The results are shown in Figure 1.

[0085] As shown in FIG. 1, it was revealed that the expression level of GLUT1 decreases with age in normal human epidermal keratinocytes and normal human dermal fibroblasts.

[0086] <Test Example 2> Screening of ingredients that increase the expression level of GLUT1 in the epidermis We screened for compounds that increase the expression level of GLUT1 in normal human epidermal keratinocytes. (1) Using HuMedia-KG2 medium (manufactured by Kurashiki Boseki Co., Ltd.), normal human epidermal keratinocytes were cultured at 5.0 × 10 4 The cells were seeded at 10 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. (2) After culturing, the medium was removed, and HuMedia-KB2 medium (manufactured by Kurashiki Boseki Co., Ltd.) containing 0.5% of the plant extract to be tested or the solvent control was added, followed by culturing for 24 hours at 37°C in a 5% CO2 environment. Hereinafter, the cells to which the medium containing the plant extract to be tested was added are referred to as the plant extract-added group, and the cells to which the medium containing the solvent control was added are referred to as the solvent control group. (3) After the culture, the cultured normal human epidermal keratinocytes were recovered and mRNA was extracted according to the protocol of the RNeasy Mini Kit (QIAGEN). (4) The expression level of GLUT1 mRNA was measured by real-time qPCR, along with the expression level of β-actin mRNA, an endogenous control. For the measurement, Fast SYBR Green Master Mix (Thermo Fisher) was used, and the primer used was Hs_SLC2A1_1_SG QuantiTect Primer Assay (QIAGEN). (5) The expression levels of GLUT1 mRNA in the plant extract group and the solvent control group were corrected by the expression level of β-actin mRNA. The corrected expression level of GLUT1 mRNA in the plant extract group was calculated by setting the corrected expression level of GLUT1 in the solvent control group to 1. The results are shown in Figure 2.

[0087] As shown in FIG. 2, it was found that Saxifrage extract, Thyme extract, and Hypericum extract increased the expression level of GLUT1 in normal human epidermal keratinocytes. From the above, it was demonstrated that the screening method of the present invention makes it possible to screen for components that increase the expression level of GLUT1 in the epidermis layer.

[0088] <Test Example 3> Screening of ingredients that increase the expression level of GLUT1 in the dermis layer We screened for compounds that increase the expression level of GLUT1 in normal human dermal fibroblasts. (1) Using DMEM medium (Sigma) containing 10% FBS, normal human dermal fibroblasts were cultured at 7.0 × 10 4 The cells were seeded at 10 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. (2) After culturing, the medium was removed, and DMEM medium containing 0.5% of the plant extract to be tested or the solvent control and 1% FBS was added, followed by culturing for 24 hours at 37°C in a 5% CO2 environment. Hereinafter, the cells to which the medium containing the plant extract to be tested was added are referred to as the plant extract-added group, and the cells to which the medium containing the solvent control was added are referred to as the solvent control group. Thereafter, the expression level of GLUT1 in normal human dermal fibroblasts was calculated in the same manner as in (3) of Test Example 2 above. The results are shown in FIG.

[0089] As shown in FIG. 3, it was found that royal jelly extract increases the expression level of GLUT1 in normal human dermal fibroblasts. From the above, it was demonstrated that the screening method of the present invention makes it possible to screen for a component that increases the expression level of GLUT1 in the dermis layer.

[0090] The anti-aging agent of the present invention can be obtained by blending the ingredients screened in Test Examples 2 and 3 as active ingredients. In addition, in the anti-aging method of the present invention, for example, an anti-aging agent containing the ingredients screened in Test Examples 2 and 3 as active ingredients is applied to the skin.

[0091] <Test Example 4> Relationship between the expression levels of GLUT1, involucrin, and occludin The relationship between the expression levels of GLUT1, involucrin, and occludin was examined.

[0092] (I) Cell culture The cells used were human fibroblasts (NHDF) and human keratinocytes (NHEK). For each cell type, cells derived from young donors (18 and 28 years old) and old donors (54 years old) were used in the study. Each cell was cultured according to the method recommended by the respective manufacturer. The cells were seeded in a cell culture flask 250 (Sumitomo Bakelite #MS-21250), and when the cells reached 60-90% confluence, they were detached using trypsin neutralizing solution (KURABO #HK-3220) and subcultured. The passage numbers were 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 a plate and cultured overnight at 37°C and 5% CO in the complete medium shown in Table 1 below until the cells became 70-90% confluent (N=3-4). In the case of a 24-well plate, 6.0-7.0 × 10 cells were cultured per well.4 Cells were seeded at 3.0-3.5 × 10 cells / 500 μL per well in a 48-well plate. 4 The cells were seeded at a concentration of 250 cells / 250 μL. Table 1 shows the combination of the cells used and the medium used for culturing the cells.

[0094] [Table 1]

[0095] (2) Lipofectamin Reagent (Invitrogen) was mixed with the dedicated medium (listed in Table 1 above) corresponding to each cell type, and diluted at a ratio of 1 to 2:50. (3) The siRNA to be delivered was prepared by diluting the ON-TARGET plus siRNA (Dharmacon) reagent with a dedicated medium and PBS (final concentration added to cells: 25 nM), and mixing with the diluted Lipofectamin Reagent at a 1:1 ratio. The mixture was then incubated at room temperature for 15 minutes to prepare a siRNA complex delivery reagent. The siRNAs used were those listed in Table 2 below.

[0096] [Table 2]

[0097] (4) The culture medium was removed from the cultured cells, and the cells were washed once with an equal volume of PBS. (5) After adding 200-400 μL of the dedicated medium per well, 50-100 μL of the siRNA complex transfection 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 cultured 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 medium from the cultured cells cultured in (II)(5) above, the cells were washed once with an equal volume of PBS to the removed medium. The cells were 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 a QIAcube (QIAGEN #9001293). The elution volume was 30 μL per sample. (3) cDNA was synthesized using the SuperScript cDNA Synthesis kit (Life Technologies #11754250) in a thermal cycler (BIO RAD #T100) according to the manufacturer's recommended protocol. Sample RNA (involucrin and occludin RNA) concentrations were 20-50ng / 20μL, and standard RNA concentrations were 50-100ng / 20μL. (4) qPCR was performed using a real-time PCR system (Applied Biosystems, QuantStudio5) by the standard method to measure the mRNA expression levels of β-actin, involucrin, and occludin. Each sample was prepared with 2.5 μL of ion-exchanged water, 0.5 μL of primer, 5 μL of SYBR Green, and 2 μL of 10-30 fold diluted cDNA sample or 5-30 fold diluted standard cDNA sample. The number of cycles was 45-50. The primers used were 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 carried out three times, and the average value of the three measurements was calculated. The results are shown in Table 4 and Figures 4 to 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 to 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). These results demonstrate that when the expression level of GLUT1 decreases in skin tissue, the expression levels of involucrin and occludin decrease.

[0103] From the above results, it became clear that components that promote the expression of involucrin or occludin can be screened using the expression level of GLUT1 as an indicator. Specifically, when tested using the methods of Test Examples 2 and 3 above, a component that promotes GLUT1 expression can be determined to be a component that promotes involucrin expression (involucrin expression-promoting component). Furthermore, in the involucrin expression promoting method of the present invention, for example, an involucrin expression promoting agent containing the ingredients screened in Test Examples 2 and 3 as active ingredients is applied to the skin.

[0104] Furthermore, when tested using the methods of Test Example 2 and Test Example 3 above, a component that promotes GLUT1 expression can be determined as a component that promotes occludin expression (occludin expression-promoting component). Furthermore, in the method for promoting occludin expression of the present invention, for example, an occludin expression promoter containing the ingredients screened in Test Examples 2 and 3 as active ingredients is applied to the skin. [Industrial Applicability]

[0105] The present invention makes it possible to screen for components that increase the expression level of GLUT1 in skin tissue. It is also possible to provide an anti-aging agent that contains, as an active ingredient, a component that increases the expression level of GLUT1 in skin tissue, and an anti-aging method that uses the anti-aging agent.

[0106] Furthermore, the present invention makes it possible to screen for components that increase the expression levels of involucrin or occludin in skin tissue. In addition, the present invention can provide an involucrin expression promoter that contains as an active ingredient a component that increases the expression level of involucrin in skin tissue, and a method for promoting involucrin expression that uses the involucrin expression promoter. It is also possible to provide an occludin expression promoter that contains as an active ingredient a component that increases the expression level of occludin in skin tissue, and a method for promoting occludin expression that uses the occludin expression promoter.

Claims

1. A screening method for screening candidates for anti-aging components using the expression level of GLUT1 in the dermis layer as an index, A screening method comprising selecting a component that increases the expression level of GLUT1 as a candidate for an anti-aging component that promotes occludin expression.

2. The screening method according to claim 1, comprising adding a test substance to a cell culture system and measuring the expression level of GLUT1 in the cells.

3. 3. The screening method according to claim 1 or 2, wherein when the expression level of Glut1 in cells cultured with the addition of a test substance is statistically significantly greater than the expression level of Glut1 in cells cultured without the addition of the test substance, the test substance is determined to be a candidate anti-aging component that promotes occludin expression.

4. The screening method according to any one of claims 1 to 3, wherein when the expression level of GLUT1 is greater than 1-fold that in cells cultured without the addition of the test substance, the test substance is selected as a candidate for an anti-aging component that promotes occludin expression.

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