Screening method for skin aging improving agents

Abstract

To search for a component effective as a skin aging improver by a new approach to skin aging symptoms, and establish a new screening method therefor.SOLUTION: A skin aging improver is screened with the activity of aquaporin in a fibroblast as an index.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for screening an agent for improving skin aging. [Background technology]

[0002] Skin aging symptoms such as wrinkles and sagging have a significant impact on the appearance of the skin, and therefore there is great interest in improving them. In recent years, there has been active development of anti-aging agents, anti-wrinkle agents, moisturizing agents, and the like, aimed at improving skin aging symptoms (Patent Documents 1 to 3, etc.).

[0003] In skin cells, aquaporins, known as water channels, are expressed on the cell membrane and are known to play a role in taking up low molecular weight substances, including water in the intercellular space, into the cells. In humans, 13 types of aquaporins (AQP0 to AQP12) are known to exist. In epidermal cells, AQP3 mainly exists and is thought to play a role in maintaining the moisture content and elasticity of the skin. For this reason, it has been proposed to promote the production of aquaporins in epidermal cells to improve the health of the skin (Patent Document 4, etc.). However, the function of aquaporins in fibroblasts is unknown. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2015-131797 A [Patent Document 2] JP 2015-174857 A [Patent Document 3] JP 2003-171225 A [Patent Document 4] JP 2015-134751 A Summary of the Invention [Problem to be solved by the invention]

[0005] Although conventional skin anti-aging agents have been shown to have a certain degree of anti-aging effect, it is difficult to say that they have achieved a fully satisfactory effect. In addition, there is a need to further elucidate the mechanism by which skin aging symptoms occur. In view of the above circumstances, the present invention aims to search for ingredients that are effective as skin aging improving agents through a new approach to skin aging symptoms, and has an objective of establishing a new screening method for this purpose. [Means for solving the problem]

[0006] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that the expression of aquaporin in dermal fibroblasts is reduced by aging, and that this reduction reduces the proliferation ability of dermal fibroblasts. They then discovered that skin aging symptoms can be improved by enhancing the expression of aquaporin, and thus completed the present invention.

[0007] That is, the present invention is as follows. [1] A method for screening agents for improving skin aging using aquaporin activity in fibroblasts as an indicator. [2] The method according to [1], wherein the activity of the aquaporin is the expression level of a gene encoding a protein that constitutes the aquaporin or the protein, and when the expression level in fibroblasts to which the test substance has been added is greater than the expression level in cells to which the test substance has not been added, the test substance is determined to have an effect of improving skin aging. [3] The method according to [1] or [2], wherein the aquaporin is aquaporin 1 and / or aquaporin 3. [4] A method for designing a composition, comprising the steps of carrying out the screening method according to any one of [1] to [3] and incorporating a substance selected by the above step. [5] The design method described in [4], wherein the composition is for improving skin aging. [6] The design method described in [4] or [5], wherein the composition is a skin topical agent or a food or beverage. Effect of the Invention

[0008] INDUSTRIAL APPLICABILITY The present invention provides a screening method capable of searching for ingredients that are effective as skin aging improving agents and that are suitable for inclusion in external skin preparations, foods and beverages. [Brief description of the drawings]

[0009] [Figure 1] Graph showing the relative expression levels of aquaporin 1 and 3 genes in human fibroblasts to which reactive oxygen species had been added. [Diagram 2] Graph showing the cell proliferation ability of human fibroblasts in which aquaporin 1 and / or 3 was knocked down. [Diagram 3] Left: Graph showing the relative expression level of aquaporin 1 gene in human fibroblast cells supplemented with Centaurea Centaurea extract. Right: Graph showing the relative expression level of aquaporin 3 gene in human fibroblast cells supplemented with Zostera marina extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The screening method of the present invention is characterized in that it uses aquaporin activity in fibroblasts as an indicator. Aquaporins are known to be water channels expressed on the cell membrane, and their expression levels in epidermal keratinocytes are known to decrease with age. The present inventors have found that the expression level of aquaporin in fibroblasts is also involved in the occurrence of skin aging symptoms. More specifically, as shown in the experimental examples described below, reactive oxygen, which is an aging factor in fibroblasts, reduces the expression level of aquaporin. Fibroblasts with reduced expression level and activity of aquaporin have a significantly reduced proliferation ability. The reduced proliferation ability of collagen-producing fibroblasts reduces the firmness and elasticity of the skin, causing skin aging symptoms. In addition, the present inventors have found that the reduced expression level of aquaporin reduces the expression of tenascin-X (TNXB), which is involved in the cross-linking of collagen fibers, type 2 collagen (COL7A1), collagen 17 (COL17), which is responsible for the function of adhering epithelial tissue to the basement membrane, and a protein (COMP) involved in the promotion of collagen formation. In this skin aging process, by enhancing the activity of aquaporin in fibroblasts, the decrease in the proliferation ability of dermal fibroblasts can be suppressed, and as a result, the onset of skin aging symptoms can be suppressed and improved. In other words, a substance that enhances the activity of aquaporin in fibroblasts can be a skin aging improving agent.

[0011] The aquaporin activity that serves as an index in the screening method of the present invention is usually the expression level of aquaporin, which refers to either the transcription level of mRNA of a gene encoding a protein constituting an aquaporin or the translation level of the protein.

[0012] In a preferred embodiment of the screening method of the present invention, if the expression level of aquaporin in fibroblasts to which a test substance has been added is greater than the expression level of aquaporin in fibroblasts to which the test substance has not been added (control), the test substance is determined to have an effect of improving skin aging.

[0013] The aquaporin used as an index in the screening method of the present invention is not particularly limited, but aquaporin 1 (AQP1) and / or aquaporin 3 (AQP3) are preferred. More preferably, a combination of AQP1 and AQP3 is used as an index. More preferably, In one embodiment, a test substance that increases the expression levels of both AQP1 and AQP3 compared to a control is selected as a skin aging improving agent.

[0014] The expression level of the gene encoding aquaporin can be measured by any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer, and quantitative detection is performed. Note that the human gene sequence encoding aquaporin is publicly available, and a person skilled in the art can design appropriate primers and subject them to PCR. Furthermore, for example, the amount of aquaporin protein on the cell membrane may be measured by a standard method, such as an immunological method using an antibody, and used as the expression level of the gene.

[0015] The cells used in the screening method of the present invention are fibroblasts, which are normally present in the dermis. As for the cell culture conditions, in addition to normal culture conditions, any culture conditions can be applied without any particular limitations as long as they do not interfere with the execution of the screening method of the present invention, specifically, do not interfere with the measurement of the expression level of aquaporin.

[0016] The test substance targeted by the screening method of the present invention may be any of a pure substance, an extract derived from an animal or plant, or a mixture thereof. The term "animal or plant-derived extract" refers to not only the extract itself derived from an animal or plant, but also a collective term for an extract fraction, a purified fraction, an extract or fraction, or a purified product after the solvent has been removed. Examples of plant-derived extracts include extracts made from wild or cultivated plants, extracts sold as herbal medicine ingredients, and commercially available extracts. The extraction operation can use the whole plant, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flowers, flower buds, and fruits, but it is preferable to crush or chop them in advance to improve the extraction efficiency. The extraction solvent can be one or more selected from polar solvents such as 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. A specific extraction method includes, for example, adding 1 to 30 parts by mass of a solvent to 1 part by mass of a part to be used for extraction of the plant body or its dried product, soaking the part for several days at room temperature or several hours at a temperature near the boiling point, cooling to room temperature, removing insoluble matter and / or solvent as desired, and fractionating and purifying the part by column chromatography or the like.

[0017] An example of the procedure in the screening method of the present invention is given below, but the present invention is not limited to the following content and can be carried out with appropriate modifications without departing from the spirit of the present invention. First, a test substance is added to fibroblasts and incubated for 24 to 48 hours. Then, mRNA is extracted from the cells, and the expression level of a gene encoding aquaporin is quantitatively measured by RT-PCR using primers that specifically detect the gene. As a control, the expression level of the gene is also measured in fibroblasts to which the test substance has not been added. If the expression level of the gene in the cells to which the test substance has been added is greater than the expression level of the gene in the cells to which the test substance has not been added (control), the test substance is determined to have a skin aging improving effect. The substance determined to have such an effect can be a skin aging improving agent that can be suitably contained in a composition for improving skin aging. The degree of change in expression level is preferably 120% or more relative to the control, more preferably 150% or more, and even more preferably 200% or more.

[0018] The test substance selected by screening has an effect of improving skin aging, for example. This can be confirmed by well-known methods, such as subjective evaluation by a subject to which a composition containing the substance has been applied, evaluation of wrinkles and sagging by image analysis, evaluation of the viscoelastic properties of the skin, evaluation of the amount of collagen produced by the skin, or evaluation of the amount of moisture retained by the skin.

[0019] A substance (skin aging improving agent) determined to have an effect of improving skin aging symptoms by the screening method of the present invention can be incorporated into a composition by any preparation method. That is, the screening method of the present invention can be suitably used for designing a composition. Suitable examples of such compositions include skin external compositions and food and beverage compositions, which can be preferably used for improving skin aging and anti-aging purposes. Substances (skin aging improving agents) determined by the screening of the present invention to have the effect of improving skin aging symptoms can be effective components of anti-aging compositions that use a new approach in that they improve skin aging symptoms through a new mechanism that targets the occurrence of skin aging caused by aquaporins in fibroblasts.

[0020] When a substance (skin aging improving agent) determined to have an effect of improving skin aging symptoms by the screening of the present invention is contained in a composition, its content (formulation amount) is usually 0.00001 mass% or more, preferably 0.0001 mass% or more, more preferably 0.001 mass% or more, and usually 15 mass% or less, preferably 10 mass% or less, more preferably 5 mass% or less. If the content (formulation amount) of the skin aging improving agent is too small, it may be difficult to obtain the desired effect, and if it is too large, not only the effect may plateau, but also the freedom of formulation of the composition may be impaired. In addition, the type of skin aging improving agent contained in the composition may be not only one type but also two or more types. Note that the formulation amount of animal and plant extracts, etc. is calculated as the solid matter amount. The composition may also contain ingredients for improving skin aging other than the substances determined to have the effect of improving skin aging symptoms by the screening of the present invention.

[0021] When the skin aging improving agent according to the present invention is incorporated into a skin external composition, ingredients that are usually used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be optionally blended therein during the production thereof. Examples of such optional components include hydrocarbons such as squalane, petrolatum, and microcrystalline wax; esters such as jojoba oil, carnauba wax, and octyldodecyl oleate; triglycerides such as olive oil, beef tallow, and coconut oil; fatty acids such as stearic acid, oleic acid, and retinoic acid; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; anionic surfactants such as sulfosuccinic acid esters and sodium polyoxyethylene alkyl sulfates; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkyl ammonium salts; nonionic surfactants such as sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, and polyoxyethylene fatty acid esters; polyhydric alcohols such as polyethylene glycol, glycerin, and 1,3-butanediol; thickeners and gelling agents; antioxidants; ultraviolet absorbing agents; coloring agents; preservatives; and powders. The composition for external use on the skin can be produced by processing and blending the above-mentioned components according to a conventional method, and can be in any form, such as a lotion, emulsion, or oil.

[0022] When the skin aging improving agent according to the present invention is to be contained in a food or drink, ingredients that are usually used in food production can be optionally blended therein during production. Such optional ingredients may include, for example, proteins, carbohydrates, fats, nutrients, seasonings, flavorings, etc. Carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextran, glycerol ... Examples of the flavoring agent include ordinary sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. In addition, additives used in the above-mentioned pharmaceutical compositions that are also added to ordinary foods may be used in the same manner.

[0023] The form of the food or drink may be liquid, paste, solid, powder, granule, etc. Tablets, liquid food, feed, etc. are also included in the form of the food or drink.

[0024] In addition, the present invention may be incorporated into other general foods and beverages, such as wheat flour products, including bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, and breadcrumbs; instant foods, including instant noodles, cup noodles, retort and cooked foods, cooked canned foods, microwave foods, instant soups and stews, instant miso soup and clear soups, canned soups, and freeze-dried foods; agricultural processed products, including canned agricultural products, canned fruit, jams and marmalades, pickles, boiled beans, dried agricultural goods, and cereals (processed grain products); marine processed products, including canned seafood, fish ham and sausages, marine paste products, marine delicacies, and tsukudani (fried foods and stews); livestock processed products, including canned livestock products and pastes, and livestock ham and sausages; dairy products, including processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, modified milk powder, cream, and other dairy products; butter, margarine, and other processed foods. fats and oils such as phosphorus and vegetable oils; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, and dessert snacks; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks containing fruit juice, fruit pulp drinks, fruit drinks containing fruit grains, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, and alcoholic drinks; and foods other than these, the skin aging improving agent of the present invention may be added.

[0025] Examples of food compositions containing the skin aging improving agent of the present invention include ordinary foods, beverages, functional foods, health functional foods such as foods for specified health uses, and supplements, with functional foods being particularly preferred. When the food composition according to the present invention is used for improving skin aging or for anti-aging purposes, the usefulness and functionality of the product may be indicated when the product is manufactured. Such "indication" acts include all acts for informing consumers of the above-mentioned uses, and any expression that can recall or infer uses such as "for improving skin aging," "for anti-aging," or "for firming and elasticity of the skin" falls under the "indication" acts of the present invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc. In addition, it is preferable that the "labeling" is done in an expression that allows consumers to directly recognize the above-mentioned use. Specifically, examples include the act of transferring, delivering, displaying for transfer or delivery, and importing a food or drink product or a product package, container, etc. on which the above-mentioned use is written, and the act of displaying or distributing the above-mentioned use in advertising materials at the sales site such as advertisements, price lists, catalogs, pamphlets, POP, etc., or in transaction documents, or the act of providing the above-mentioned use in information containing the above-mentioned use by electromagnetic (Internet, etc.) methods. In addition, when the food composition of the present invention is approved based on various systems established by the government such as health functional foods and is implemented under such approval, it is preferable to label it in a manner based on the approval.

[0026] The preferred intake amount of the food composition containing the skin aging improving agent according to the present invention for adults is preferably 0.001 to 1000 mg / kg per day in terms of solid matter of the skin aging improving agent, from the viewpoint of fully exerting the effects, and more preferably 0.01 to 100 mg / kg per day. g, and more preferably 0.1 to 10 mg / kg. This daily amount can be taken all at once or in several divided doses. In addition to taking it once, it is preferable to take it continuously or intermittently for several weeks to several months. EXAMPLES

[0027] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples as long as it does not depart from the gist of the present invention.

[0028] <Reference Example 1> Confirmation of the effect of reactive oxygen species on aquaporins in fibroblasts Human fibroblasts were cultured in 24-well plates at 2 × 10 in DMEM medium containing 10% FBS. 4 Cells / well were seeded and cultured for 24 hours at 37℃ and 5% CO2. After culture, the medium was removed and replaced with DMEM medium containing 1% FBS, and cultured for another 24 hours. 50μM reactive oxygen was added to each well, and cultured for 3 hours, after which the reactive oxygen was removed and cultured for another 21 hours. The mRNA of the above fibroblasts was extracted using RNeasy Mini Kit (QIAGEN), cDNA was synthesized using Superscript VILO DNA synthesis Kit (Lifetechnologies), and the mRNA expression levels of AQP1 and AQP3 were measured by real-time qPCR using primers (AQP1: QT00013237, QIAGEN, AQP3: QT00212996, QIAGEN). The mRNA expression level of GAPDH, an endogenous control gene, was also measured. For the measurement, QuantiFact SYBR GREEN PCR kit (QIAGEN) was used. The expression levels of AQP1 or AQP3 mRNA in the active oxygen-added group and non-added group were corrected by the expression level of GAPDH, and the relative expression level in the active oxygen-added group was calculated by setting the expression level in the non-added group at 100. The results are shown in Figure 1. It can be seen that the addition of reactive oxygen species significantly reduced the mRNA expression levels of both AQP1 and AQP3 in fibroblasts.

[0029] <Reference Example 2> Confirmation of the effect of aquaporin expression reduction on the proliferation ability of fibroblasts Human fibroblasts were cultured in 24-well plates at 5 × 10 cells in DMEM medium containing 10% FBS. 3Cells were seeded per well and cultured for 24 hours under 37℃ and 5% CO2. siRNA (AQP1: s1516, Life Technologies; AQP3: s1521, Life Technologies) was introduced using a lipofection reagent to knock down AQP1 and / or AQP3, and the cells were further cultured for 72 hours. Non-coding siRNA was used as a control. After culture, the medium was removed from the plate and washed with PBS(-), and 1000μL / well of tetrazolium salt WST-8 reagent (Cell Counting Kit-8; Dojindo Laboratories) diluted 20-fold with the medium was added to the wells, and the reaction was allowed to proceed for 2 hours in a CO2 incubator. The absorbance at 450nm and 650nm was measured using a plate reader, and the difference in the measured values ​​(Abs.450-Abs.650) was calculated as the cell number, relative to the control, which was set as 1. The results are shown in Figure 2. It can be seen that the proliferation ability of fibroblasts in which AQP1 and / or AQP3 were knocked down was significantly reduced. In particular, when both AQP1 and AQP3 were knocked down, the cell proliferation ability was significantly reduced.

[0030] <Example 1> Screening of skin aging improvement agents Screening of skin aging improving agents was carried out using aquaporin gene expression as an indicator according to the following procedure. DMEM medium (Sigma) was used to culture human fibroblasts at 2 × 10 4 Cells were seeded at 1000 cells / well and cultured for 24 hours at 37°C and 5% CO2. After culture, the medium was removed and the cells were washed with PBS. Maintenance medium containing 0.2% by weight (final concentration) of cornflower extract (manufactured by Koei Kogyo Co., Ltd.), 0.2% by weight (final concentration) of eelgrass extract (manufactured by Technoble Co., Ltd.), or solvent control (PBS, 1.5 μL / mL medium) was added and cultured for 24 hours at 37°C and 5% CO2. After culture, the medium was removed and the cells were washed with PBS. Then, human fibroblast mRNA was extracted using QIAzol Lysis Reagent (QIAGEN), cDNA was synthesized using Superscript VILO DNA synthesis Kit (Lifetechnologies), and the mRNA expression levels of AQP1 or AQP3 were measured by real-time qPCR using primers (AQP1: QT00013237, QIAGEN, AQP3: QT00212996, QIAGEN). In addition, the mRNA expression level of GAPDH, an endogenous control gene, was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (QIAGEN) was used. The mRNA expression levels of AQP1 or AQP3 in the extract-added group and the solvent control group were corrected by the expression level of GAPDH, and the relative expression level of the extract-added group was calculated when the expression level of the solvent control group was set to 100. The results are shown in Figure 3. It can be seen that the centaurea cyanus extract increased the expression level of AQP1 in fibroblasts, and the eelgrass extract increased the expression level of AQP3, respectively, and they may be effective ingredients in an agent for improving skin aging. [Industrial Applicability]

[0031] The screening method of the present invention makes it possible to search for ingredients that are effective as skin aging improving agents. Such skin aging improving agents are industrially very useful because they can be suitably contained in skin external preparations and foods and beverages for improving skin aging.

Claims

1. A method for screening a skin aging improving agent using aquaporin activity in fibroblasts as an index, comprising: the aquaporin is aquaporin 1 and aquaporin 3; The skin aging improving agent improves skin aging by inhibiting a decrease in the proliferation ability of fibroblasts.

2. The method according to claim 1, wherein the activity of the aquaporin is the expression level of a gene encoding a protein that constitutes the aquaporin or the protein, and the test substance is determined to have an effect of improving skin aging when the expression level in fibroblasts to which the test substance has been added is greater than the expression level in fibroblasts to which the test substance has not been added.

3. A method for designing a composition, comprising the steps of: carrying out the screening method according to claim 1 or 2; and incorporating a substance selected by said step.

4. The method according to claim 3 , wherein the composition is for improving skin aging.

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