JP7251036B2 - Screening method for skin aging improving agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for screening skin aging improving agents.

Since skin aging symptoms such as wrinkles and saggy skin have a great impact on appearance, there is a great deal of interest in their improvement. In recent years, anti-aging agents, anti-wrinkle agents, moisturizing agents and the like have been actively developed for the purpose of improving skin aging symptoms. They mainly target dermal fibroblasts that produce substances such as collagen and elastin that maintain skin tension and elasticity (Patent Documents 1 and 2, etc.) and epidermal basal cells. It was a thing (for example, patent document 3 etc.).

JP 2015-131797 A JP 2015-174857 A Japanese Patent Application Laid-Open No. 2003-171225

Although conventional skin anti-aging agents are recognized to have anti-aging effects to some extent, it is difficult to say that they are sufficiently satisfactory. There is also a need for further elucidation of the mechanism by which skin aging symptoms occur.
In view of this situation, the present invention aims to search for ingredients effective as skin aging improving agents by a new approach to skin aging symptoms, and to establish a new screening method for that purpose.

As a result of intensive research to solve the above problems, the present inventors focused on chemerin, which is one of the adipocytokines, and found that chemerin secreted from subcutaneous adipocytes affects dermal fibroblasts and the like. rice field. Then, the present invention was completed based on the finding that skin aging symptoms can be improved by inactivating chemerin.

That is, the present invention is as follows.
[1] A method of screening a skin aging improving agent using the activity of chemerin in subcutaneous adipocytes as an indicator.
[2] The activity of the chemerin is a gene encoding a protein constituting the chemerin or the expression level of the protein, and the expression level in subcutaneous adipocytes to which the test substance has been added is the cell to which the test substance has not been added. The method according to [1], wherein the test substance is determined to have a skin aging-improving effect when the expression level is small compared to the expression level in [1].
[3] A method of designing a composition, comprising the step of performing the screening method of [1] or [2], and the step of incorporating the substance selected in the above step.
[4] The designing method according to [3], wherein the composition is for improving skin aging.
[5] The designing method according to [3] or [4], wherein the composition is food or drink or an external preparation for skin.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a screening method capable of searching for a component effective as an agent for improving skin aging, which is suitable for inclusion in foods, beverages, and external preparations for skin.

Graph showing the relative expression levels of the versican gene in human fibroblasts supplemented with chemerin. Graph showing the relative expression level of the mimecan gene in human tendon cells supplemented with chemerin. A graph showing the relative expression level of the hyaluronic acid synthase (HAS3) gene in human epidermal keratinocytes to which chemerin was added. A graph showing the relative expression level of the chemerin gene in subcutaneous adipocytes to which walnut polyphenol extract was added.

The screening method of the present invention is characterized by using the activity of chemerin in subcutaneous adipocytes as an indicator.
Chemerin is a kind of adipocytokine secreted from subcutaneous adipocytes, and its secretion is known to increase due to aging, obesity, hyperlipidemia, and the like. The present inventors have found that when the expression level of chemerin increases, its activity is enhanced, and phenomena involved in the occurrence of skin aging symptoms, such as a decrease in the amount of hyaluronic acid, the amount of versican, and the skin support structure (RC structure), etc. found to be triggered. More specifically, the activation of chemerin reduces the amount of hyaluronic acid or versican due to a decrease in the enzyme that synthesizes hyaluronic acid or versican, or reduces the amount of protein that constitutes the network structure of the subcutaneous supportive band below the subcutaneous tissue. The amount of expression decreases, causing the network structure to become sparse. As a result, skin aging symptoms such as wrinkles and saggy skin appear in the dermis. In such a skin aging process, by attenuating the activity of chemerin in subcutaneous adipocytes, it is possible to suppress and improve skin aging symptoms. In other words, a substance that suppresses the activity of chemerin in subcutaneous adipocytes can be a skin aging improving agent.

Hyaluronic acid plays a role of water storage inside the skin, and generally aging causes a decrease in hyaluronic acid synthase and an increase in hyaluronan degrading enzyme, which causes a decrease in the amount of hyaluronic acid. When the amount of hyaluronic acid decreases, the water retention capacity of the skin becomes poor, causing dryness, wrinkles, roughness of the texture, and the like, leading to aging of the skin.
Versican is a proteoglycan that serves as a scaffold for fibroblasts to act when the dermis is created. It is known to decrease with age, leading to skin aging and reduced elasticity (Fragrance Journal 44(1)). : 88-88, 2016). As shown in the examples below, activation of chemerin causes a decrease in the amount of versican, which is thought to be due to a decrease in enzymes involved in versican production and an increase in versican-degrading enzymes. be done.
The skin support structure (RC structure) refers to a network structure called a subcutaneous support band (retinacula cutis; RC) under the subcutaneous tissue, and is mainly composed of type I collagen and a glycoprotein called mimecan. When the structure becomes sparse, the elasticity of the deep skin decreases, causing sagging (Fragrance Journal 44(2), 23-27, 2016). As shown in Examples below, when chemerin is activated, the expression levels of proteins that compose the RC structure are reduced, resulting in weakening of the RC structure (sparse network structure).

The activity of chemerin used as an index in the screening method of the present invention is usually the expression level of chemerin. Here, the expression level refers to either the transcription level of the mRNA of the gene encoding the protein constituting chemerin or the translation level of the protein.

In a preferred embodiment of the screening method of the present invention, when the expression level of chemerin in subcutaneous adipocytes to which the test substance is added is lower than the expression level of chemerin in subcutaneous adipocytes to which no test substance is added (control) In addition, the test substance is determined to have an action of improving skin aging.

The expression level of the gene encoding chemerin can be measured using 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. The human gene sequence encoding chemerin is open to the public, and those skilled in the art can appropriately design primers and subject them to PCR.
Alternatively, for example, the amount of chemerin protein on the cell membrane may be measured by a conventional method, such as an immunological method using an antibody, and the expression level of the gene may be determined.

Subcutaneous adipocytes are used as the cells used in the screening method of the present invention.
The conditions for culturing the cells are not particularly limited, in addition to normal culture conditions, as long as the culture conditions do not interfere with the execution of the screening method of the present invention, specifically, the measurement of the expression level of chemerin. be able to.

A test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals or plants, or a mixture thereof.
Animal and plant-derived extracts shall mean not only animal- or plant-derived extracts themselves, but also fractions of extracts, purified fractions, extracts or fractions, and solvent-removed purified products. Plant-derived extracts include wild or grown plants, extracts using those sold as raw materials for herbal medicines, commercially available extracts, and the like.
In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the flower, the flower bud, the fruit, etc. can be used. Alternatively, it is preferable to cut into small pieces to improve the extraction efficiency. Extraction solvents include 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. One or more selected from polar solvents such as polar solvents can be exemplified as suitable. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of the part used for extraction of a plant or the like or its dried product, and at room temperature for several days, at a temperature near the boiling point. If possible, a method of immersing for several hours, cooling to room temperature, optionally removing insoluble matter and/or solvent, and fractionating and purifying by column chromatography or the like can be mentioned.

An example of the procedure in the screening method of the present invention is shown below, but it is not limited to the following content as long as it does not deviate from the gist of the present invention, and can be carried out with appropriate modifications.
First, a test substance is added to subcutaneous adipocytes and incubated for 24 to 48 hours. Thereafter, mRNA is extracted from the cells, and the expression level of the gene encoding chemerin 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 subcutaneous adipocytes to which no test substance was added. When the expression level of the gene in the cells to which the test substance was added is smaller than the expression level of the gene in the cells to which the test substance was not added (control), the test substance is determined to have skin aging-improving action. . The determined substance can be a skin aging improving agent that can be preferably contained in a composition for improving skin aging.
The degree of variation in expression level is preferably 80% or less, more preferably 65% or less, and even more preferably 50% or less relative to the control.

The fact that a test substance selected by screening has an action to improve skin aging is, for example, subjective evaluation by subjects who applied a composition containing the substance, evaluation of wrinkles and sagging by image analysis, and skin viscoelastic physical properties. It can be confirmed by a well-known method such as evaluation, evaluation of water retention amount of skin, and the like.

Substances (skin aging improving agents) determined to have an action to improve skin aging symptoms by the screening method of the present invention can be incorporated into the composition by any preparation method. That is, the screening method of the present invention can be suitably used for designing compositions. Suitable examples of such compositions include food and drink compositions, external skin compositions, and the like, and these can be preferably applied to skin aging improving applications and antiaging applications.
A substance (skin aging improving agent) determined to have an action to improve skin aging symptoms by the screening of the present invention is a new mechanism that targets the occurrence of skin aging caused by chemerin secreted by subcutaneous adipocytes. In terms of improving skin aging symptoms, it can be a compounding component of an effective anti-aging composition based on a new approach.

When a substance (skin aging improving agent) determined by the screening of the present invention to have an action to improve skin aging symptoms is included in the composition, the content (blended amount) is usually 0.00001% by mass or more. , preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, and usually 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less. If the content (blended amount) of the skin aging improving agent is too small, it may be difficult to obtain the desired effect. be. Moreover, the number of skin aging improving agents to be contained in the composition may be one or two or more.
The composition may also contain a skin aging improving component other than the substance determined by the screening of the present invention to have an action to improve skin aging symptoms.

When the skin aging improving agent according to the present invention is contained in a composition for external use on the skin, it is possible to arbitrarily add components that are commonly used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc. during the production thereof. .
Examples of such optional ingredients 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, stearic acid, Fatty acids such as 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 alkylsulfate, amphoteric surfactants such as alkyl betaine salts cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene fatty acid esters, polyethylene glycol , glycerin, polyhydric alcohols such as 1,3-butanediol, thickeners/gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, and the like can be arbitrarily blended.
The composition for external use on the skin can be produced by treating and blending the aforementioned ingredients according to a conventional method. Moreover, the form can be, for example, a lotion type, an emulsifier type, an oil type, or any other dosage form.

When the skin aging improving agent according to the present invention is contained in a food or drink, ingredients that are commonly used in food production can be arbitrarily blended in the production thereof.
Such optional ingredients may include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; sugar alcohols such as erythritol; As flavoring agents, natural flavoring agents (thaumatin, stevia extract, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. In addition, additives that are used in the pharmaceutical compositions described above and that are usually added to foods may also be used.

The form of food and drink may be liquid, paste, solid, powder, granules, or the like. In addition, tablets, liquid foods, feeds, etc. are also included in the mode of food and drink.

In addition, it may be contained in other general food and drink products, such as bread, macaroni, spaghetti, noodles, cake mixes, fried chicken flour, bread crumb flour products; instant noodles, cup noodles, retort and cooked foods , Cooked canned food, Microwave food, Instant soup/stew, Instant miso soup/Soup, Canned soup, Instant food such as freeze-dried food; Agricultural processed products such as cereals (processed grain products); Processed marine products such as canned marine products, fish ham and sausages, fish paste products, marine delicacies, and tsukudani products; Milk and dairy products such as processed milk, milk drinks, yogurts, lactic acid beverages, cheese, ice creams, formulated milk powders, cream, and other dairy products; Oils and fats such as butter, margarine, and vegetable oil; Soy sauce, Basic seasonings such as miso, sauces, processed tomato seasonings, mirin, and vinegar; complex seasonings such as cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings Frozen foods such as frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramels, candies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets , Confectionery such as desserts; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks containing fruit juice, pulp drinks, fruit drinks containing fruit, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrates The agent for improving skin aging according to the present invention may be added to beverages such as beverages, sports drinks, nutritional drinks, and alcoholic beverages; and foods other than these.

Embodiments of the food composition containing the skin aging improving agent according to the present invention include ordinary foods, beverages, foods with function claims, foods with health claims such as foods for specified health uses, supplements, etc., particularly functional claims. Food is preferred.
When the food composition according to the present invention is used for improving skin aging or anti-aging, the usefulness and functionality of the food composition may be indicated at the time of commercialization.
Such "indication" acts include all acts to inform consumers of the above uses, and expressions that can remind or analogize uses such as "improving skin aging" and "anti-aging" If so, regardless of the purpose of the display, the content of the display, the object/medium to be displayed, etc., all of them fall under the act of "display" of the present invention.
In addition, it is preferable that the "display" be performed in an expression that allows the consumer to directly recognize the use. Specifically, the act of transferring, handing over, displaying for the purpose of transfer or delivery, importing products related to food and beverages or packaging, containers, etc. of products with the above-mentioned use, advertisements related to products, price lists, Exhibiting or distributing the above-mentioned use in advertising materials at sales sites such as catalogs, pamphlets, POPs, etc., or transaction documents, or describing the above-mentioned use in information containing these and electromagnetically (Internet, etc.) ) act of providing by the method. In addition, when the food composition of the present invention is approved under various governmental systems such as foods with health claims and is implemented under such approval, it is preferable to display in a manner based on the approval.

The preferable intake of the food composition containing the skin aging improving agent according to the present invention is 0.001 to 1000 mg/kg per day in terms of solid matter of the skin aging improving agent when ingested by adults. It is preferable from the viewpoint of sufficiently exhibiting the effect, more preferably 0.01 to 100 mg/kg, particularly preferably 0.1 to 10 mg/kg. This daily amount can be taken at once or divided into several times. In addition to single ingestion, continuous or intermittent ingestion for several weeks to several months is preferred.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

<Reference Example 1> Confirmation of the effect of chemerin on versican production in fibroblasts
Normal human dermal fibroblasts were plated in a 24-well plate using DMEM medium (manufactured by SIGMA).
0×10 ⁴ cells/well were seeded and cultured for 2 days under 37° C./5% CO ₂ environment. After culturing, remove the medium, wash the cells with PBS, chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or solvent control (PBS, 1.5 μL / mL A DMEM medium containing medium) was added, and the cells were cultured for 24 hours in a 37°C/5% _CO2 environment.
RNeasy Mini Kit (manufactured by QIAGEN) was used to extract mRNA from the fibroblasts, cDNA was synthesized using Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies), and primers (QT00064064, manufactured by QIAGEN) were used to synthesize versican mRNA. The expression level was measured by the real-time qPCR method. In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was similarly measured. QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used for the measurement.
The expression level of versican mRNA in the chemerin-added group and the solvent control group was corrected by the expression level of ACTB, and the relative expression level of the chemerin-added group was calculated when the expression level of the solvent control group was set to 1. The results are shown in FIG.

<Reference Example 2> Confirmation of the effect of chemerin on the production of RC constituent proteins in subcutaneous tendon cells
Normal human tendon cells were seeded in a 24-well plate at 2.0×10 ⁴ cells/well using Tenocyte Culture Medium (manufactured by Ds Pharma Biomedical) and cultured for 1 day at 37° C. and 5% CO ₂ environment. After culturing, remove the medium, wash the cells with PBS, chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or solvent control (PBS, 1.5 μL / mL Medium) was added, and the cells were cultured for 48 hours at 37°C and 5% _CO2 .
The tendon cell mRNA was extracted using the RNeasy Mini Kit (manufactured by QIAGEN), cDNA was synthesized using the Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies), and mimecan mRNA was expressed using a primer (QT00000483, manufactured by QIAGEN). Quantities were measured by real-time qPCR method. In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was similarly measured. QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used for the measurement.
The mimecan mRNA expression levels in the chemerin-added group and the solvent control group were corrected by the expression level of ACTB, and the relative expression level in the chemerin-added group was calculated when the expression level in the solvent control group was set to 1. The results are shown in FIG.

<Reference Example 3> Confirmation of the effect of chemerin on hyaluronic acid synthase production in epidermal keratinocytes
Human adult epidermal keratinocytes (NHEK, manufactured by Kurabo Industries) were cultured using Humedia-KG2 medium (manufactured by Kurabo Industries) and seeded in a 24-well plate at 7.0×10 ⁴ cells/well, under a 37° C./5% CO ₂ environment. was cultured for 1 day. After culturing, remove the medium, wash the cells with PBS, chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or solvent control (PBS, 1.5 μL / mL medium) is added, 37 ° C., 5% CO
₂ environment, and cultured for 48 hours.
RNeasy Mini Kit (manufactured by QIAGEN) was used to extract mRNA from the above keratinocytes, and Superscript
After synthesizing cDNA using VILO DNA synthesis Kit (manufactured by Lifetechnologies), mRNA expression level of HAS3 was measured by real-time qPCR method using a primer (QT00014903, manufactured by QIAGEN). In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was similarly measured. QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used for the measurement.
The HAS3 mRNA expression level in the chemerin-added group and the solvent control group was corrected by the expression level of ACTB, and the relative expression level in the chemerin-added group was calculated when the expression level in the solvent control group was set to 1. The results are shown in FIG.

<Example 1> Screening of Skin Aging Agent A skin aging improving agent was screened according to the following procedure using gene expression of chemerin as an indicator.
Using a growth medium (manufactured by CELL), normal human subcutaneous adipocytes were seeded in a 24-well plate at 1.0×10 ⁵ cells/well and cultured at 37° C., 5% CO ₂ for 2 days. After culturing, the medium was removed, the cells were washed with PBS, replaced with a differentiation medium (manufactured by CELL), and cultured for 10 days at 37° C. and 5% CO ₂ . After culturing, the medium was removed, the cells were washed with PBS, and maintenance medium containing 10 μg/mL (final concentration) of walnut polyphenol extract (manufactured by R&D SYSTEMS) or a solvent control (PBS, 1.5 μL/mL medium) was added. , 37°C, 5% CO ₂ environment for 48 hours. After culturing, the medium was removed, the cells were washed with PBS, mRNA was extracted from the normal human subcutaneous adipocytes using QIAzol Lysis Reagent (manufactured by QIAGEN), and Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies) was used. After cDNA was synthesized using the primer (QT00091945, manufactured by QIAGEN), the mRNA expression level of chemerin was measured by real-time qPCR method. In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was similarly measured. QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used for the measurement. The chemerin mRNA expression levels in the extract-added group and the solvent control group were corrected by the expression level of ACTB, and the relative expression level in the extract-added group was calculated when the expression level in the solvent control group was set to 1. The results are shown in FIG.

By the screening method of the present invention, it is possible to search for ingredients that are effective as agents for improving skin aging. Such an agent for improving skin aging is industrially very useful because it can be suitably contained in foods and drinks for improving skin aging and external skin preparations.

Claims (4)

A method for screening a skin aging improving agent using the activity of chemerin in subcutaneous adipocytes as an index,
The activity of the chemerin is a gene encoding a protein constituting the chemerin or the expression level of the protein, and the expression level in subcutaneous adipocytes to which the test substance is added is the subcutaneous adipocyte to which the test substance is not added. A method, wherein the test substance is determined to have skin aging-improving action when the expression level is small compared to the expression level . A method of designing a composition, comprising the steps of performing the screening method according to claim 1 and incorporating a substance selected by said steps. 3. The designing method according to claim 2 , wherein the composition is for improving skin aging. 4. The designing method according to claim 2 or 3 , wherein the composition is a food or drink or an external preparation for skin.

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