JP2020014413A - Skin aging improver screening method - Google Patents

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

  The present invention relates to a method for screening a skin aging improving agent.

  Since skin aging symptoms such as wrinkles and tarmi have a great effect on the appearance impression, there is a high interest in improving them. In recent years, anti-aging agents, anti-wrinkle agents, moisturizing agents, and the like for the purpose of improving skin aging symptoms have been actively developed (Patent Documents 1 to 3, and the like).

  In skin cells, aquaporins known as water channels are known to be expressed on cell membranes and play a role in taking in low-molecular substances such as intercellular water into cells. In humans, the existence of 13 types of aquaporins (AQP0 to AQP12) is known. AQP3 is mainly present in epidermal cells, and is considered to play a role in maintaining the water content and elasticity of the skin. Therefore, it has been proposed to promote the production of aquaporin in epidermal cells to improve the soundness of the skin (Patent Document 4 and the like). However, the function of aquaporins in fibroblasts is not known.

JP-A-2013-131797 JP 2015-174857 A JP 2003-171225 A JP 2015-134751 A

Conventional anti-aging agents for skin have some anti-aging effects, but it is hard to say that satisfactory effects have been obtained. There is also a need for further elucidation of the mechanism by which skin aging occurs.
In view of such circumstances, an object of the present invention is to search for a component effective as a skin aging improving agent by a new approach to skin aging symptoms, and to provide a new screening method therefor.

  The present inventors have conducted intensive studies in order to solve the above-mentioned problems. I found it. Then, the present inventors have found that the skin aging symptom can be improved by enhancing the expression of aquaporin, and have completed the present invention.

That is, the present invention is as follows.
[1] A method of screening for an agent for improving skin aging by using aquaporin activity in fibroblasts as an index.
[2] The activity of the aquaporin is a gene encoding a protein constituting the aquaporin or the expression level of the protein, and the expression level in a fibroblast to which a test substance is added is a cell to which no test substance is added. The method according to [1], wherein the test substance is determined to have a skin aging-improving activity when the expression level is larger than the expression level in the above.
[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 a step of performing the screening method according to any one of [1] to [3], and a step of including a substance selected by the step.
[5] The design method according to [4], wherein the composition is for improving skin aging.
[6] The design method according to [4] or [5], wherein the composition is an external preparation for skin or a food or drink.

  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 being contained in a skin external preparation or food or drink.

The graph which shows the relative expression level of each gene of aquaporin 1 and 3 in the human fibroblast to which active oxygen was added. 4 is a graph showing the cell proliferation ability of human fibroblasts in which aquaporins 1 and / or 3 have been knocked down. Left: Graph showing the relative expression level of aquaporin 1 gene in human fibroblasts to which cornflower extract was added. Right: Graph showing the relative expression level of aquaporin 3 gene in human fibroblasts to which eelgrass extract was added.

The screening method of the present invention is characterized by using aquaporin activity in fibroblasts as an index.
Aquaporin is known as a water channel expressed on the cell membrane, and it is known that the expression level of epidermal keratinocytes decreases with aging.
The present inventors have found that the expression level of aquaporin is also involved in the development of skin aging symptoms in fibroblasts. More specifically, as shown in the experimental examples described below, active oxygen, which is an aging factor in fibroblasts, reduces the expression level of aquaporin. Fibroblasts in which the expression level of aquaporin has been reduced and its activity has been reduced have significantly reduced proliferative ability. Decreased proliferation ability of fibroblasts that produce collagen, ie, reduces skin firmness and elasticity, causes skin aging symptoms. In addition, the present inventors have found that a decrease in the expression level of aquaporin is caused by tenascin X (TNXB) involved in collagen fiber cross-linking, type 2 collagen (COL7A1), and collagen 17 which functions to adhere epithelial tissue to the basement membrane. (COL17) and the expression of a protein (COMP) involved in promoting collagen formation.
In such a skin aging flow, it is possible to suppress a decrease in the proliferation ability of skin fibroblasts by enhancing the activity of aquaporin in fibroblasts, thereby suppressing and improving the occurrence of skin aging symptoms. it can. In other words, a substance that enhances the activity of aquaporin in fibroblasts can be an agent for improving skin aging.

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

  In a preferred embodiment of the screening method of the present invention, when the expression level of aquaporin in fibroblasts to which the test substance is added is larger than the expression level of aquaporin in fibroblasts to which no test substance is added (control) Then, the test substance is determined to have a skin aging improving effect.

The aquaporin used as an index in the screening method of the present invention is not particularly limited, but preferably includes aquaporin 1 (AQP1) and / or aquaporin 3 (AQP3). More preferably, a combination of AQP1 and AQP3 is used as an index. In a more preferred embodiment, a test substance in which the expression level of both AQP1 and AQP3 is higher than that of a control is selected as a skin aging improving agent.

The expression level of the gene encoding aquaporin 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 to perform quantitative detection. The human gene sequence encoding aquaporin has been published, and those skilled in the art can appropriately design primers and use them for PCR.
Alternatively, for example, the amount of aquaporin protein on the cell membrane may be measured by a conventional method, for example, by an immunological technique using an antibody, etc., and used as the gene expression level.

Fibroblasts are used as cells used in the screening method of the present invention, and fibroblasts usually exist in the dermis.
As the conditions for culturing the cells, other than ordinary culture conditions, any culture conditions that do not hinder the execution of the screening method of the present invention and specifically do not hinder the measurement of the aquaporin expression level can be applied without particular limitation. be able to.

The test substance targeted by the screening method of the present invention may be any of a pure substance, an extract derived from animals and plants, or a mixture thereof.
An extract derived from animals and plants means not only an extract derived from an animal or a plant itself, but also a fraction of an extract, a purified fraction, an extract or a fraction, a general term for a solvent-extracted product of a purified product, Examples of plant-derived extracts include native or grown plants, extracts using those sold as raw materials of Chinese herbal medicines, and commercially available extracts.
For the extraction operation, in addition to using the whole plant part, plant parts, above-ground parts, rhizomes, tree trunks, leaves, stems, flowers, flower buds, fruits and the like can be used, but these are crushed in advance. Alternatively, it is preferable to improve the extraction efficiency by shredding. Examples of the extraction solvent 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 two or more selected from polar solvents such as the above can be exemplified as suitable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a site used for extraction of a plant or the like or a dried product thereof, and at room temperature for several days, at a temperature near the boiling point. If so, a method of immersing for several hours, cooling to room temperature, removing insolubles and / or solvent if desired, 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 will be described below, but the present invention is not limited to the following contents without departing from the gist of the present invention, and can be appropriately modified and carried out.
First, a test substance is added to fibroblasts and incubated for 24 to 48 hours. Thereafter, mRNA is extracted from the cells, and the expression level of the 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 no test substance has been added. When the expression level of the gene in the cells to which the test substance is added is larger than the expression level of the gene in the cells to which the test substance is not added (control), it is determined that the test substance has a skin aging improving effect. . The determined substance can be a skin aging improving agent that can be suitably contained in a composition for improving skin aging.
The degree of the change in the expression level is preferably 120% or more, more preferably 150% or more, and even more preferably 200% or more with respect to the control.

That the test substance selected by screening has a skin aging-improving effect is, for example, the subjective evaluation of the subject to whom the composition containing the substance has been applied, the evaluation of wrinkles and tarmi by image analysis, the viscoelasticity of the skin It can be confirmed by a well-known method such as evaluation, evaluation of the amount of collagen produced by the skin, and evaluation of the amount of water retained in the skin.

The substance (skin aging improving agent) determined to have the effect of improving skin aging symptoms by the screening method of the present invention can be contained in the composition by any preparation method. That is, the screening method of the present invention can be suitably used for designing a composition. As such a composition, for example, a composition for external use on the skin, a composition of food and drink, and the like are suitably mentioned, and these can be preferably applied to a use for improving skin aging and an anti-aging use.
The substance (skin aging improving agent) determined to have the action of improving skin aging symptoms by the screening of the present invention is a new mechanism targeting skin aging caused by aquaporin in fibroblasts. In terms of improving symptoms, it can be an effective anti-aging composition component by a new approach.

When a substance (skin aging improver) determined to have an effect of improving skin aging symptoms by the screening of the present invention is contained in the composition, the content (blending amount) is usually 0.00001% by mass or more. , Preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, usually 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less. If the content (blending amount) of the skin aging improver is too small, the desired effect may not be easily obtained, and if it is too large, the effect may not only reach a plateau but also impair the flexibility of formulation of the composition. is there. The type of the skin aging improver to be contained in the composition is not limited to one type, and may be two or more types. In addition, about the extract of animals and plants, the compounding quantity shall be a solid equivalent.
In addition, the composition may also contain a skin aging improving component other than the substance determined to have an effect of improving skin aging symptoms by the screening of the present invention.

When the skin aging improver according to the present invention is contained in a composition for external use on the skin, at the time of its production, ingredients commonly used in the preparation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be arbitrarily blended. .
Such optional components include, for example, hydrocarbons such as squalane, petrolatum, microcrystalline wax, jojoba oil, carnauba wax, esters such as octyldodecyl oleate, olive oil, tallow, triglycerides such as 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 sulfosuccinates and sodium polyoxyethylene alkyl sulfate; and amphoteric surfactants such as alkyl betaine salts , Surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, polyoxy Nonionic surfactants such as tylene fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin and 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, coloring agents, preservatives , Powder and the like can be arbitrarily compounded.
The composition for external use on the skin can be produced by treating and blending the above-mentioned components according to a conventional method. In addition, the form can be an arbitrary form such as a lotion form, an emulsifier form, and an oil form.

When the skin aging improver according to the present invention is contained in foods and drinks, components that are usually used in the production of foods can be arbitrarily mixed during the production.
As such optional components, for example, proteins, carbohydrates, fats, nutrients, seasonings, flavors and the like can be used. Carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as ordinary sugars such as dextrin, cyclodextrin and the like, and xylitol, sorbitol, Sugar alcohols such as erythritol; As the flavor, a natural flavor (eg, thaumatin, stevia extract) and a synthetic flavor (eg, saccharin, aspartame) can be used. In addition, additives that are used in the above-mentioned pharmaceutical compositions and that are usually added to foods may also be used.

  The form of the food or drink may be any form such as a liquid, a paste, a solid, a powder, and a granule. Tablet confectionery, liquid food, feed, etc. are also included in the form of food and drink.

  In addition, other forms may be included in general foods and drinks, for example, bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread flour products; instant noodles, cup noodles, retort / cooked foods , Cooking cans, microwave foods, instant soups and stews, instant miso soups and soups, canned soups, frozen and dried foods, and other instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, Agricultural products such as cereals (processed cereals); Canned fish products, fish ham / sausage, fish paste products, fishery delicacies, fishery processed products such as boiled tsukudani; livestock canned products / pastes, processed livestock products such as meat ham / sausage ; Processed milk, milk drinks, yogurt, lactic acid drinks, cheese, ice cream, powdered milk, cream, etc. Milk and dairy products such as dairy products; butters, margarines, vegetable oils and other fats and oils; soy sauce, miso, sauces, tomato processing seasonings, mirins, vinegars and other basic seasonings; cooking mixes, curry bases , Sauces, dressings, noodle soups, spices and other complex seasonings and other complex seasonings and foods; frozen foods such as frozen foods, semi-cooked frozen foods and cooked frozen foods; caramels, candy and chewing gum , Chocolate, cookies, biscuits, cakes, pies, snacks, crackers, sweets such as Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery; carbonated beverages, natural juices, fruit juice beverages, fruit juice soft drinks, fruit juice beverages, and fruit grains Fruit drinks, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, etc. Beverages; to foods other than the above, the skin aging improving agent according to the present invention may be added.

Examples of the food composition containing the skin aging improver according to the present invention include normal foods, beverages, functionally labeled foods, health functional foods such as foods for specified health use, supplements, and the like. Food is preferred.
When the food composition according to the present invention is used for improving skin aging or for anti-aging, it may be provided with an indication regarding its usefulness and functionality when commercialized.
Such "display" acts include all acts for informing the consumer of the use, and include uses such as "for improving skin aging", "for anti-aging", and "for skin firmness and elasticity". Any expression that can be recalled or analogized corresponds to the “display” act of the present invention, regardless of the purpose of display, the content of the display, the object / medium to be displayed, and the like.
In addition, “display” is preferably performed in an expression that allows the consumer to directly recognize the use. To be specific, the goods related to food and drink or the packaging, containers, etc. of the goods described above are transferred, delivered, displayed for transfer or delivery, and imported, acts related to goods, advertising, price list, Catalogs, pamphlets, POPs and other advertising materials at the point of sale, or business documents, exhibiting or distributing the above-mentioned applications, or describing the above-mentioned applications in information containing these, and electromagnetically (such as the Internet) ) Act provided by the method. In addition, when the food composition of the present invention is approved based on various systems determined by the administration of health functional foods and the like, and is implemented under the approval, it is preferable to display in a form based on the approval.

The preferred intake of the food composition containing the skin aging improver according to the present invention, when taken by an adult, may be 0.001 to 1000 mg / kg per day in terms of solids of the skin aging improver. It is preferable from the viewpoint that the effect is sufficiently exerted, and further 0.01 to 100 mg / k.
g, particularly preferably 0.1 to 10 mg / kg. This daily dose can be taken at once or in several portions. In addition to a single ingestion, it is preferable to ingest it continuously or intermittently for several weeks to several months.

  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 unless it exceeds the gist thereof.

<Reference Example 1> Confirmation of the effect of active oxygen on aquaporin in fibroblasts Using a DMEM medium containing 10% FBS, human fibroblasts were seeded in a 24-well plate at 2 × 10 ⁴ cells / well, and 5% at 37 ° C. Culture was performed for 24 hours in a CO ₂ environment. After the culture, the medium was removed, replaced with a DMEM medium containing 1% FBS, and the cells were further cultured for 24 hours. After adding 50 μM of active oxygen to each well and culturing for 3 hours, the active oxygen was removed and the cells were further cultured for 21 hours.
The mRNA of the above fibroblasts is extracted using the RNeasy Mini Kit (manufactured by QIAGEN), and cDNA is synthesized using the Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies). The mRNA expression levels of AQP1 and AQP3 were measured by real-time qPCR using QIAGEN. Also, the mRNA expression level of GAPDH, which is an endogenous control gene, was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used.
With respect to the mRNA expression levels of AQP1 or AQP3 in the active oxygen-added group and the non-added group, the relative expression levels of the active oxygen-added group were corrected by correcting the expression level of GAPDH with the expression level of the non-added group as 100. Calculated.
The results are shown in FIG. It can be seen that the addition of active oxygen significantly reduces the expression levels of AQP1 and AQP3 mRNA in fibroblasts.

<Reference Example 2> Confirmation of influence of decrease in aquaporin expression level on proliferation ability of fibroblasts Human fibroblasts were seeded at 5 × 10 ³ cells / well in a 24-well plate using DMEM medium containing 10% FBS, and then incubated at 37 ° C. and cultured for 24 hours under · 5% CO ₂ environment. Using a lipofection reagent, siRNA (AQP1: s1516, manufactured by Life technologies, AQP3: s1521, manufactured by Life technologies) was introduced, AQP1 and / or AQP3 were knocked down, and the cells were further cultured for 72 hours. Non-coding siRNA was used as a control. After culturing, the medium was removed from the plate, washed with PBS (-), and the well was diluted with 20-fold tetrazolium salt WST-8 reagent (Cell Counting Kit-8; Dojindo Laboratories) using the medium in the wells. 1000 μL / well was added and reacted for 2 hours in a CO ₂ incubator. The absorbance at 450 nm and 650 nm was measured with a plate reader, and the difference between the measured values (Abs.450-Abs.650) was calculated as the number of cells, and calculated as the relative amount when the control was set to 1.
The results are shown in FIG. It can be seen that the proliferative ability of AQP1 and / or AQP3 knockdown fibroblasts was significantly reduced. In particular, when both AQP1 and AQP3 were knocked down, the cell proliferation ability was significantly reduced.

<Example 1> Screening of skin aging improving agent A skin aging improving agent was screened by the following procedure using aquaporin gene expression as an index.
Using a DMEM medium (manufactured by Sigma), human fibroblasts were seeded at 2 × 10 ⁴ cells / well in a 24-well plate and cultured at 37 ° C. in a 5% CO ₂ environment for 24 hours. After the culture, the medium was removed, and the cells were washed with PBS. Then, cornflower extract (manufactured by Koei Kogyo Co., Ltd.) 0.2% by weight (final concentration), eelgrass extract (manufactured by Technoble Co., Ltd.) 0.2% % (Final concentration) or a maintenance medium containing a solvent control (PBS, 1.5 μL / mL medium) was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ environment for 24 hours. After culturing, the medium is removed, the cells are washed with PBS, and the mRNA of human fibroblasts is extracted using QIAzol Lysis Reagent (manufactured by QIAGEN), and then extracted using Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies). After synthesizing the cDNA, the mRNA expression level of AQP1 or AQP3 was measured by a real-time qPCR method using primers (AQP1: QT00013237, manufactured by QIAGEN, AQP3: QT00212996, manufactured by QIAGEN). Also, the mRNA expression level of GAPDH, which is an endogenous control gene, was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used. The mRNA expression level of AQP1 or AQP3 in the extract-added group and the solvent control group was 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 100.
The results are shown in FIG. It can be seen that the cornflower extract increases the expression level of AQP1 and the eel extract increases the expression level of AQP3 in fibroblasts, which can be effective ingredients of the skin aging improving agent.

  By the screening method of the present invention, a component effective as a skin aging improving agent can be searched for. Such an agent for improving skin aging can be suitably contained in a skin external preparation for improving skin aging, a food or drink, and is very useful in industry.

Claims (6)

  A method for screening an agent for improving skin aging by using aquaporin activity in fibroblasts as an index.   The activity of the aquaporin is the expression level of a gene encoding the protein constituting the aquaporin or the protein, and the expression level in a fibroblast to which a test substance is added, in a fibroblast to which no test substance is added. The method according to claim 1, wherein the test substance is determined to have a skin aging ameliorating effect when the expression level is larger than the expression level.   The method according to claim 1 or 2, wherein the aquaporin is aquaporin 1 and / or aquaporin 3.   A method for designing a composition, comprising a step of performing the screening method according to any one of claims 1 to 3, and a step of containing the substance selected in the step.   The design method according to claim 4, wherein the composition is for improving skin aging.   The design method according to claim 4, wherein the composition is a food or drink or an external preparation for skin.