FGF21 receptor expression enhancer

Abstract

To provide a technique of exerting a function of Fibroblast Growth Factor 21 (FGF21) in a fibroblast or a melanocyte.SOLUTION: Expression of an FGF21 receptor in a fibroblast or a melanocyte is enhanced by using specific plant extract. Extract of Myrtaceae plant and / or extract of Asteraceae plant are used as an active ingredient of an FGF21 receptor expression enhancer in a fibroblast. Such an agent can be preferably blended into anti-wrinkle composition. The extract of Myrtaceae plant and / or extract of Leguminosae plant are used as an active ingredient of the FGF21 receptor expression enhancer in a melanocyte. Such an agent can be preferably blended into a whitening composition. In a preferable aspect, the extract of Myrtaceae plant is Syzygium aromaticum extract, the extract of Asteraceae plant is Achillea millefolium Linne extract, and the extract of Leguminosae plant is Aspalathus linearis extract.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to Fibroblast Growth Factor 21 (hereinafter referred to as FGF21 The present invention relates to a receptor expression enhancer, and further to an anti-wrinkle composition or a whitening composition containing the same. [Background technology]

[0002] Fibroblast growth factor (FGF) is a multifunctional intercellular signaling factor that exhibits a variety of actions, such as proliferation activity and differentiation induction, on various cells including fibroblasts. There are 22 types of FGF in humans, and among them, FGF21 has attracted attention as a candidate factor for therapeutic drugs against obesity and metabolic disorders caused by obesity. FGF21 is mainly secreted from the liver and is known as a multifunctional "anti-obesity factor" that promotes fat metabolism and suppresses appetite for sugar (Non-Patent Document 1). It has also been reported that FGF21 has the effect of suppressing melanin production (Non-Patent Document 2).

[0003] Like many other FGFs, FGF21 exhibits activity by binding to receptors present on the cell surface. Specifically, FGF21 binds at its N-terminus to FGR1 on the surface of target cells, and at its C-terminus to its co-receptor βKlotho (KLB), transducing a signal into the cell (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Itoh, N., Cell Tissue Res., 342, 1-11 (2010) [Non-Patent Document 2] R. Wang, et al., Biochem Biophys Res Commun. 19;490(2):466-471 (2017) Summary of the Invention [Problem to be solved by the invention]

[0005] Skin conditions caused by pigmentation such as age spots and freckles have a significant impact on the impression of the face, so there is a great demand for skin whitening agents and cosmetics for skin whitening. One of the causes of pigmentation is the increased melanin production in pigment cells (melanocytes), and as mentioned above, FGF21 inhibits the production of melanin in melanocytes, so it is expected that the function of FGF21 will have a whitening effect.

[0006] In addition, wrinkles also greatly affect the impression of the face, so there is a great demand for anti-wrinkle agents and anti-wrinkle cosmetics. The mechanisms by which wrinkles form are complex and diverse, but it is known that one of the causes is a decrease in collagen fibers in the skin. The present inventors have newly discovered that FGF21 enhances gene expression of decorin in fibroblasts. Decorin, a type of proteoglycan, plays a role in protecting collagen from degradation by associating with and binding to collagen fibers. Therefore, it is expected that anti-wrinkle effects can be obtained by promoting the production of decorin through the function of FGF21.

[0007] In this situation, an objective of the present invention is to provide a technique for enhancing the function of FGF21 in fibroblasts or melanocytes. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to solve the above problems, it was found that FGF binds to FGF receptors. The present inventors found that the expression level of FGF21 receptor is decreased by hydrogen peroxide generated by various life factors. Therefore, they focused on the fact that the function of FGF21 can be more effectively exerted by enhancing the expression level of FGF21 receptor. They then found plant extracts that have the effect of enhancing the expression of FGF21 receptor, and came to the conclusion that these can be used as active ingredients that bring about anti-wrinkle effects and whitening effects, thus completing the present invention. Furthermore, based on the new finding that anti-wrinkle effects are achieved by promoting the production of decorin through the function of FGF21, as described above, the inventors have completed a method for screening active ingredients of anti-wrinkle agents using the expression level of the FGF21 receptor as an indicator.

[0009] That is, the present invention is as follows. [1] A Fibroblast Growth Factor 21 (FGF21) receptor expression enhancer comprising one or more extracts selected from the group consisting of extracts of Myrtaceae plants, extracts of Asteraceae plants, and extracts of Legume plants. [2] An FGF21 receptor expression enhancer described in [1], which contains an extract of a Myrtaceae plant and / or an extract of a Asteraceae plant and enhances the expression of the FGF21 receptor in fibroblasts. [3] The FGF21 receptor expression enhancer according to [2], wherein the extract of the Myrtaceae plant is clove extract, and the extract of the Asteraceae plant is yarrow extract. [4] An anti-wrinkle composition comprising the FGF21 receptor expression enhancer according to [2] or [3]. [5] An FGF21 receptor expression enhancer described in [1], which contains an extract of a Myrtaceae plant and / or an extract of a legume plant and enhances the expression of the FGF21 receptor in melanocytes. [6] The FGF21 receptor expression enhancer described in [5], wherein the extract of the Myrtaceae plant is clove extract and the extract of the legume plant is rooibos extract. [7] A whitening composition comprising the FGF21 receptor expression enhancer described in [5] or [6]. [8] The composition described in [4] or [7], which is a cosmetic. [9] A method for screening anti-wrinkle agents using the expression level of FGF21 receptor in fibroblasts as an indicator.

[10] adding a test sample to fibroblasts; and The method according to [9], comprising a step of measuring the expression level of FGF21 receptor in the fibroblasts.

[11] The method according to

[10] , wherein the test sample is judged to have an anti-wrinkle effect when the expression level is greater than the expression level in fibroblasts to which the test sample has not been added. Effect of the Invention

[0010] The present invention provides a component that enhances the expression of FGF21 receptor in fibroblasts or melanocytes. In addition, by including such a component in a composition, it can be made into an anti-wrinkle composition or a whitening composition. Furthermore, the present invention provides a method for screening components having anti-wrinkle activity. [Brief description of the drawings]

[0011] [Figure 1] A diagram showing the expression of the KLB gene, an FGF21 receptor, in fibroblasts incubated with or without the addition of hydrogen peroxide. (A) Graph showing the relative expression level of the KLB gene when the control is set at 1. (B) Fluorescence micrograph of immunostained fibroblasts. The red color indicates the KLB protein. [Diagram 2] Graph showing the expression level of FGF21 receptor gene in fibroblasts incubated with or without the addition of plant extracts. The expression level is the relative level with the control set at 1. (A) Clove extract, FGFR1 gene. (B) Achillea millefolium extract, KLB gene. [Diagram 3] Graph showing the expression level of the decorin gene in fibroblasts when incubated with or without the addition of FGF21. The expression level is the relative level when the control is set to 1. [Figure 4] 1 is a graph showing the expression level of the decorin gene in fibroblasts when incubated with or without FGF21, or FGF21 and a plant extract. The expression level is the relative level when the control is set to 1. [Diagram 5] Graph showing the expression level of FGF21 receptor gene in melanocytes incubated with or without hydrogen peroxide. The expression level is the relative level with the control set at 1. (A) FGFR1 gene. (B) KLB gene. [Figure 6] Graph showing the expression level of FGF21 receptor gene in melanocytes incubated with or without the addition of plant extracts. The expression level is the relative level with the control set at 1. (A) Clove extract, FGFR1 gene. (B) Rooibos extract, KLB gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <1> FGF21 receptor expression enhancer The agent of the present invention contains one or more extracts selected from the group consisting of extracts of Myrtaceae plants, extracts of Asteraceae plants, and extracts of Leguminosae plants. As the Myrtaceae plant, the Myrtaceae genus plant is preferable, and Paeonia lactiflora is more preferable. As the Myrtaceae plant extract, clove extract is particularly preferable. Note that the clove extract is an extract of the flower buds (cloves) of Paeonia lactiflora of the Myrtaceae family. As the Asteraceae plant, the genus Yarrow is preferable, and Achillea millefolium Linne is more preferable. As the extract of the Asteraceae plant, Yarrow extract, which is an extract of Yarrow millefolium, is particularly preferable. As the legume plant, the Aspalathus genus plant is preferable, and rooibos (Aspalathus linearis) is more preferable. As the extract of the legume plant, rooibos extract is rooibos Extracts are particularly preferred.

[0013] The above-mentioned extract is a general term not only for the extract itself, but also for a fraction of the extract, a purified fraction, an extract or fraction, or a product obtained by removing the solvent from the purified product.

[0014] The extract may be any extract that is normally used in skin external preparations such as cosmetics and pharmaceuticals, or in compositions for oral ingestion, and may be extracted from a plant body by a conventional method. As the source of extraction, for example, the whole plant is used, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flowers, flower buds, fruits, and fruit juice are used for the extraction operation.

[0015] As the extraction solvent, one or more selected from polar solvents such as 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. are preferably used.

[0016] A specific example of the extraction method is to add 1 to 30 parts by mass of a solvent to 1 part by mass of the part of the plant body or the like used for extraction or its dried matter, immerse the part for several days at room temperature or several hours at a temperature near the boiling point, cool to room temperature, and then remove insoluble matter and / or the solvent as desired, and fractionate and purify the part by column chromatography or the like.

[0017] The content of one or more plant extracts selected from extracts of Myrtaceae plants, extracts of Asteraceae plants, and extracts of Legumes plants in the agent of the present invention is preferably 0.00001 to 0.09 mass%, more preferably 0.00005 to 0.03 mass%, and even more preferably 0.0001 to 0.003 mass%, calculated as solid matter relative to the total composition. By setting the content within the above range, the desired effect can be easily obtained and the freedom in formulation design can be ensured. The above content can be appropriately adjusted according to the administration route described below and the form of the composition to be contained.

[0018] Extracts from plants of the Myrtaceae family, Asteraceae family, and Leguminosae family all have the effect of enhancing the expression of the FGF21 receptor gene, i.e., each of these extracts is an active ingredient of an FGF21 receptor expression enhancer. Specifically, the extract of Myrtaceae plants and the extract of Asteraceae plants each have the effect of enhancing the expression of FGF21 receptor in fibroblasts.More specifically, the extract of Myrtaceae plants, particularly preferably Clove extract, has the effect of enhancing the expression of FGFR1 in fibroblasts, and the extract of Asteraceae plants, particularly preferably Achillea millefolium extract, has the effect of enhancing the expression of βKlotho (KLB) in fibroblasts. In addition, the extract of Myrtaceae plants and the extract of Leguminosae plants each have the effect of enhancing the expression of FGF21 receptor in melanocytes. More specifically, the extract of Myrtaceae plants, particularly preferably clove extract, has the effect of enhancing the expression of FGFR1 in melanocytes, and the extract of Leguminosae plants, particularly preferably rooibos extract, has the effect of enhancing the expression of KLB in melanocytes.

[0019] The FGF21 receptor expression enhancing effect of any substance can be confirmed by the fact that the expression level of FGF21 receptor in cells to which the substance has been added is greater than that in cells to which the substance has not been added, usually 110% or more, preferably 120% or more, more preferably 130% or more. The expression level of FGF21 receptor can be measured, for example, by carrying out PCR using a DNA fragment having a sequence that specifically binds to the sequence of the FGF21 receptor gene, specifically the FGFR1 gene or the KLB gene, as a primer, and measuring the amount of mRNA. In addition, for example, the intracellular amount of the protein encoded by the FGF21 receptor gene, specifically the FGFR1 gene or the KLB gene, can be quantitatively measured by a conventional method to determine the expression level of FGF21 receptor.

[0020] As shown in the examples below, it has been found that FGF21 enhances the gene expression of decorin in fibroblasts. It has also been found that when the expression of FGF21 receptor is enhanced in fibroblasts, the effect of enhancing the expression of decorin gene by FGF21 is further enhanced. Decorin protects collagen from degradation by associating with and binding to collagen fibers. A decrease in collagen fibers is one of the factors that cause wrinkles. Therefore, it is believed that enhancing the expression of FGF21 receptor in fibroblasts leads to the inhibition of collagen degradation through an increase in decorin, which in turn leads to the effect of inhibiting the occurrence or worsening of wrinkles. Therefore, the FGF21 receptor expression promoter in fibroblasts in the present invention can be suitably contained in an anti-wrinkle composition.

[0021] In addition, FGF21 is known to have the effect of suppressing melanin production in melanocytes. By enhancing the expression of FGF21 receptor, FGF21 becomes more active and functions in melanocytes. Therefore, the FGF21 receptor expression promoter in melanocytes of the present invention can be suitably contained in a whitening composition. The term "whitening" as used herein includes suppressing melanin production, suppressing pigmentation in the skin, and suppressing skin darkening.

[0022] The route of administration of the agent of the present invention is not particularly limited and may be transdermal, oral, nasal, intravenous, etc., but transdermal administration is preferred. Here, "administration" may be substituted with "ingestion". The dosage is not particularly limited, but taking into consideration the desired effect and safety, it is preferable to take 0.3 to 300 μg / day in solid terms of one or more plant extracts selected from the extracts of Myrtaceae plants, the extracts of Asteraceae plants, and the extracts of Leguminosae plants in one or several divided doses. In addition to taking it once, it is also preferable to take it continuously or intermittently for several weeks to several months.

[0023] When the agent of the present invention is to be ingested by transdermal administration, it is preferably formulated as a composition for external application to the skin. The form of the composition for external use on the skin is not particularly limited as long as it is applied externally to the skin, but preferred examples include cosmetics (including quasi-drugs) and pharmaceuticals, with cosmetics being more preferred. When an extract of a plant of the Myrtaceae family and / or an extract of a plant of the Asteraceae family, particularly preferably clove extract and / or yarrow extract, is contained, it is preferable to use it as an anti-wrinkle cosmetic.When an extract of a plant of the Myrtaceae family and / or an extract of a plant of the Fabaceae family, particularly preferably clove extract and / or rooibos extract, is contained, it is preferable to use it as a whitening cosmetic. The area to which the topical skin composition is applied is not particularly limited, but is usually the face, limbs, neck, and décolleté.

[0024] Examples of the dosage form of the external skin composition include, but are not limited to, lotion dosage forms, emulsion dosage forms such as milky lotion and cream, oil dosage forms, gel dosage forms, packs, cleansers, and the like. The composition for external use on the skin may be either a leave-on type or a leave-off type.

[0025] When the composition of the present invention is in the form of a composition for external use on the skin, ingredients that are usually used in the formulation of cosmetics, quasi-drugs, pharmaceuticals and the like can be optionally blended in the preparation 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.

[0026] <2> Screening Methods The screening method of the present invention screens for anti-wrinkle agents using the expression level of FGF21 receptor in fibroblasts as an index.

[0027] As mentioned above, enhancing the expression of FGF21 receptors in fibroblasts leads to anti-wrinkle effects through an increase in decorin. Therefore, ingredients that enhance the expression of FGF21 receptors in fibroblasts could be effective ingredients in anti-wrinkle agents.

[0028] In the screening method of the present invention, the expression level of the FGF21 receptor is determined by the expression level of the FGF21 receptor gene, specifically the FGFR1 gene or the KLB gene, or the expression level of the FGF21 receptor gene. The amount of a gene that is encoded by the gene may be the translation amount or abundance of the protein encoded by the gene. A preferred embodiment of the screening method of the present invention comprises the steps of adding a test sample to fibroblasts and culturing them, and measuring the expression level of FGF21 receptor in the fibroblasts. The expression level of FGF21 receptor can be measured by any method. For example, the expression level of FGF21 receptor can be quantified by performing PCR using a DNA fragment having a sequence that specifically binds to the sequence of FGF21 receptor gene, specifically FGFR1 gene or KLB gene, as a primer, and measuring the amount of mRNA. The base sequences of the genes that code for proteins contained in known FGF21 receptors are each publicly available, and those skilled in the art can design appropriate primers and subject them to PCR. Furthermore, for example, the amount of a protein encoded by an FGF21 receptor gene, specifically an FGFR1 gene or a KLB gene, present in a cell may be quantitatively measured by a standard method and used as the expression level of the FGF21 receptor.

[0029] In the screening method of the present invention, if the expression level of FGF21 receptor in fibroblasts cultured with the addition of a test sample is greater than the expression level when no test sample is added (control), the test sample is determined to have an anti-wrinkle effect. The degree of high expression level is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more, relative to the control. However, the test sample should be added within a concentration range that does not cause significant cytotoxicity. The expression level of at least one protein contained in the FGF21 receptor, such as FGFR1 or KLB, may be enhanced, and preferably the expression levels of two or more proteins are enhanced.

[0030] The fibroblasts used in the screening method of the present invention may be human-derived fibroblasts or fibroblasts derived from mammals such as rats, mice, and rabbits, without any particular limitations. In general, however, it is preferable to use normal cells, and it is more preferable to use normal human-derived fibroblasts. The cell culture conditions can be ordinary culture conditions, and are not particularly limited as long as they do not interfere with the implementation of the screening method of the present invention.

[0031] The test sample 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.

[0032] 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 sample is added to pre-cultured fibroblasts and incubated at 37°C for 24 to 72 hours. Thereafter, the expression level of the gene encoding the FGF21 receptor or the protein in the fibroblasts is measured by a conventional method. As a control, the expression level is also measured in fibroblasts cultured in the same manner without adding the test sample. If the expression level in the fibroblasts to which the test sample has been added is greater than the expression level in the fibroblasts to which the test sample has not been added (control), the test sample is determined to have an anti-wrinkle effect.

[0033] The component determined to have an anti-wrinkle effect 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 when designing an anti-wrinkle composition. For example, such a composition is preferably a skin topical composition, more preferably a cosmetic, and preferably an anti-wrinkle cosmetic.

[0034] When a component (active ingredient) determined to have anti-wrinkle action by the screening of the present invention is contained in a composition, its content (formulation amount) is usually 0.000001 mass% or more, preferably 0.00001 mass% or more, more preferably 0.0001 mass% or more of the total composition, and usually 15 mass% or less, preferably 10 mass% or less, more preferably 5 mass% or less. If the content (formulation amount) of the active ingredient 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 active ingredient contained in the composition may be not only one type but also two or more types. In addition, the formulation amount of animal and plant extracts, etc. is the amount converted into solid matter.

[0035] When an ingredient (active ingredient) determined to have an anti-wrinkle effect by the screening of the present invention is to be contained in an anti-wrinkle composition, ingredients that are usually used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be optionally blended in the composition during production. <1> The explanation given above can be followed. EXAMPLES

[0036] 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.

[0037] <Reference example 1> The various extracts were prepared according to the following procedure. Clove extract: Dried cloves (flower buds of the Myrtaceae family, Myrtaceae) are mixed with 10 times the amount of 30% ethanol aqueous solution, and the liquid extracted by reflux is freeze-dried to powder. Achillea millefolium extract: Dried whole plant of Achillea millefolium of the Asteraceae family was crushed, and 10 times the amount of 30% ethanol solution was added, and the liquid extracted by refluxing was freeze-dried to powder. Rooibos extract: Dried rooibos leaves from the Aspalathus genus of the legume family are crushed, 10 times the amount of 30% ethanol solution is added, and the liquid extracted by reflux is freeze-dried to powder.

[0038] <Reference Example 2> Examination of the effect of hydrogen peroxide on the expression level of FGF21 receptor gene in fibroblasts The expression level of the FGF21 receptor gene in fibroblasts to which hydrogen peroxide had been added was measured by the following procedure. Human normal fibroblasts were cultured in 24-well plates at 3 × 10 in DMEM medium containing 10% FBS. 4 Cells were seeded per well and cultured overnight at 37°C under 5% CO2. After culture, the medium was removed and replaced with DMEM medium containing 1% FBS and 30 μM hydrogen peroxide. The cells were further cultured for 3 hours. After the culture, the cells were washed with PBS, replaced with DMEM medium containing 10% FBS, and further cultured for 24 hours. After the culture, the cells were washed with PBS and collected, and RNA was extracted with QIAcube (QIAGEN) using QIAshredder and RNeasy Mini Kit (QIAGEN). The extracted RNA was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrigen), and RT-qPCR was performed using SYBR (QIAGEN) with QuantStudio 7 (Applied Biosystems) to analyze the expression level of the KLB gene. The β-Actin gene was used as an endogenous control. In addition, the cells collected after the culture were fixed using 4% paraformaldehyde by standard methods. KLB protein was immunostained using an antibody (Abcam) and observed under a fluorescent microscope. did.

[0039] FIG. 1 shows the expression level of KLB gene mRNA in fibroblasts to which hydrogen peroxide had been added, expressed as a relative value when the expression level of KLB gene mRNA in fibroblasts from the solvent control was set at 1. In fibroblasts treated with hydrogen peroxide, the expression levels of each of the KLB gene mRNAs were significantly decreased compared to the control, and the amount of KLB protein was also decreased compared to the control.

[0040] <Test Example 1> Examination of the effect of various extracts on the expression level of FGF21 receptor gene in fibroblasts The expression level of the FGF21 receptor gene in fibroblasts to which the test extract had been added was measured according to the following procedure. Human normal fibroblasts were cultured in 24-well plates at 3 × 10 in DMEM medium containing 10% FBS. 4Cells were seeded per well and cultured overnight at 37°C and 5% CO2. After culture, the medium was removed and replaced with 1% FBS-containing DMEM medium containing 0.1% by weight of the test extract adjusted to a solid content concentration of 1.0% by mass, and cultured for another 48 hours. After culture, the cells were washed with PBS and collected, and RNA was extracted using QIAshredder, RNeasy Mini Kit (QIAGEN) and QIAcube (QIAGEN). The extracted RNA was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrigen), and RT-qPCR was performed using SYBR (QIAGEN) and QuantStudio 7 (Applied Biosystems) to analyze the expression levels of the FGFR1 gene and KLB gene. The β-Actin gene was used as an endogenous control. The test extracts used were all prepared according to the method described in the Reference Example.

[0041] FIG. 2 shows the mRNA expression levels of the FGFR1 gene and the KLB gene in fibroblasts to which each extract was added, expressed as relative values ​​when the mRNA expression level of each gene in fibroblasts in the solvent control was set at 1. In fibroblasts to which any of the extracts had been added, it was found that the mRNA expression levels of the FGFR1 gene and the KLB gene were significantly increased compared to the control.

[0042] <Test Example 2> Examination of the effect of FGF21 on decorin gene expression in fibroblasts The expression level of the decorin gene in fibroblasts to which FGF21 had been added was measured by the following procedure. Human normal fibroblasts were cultured in 24-well plates at 4 × 10 in DMEM medium containing 10% FBS. 4Cells were seeded per well and cultured for 8 hours at 37℃ and 5% CO2. After culture, the medium was removed and replaced with DMEM medium containing 0.1% FBS and cultured overnight. After culture, the cells were washed with PBS, and the medium was replaced with DMEM medium containing 0.1% FBS containing 100 pg / mL FGF21 and cultured for a further 72 hours. After culture, the cells were washed with PBS and collected, and RNA was extracted using QIAshredder and RNeasy Mini Kit (QIAGEN) on a QIAcube (QIAGEN). The extracted RNA was analyzed using a SuperScript VILO cDNA Synthesis Kit (Invitrigen). The cDNA was generated using SYBR (QIAGEN) and analyzed by QuantStudio 7 (Applied Biosystems). The expression levels of the decorin gene were analyzed by RT-qPCR. used the β-Actin gene.

[0043] FIG. 3 shows the mRNA expression level of the decorin gene in fibroblasts to which FGF21 had been added, expressed as a relative value when the mRNA expression level of the decorin gene in fibroblasts in the solvent control was set at 1. In fibroblasts treated with FGF21, the mRNA expression level of the decorin gene was significantly increased compared to the control, indicating that the presence of FGF21 enhances the expression of the decorin gene. Because decorin protects collagen from degradation, it is speculated that it inhibits collagen degradation, leading to the effect of preventing wrinkles from occurring or slowing their worsening.

[0044] <Test Example 3> Examination of the effect of FGF21 receptor expression enhancer on decorin gene expression level in fibroblasts The amount of decorin gene expression was measured in fibroblasts to which an extract that had been confirmed to enhance FGF21 receptor expression in the presence of FGF21 was added, using the following procedure. Human normal fibroblasts were cultured in 24-well plates at 4 × 10 in DMEM medium containing 10% FBS. 4 Cells / well were seeded and cultured for 8 hours at 37°C and 5% CO2. After culture, the medium was removed and replaced with DMEM medium containing 0.1% FBS and cultured overnight. After culture, the cells were washed with PBS and replaced with DMEM medium containing 0.1% FBS containing 100 pg / mL FGF21, or DMEM medium containing 1% FBS containing 100 pg / mL FGF21 and 0.1% by weight of clove extract and 0.1% by weight of yarrow extract adjusted to a solid content concentration of 1.0 mass%, and cultured for another 72 hours. After culture, the cells were washed with PBS and collected, and RNA was extracted using QIAshredder, RNeasy Mini Kit (QIAGEN) and QIAcube (QIAGEN). The extracted RNA was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrigen), and analyzed using SYBR (QIAGEN) with QuantStudio 7 (Applied The expression levels of each decorin gene were analyzed by RT-qPCR using a RT-PCR kit manufactured by Biosystems. The β-Actin gene was used as an endogenous control. The extracts used were all prepared according to the method of Reference Example.

[0045] Figure 4 shows the mRNA expression level of the decorin gene in fibroblasts to which FGF21 or FGF21 and two types of extracts were added, expressed as a relative value when the mRNA expression level of each gene in fibroblasts treated with the solvent (control) was set to 1. In fibroblasts treated with FGF21, the mRNA expression level of the decorin gene was significantly increased compared to the control. In fibroblasts treated with FGF21 and two kinds of extracts, the mRNA expression level of the decorin gene was significantly increased compared to both the control and FGF21-added groups. From these results, it can be seen that the expression of FGF21 receptor was enhanced by clove extract and yarrow extract, which made it easier for FGF21 bound to the receptor to be activated, and thus further enhanced the effect of FGF21 in enhancing decorin expression. Therefore, it is speculated that these extracts, which are the active ingredients of the FGF21 receptor expression enhancer, contribute to the anti-wrinkle effect by further promoting the enhancement of decorin expression.

[0046] <Reference Example 3> Examination of the effect of hydrogen peroxide on the expression level of FGF21 receptor gene in melanocytes The expression level of the FGF21 receptor gene in melanocytes to which hydrogen peroxide had been added was measured by the following procedure. Human normal melanocytes were cultured in 24-well plates at 1 × 10 in Medium 254 medium supplemented with HMGS. 4 Cells were seeded per well and allowed to grow to confluence at 37℃ in a 5% CO2 environment. After culturing, the medium was removed and replaced with HMGS-supplemented Medium 254 medium containing 30 μM hydrogen peroxide, and cultured for a further 24 hours. After culturing, the cells were washed with PBS, and the medium was replaced with HMGS-supplemented Medium 254 medium, and cultured for a further 48 hours. After culturing, the cells were washed with PBS and collected, and RNA was extracted with QIAcube (QIAGEN) using QIAshredder and RNeasy Mini Kit (QIAGEN). The extracted RNA was analyzed using SuperScript VILO cDNA The cDNA was generated using a Synthesis Kit (Invitrigen), and RT-qPCR was performed using SYBR (QIAGEN) and QuantStudio 7 (Applied Biosystems) to analyze the expression levels of the FGFR1 gene and the KLB gene. The β-Actin gene was used as an endogenous control.

[0047] FIG. 5 shows the mRNA expression levels of the FGFR1 gene and the KLB gene in melanocytes to which hydrogen peroxide had been added, expressed as relative values ​​when the mRNA expression level of each gene in melanocytes of the solvent control was set at 1. In melanocytes to which hydrogen peroxide had been added, it was found that the mRNA expression levels of the FGFR1 gene and the KLB gene were significantly decreased compared to the control.

[0048] <Test Example 4> Examination of the effect of various extracts on the expression level of FGF21 receptor gene in melanocytes The expression level of the FGF21 receptor gene in melanocytes to which the test extract was added was measured by the following procedure. Human normal melanocytes were cultured in 24-well plates at 1 × 10 in Medium 254 medium supplemented with HMGS. 4 Cells were seeded per well and cultured at 37°C and 5% CO2 until confluent. After culture, the medium was removed and replaced with HMGS-added Medium 254 medium containing 0.1% by weight of the test extract adjusted to a solid content concentration of 1.0% by mass, and cultured for another 48 hours. After culture, the cells were washed with PBS and collected, and RNA was extracted using QIAshredder, RNeasy Mini Kit (QIAGEN) and QIAcube (QIAGEN). The extracted RNA was converted to cDNA using SuperScript VILO cDNA Synthesis Kit (Invitrigen), and RT-qPCR was performed using SYBR (QIAGEN) and QuantStudio 7 (Applied Biosystems) to analyze the expression levels of the FGFR1 gene and KLB gene. The β-Actin gene was used as an endogenous control. The test extracts used were all prepared according to the method described in the Reference Example.

[0049] FIG. 6 shows the mRNA expression levels of the FGFR1 gene and the KLB gene in melanocytes to which each extract was added, expressed as relative values ​​when the mRNA expression level of each gene in melanocytes of the solvent control was set at 1. In melanocytes to which any of the extracts was added, it was found that the mRNA expression levels of the FGFR1 gene and the KLB gene were significantly increased compared to the control. [Industrial Applicability]

[0050] The present invention provides a component that enhances the expression of FGF21 receptor in fibroblasts or melanocytes. In addition, by incorporating such a component into a composition, it can be made into an anti-wrinkle composition or a whitening composition. Furthermore, the present invention provides a method for screening components having anti-wrinkle activity. These inventions contribute to development in the beauty industry and meet consumer demand, and are very useful industrially.

Claims

1. A Fibroblast Growth Factor 21 (FGF21) receptor expression enhancer comprising one or more extracts selected from the group consisting of extracts of Myrtaceae plants, extracts of Asteraceae plants, and extracts of Leguminosae plants.

2. The FGF21 receptor expression enhancer according to claim 1, which contains an extract of a Myrtaceae plant and / or an extract of a Asteraceae plant and enhances the expression of the FGF21 receptor in fibroblasts.

3. 3. The FGF21 receptor expression enhancer according to claim 2, wherein the extract of the Myrtaceae plant is clove extract and the extract of the Asteraceae plant is yarrow extract.

4. An anti-wrinkle composition comprising the FGF21 receptor expression enhancer according to claim 2 or 3.

5. The composition described in claim 4, which is a cosmetic.

6. The FGF21 receptor expression enhancer according to claim 1, which contains an extract of a Myrtaceae plant and / or an extract of a Leguminosae plant and enhances the expression of the FGF21 receptor in melanocytes.

7. The FGF21 receptor expression enhancer according to claim 6, wherein the extract of the Myrtaceae plant is clove extract and the extract of the Leguminosae plant is rooibos extract.

8. A whitening composition comprising the FGF21 receptor expression enhancer according to claim 6 or 7.

9. The composition according to claim 8, which is a cosmetic.

10. A method for screening anti-wrinkle agents using the expression level of FGF21 receptor in fibroblasts as an index.

11. adding a test sample to fibroblasts; and The method according to claim 10, further comprising measuring the expression level of FGF21 receptor in the fibroblasts.

12. The method according to claim 11, wherein the test sample is determined to have an anti-wrinkle effect when the expression level is greater than the expression level in fibroblasts to which the test sample has not been added.

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