RBMS3 gene expression enhancer

Abstract

To provide a technology for enhancing the expression of RBMS3 gene.SOLUTION: Provided is a technology for enhancing the expression of RBMS3 gene with an RBMS3 gene expression enhancer containing at least one selected from the group consisting of Althaea officinalis extract, Sambucus nigra extract, Tilia cordata Mill. extract, Arnica montana extract, Alpinia zerumbet extract, Eucalyptus globulus subsp. extract, Cymbopogon citratus extract, and Origanum majorana extract.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent for enhancing expression of the RNA-binding motif single-stranded-interacting protein 3 (hereinafter referred to as RBMS3) gene, and further to a composition for external application to the skin containing the same for enhancing expression of the RBMS3 gene. [Background technology]

[0002] The RBMS3 gene is known as a gene that encodes an RNA-binding protein (RBMS3) belonging to the c-myc gene single-stranded binding protein family, and has traditionally been known as a suppressor gene for various cancers, such as nasopharyngeal carcinoma (Non-Patent Document 1) and breast cancer (Non-Patent Document 2, Non-Patent Document 3).

[0003] On the other hand, the uses of the plant extracts used in the present invention have been reported as follows: for example, Altea extract is used as an elastin production promoter (Patent Document 1), Elderberry extract is used as a GLUT1 expression promoter (Patent Document 2), and Tilia cordata extract is used as a sebum secretion promoter (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2012-056933 A [Patent Document 2] Patent Publication No. 2021-091652 [Patent Document 3] JP 2017-066106 A [Non-patent literature]

[0005] [Non-Patent Document 1] Chen J, et al., PLOS ONE, volume 7, 1-12 (2012) [Non-Patent Document 2] Wang C. et al., Technology in Cancer Research & Treatment, volume 20, 1-9 (2021) [Non-Patent Document 3] C. James Block et al., Oncogene, volume 40, 6430-6442 (2021) Summary of the Invention [Problem to be solved by the invention]

[0006] However, it is not known that plant extracts such as Althea extract have the effect of enhancing the expression of the RBMS3 gene. Therefore, an objective of the present invention is to provide a technique for enhancing the expression of the RBMS3 gene. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that althea extract, elder extract, Tilia linden extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract enhance the expression of the RBMS3 gene, thereby completing the present invention.

[0008] That is, the present invention is as follows. [1] An RBMS3 gene expression enhancer containing at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract. [2] A composition for external application to the skin for enhancing RBMS3 gene expression, comprising at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract. thing. Effect of the Invention

[0009] The present invention provides a component that enhances the expression of the RBMS3 gene. Furthermore, the present inventors have found that the generation of age spots and freckles can be suppressed when the expression of the RBMS3 gene is enhanced, and have found that the present invention can be used for preventing age spots and freckles. [Brief description of the drawings]

[0010] [Figure 1] Graph showing the expression level of RBMS3 gene in fibroblasts incubated with or without the addition of Althea extract. The expression level is the relative level when the solvent control is taken as 100. [Diagram 2] Graph showing the expression level of RBMS3 gene in fibroblasts incubated with or without the addition of elderberry extract. The expression level is the relative level when the solvent control is taken as 100. [Diagram 3] Graph showing the expression level of RBMS3 gene in fibroblasts incubated with or without Tilia cordata extract. The expression level is the relative level when the solvent control is taken as 100. [Figure 4] Graph showing the expression level of RBMS3 gene in fibroblasts incubated with or without Arnica extract. The expression level is the relative level with the solvent control taken as 100. [Diagram 5] Graph showing the expression level of the RBMS3 gene in fibroblasts when incubated with or without the addition of Shell Ginger extract. The expression level is the relative level when the solvent control is taken as 100. [Figure 6] Graph showing the expression level of the RBMS3 gene in fibroblasts incubated with or without Eucalyptus extract. The expression level is the relative level when the solvent control is taken as 100. [Figure 7] Graph showing the expression level of the RBMS3 gene in fibroblasts incubated with or without the addition of lemongrass extract. The expression level is the relative level when the solvent control is taken as 100. [Figure 8]Graph showing the expression level of the RBMS3 gene in fibroblasts incubated with or without the addition of marjoram extract. The expression level is the relative level with the solvent control taken as 100. [Figure 9] A graph showing the change in melanin amount when culture supernatant of fibroblasts in which the RBMS3 gene was knocked down was added to melanocytes. The amount of melanin is relative to the control, which is set at 100. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The RBMS3 gene expression enhancer of the present invention contains at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract as an active ingredient. Althea extract is an extract of Athaea officinalis, a member of the Malvaceae family. Elderberry extract is an extract of Sambucus nigra, which belongs to the Elderberry genus of the family Sambucusaceae. Tilia cordata extract is a plant of the Tilia genus of the Tilia family. It is an extract. Arnica extract is an extract of Arnica montana, a plant of the Asteraceae family. Shell ginger extract is an extract of Alpinia zerumbet, a plant in the ginger family. Eucalyptus extract is an extract of Eucalyptus globulus, a member of the Myrtaceae family. It is. Lemongrass extract is made from the grass family Cymbopogon citratus. It is an extract. Marjoram extract is an extract of Origanum majorana, a plant of the Lamiaceae family. It is. The above extracts may be used alone or in combination of two or more.

[0012] 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. The extract may be any extract that is normally used in skin external preparations such as cosmetics or pharmaceuticals, or in compositions for oral ingestion, and may be extracted from a plant body by a conventional method.

[0013] As the extraction solvent, one or more selected from polar solvents such as water, alcohols such as ethanol, isopropyl alcohol, butanol, etc., polyhydric alcohols such as 1,3-butylene glycol, polypropylene glycol, etc., ketones such as acetone, methyl ethyl ketone, etc., ethers such as diethyl ether, tetrahydrofuran, etc. are preferably used.

[0014] 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.

[0015] Constituent parts of the plant used for extraction include, for example, leaves, stems, flowers, buds, fruits, peels, pits, roots, or mixtures thereof, but in the case of Althaea extract, the roots are preferred, in the case of Sambucus nigra extract, the flowers are preferred, in the case of Tilia cordata extract, the flowers are preferred, in the case of Arnica extract, the flowers are preferred, in the case of Shell Ginger extract, the leaves are preferred, in the case of Eucalyptus extract, the leaves are preferred, in the case of Lemongrass extract, the leaves and / or stems are preferred, and in the case of Marjoram extract, the leaves are preferred.

[0016] The content of the althea extract, elder extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, or marjoram extract 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. Furthermore, when two or more of these extracts are used in combination, the total content of the two or more extracts in the agent of the present invention is preferably 0.0001 to 0.9 mass%, more preferably 0.0005 to 0.3 mass%, and even more preferably 0.001 to 0.03 mass%, calculated as solid matter relative to the entire 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.

[0017] Althea extract, Elder extract, Tilia cordata extract, Arnica extract, Shell ginger extract, Eucalyptus extract, Lemongrass extract, and Marjoram extract all have the effect of enhancing the expression of the RBMS3 gene. In other words, these extracts are each active ingredients of RBMS3 gene expression enhancers.

[0018] The RBMS3 expression enhancing effect of any substance can be confirmed by the fact that the expression level of the RBMS3 gene in cells to which the substance of interest 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, and more preferably 130% or more. The expression level of the RBMS3 gene can be measured, for example, by performing PCR using a DNA fragment having a sequence that specifically binds to the sequence of the RBMS3 gene as a primer, and measuring the amount of mRNA. Alternatively, for example, the amount of protein encoded by the RBMS3 gene present in cells may be quantitatively measured by a conventional method to determine the expression level of the RBMS3 gene.

[0019] In another aspect, the present specification also provides a composition for external application to the skin for enhancing expression of the RBMS3 gene, which contains at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract.

[0020] Furthermore, as shown in the Examples below, knocking down the RBMS3 gene in fibroblasts increases melanin production. This suggests that enhancing expression of the RBMS3 gene in fibroblasts suppresses melanin production. Therefore, the RBMS3 gene expression enhancer of the present invention can suppress melanin production, and is expected to have the effect of suppressing the occurrence of age spots and freckles. Therefore, the RBMS3 gene expression enhancer of the present invention can be an active ingredient of an external skin composition for preventing and / or improving age spots and / or freckles.

[0021] 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 of the agent and composition of the present invention 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 as the total amount of Althaea extract, Elderberry extract, Tilia cordata extract, Arnica extract, Shell ginger extract, Eucalyptus extract, Lemongrass extract, or Marjoram extract (when two or more of these extracts are used, the total amount of the two or more extracts) once or several times. In addition to taking it once, it is preferable to take it continuously or intermittently for several weeks to several months.

[0022] The form of the topical skin composition of the present invention is not particularly limited as long as it is applied topically to the skin, but preferred examples include cosmetics (including quasi-drugs) and pharmaceuticals, with cosmetics being more preferred. The site to which the topical skin composition of the present invention is applied is not particularly limited, but is usually the face, limbs, neck, and décolleté.

[0023] Examples of the dosage form of the composition for external use on the skin include lotion dosage forms, emulsion dosage forms such as milky lotion and cream, oil dosage forms, gel dosage forms, packs, cleansers, and the like, and are not particularly limited. The composition for external use on the skin may be either a leave-on type or a leave-off type.

[0024] In producing the composition for external use on the skin of the present invention, any ingredients that are usually used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be blended. 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; oleyl alcohol, stearyl alcohol, and octyldodecyl oleate. Any of the following may be blended: higher alcohols such as decanol, anionic surfactants such as sulfosuccinate 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, thickening / gelling agents, antioxidants, ultraviolet absorbing agents, colorants, preservatives, powders, and the like. EXAMPLES

[0025] 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.

[0026] <Reference example 1> The various extracts were prepared according to the following procedure. Althaea extract: Dried roots of Althaea (Mallow family, Malvaceae) were crushed, 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed and freeze-dried to powder. The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution. Elderberry extract: Dried flowers of Elderberry genus of the family Elderflower were crushed, and 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the liquid extracted by refluxing was freeze-dried to powder.The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution. Tilia cordata extract: Dried Tilia cordata flowers were crushed, 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed. The liquid was freeze-dried to powder, and the powder was then dissolved in 100% 1,3-butylene glycol aqueous solution. Arnica extract: Dried flowers of Arnica spp. (Asteraceae) were crushed, and 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed. The liquid was powdered by freeze-drying. The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution. Shell Ginger Extract: Dried shell ginger (Zingiberaceae, Alpinia genus) leaves (Shell Ginger leaves) were crushed, 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the liquid extracted by reflux was freeze-dried to powder.The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution. Eucalyptus extract: Dried leaves of Eucalyptus genus of the Myrtaceae family were crushed, and 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed and freeze-dried to powder.The powder was then dissolved in 100% 1,3-butylene glycol aqueous solution. Lemongrass extract: Dried leaves and stems of lemongrass (Poaceae, Cymbopogon genus) were crushed, and 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed and freeze-dried to powder. The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution. Marjoram extract: Dried leaves of Origanum vulgare of the Lamiaceae family were crushed, and 10 times the amount of 30% 1,3-butylene glycol aqueous solution was added, and the extract was refluxed and freeze-dried to powder.The powder was then dissolved in 50% 1,3-butylene glycol aqueous solution.

[0027] <Test Example 1> Examination of the effect of the test extract on the RBMS3 gene expression level in fibroblast cells Normal human dermal fibroblasts (NHDF: Normal Human Dermal Fibroblasts, neonatal, male, Asian / Caucasian, Kurabo, Lot: 05884) were cultured in Dulbecco's Modified Eagle Medium (high glucose) medium (DMEM) supplemented with 10% FBS and 1% Antibiotic-Antimycotic. 4.0×10 4 The cells were seeded onto a 24-well plate at 20 cells / well and cultured at 37°C under 5% CO2 for 24 hours. After cultivation, the medium was removed and replaced with DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic containing the test extracts, or with DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic containing 1,3-butylene glycol instead of the test extracts, so that the final concentrations were 0.1% by mass for Althaea extract, 0.1% by mass for Elder extract, 0.05% by mass for Tilia cordata extract, 0.1% by mass for Arnica extract, 0.1% by mass for Shell Ginger extract, 0.05% by mass for Eucalyptus extract, 0.05% by mass for Lemongrass extract, and 0.1% by mass for Marjoram extract, and cultivated for a further 24 hours. The final concentration of 1,3-butylene glycol in each medium containing the test extract was 0.025% for the medium containing lemongrass extract and 0.05% for each medium containing the other extracts, and the final concentration of 1,3-butylene glycol in each medium containing 1,3-butylene glycol instead of the test extract was the same as that in each medium containing the test extract. After culturing, the cells were washed with PBS(-) and collected, treated with QIAshredder and RNeasy Mini Kit (QIAGEN), and RNA was extracted using 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 each RBMS3 gene. The β-Actin gene was used as an endogenous control. The test extracts used were all prepared by the method described in Reference Example 1.

[0028] 1 to 8 show the mRNA expression level of the RBMS3 gene in fibroblasts to which each extract was added, expressed as a relative value when the mRNA expression level of each gene in fibroblasts in the solvent control was set at 100. In epidermal keratinocytes to which any of the extracts had been added, the expression levels of each mRNA of the RBMS3 gene were found to be significantly increased compared to the control, indicating that each extract has the effect of enhancing RBMS3 gene expression.

[0029] <Test Example 2> Verification of the relationship between RBMS3 gene expression and pigmentation (1) Effect on the expression level of melanocyte stimulating factor We measured the expression level of melanocyte stimulating factor when the RBMS3 gene was knocked down in fibroblasts using siRNA.

[0030] The specific steps are as follows: (I) Cell culture Human fibroblasts (NHDF: Normal Human Dermal Fibroblasts, 18 years old, female, Caucasian, Kurabo, Lot. 00967) were added at 3.0 × 10 4The cells were seeded in a 24-well plate at 100 cells / well, and then incubated in Medium 254 medium (Thermo Fisher Scientific) (450 The cells were cultured overnight at 37°C in a 5% CO2 environment at 1 μL / well.

[0031] (II) Preparation of siRNA mix The siRNA mix was prepared as follows. (1) Opti-MEM (trademark) (manufactured by Thermo Fisher Scientific) and Lipofectamine 3000 were mixed so that the total volume was 2 μL of Lipofectamine 3000 (manufactured by Thermo Fisher Scientific) per 50 μL of Opti-MEM (trademark) (manufactured by Thermo Fisher Scientific). (2) Opti-MEM was mixed with Lipofectamine 3000 so that 3 μL of siRNA (20 μM) was used per 50 μL of Opti-MEM. Hs_RBMS3_2_ Flexi Tube siRNA (QIAGEN, Cat No. SI00700042) was used to suppress the expression of the RBMS3 gene, and Allstars Negative Control siRNA (QIAGEN, Cat No. SI0365031) was used as a control. (3) Equal amounts of the mixtures in (1) and (2) were mixed and allowed to stand at room temperature for 5 minutes.

[0032] (III) Knockdown of the RBMS3 gene by siRNA transfection After incubation, 50 μL of the prepared siRNA mix was added to each well, and the cells were incubated for an additional 48 hours at 37°C and 5% CO2.

[0033] (IV) RNA purification and microarray analysis After the culture, the cells were washed with Buffer RLT (QIAGEN), collected, treated with QIAshredder and RNeasy Mini Kit (QIAGEN), and then RNA was extracted using QIAcube (QIAGEN). The purity and concentration of the extracted RNA were confirmed using NanoDrop (Thermo Fisher Scientific). Microarray analysis was performed on the extracted mRNA using the SurePrint G3 Human GE Microarray 8x60K Ver. 2.0 (Agilent Technologies). The expression levels of the genes were measured.

[0034] The expression level of each gene in the group in which the RBMS3 gene was knocked down is shown in Table 1 as a relative value when the expression level in the control group was set at 100%. In fibroblasts in which the RBMS3 gene was knocked down, it was confirmed that the gene expression of basic fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factors A, B, and D, and insulin-like growth factor 1 was increased. These factors are known as melanocyte stimulating factors. These results indicated that the expression of the RBMS3 gene is associated with the occurrence of pigmentation.

[0035] [Table 1]

[0036] (2) Effect on melanin production The RBMS3 gene was knocked down in fibroblasts using siRNA, and the amount of melanin produced was measured when the culture supernatant of the fibroblasts was added to melanocytes. The amount of melanin was measured using 2-[2- 14 Thiouracil is known to bind to melanin intermediates during the melanin production process and is specifically incorporated into melanin (Whittaker JR., J Biol Chem. 1971 (See, e.g., Dencker L., et al., Acta Pharmacol Toxicol (Copenh). 1981 Aug;49(2):141-9., Palumbo A. et al., Biochim Biophys Acta. 1990 Dec 6;1036(3):221-7., and Palumbo A. et al., Biochim Biophys Acta. 1994 Aug 18;1200(3):271-6.) Therefore, the amount of thiouracil incorporated into melanin is proportional to the amount of melanin produced. Using this, the method of Broxterman et al. HJ et al., Cancer Res. 1983 Mar;43(3):1316-20.) 2-[2- 14 C] thiouracil was added and cultured with fibroblasts to produce melanin. 2-[2- 14 The amount of melanin produced was determined by measuring the radioactivity of [C]thiouracil.

[0037] The specific steps are as follows: (I) Cell culture Human fibroblasts (NHDF: Normal Human Dermal Fibroblasts, 18 years old, female, Caucasian, Kurabo, Lot. 00967) were added at 6.0 × 10 4 The cells were seeded onto a 12-well plate at 100 cells / well and cultured overnight in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FBS (900 μL / well) at 37°C and 5% CO2.

[0038] (II) Preparation of siRNA mix The siRNA mix was prepared as follows. (1) Opti-MEM (trademark) (manufactured by Thermo Fisher Scientific) and Lipofectamine 3000 were mixed so that the total volume was 2 μL of Lipofectamine 3000 (manufactured by Thermo Fisher Scientific) per 50 μL of Opti-MEM (trademark) (manufactured by Thermo Fisher Scientific). (2) Opti-MEM and Lipofectamine 3000 were mixed so that 50 μL of Opti-MEM contained 3 μL of siRNA (20 μM). Hs_RBMS3_2_Flexi Tube siRNA (QIAGEN, Cat No. SI00700042) and control The roll used was Allstars Negative Control siRNA (QIAGEN, Cat No. SI0365031). (3) Equal amounts of the mixtures in (1) and (2) were mixed and allowed to stand at room temperature for 5 minutes.

[0039] (III) Knockdown of the RBMS3 gene by siRNA transfection After incubation, 100 μL of the prepared siRNA mix was added to each well, and the cells were further incubated overnight at 37°C and 5% CO2.

[0040] (IV) Recovery of fibroblast culture supernatant After the culture, the medium was replaced with Medium 254 medium (Thermo Fisher Scientific) (1.1 mL / well). The medium was replaced with ll) and cultured for 2 days, after which the culture supernatant was collected.

[0041] (V) Addition of fibroblast culture supernatant to melanocytes and calculation of melanin content Normal human epidermal melanocytes (NHEM, newborn, male, dark / African American, Life Technologies, Lot. 1223150) were cultured at 8.0 × 10 4The cells were seeded in a 24-well plate at 500 μL / well and cultured overnight in Medium 254 medium (Thermo Fisher Scientific) (900 μL / well) supplemented with HMGS (Thermo Fisher Scientific) at 37°C and 5% CO2. . After incubation, fibroblast culture supernatant was added (500 μL / well), and 2-[2- 14 C]Thiouracil was added (0.5 μCi / well) and cultured for 72 hours. After culturing, use tetrazolium salt WST-8 reagent (Cell Counting Kit-8; Dojindo Laboratories) 50 μL / well was added and reacted in a CO incubator for 2 hours. The absorbance at 450 nm and 650 nm was measured using a plate reader, and the difference between the measured values ​​(Abs.450-Abs.650) was calculated as the cell number relative to the control, which was set at 100. After that, the medium was removed from the plate, and the plate was washed with PBS. Then, 100% T CA was added at 120 μL to lyse the cells. Then, ice-cold water was added at 500 μL / well, and the cells were collected in a 1.5 mL tube. Ice-cold water was added at 500 μL / well to the wells after cell collection, and the cells were further collected in the 1.5 mL tube, mixed, and left to stand at 4°C for 30 minutes. After centrifugation at 4°C and 15,000 rpm for 5 minutes, the supernatant was removed, 10% TCA was added at 1 mL each, and the cells were left to stand at 4°C for 30 minutes. Next, centrifugation was performed at 4°C and 15,000 rpm for 5 minutes, and the supernatant was Remove the samples and add 1 mL of liquid scintillation cocktail AQUASOL-2 to each 1.5 mL tube. After adding and mixing, radioactivity was measured using a liquid scintillation counter, and the amount of melanin produced was calculated as a relative amount when the control was set at 100.

[0042] The results of melanin production per cell number are shown in Figure 9. It was confirmed that when the supernatant of fibroblasts in which the RBMS3 gene was knocked down was added to melanocytes, the amount of melanin production increased. The results of Test Example 2 showed that the expression of the RBMS3 gene is associated with the occurrence of age spots. Taking into consideration the results of Test Example 1, it was suggested that the RBMS3 gene expression enhancing effect of each extract, such as Althea extract, suppresses melanin production and the occurrence of age spots and freckles.

Claims

1. An RBMS3 gene expression enhancer comprising at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract.

2. A skin topical composition for enhancing RBMS3 gene expression, comprising at least one selected from the group consisting of althea extract, elderberry extract, tilia cordata extract, arnica extract, shell ginger extract, eucalyptus extract, lemongrass extract, and marjoram extract.

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