Screening method for PLA2G2F expression enhancers, agents for improving or preventing rough skin, and agents for skin whitening.

Abstract

The objective is to provide a technology that enhances the expression of PLA2G2F. [Solution] The expression of PLA2G2F is enhanced using a specific plant. One or more substances selected from the group consisting of wild rose extract and yarrow extract are used as the active ingredient of the PLA2G2F expression enhancer. Such agents can be suitably incorporated into compositions for improving or preventing rough skin, and for whitening. Furthermore, agents for improving or preventing rough skin, and for whitening are screened using the expression level of PLA2G2F in epidermal keratinocytes as an indicator.

Description

Technical Field

[0001] The present invention relates to a PLA2G2F expression enhancer and a method for screening agents for improving or preventing skin roughness and for whitening.

Background Art

[0002] In the upper layer of the stratum corneum of healthy skin, the upper and lower stratum corneum cells are not in close contact with each other and have a spatial structure. Such a structure is also called a Basket weave structure. Due to this spatial structure, smooth exfoliation of the surface stratum corneum is promoted, or the space is filled with intercellular lipids such as ceramides, fatty acids, and cholesterol, thereby improving the barrier function (Non-Patent Document 1). In order to form such a spatial structure, it is necessary for desmoglein (Dsg) 1, which is the main component of corneodesmosomes that adhere stratum corneum cells to each other, to be decomposed in the upper layer of the stratum corneum (Non-Patent Document 2). It is known that Dsg1 in the upper layer of the stratum corneum is decreased by various fatty acids (stearic acid, linolenic acid, methyl linoleate, ricinoleic acid, methyl ricinoleate, or methoprene) (Patent Document 1).

[0003] Phospholipase A2 group IIF (also known as PLA2G2F) is the most highly expressed phospholipase in the epidermis of the skin. PLA2G2F expression is induced by keratinocyte differentiation and is mainly localized in the upper layers above the spinous stratum. When secreted extracellularly, PLA2G2F degrades phospholipids contained in lamellar granules and other structures, producing fatty acids and lysophospholipids (Non-Patent Literature 3, 4). In the skin, PLA2G2F preferentially degrades diacyl and plasmalogen-type phosphatidylethanolamines with acyl groups derived from docosahexaenoic acid, but it also degrades a wide range of phospholipids, including phosphatidylethanolamines and phosphatidylcholine with acyl groups derived from stearic acid and oleic acid (Non-Patent Literature 3). The fatty acids produced by the degradation of these phospholipids are thought to be components of intercellular lipids (Non-Patent Literature 4-8). Furthermore, PLA2G2F has also been reported to be involved in differentiation promotion and inflammation (Non-patent documents 3, 9).

[0004] Forkhead box protein N1 (also known as FOXN1) is involved in the proliferation of melanocytes and melanocytes. FOXN1 is a transcription factor of fibroblast growth factor-2 (FGF2), which promotes melanin synthesis, and is known to promote pigmentation by recruiting melanocytes and receiving melanin. FOXN1 is partially expressed in keratinocytes in the basal layer of human epidermis, and is uniformly expressed from the first layer upwards (Non-Patent Literature 10).

[0005] Syntaxin3 (also known as STX3) and synaptosome-associated protein of 23 (SN AP23 (also known as AP23) is a protein belonging to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family and is a protein necessary for the exocytosis-mediated melanosome efflux mechanism (Non-Patent Literature 11). [Prior art documents] [License]

[0006] [License 1] Special Announcement No. 2022-082367 [Non-licensed literature]

[0007] [Non-licensed Document 1] Goto, H. et al., The British journal of dermatology, 2020, 182(2), 364-372 [Non-licensed Document 2] Kitajima, Y., Dermatologica Sinica, 2015, 33, 58-63 [Non-licensed Document 3] Yamamoto, K. et al., The Journal of Experimental Medicine, 2015, 212(11), 1901-1919 [Non-licensed Document 4] Ilic, D. et al., Biochimica et Biophysica Acta, 2014, 1841(3), 416-421 [Non-licensed Document 5] Merleev, AA et al., JCI Insight, 2022, 7(16) [Non-licensed Document 6] Chan, A. et al., Dermato-Endocrinology, 2011, 3(2), 84-90 [Non-licensed Document 7] Mao-Qiang, M., et al., Journal of Lipid Research, 1995, 36(9), 1925-1935 [Non-licensed Document 8] Fluhr, JW et al., Journal of Investigative Dermatology, 2001, 117(1), 44-51 [Non-Patent Document 9] Shao, S. et al. JCI insight, 2021, 6(20) [Non-Patent Document 10] Weiner, L. et al., Cell, 2007, 130(5), 932-942 [Non-Patent Document 11] Hongo et al., Fragrance Journal, Specialized Journal of Fragrance Science Research and Development, 2018, 46, 24-29. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] As mentioned above, since the breakdown of phospholipids by PLA2G2F generates fatty acids that are components of intercellular lipids, it is expected that increasing the expression of PLA2G2F will promote the formation of spatial structures in the stratum corneum, improving the skin's barrier function and potentially leading to improvements in and prevention of rough skin.

[0009] Furthermore, skin conditions caused by pigmentation such as age spots and freckles significantly affect facial appearance, leading to a high demand for whitening agents and cosmetics. One of the causes of pigmentation is increased melanin production by pigment cells (melanocytes). The inventors have newly discovered that increasing the expression of PLA2G2F can increase the expression of the aforementioned proteins STX3 and SNAP23, which are involved in the excretion of melanosomes, and can suppress the expression of the aforementioned protein FOXN1, which is involved in promoting melanin synthesis. Therefore, there is a prospect that increasing the expression of PLA2G2F will yield a whitening effect.

[0010] In this context, the present invention aims to provide a technology for enhancing the expression of PLA2G2F. [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors have found a plant extract having an action of enhancing the expression of PLA2G2F, and have conceived that they serve as active ingredients that bring both the effects of improving and preventing rough skin and the whitening effect, and have completed the present invention. Furthermore, as described above, the present inventors have conceived that the improvement or prevention effect of rough skin and the whitening effect can be obtained by enhancing the expression of PLA2G2F, and have completed a method for screening active ingredients of agents for improving or preventing rough skin and for whitening.

[0012] That is, the present invention is as follows. [1] A PLA2G2F (phospholipase A2 group IIF) expression enhancer comprising one or more selected from the group consisting of Rosa multiflora extract and Achillea millefolium extract as an active ingredient. [2] The PLA2G2F expression enhancer according to [1], which enhances the expression of PLA2G2F in epidermal keratinocytes. [3] A composition for suppressing FOXN1 expression, containing the PLA2G2F expression enhancer according to [1] or [2]. [4] A composition for enhancing the expression of one or more selected from the group consisting of STX3 and SNAP23, containing the PLA2G2F expression enhancer according to [1] or [2]. [5] A composition for improving or preventing rough skin and for whitening, containing the PLA2G2F expression enhancer according to [1] or [2]. [6] The composition according to any one of [3] to [5], which is a cosmetic. [7] A screening method for screening agents for improving or preventing rough skin and for whitening, using the expression level of PLA2G2F in epidermal keratinocytes as an index. [8] A step of adding a test sample to epidermal keratinocytes, and <The method according to [7], comprising the step of measuring the expression level of PLA2G2F in the epidermal keratinocytes. [9] The method according to [8], wherein if the expression level is greater than the expression level in epidermal keratinocytes to which the test sample was not added, the test sample is determined to have an effect of improving or preventing rough skin, as well as a whitening effect. [Effects of the Invention]

[0013] The present invention provides a component that enhances the expression of PLA2G2F. Furthermore, by incorporating such a component into a composition, a composition for improving or preventing rough skin and for whitening can be created. Furthermore, the present invention provides a method for screening ingredients that have skin-improving or preventive effects, as well as skin-whitening effects. [Brief explanation of the drawing]

[0014] [Figure 1A] A graph showing the expression level of the SNAP23 gene in epidermal keratinocytes in which the PLA2G2F gene has been knocked down. [Figure 1B] A graph showing the expression level of the STX3 gene in epidermal keratinocytes in which the PLA2G2F gene has been knocked down. [Figure 1C] A graph showing the expression level of the FOXN1 gene in epidermal keratinocytes in which the PLA2G2F gene has been knocked down. [Figure 2A] A graph showing the expression level of the PLA2G2F gene in epidermal keratinocytes cultured with wild rose extract. [Figure 2B] A graph showing the expression level of the PLA2G2F gene in epidermal keratinocytes cultured with yarrow extract. [Modes for carrying out the invention]

[0015] <1> PLA2G2F expression enhancer The agent of the present invention contains one or more active ingredients selected from the group consisting of Rosa multiflora extract and Achillea millefolium extract.

[0016] Wild rose extract and yarrow extract include not only the extracts derived from wild rose and yarrow themselves, but also fractions of these extracts, purified fractions, and extracts. Alternatively, it may refer to the general term for fractions, purified products, and solvent-removed products.

[0017] Furthermore, the extracts derived from wild rose and yarrow can be any extracts commonly used in topical skin preparations such as cosmetics and pharmaceuticals, or in oral compositions, and can be those extracted from plants by conventional methods. For extraction, the entire plant may be used, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flowers, flower buds, fruits, peels, seeds, and roots may be used in the extraction process.

[0018] As the extraction solvent, one or more polar solvents selected from 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 are preferably used. In particular, ethanol is preferably used as the extraction solvent for rosehip extract, and 1,3-butylene glycol is preferably used as the extraction solvent for yarrow extract.

[0019] Specific extraction methods include, for example, adding 1 to 30 parts by mass of solvent to 1 part by mass of the plant body or its dried product to be used for extraction, immersing it for several days at room temperature or for several hours at a temperature near its boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography or the like.

[0020] The content of one or more selected from the group consisting of wild rose extract and yarrow extract in the agent of the present invention is preferably 0.00001 to 0.09% by mass, more preferably 0.00005 to 0.05% by mass, and even more preferably 0.0001 to 0.003% by mass, on a solid basis. By setting the content within the above range, the desired effect can be easily obtained, and flexibility in formulation design can be ensured. The above-mentioned content can be appropriately adjusted according to the administration route and the form of the composition to be contained, as described later.

[0021] Both wild rose extract and yarrow extract have the effect of enhancing the expression of the PLA2G2F gene. In other words, these extracts are active ingredients in PLA2G2F expression enhancers. Specifically, wild rose extract and yarrow extract each have the effect of enhancing the expression of the PLA2G2F gene in epidermal keratinocytes.

[0022] The PLA2G2F expression-enhancing effect can be confirmed by the fact that the PLA2G2F expression level in cells treated with the target substance is higher than that in cells not treated with the substance, usually 110% or more, preferably 120% or more, and more preferably 130% or more. The PLA2G2F expression level can be determined, for example, by performing PCR using a DNA fragment having a sequence that specifically binds to the PLA2G2F gene sequence as a primer, and measuring the amount of mRNA. Alternatively, for example, the intracellular abundance of the protein encoded by the PLA2G2F gene may be quantitatively measured by a conventional method and used as the PLA2G2F expression level.

[0023] As mentioned above, the degradation of phospholipids by PLA2G2F generates fatty acids that are components of intercellular lipids. Therefore, by increasing the expression of PLA2G2F, the formation of spatial structures in the stratum corneum is promoted, improving the skin's barrier function and potentially leading to improved or preventive effects on rough skin. Accordingly, the PLA2G2F expression enhancer in this invention is intended for use in improving or preventing rough skin. It can be suitably incorporated into the finished product. In this context, "skin roughness" refers to a condition in which the stratum corneum barrier function is weakened, the balance between skin moisture and sebum is disrupted, inflammation such as acne occurs, and the smoothness and texture of the skin are lost. "Improvement or prevention of skin roughness" refers to improving or preventing such a condition.

[0024] Furthermore, as shown in the examples described later, it was found that knockdown of the PLA2G2F gene in epidermal keratinocytes reduced the expression levels of the STX3 and SNAP23 genes. This suggests that upregulation of PLA2G2F expression promotes the expression of the STX3 and SNAP23 genes. Therefore, the PLA2G2F expression enhancer in the present invention can be suitably included in a composition for enhancing the expression of one or more substances selected from the group consisting of STX3 and SNAP23.

[0025] The STX3 expression-enhancing effect can be confirmed by the fact that the STX3 expression level in cells to which the target substance was added is higher than that in cells without the substance, usually 110% or more, preferably 120% or more, and more preferably 130% or more. The STX3 expression level can be determined, for example, by performing PCR using a DNA fragment having a sequence that specifically binds to the STX3 gene sequence as a primer and measuring the amount of mRNA. Alternatively, for example, the intracellular abundance of the protein encoded by the STX3 gene may be quantitatively measured by a conventional method and used as the STX3 expression level. The same applies to the SNAP23 expression-enhancing effect.

[0026] Furthermore, as shown in the examples described later, it was found that knockdown of the PLA2G2F gene in epidermal keratinocytes increased the expression level of the FOXN1 gene. This suggests that upregulation of PLA2G2F suppresses FOXN1 gene expression. Therefore, the PLA2G2F expression enhancer in the present invention can be suitably included in a composition for suppressing FOXN1 expression.

[0027] The inhibitory effect on FOXN1 expression can be confirmed by the fact that the FOXN1 expression level in cells to which the target substance was added is lower than that in cells without the substance, usually 90% or less, preferably 80% or less, and more preferably 70% or less. The FOXN1 expression level can be determined, for example, by performing PCR using a DNA fragment having a sequence that specifically binds to the FOXN1 gene sequence as a primer and measuring the amount of mRNA. Alternatively, for example, the intracellular abundance of the protein encoded by the FOXN1 gene may be quantitatively measured by a conventional method and used as the FOXN1 expression level.

[0028] As mentioned above, STX3 and SNAP23 are known to be involved in the melanosome efflux mechanism. Also, as mentioned above, FOXN1 is known to promote pigmentation by receiving melanin. Therefore, it is thought that enhancing the expression of PLA2G2F in epidermal keratinocytes leads to increased expression of the SNX3 and SNAP23 genes, and suppression of the FOXN1 gene, which in turn leads to a whitening effect. Accordingly, the PLA2G2F expression enhancer in the present invention can be suitably included in a whitening composition. In this context, "skin whitening" includes suppressing melanin production, suppressing pigmentation in the skin, and suppressing skin darkening.

[0029] The administration route of the agent of the present invention is not particularly limited and can be transdermal, oral, nasal, intravenous injection, etc., but transdermal administration is preferred. Here, "administration" may be replaced with "ingestion". The dosage is not particularly limited, but considering the desired effect and safety, the total amount of one or more plant extracts selected from the group consisting of wild rose extract and yarrow extract is 0.3 to 300 μg / day in solid matter terms, taken in one or several divided doses. This is preferable. In addition to single doses, it is also preferable to take it continuously or intermittently for several weeks to several months.

[0030] When the agent of the present invention is administered transdermally, it is preferable to use it as a topical skin composition. The form of the topical skin composition is not particularly limited as long as it is applied topically to the skin, but cosmetics (including quasi-drugs) and pharmaceuticals are preferred, with cosmetics being more preferred. The areas to which the topical skin composition is applied are not particularly limited, but are usually the face, limbs, neck, and décolleté.

[0031] Examples of dosage forms for topical skin compositions include, but are not limited to, lotions, emulsifiers such as emulsions and creams, oils, gels, packs, and cleansers. Furthermore, the composition for topical application to the skin may be either a leave-on type or a leave-off type.

[0032] When the composition of the present invention is in the form of a topical skin composition, during its manufacture, ingredients commonly used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc., may be arbitrarily incorporated. Such optional components may include, for example, 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 sulfosuccinate esters and sodium polyoxyethylene alkyl sulfate; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkylammonium salts; 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 / gelling agents; antioxidants; UV absorbers; colorants; preservatives; powders, etc., which can be optionally blended.

[0033] <2> Screening method The present invention's screening method uses the expression level of PLA2G2F in epidermal keratinocytes as an indicator to screen for agents for improving or preventing rough skin and for skin whitening.

[0034] As mentioned above, enhancing the expression of PLA2G2F in epidermal keratinocytes leads to the improvement or prevention of rough skin and the exertion of whitening effects. Therefore, ingredients that enhance the expression of PLA2G2F in epidermal keratinocytes can be active ingredients in agents for improving or preventing rough skin and for whitening.

[0035] In the screening method of the present invention, the expression level of PLA2G2F may be the expression level of the PLA2G2F gene, or the translation level or abundance of the protein encoded by the gene. A preferred embodiment of the screening method of the present invention includes the steps of adding a test sample to epidermal keratinocytes and culturing them, and measuring the expression level of PLA2G2F in the epidermal keratinocytes. The expression level of PLA2G2F can be measured using any method. For example, the expression level of PLA2G2F can be quantified by performing PCR using a DNA fragment having a sequence that specifically binds to the PLA2G2F gene sequence as a primer, and measuring the amount of mRNA. The nucleotide sequences of known PLA2G2F genes are publicly available, and those skilled in the art can design appropriate primers for PCR. Alternatively, for example, the intracellular abundance of the protein encoded by the PLA2G2F gene may be quantitatively measured using a conventional method and used as the expression level of PLA2G2F.

[0036] In the screening method of the present invention, if the expression level of PLA2G2F in epidermal keratinocytes cultured with the test sample is higher than the expression level when the test sample is not added (control), the test sample is determined to have an effect of improving or preventing rough skin, as well as a whitening effect. The degree of high expression is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more compared to the control. However, the test sample should be added within a concentration range that does not cause significant cytotoxicity.

[0037] The epidermal keratinocytes used in the screening method of the present invention can be human-derived epidermal keratinocytes, or epidermal keratinocytes derived from mammals such as rats, mice, or rabbits, without any particular limitations. However, it is generally preferable to use normal cells, and it is more preferable to use normal human-derived epidermal keratinocytes. As for the conditions for culturing cells, in addition to normal culture conditions, any culture conditions that do not hinder the execution of the screening method of the present invention can be applied without any particular limitations.

[0038] The test sample targeted by the screening method of the present invention may be a pure substance, an extract derived from plants or animals, or a mixture thereof. Extracts derived from animals or plants refer not only to the extracts themselves, but also to fractions of the extracts, purified fractions, and solvent-removed extracts or fractions or purified products. Plant-derived extracts include extracts made from plants that grow naturally or are cultivated, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts. The extraction process can use the whole plant, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flowers, flower buds, and fruits. However, it is preferable to crush or finely chop these parts beforehand to improve extraction efficiency. Suitable extraction solvents include one or more polar solvents selected from 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 may involve adding 1 to 30 parts by mass of solvent to 1 part by mass of the plant body or its dried product, immersing it for several days at room temperature or for several hours at a temperature near its boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography or the like.

[0039] An example of the procedure in the screening method of the present invention is given below, but the invention is not limited to the following and can be modified as appropriate without departing from the spirit of the invention. First, the test sample is added to pre-cultured epidermal keratinocytes and incubated at 37°C for 24 to 72 hours. Subsequently, the expression level of the PLA2G2F gene or the protein encoded by the said gene in the epidermal keratinocytes is measured by a standard method. As a control, the expression level is also measured in epidermal keratinocytes cultured in the same manner without the addition of the test sample. If the expression level in epidermal keratinocytes to which the test sample was added is higher than the expression level in epidermal keratinocytes without the test sample (control), the test sample is judged to have an effect of improving or preventing rough skin, as well as a whitening effect.

[0040] Ingredients determined to have skin-improving or preventive effects, as well as whitening effects, 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 allows for the creation of compositions for improving or preventing skin roughness, and for whitening. Such compositions can be suitably used in the design process. Preferred examples of such compositions include external skin compositions, more preferably cosmetics, and particularly suitable examples include cosmetics for improving or preventing rough skin, and for whitening.

[0041] When a composition contains an active ingredient determined to have skin-improving or preventive effects, as well as a whitening effect, through screening according to the present invention, its content (amount) is usually 0.000001% by mass or more of the total composition, preferably 0.00001% by mass or more, more preferably 0.0001% by mass or more, and usually 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less. This is because if the content (amount) of the active ingredient is too low, the desired effect may not be easily obtained, and if it is too high, the effect may plateau and the freedom of formulation of the composition may be impaired. Furthermore, the composition may contain not only one type of active ingredient but also two or more types. For animal and plant extracts, the amount is expressed on a solid basis.

[0042] When incorporating an active ingredient (active ingredient) determined to have skin-improving or preventive effects, as well as whitening effects, into a composition for improving or preventing skin roughness and for whitening, the composition may optionally include ingredients commonly used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc., during its manufacture. <1> The explanation given above can be applied. [Examples]

[0043] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the essence of the invention.

[0044] <Reference example 1> Various extracts were prepared using the following procedure. Wild rose extract: Extracted from the fruit of wild rose (Rosa multiflora), a species of the Rosaceae family, using a 30% ethanol aqueous solution. Yarrow extract: The flower heads or whole plants of Achillea millefolium, a member of the Asteraceae family, were extracted with a 30% aqueous solution of 1,3-butylene glycol.

[0045] <Test Example 1> Relationship between PLA2G2F expression levels and FOXN1, SNAP23, and STX3 expression levels The relationship between PLA2G2F expression levels and the expression levels of FOXN1, SNAP23, and STX3 was examined using cells in which the PLA2G2F gene was knocked down, following the procedure below.

[0046] (1) Perform reverse transfection Following standard procedures, Opti-MEM I Reduced Serum Medium (Gibco) and Lipofectamin RNAiMAX were used. Transfection Reagent (Thermo Fisher Scientific) and PLA2G2F siRNA or negative A solution mixed with the active control siRNA was placed in a 24-well plate at a rate of 100 μL / well. The mixture was added and allowed to stand at room temperature for 10-20 minutes.

[0047] (2) Cell seeding Add the mixed reagents described in (1) above to the 24-well plate and add Humedia-KG2 medium. Using (Kurabo Industries Ltd., KK-2150S), normal human epidermal keratinocytes (NHEK, Kurabo Industries Ltd., neonatal, male, lightly pigmented) were subjected to 8 ×10 4 Seeds were seeded to a concentration of 500 μL per well and incubated at 37°C and 5% CO2 for 48 hours.

[0048] (3) Differentiation induction of epidermal keratinocytes and RT-qPCR After culturing as described in (2) above, remove the culture medium, wash with 1 mL of PBS, add 500 μL / well of assay medium (Japan Tissue Engineering Co., Ltd., 402250), and then culture at 37°C. The cells were cultured for 24 hours in a 5% CO2 environment. After culturing, the cells were harvested and RNA was extracted using a standard method. The extracted RNA was used to synthesize cDNA using a conventional method, and Fast SYBR Green Master Mix (Applied Using a Biosystems IV system, the mRNA expression levels of the FOXN1, SNAP23, and STX3 genes were analyzed by RT-qPCR. The GAPDH gene was used as an endogenous control.

[0049] Figures 1A to 1C show the mRNA expression levels of the FOXN1, SNAP23, and STX3 genes in the group in which the PLA2G2F gene was knocked down, as relative values ​​with the mRNA expression level in the negative control group (in which none of the genes were knocked down) set to 1.

[0050] As shown in Figures 1A to 1C, compared to the negative control group, the group in which the PLA2G2F gene was knocked down showed decreased mRNA expression levels of SNAP23 and STX3, and increased mRNA expression levels of FOXN1. From the above findings, it was revealed that in epidermal keratinocytes, a decrease in PLA2G2F expression leads to a decrease in SNAP23 and STX3 expression, while increasing FOXN1 expression.

[0051] <Test Example 2> Examination of the effects of various extracts on PLA2G2F gene expression levels in epidermal keratinocytes. The expression level of the PLA2G2F gene in epidermal keratinocytes to which the test extract was added was measured using the following procedure. Using HuMedia-KG2 medium (Kurabo Industries Ltd., KK-2150S), normal human epidermal keratinocytes (NHEK, Kurabo Industries Ltd., neonatal, male, lightly pigmented) were plated in 24-well plates in 4 × 10⁶ layers. 4 Cells were seeded per well and cultured overnight at 37°C and 5% CO2. After culturing, the medium was removed and replaced with HuMedia-KG2 medium containing 0.1% by weight of the test extract in solid content, and cultured for another 24 hours at 37°C and 5% CO2. The cells were cultured for a specified time. After culturing, the cells were washed with PBS(-) and harvested, and RNA was extracted using a standard method. cDNA was synthesized from the extracted RNA using a conventional method, and the expression levels of each PLA2G2F gene were analyzed by RT-qPCR using Fast SYBR Green Master Mix (Applied Biosystems). The GAPDH gene was used as an endogenous control. The test extracts used were all prepared using the method described in the reference example.

[0052] Figures 2A and 2B show the mRNA expression levels of the PLA2G2F gene in epidermal keratinocytes treated with each extract, expressed as relative values ​​with the mRNA expression level of the PLA2G2F gene in solvent-controlled epidermal keratinocytes set to 1. In epidermal keratinocytes treated with any of the extracts, a significant increase in mRNA expression of the PLA2G2F gene was observed compared to the control group. [Industrial applicability]

[0053] This invention provides a component that enhances the expression of PLA2G2F. Furthermore, by incorporating such a component into a composition, a composition for improving or preventing rough skin and for whitening can be created. Moreover, this invention provides a method for screening components that have skin-improving or preventive effects and whitening effects. These inventions contribute to development in the beauty industry and meet consumer demand, making them highly useful industrially.

Claims

1. A PLA2G2F (phospholipase A2 group IIF) expression enhancer comprising one or more active ingredients selected from the group consisting of wild rose (Rosa multiflora) extract and yarrow (Achillea millefolium) extract.

2. A PLA2G2F expression enhancer according to claim 1, which enhances the expression of PLA2G2F in epidermal keratinocytes.

3. A composition for inhibiting FOXN1 expression, comprising the PLA2G2F expression enhancer described in claim 1 or 2.

4. A composition for enhancing expression, selected from the group consisting of STX3 and SNAP23, containing the PLA2G2F expression enhancer described in claim 1 or 2.

5. A composition for improving or preventing rough skin and for whitening, comprising the PLA2G2F expression enhancer described in claim 1 or 2.

6. The composition according to claim 3, which is a cosmetic.

7. A screening method for screening agents for improving or preventing rough skin, as well as for skin whitening, using the expression level of PLA2G2F in epidermal keratinocytes as an indicator.

8. The process of adding the test sample to epidermal keratinocytes, and The method according to claim 7, comprising the step of measuring the expression level of PLA2G2F in the epidermal keratinocytes.

9. The method according to claim 8, wherein if the expression level is greater than that in epidermal keratinocytes to which the test sample was not added, the test sample is determined to have an effect of improving or preventing rough skin, as well as a whitening effect.

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