Screening method for skin whitening agents

Abstract

To provide a method for screening a substance useful as a whitening agent by utilizing a mechanism involved in suppressing differentiation and proliferation of melanocytes, a BMP-4 inhibitor as a whitening agent whose usefulness is confirmed by the screening method, and a cosmetic comprising the whitening agent whose usefulness is confirmed by the screening method.SOLUTION: A method of screening for whitening agents uses as an index, decrease in an expression level of melanocyte differentiation-inducing factor in cells exposed to a whitening agent candidate substance relative to the expression level of melanocyte differentiation-inducing factor in a control group in which cells are not exposed to the whitening agent candidate substance. A BMP-4 inhibitor includes at least one of a group consisting of a burnet extract, an aloe vera leaf extract, soymilk fermented liquid, a moth bean seed extract, a horse chestnut extract, a glycyrrhiza extract, and an Angelica keiskei leaf / stem extract. A cosmetic comprises a whitening agent candidate substance whose expression level of melanocyte differentiation-inducing factor is confirmed to be reduced by the whitening agent screening method.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for screening skin whitening agents. By law Regarding. [Background technology]

[0002] Conventionally, whitening cosmetics containing various whitening active ingredients such as placenta, ellagic acid, kojic acid, tranexamic acid, cetyl tranexamate, and linoleic acid have been provided to prevent skin pigmentation and maintain white and bright skin. For example, Patent Document 1 below discloses a cosmetic containing a specific cysteine derivative or a salt of a cysteine derivative and a specific whitening agent, and also discloses a number of plant extracts as the specific whitening agent.

[0003] Melanin, the dark pigment in skin, is produced by pigment-producing cells called melanocytes. Patent Document 2 below discloses a method for promoting the induction of melanocyte differentiation for the purpose of treating diseases such as vitiligo vulgaris, hereditary symmetrical pigmentary disorder, and albinism, which are caused by abnormally suppressed melanin production or a deficiency of melanocytes. Specifically, the method disclosed includes culturing human pluripotent stem cells in the presence of a component that induces epidermal cell differentiation and a component that induces terminal differentiation into keratinocytes.

[0004] Patent Document 3 listed below discloses a method for screening for differentiation inducers of pluripotent stem cells, and lists numerous factors belonging to the so-called TGF-β (transforming growth factor-β) superfamily as stem cell differentiation inducers, one of which is BMP-4 (Bone Morphogenetic Protein-4). BMP-4 has been reported to be involved in the differentiation of stem cells into retinal pigment epithelium (Patent Document 4 listed below) and in promoting hair growth (Patent Document 5 listed below). Patent Document 6 listed below also reports that BMP-4 reduces melanin production in melanocytes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 081147 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-146803 [Patent Document 3] Special Publication No. 2005-500847 [Patent Document 4] Japanese Patent Application Laid-Open No. 2009-226069 [Patent Document 5] Special Publication No. 2008-534607 [Patent Document 6] Special Publication No. 2006-508026 Summary of the Invention [Problem to be solved by the invention]

[0006] Skin pigmentation is caused by the differentiation and proliferation of melanocytes from stem cells, which in turn increases melanin production. Therefore, substances that can inhibit the differentiation and proliferation of melanocytes are considered to be useful as skin whitening agents.

[0007] The present disclosure aims to screen for substances useful as whitening agents by utilizing mechanisms involved in the inhibition of melanocyte differentiation and proliferation, to provide a BMP-4 inhibitor as a whitening agent whose usefulness has been confirmed through screening, and to provide a cosmetic product containing the whitening agent whose usefulness has been confirmed through screening, and a method for producing the cosmetic product. [Means for solving the problem]

[0008] Specific means for solving the above problems include the following embodiments. [1] A method for screening whitening agents, which uses as an indicator a decrease in the expression level of a melanocyte differentiation-inducing factor when cells are exposed to a candidate whitening agent, compared to the expression level of the melanocyte differentiation-inducing factor in a control group in which the cells are not exposed to the candidate whitening agent.

[0009] [2] The method for screening a skin whitening agent according to [1], wherein the melanocyte differentiation promoter is bone morphogenetic protein 4.

[0010] [3] The method for screening a skin whitening agent according to [1] or [2], wherein the cells are normal human cells.

[0011] [4] The method for screening a skin whitening agent according to [4], wherein the normal human cells are epidermal keratinocytes.

[0012] [5] The method for screening a whitening agent according to [5], wherein the normal human cells are fibroblasts.

[0013] [6] A bone morphogenetic protein 4 inhibitor comprising at least one selected from the group consisting of burnet extract, aloe vera leaf extract, fermented soy milk broth, moth bean seed extract, horse chestnut extract, licorice extract, and angelica tree leaf / stem extract.

[0014] [7] A bone morphogenetic protein 4 inhibitor according to [6], which contains particles containing Sanguisorba officinalis extract, and the particles have a median diameter of less than 1 μm.

[0015] [8] A cosmetic preparation containing the bone morphogenetic protein 4 inhibitor of [6] or [7] as a whitening agent.

[0016] [9] A cosmetic product containing, as a whitening agent, a candidate substance for a whitening agent that has been confirmed to have a reduced gene expression level of a melanocyte differentiation induction promoting factor by any of the screening methods for whitening agents set forth in [1] to [5].

[0017]

[10] A method for producing a cosmetic, comprising: a step of determining, as a whitening agent, a candidate whitening agent for which a reduced expression level of a melanocyte differentiation induction promoting factor has been confirmed by any one of the screening methods for whitening agents set forth in [1] to [5]; and a step of adding the whitening agent to cosmetic ingredients. [Effects of the Invention]

[0018] The technology disclosed herein makes it possible to screen for substances useful as skin whitening agents by utilizing the mechanisms involved in the inhibition of melanocyte differentiation and proliferation. Furthermore, it is possible to provide a BMP-4 inhibitor as a skin whitening agent whose usefulness has been confirmed by screening, as well as a cosmetic product containing the skin whitening agent whose usefulness has been confirmed by screening, and a method for producing the cosmetic product. [Brief explanation of the drawings]

[0019] [Figure 1] Graph showing the melanin production effect of BMP-4. [Figure 2] Graph showing the results of screening candidate substances for skin whitening agents. [Figure 3] Graph showing the effect of the presence or absence of Sanguisorba officinalis extract on melanin production. [Figure 4] Graph showing the effect of nano-sizing of Sanguisorba officinalis extract on melanin production. DETAILED DESCRIPTION OF THE INVENTION

[0020] (1) Screening method for skin whitening agents The first embodiment of the screening method for whitening agents uses as an indicator the reduction in the expression level of a melanocyte differentiation-inducing factor when cells are exposed to a candidate whitening agent substance relative to the expression level of a melanocyte differentiation-inducing factor in a control group in which the cells are not exposed to the candidate whitening agent substance.

[0021] In this embodiment, when a substance is a candidate for a whitening agent, the expression level of a melanocyte differentiation-inducing factor is examined in an experimental group in which cells are exposed to the substance and in a control group in which cells are not exposed to the substance. Whether the expression level in the experimental group is reduced relative to the expression level in the control group is used as an indicator for screening as a whitening agent. Here, a "candidate whitening agent" refers to a candidate substance that has the potential to become a whitening agent and is a test substance for screening. Furthermore, a "whitening agent" refers to a substance among candidate whitening agents that has been confirmed through screening to have the ability to reduce the expression level of a melanocyte differentiation-inducing factor.

[0022] Melanocyte differentiation-promoting factors are proteins that promote the differentiation of pluripotent stem cells (e.g., iPS cells (induced pluripotent stem cells)) or melanocyte stem cells into melanocytes. Proteins that function as melanocyte differentiation-promoting factors include, for example, BMP-2, BMP-4, BMP-7, stem cell factor (SCF), endothelin-1, endothelin-3, and Wnt3a.

[0023] Here, the expression level of the melanocyte differentiation-inducing factor may be confirmed by detecting the melanocyte differentiation-inducing factor itself as a produced protein, or by detecting the expression of the corresponding gene when the melanocyte differentiation-inducing factor is produced. That is, the expression level of the melanocyte differentiation-inducing factor may be either the gene expression level or the protein expression level, or both.

[0024] Methods for confirming gene expression levels include, for example, PCR (polymerase chain reaction), RT-PCR (reverse transcription PCR), a method for detecting gene quantity using an RNA probe (microarray method), a method for sequencing RNA sequences using a next-generation sequencer to detect expression levels, and a method for detecting expression levels using nucleic acid hybridization (in situ hybridization method).

[0025] Methods for confirming protein expression levels include semi-quantitative protein detection using Western blotting, quantitative protein detection using ELISA, measuring the intensity of staining such as fluorescence in images of immunostained specimens, and proteome analysis for protein detection.

[0026] A decrease in the expression level of the melanocyte differentiation induction promoter in the experimental group may be determined by whether the expression level in the experimental group is even slightly lower than that in the control group, or preferably by whether the reduction rate of the expression level in the experimental group relative to that in the control group (i.e., the rate calculated by "(AX) / A", where A is the expression level in the control group and X is the expression level in the experimental group) is 5% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 50% or more. Furthermore, regardless of the reduction rate, it is desirable that the difference between the expression level in the experimental group and the expression level in the control group is statistically significant. The risk level for determining statistical significance can be 5% or less.

[0027] According to this embodiment, candidate whitening agents that may be useful as whitening agents can be screened using a decrease in the gene expression level of a melanocyte differentiation-inducing factor as an indicator, which is a mechanism involved in the inhibition of melanocyte differentiation and proliferation, and the substances obtained by screening can be used as whitening agents.

[0028] In the method for screening a skin whitening agent of the second embodiment, the melanocyte differentiation promoter of the first embodiment is bone morphogenetic protein 4.

[0029] Among the factors that can serve as indicators in the screening method for whitening agents according to the first embodiment as factors that promote melanocyte differentiation induction, candidate whitening agents can be clearly screened by using a decrease in the gene expression level of bone morphogenetic protein 4 (BMP-4) as an indicator.

[0030] In the screening method for a skin whitening agent of the third embodiment, the cells are normal human cells in the first or second embodiment.

[0031] The screening method for a skin whitening agent of the fourth embodiment is the same as that of the third embodiment, except that the normal human cells are epidermal keratinocytes.

[0032] The screening method for a skin whitening agent of the fifth embodiment is the same as that of the third embodiment, except that the normal human cells are fibroblasts.

[0033] Melanin-producing melanocytes differentiate from melanocyte stem cells. It is believed that melanocyte differentiation-promoting factors secreted by cells present in the skin surrounding melanocyte stem cells (e.g., human normal cells such as epidermal keratinocytes, fibroblasts, Langerhans cells, neurons, T cells, Merkel cells, and melanocytes; hereinafter referred to as "surrounding cells") are involved in the differentiation of melanocyte stem cells into melanocytes. Therefore, if the secretion of melanocyte differentiation-promoting factors from surrounding cells can be inhibited, it is believed that the differentiation of melanocyte stem cells into melanocytes can also be inhibited, and as a result, melanin production by melanocytes can also be inhibited. Therefore, if human normal cells, particularly epidermal keratinocytes or fibroblasts, more preferably epidermal keratinocytes, are exposed to a candidate whitening agent, and the resulting decrease in the expression level of melanocyte differentiation-promoting factors, such as BMP-4, in the exposed cells indicates that the candidate whitening agent exposed to the cells has the effect of suppressing melanocyte differentiation-promoting factors. It is believed that the candidate substance for a whitening agent exposed to cells inhibits the differentiation of melanocyte stem cells into melanocytes through its effect of suppressing melanocyte differentiation-inducing factors, resulting in the suppression of melanin production. That is, in the third to fifth embodiments, screening for whitening effects is performed by exposing candidate substances for a whitening agent to human normal cells, particularly epidermal keratinocytes or fibroblasts, and more preferably epidermal keratinocytes, and observing the expression level of melanocyte differentiation-inducing factors. Note that epidermal keratinocytes are considered more preferable as normal human cells because they are located near melanocytes and therefore more likely to affect melanocytes.

[0034] (2) Bone morphogenetic protein 4 (BMP-4) inhibitor The BMP-4 inhibitor of the sixth embodiment comprises at least one of the group consisting of burnet extract, aloe vera leaf extract, fermented soy milk broth, moth bean seed extract, horse chestnut extract, licorice root extract, and angelica tree leaf / stem extract.

[0035] When various candidate substances for skin whitening agents were exposed to epidermal keratinocytes (normal human cells), Burnettsia extract, Aloe vera leaf extract, fermented soy milk broth, Moss bean seed extract, Horse chestnut extract, Licorice root extract, and Angelica keiskei leaf / stem extract significantly suppressed the BMP-4 gene expression level, and therefore each of the substances was determined to be effective as a BMP-4 inhibitor. Thus, by reducing the BMP-4 gene expression level, it is expected that melanin production can be suppressed, and a skin whitening effect can be expected through the suppression of melanin production. While Patent Document 6 reports that BMP-4 reduces melanin production in melanocytes, the sixth embodiment suppresses melanin production by using a BMP-4 inhibitor to reduce the BMP-4 expression level. Here, the BMP-4 inhibitor may be any of the substances listed above themselves, or a composition in which the substances listed above are dissolved in an appropriate solvent. The composition may also be a composition containing particles of any size obtained by micronizing each of the above substances alone or in combination with two or more of them together with an appropriate solvent or any additive, or may further be a composition containing each of the above substances and emulsifying or dispersing them.

[0036] Burnett's burnet extract is an extract extracted from the roots and rhizomes of the great burnet (Sanguisorba officinalis), and is available, for example, as "Jiyu Extract Powder" (Maruzen Pharmaceutical Co., Ltd.). Aloe vera leaf extract is an extract extracted from the sap of aloe vera (Aloe vera) leaves, and is available, for example, as "Aloe Extract Vera Miyakojima" (Ichimaru Pharcos Co., Ltd.). Fermented soy milk filtrate is a liquid obtained by fermenting and filtering soy milk, and is available, for example, as a product from Technoble Co., Ltd. Moss bean seed extract is an extract extracted from the seeds of moth bean (Vigna aconitifolia), and is available, for example, as "VIT-A-LIKE PW LS 9898" (Yamakawa Trading Co., Ltd.). Horse chestnut extract is an extract extracted from the seeds of the horse chestnut tree (scientific name: Aesculus hippocastanum) and is available, for example, as a product from Sabinsa Japan Corporation. Licorice root extract is an extract extracted from the root of licorice (scientific name: Glycyrrhiza glabra) and is available, for example, as "Licorice Extract (Maruzen Pharmaceutical Co., Ltd.)." Angelica keiskei leaf / stem extract is an extract extracted from either or both of the leaves and stems of Angelica keiskei (scientific name: Angelica keiskei) and is available, for example, as "Angelica keiskei Extract B (Maruzen Pharmaceutical Co., Ltd.)." However, the above acquisition methods are not limited.

[0037] The BMP-4 inhibitor of the seventh embodiment is the sixth embodiment, which contains particles containing a Sanguisorba officinalis extract, and the particles have a median diameter of less than 1 μm.

[0038] Hereinafter, the BMP-4 inhibitor of the seventh embodiment will be referred to as a nanosized burnet composition. The nanosized burnet composition is a burnet composition containing a burnet extract, and the particles containing the burnet extract have a median diameter of less than 1 μm.

[0039] Here, the median diameter is defined as the diameter at which the larger and smaller particles are equal when the powder is divided into two parts by particle diameter. The median diameter can be measured by applying the principle of dynamic light scattering. For example, the median diameter (d50) of dispersed particles can be measured using a particle size analyzer (FPAR-1000, Otsuka Electronics Co., Ltd.).

[0040] The nanosized burnet composition of the seventh embodiment can improve the melanin production inhibitory effect compared to a dispersion containing particles with a median diameter of 1 μm or more. The median particle diameter is preferably less than 200 nm, more preferably less than 100 nm, and even more preferably less than 80 nm. The lower limit of the median particle diameter may be 10 nm.

[0041] The particles contained in the nanoized burnet extract composition used in the seventh embodiment may be produced by any process, such as an emulsification process or a dispersion process, as long as they are in the form of particles with a median diameter of less than 1 μm. The particles contained in the nanoized burnet extract composition may contain oily substances such as hydrogenated polyisobutene, squalane, isotridecyl isononanoate, and caprylic / caprylic triglyceride.

[0042] The nanosized burnet composition of the seventh embodiment can be produced as oil droplets in an oil-in-water emulsion, for example, by suspending the burnet extract in an oil phase and then emulsifying and dispersing it together with an aqueous phase.

[0043] The amount of oil phase in the nanosized burnet composition is preferably 1% by mass to 20% by mass, more preferably 5% by mass to 17.5% by mass. An oil phase amount of 1% by mass or more is advantageous in terms of exerting the BMP-4 inhibitory effect. Furthermore, an oil phase amount of 20% by mass or less is advantageous in terms of dispersing the burnet extract in the BMP-4 inhibitor.

[0044] The BMP-4 inhibitor of the present disclosure can be added to, for example, cosmetics, quasi-drugs, pharmaceuticals, and the like.

[0045] (3) Cosmetics and their manufacturing methods The cosmetic material of the eighth embodiment contains the BMP-4 inhibitor of the sixth embodiment or any of the embodiments as a whitening agent.

[0046] When human normal cells, particularly epidermal keratinocytes or fibroblasts, are exposed to the BMP-4 inhibitor of the sixth or seventh embodiment, the expression level of BMP-4 is reduced compared to a control group that is not exposed. Therefore, the BMP-4 inhibitor of the sixth or seventh embodiment is useful as a skin-whitening agent that suppresses melanin production resulting from a decrease in the expression level of BMP-4. Therefore, by incorporating the BMP-4 inhibitor into a cosmetic product, a cosmetic product with a skin-whitening effect can be provided.

[0047] The cosmetic of the ninth embodiment contains as a whitening agent a candidate whitening agent substance that has been confirmed to have a reduced gene expression level of a melanocyte differentiation induction promoting factor using any of the screening methods for whitening agents of the first to fifth embodiments.

[0048] The content of a candidate substance for a whitening agent (e.g., Sanguisorba officinalis extract) in a cosmetic is preferably 0.0001% to 0.05% by mass, and more preferably 0.001% to 0.025% by mass. A content of the candidate substance for a whitening agent of 0.0001% by mass or more is advantageous in that a decrease in the gene expression level of a melanocyte differentiation-inducing factor is observed. Furthermore, a content of 0.05% by mass or less is advantageous in that the candidate substance for a whitening agent can be dispersed in the cosmetic.

[0049] The method for producing a cosmetic product according to the present disclosure is not particularly limited, as long as it includes a step of adding a candidate whitening agent, which has been screened using the method for screening whitening agents according to the present disclosure, to the cosmetic product as a whitening agent. The cosmetic product components are described below. The cosmetic product according to the present disclosure can be obtained, for example, by mixing a specific amount of a thickener, a specific amount of a moisturizing component, a specific amount of a surfactant, water, and, if necessary, other components to form the cosmetic product components, and then adding a specific amount of a BMP-4 inhibitor as a whitening agent, according to a known production method.

[0050] In particular, the cosmetic of the present disclosure is suitably produced by the cosmetic production method of embodiment 10. The cosmetic production method of embodiment 10 includes a step of determining, as a whitening agent, a candidate substance for a whitening agent that has been confirmed to have a reduced expression level of a melanocyte differentiation induction promoter by the screening method for whitening agents of any of embodiments 1 to 5 (hereinafter, the candidate substance determination step), and a step of adding the whitening agent to cosmetic ingredients (hereinafter, the addition step).

[0051] In the candidate substance determination step, a candidate substance for a whitening agent is determined that has been confirmed to have a reduced expression level of a melanocyte differentiation induction promoter by the method for screening a whitening agent according to any one of the above-described first to fifth embodiments. Details of the first to fifth embodiments have been described above, so a detailed description thereof will be omitted here.

[0052] In the addition step, the candidate substance for a whitening agent is added to the cosmetic ingredients. The addition can be carried out by mixing the candidate substance for a whitening agent with the cosmetic ingredients. The mixing can be carried out by a known method such as an emulsification dispersion method. When the emulsification dispersion method is used and the candidate whitening substance is oil-soluble, for example, an oil phase containing at least the candidate whitening substance and an aqueous phase are mixed and emulsified to obtain an oil-in-water or water-in-oil emulsion.

[0053] The oil phase may contain a candidate substance for a whitening agent, and if necessary, an emulsifier, as well as other additives, an organic solvent, etc. The candidate substance for a whitening agent is as described above, and other cosmetic ingredients will be described later. Examples of emulsifiers include lecithin, glycerin fatty acid esters (for example, higher fatty acid esters such as polyglyceryl oleate), saponin, and sucrose fatty acid esters (for example, higher fatty acid esters such as sucrose stearate).

[0054] The aqueous phase may contain water, an emulsifier, an organic solvent, and, if necessary, other cosmetic ingredients. The candidate substances for the whitening agent are as described above, and the other cosmetic ingredients will be described later. Examples of emulsifiers include lecithin, glycerin fatty acid esters, saponin, and sucrose fatty acid esters. The organic solvent is preferably a water-soluble solvent, and examples thereof include ethanol and polyhydric alcohols (butylene glycol, dipropylene glycol, glycerin, etc.).

[0055] The emulsification can be carried out in a temperature range of 20°C to 90°C. Emulsification can be carried out using a stirring device such as an ultra-high speed homogenizer or an ultrasonic homogenizer (for example, ultrasonic homogenizer US-600AT manufactured by Nippon Seiki Seisakusho Co., Ltd.).

[0056] The cosmetic composition of the present disclosure obtained may be in any form, such as a lotion, emulsion, cream, gel, wax, facial cleanser, or facial foam.

[0057] The method for screening for whitening agents of the present disclosure can select candidate substances for whitening agents useful as whitening agents using as an index the phenomenon that leads to a whitening effect, i.e., a decrease in the gene expression level of melanocyte differentiation-inducing factors (especially BMP-4).Furthermore, by incorporating the candidate substances for whitening agents selected by the method for screening for whitening agents of the present disclosure into cosmetics, cosmetics having a whitening effect can be provided.

[0058] Examples of uses of the cosmetic material of the present disclosure include use as cosmetics (for example, skin care cosmetics such as lotions and serums, particularly skin whitening skin care cosmetics). Examples of the cosmetic material include, in particular, skin care cosmetics (lotions, emulsions, serums, etc.), body cosmetics (body lotions, etc.), scalp cosmetics, etc. However, the uses of the cosmetic material of the present disclosure are not limited to the above.

[0059] The cosmetic preparation of the present disclosure may contain, as cosmetic ingredients, additives commonly used in cosmetics. Examples of additives include functional ingredients (e.g., γ-oryzanol, hydrogenated polyisobutene, etc.) that exhibit useful cosmetic effects (e.g., moisturizing effect, skin conditioning effect, etc.) when used in cosmetics. Other additives include, for example, solubilizers such as polyoxyethylene polyoxypropylene decyl tetradecyl ether (cosmetic ingredient name: PPG-6 decyltetradeceth-20) and polyoxyethylene hydrogenated castor oil, surfactants other than specific surfactants, astaxanthin, carotenoids other than astaxanthin (e.g., lycopene, carotene), preservatives such as phenoxyethanol and ethylhexylglycerin, colorants, thickeners, pH adjusters such as sodium hydroxide and hydrochloric acid, buffers, fragrances, antibacterial agents, ultraviolet absorbers, active oxygen scavengers, antimicrobial agents, anti-inflammatory agents, minerals, etc. [Example]

[0060] In the following examples, the melanin production effect of BMP-4 was evaluated, and candidate substances for skin whitening agents were screened based on the evaluation results. Among the candidate substances, the burnet extract, which exhibited the most excellent effect as a BMP-4 inhibitor, was examined for its melanin production inhibitory effect, and further, the melanin production inhibitory effect of nanoparticles of the burnet extract was examined. Note that the following examples show one example of an embodiment, and the present disclosure is not limited to the examples in any way.

[0061] (1) Evaluation of the melanin production effect of BMP-4 Proteins that function as melanocyte differentiation-promoting factors include BMP-2, BMP-4, BMP-7, SCF, endothelin-1, endothelin-3, Wnt3a, etc. Of these proteins, we examined the melanin production effect of BMP-4 in inducing differentiation of iPS cells into melanocytes.

[0062] Specifically, we created a system for inducing melanocyte differentiation from iPS cells and examined the effect of BMP-4 on the process of inducing differentiation into melanocytes.

[0063] iPS cells (Cellular Dynamics International) were seeded onto a 6-well plate. When they reached confluence, they were switched to neural crest cell-inducing medium prepared according to a method described in a published literature (Cell Reports 3, 1140-1152, April 25, 2013) to obtain melanocyte stem cells (SOX10 and c-kit positive cells). iPS cells were maintained in mTeSR1 medium (STEMCELL Technology) at 37°C in a 5% CO2 incubator. The melanocyte stem cells obtained by the above method were further cultured in a confluent state, and the cultured melanocyte stem cells were cultured in a differentiation-inducing medium containing Neurobasal Medium (Gibco) supplemented with N2 and B27 supplements (Gibco) at the recommended concentrations, followed by 1% by mass of GlutaMax (Gibco), 100 ng / mL SCF, 100 ng / mL Wnt3a, 250 ng / mL EDN3 (Endothelin-3), 25 ng / mL BMP-4, 3 μM CHIR99021, and 500 μM dbcAMP (dibutylyl circlic adenocine monophosphate), to induce differentiation of the stem cells into melanocytes.

[0064] Microscopic observation confirmed the appearance of melanocytes around the colonies around day 12. By day 21 after induction, the emerged melanocytes were observed to further synthesize melanin. These findings confirmed that this induction system can reproduce the entire process of melanocyte differentiation and melanin synthesis from stem cells. Based on these results, we investigated the effect of the presence or absence of BMP-4 on final melanin production in this induction system. As a control, cells were cultured under the same conditions using a differentiation induction medium of the same composition, except that BMP-4 was not added.

[0065] Using the above differentiation induction system, the number of viable cells was measured on day 21 after induction using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) reagent (MTT-100JP, Kurabo). After measuring the number of viable cells, a melanin dissolving solution (aqueous solution containing 1 mol / L NaOH and 10% by mass DMSO (dimethyl sulfoxide)) was added to each well, and the melanin was completely dissolved by heating and ultrasonicating at 50°C for 20 minutes.

[0066] After dissolution, 200 μL of the melanin solution from each well was transferred to a 96-well plate, and the absorbance at 400 nm was measured at room temperature. The absorbance was corrected for the number of viable cells to determine the final melanin content. The results are shown in Figure 1. The vertical axis of Figure 1 indicates the relative melanin production, with the melanin production in the control group set at 1. The "(-)" bar indicates the control group, which used differentiation-inducing medium without BMP-4, and the "(+)" bar indicates the experimental group, which used differentiation-inducing medium with a BMP-4 concentration of 10 ng / mL. Note that the bar in each group represents the average value of three samples, and the error bars indicate the standard deviation.

[0067] As a result of the measurements, as shown in Figure 1, the experimental group showed more than twice the amount of melanin produced as the control group. Next, a homogeneity of variance test was performed using an F-test between the control and experimental groups, and after confirming that the variances were homogeneous, a Student's t-test was performed. In Figure 1, "**" indicates that the significance level in the Student's t-test was 1% or less. In other words, from the results of Figure 1, it can be concluded that the presence of BMP-4 significantly increased the amount of melanin produced by melanocytes.

[0068] The results of the measurements showed that BMP-4 promotes increased melanin production during differentiation. Therefore, by suppressing the gene expression level of BMP-4 in the system where melanocytes produce melanin, the amount of BMP-4 in the system is reduced, suggesting that melanin production can be suppressed, as shown in the results of the control group compared to the experimental group in Figure 1.

[0069] (2) Screening of candidate substances for skin whitening agents Next, we screened for candidate substances for skin whitening agents using a decrease in the gene expression level of BMP-4 as an index. Specifically, we screened the following candidate substances 1 to 11, which are commonly believed to have skin whitening effects. Candidate substance 1: Loquat leaf extract (product name: Loquat Leaf Extract CA, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 2: Clove extract (trade name: Clove Extract-J, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 3: Acacia extract (trade name: Acacia extract, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 4: Arnica flower extract (product name: Arnica Extract BG, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 5: Aloe vera leaf extract (product name: Aloe Extract Vera Miyakojima, Ichimaru Pharcos Co., Ltd.) Candidate substance 6: Fermented soy milk (Technoble Co., Ltd.) Candidate substance 7: Moss bean seed extract (trade name: VIT-A-LIKE PW LS 9898, Yamakawa Trading Co., Ltd.) Candidate substance 8: Horse chestnut extract (Sabinsa Japan Corporation) Candidate substance 9: Licorice root extract (trade name: Licorice extract, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 10: Angelica keiskei leaf / stem extract (product name: Angelica keiskei extract BG, Maruzen Pharmaceutical Co., Ltd.) Candidate substance 11: Sanguisorba officinalis extract (trade name: Jiyu Extract Powder, Maruzen Pharmaceutical Co., Ltd.)

[0070] Candidate substance 1 is an extract extracted from the leaves of loquat (scientific name: Eriobotrya japonica), candidate substance 2 is an extract extracted from the flower buds of clove (scientific name: Syzygium aromaticum), candidate substance 3 is an extract extracted from the leaves and young branches of gambir tree (scientific name: Uncaria gambir), and candidate substance 4 is an extract extracted from the flowers of arnica (scientific name: Arnica montana).

[0071] Each of the above candidate substances was dissolved or suspended in a solution made by mixing DMSO and ultrapure water obtained using Milli-Q (Merck Millipore) in a volume ratio of 1:1 to give a candidate substance solution with a solid content of 1 mg / mL.

[0072] Human fetal keratinocytes (nHEK, Thermo Fisher Scientific) were harvested by trypsinization and then suspended in 1% PSN (penicillin-streptomycin-neomycin)-containing medium (EpiLife, Gibco). After centrifugation (300 g, 3 minutes, room temperature), the supernatant was removed, and the cells were resuspended in the above medium. 1.5 × 10 cells were placed in each well of a 12-well plate. 5 cells / cm 2 The amount of medium in each well was 1.0 mL. The day after seeding, 10 μL of each of the candidate substance solutions was added, mixed well, and allowed to stand in a CO2 incubator (37°C). The final concentration of each candidate substance was 10 ppm.

[0073] Control groups were also prepared in which nHEK cells were cultured under the same conditions but without any of the candidate substances. Three samples were prepared for each candidate substance group and the control group, and the following measurements were performed.

[0074] One day after adding the candidate substance, the supernatant was removed from each well and washed twice with phosphate-buffered saline (PBS). Total RNA was extracted using an RNA extraction reagent (RNeasy Mini Kit, QIAGEN) according to the protocol. After measuring the RNA concentration using a spectrophotometer, Ct values for BMP-4 were measured using an RT-PCR system (Mx3000P, Agilent Technologies) with 30 μg / μL of RNA as the template. Based on the measured Ct values, the amount of BMP-4 mRNA was measured using the relative quantification method (ΔΔCt method) with RT-PCR (PrimeScript One Step RT-PCR Kit, Takara Bio).

[0075] The nucleotide sequence of the forward primer for the BMP-4 gene, which was the target substance of this RT-PCR, was the nucleotide sequence 1 shown below, and the nucleotide sequence of the reverse primer was the nucleotide sequence 2 shown below. Base sequence 1: 5'-GTCCTGCTAGGAGGCGCGAG-3' Base sequence 2: 5'-GTTCTCCAGATGTTCTTTCG-3'

[0076] In addition, as a housekeeping gene for relative quantification by RT-PCR, the nucleotide sequence of the forward primer for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was the following nucleotide sequence 3, and the nucleotide sequence of the reverse primer was the following nucleotide sequence 4. Base sequence 3: 5'-GCACCGTCAAGGCTGAGAAC-3' Base sequence 4: 5'-TGGTGAAGACGCCAGTGGA-3'

[0077] Figure 2 shows the relative expression levels of BMP-4 gene for each candidate substance (three samples were measured for each candidate substance, and the mean and standard deviation of the three samples are shown). The vertical axis of the graph represents the relative expression level of BMP-4 gene, and the horizontal axis represents the number of each candidate substance. Each bar represents the mean value of three samples for each candidate substance, with the error bars indicating the standard deviation. The leftmost bar, labeled "none," represents the mean value for the control group, with the error bars indicating the standard deviation. Furthermore, a homogeneity of variance test was performed using an F-test for the control group and the groups exposed to each candidate substance. After verifying that the variances between the control group and the groups exposed to each candidate substance were not unequal, a Student's t-test was performed. In Figure 2, "*" indicates a risk level greater than 1% and less than 5%, "**" indicates a risk level greater than 0.5% and less than 1%, and "***" indicates a risk level less than 0.1%.

[0078] As shown in Figure 2, the following substances significantly reduced BMP-4 gene expression levels compared to the control group: candidate substance 5 (aloe vera leaf extract), candidate substance 6 (fermented soy milk broth), candidate substance 7 (moth bean seed extract), candidate substance 8 (horse chestnut extract), candidate substance 9 (licorice extract), candidate substance 10 (Angelica angelica leaf / stem extract), and candidate substance 11 (sanguisorba officinalis extract). Therefore, candidate substances 5 to 11 are useful as BMP-4 inhibitors that reduce BMP-4 gene expression levels in epidermal keratinocytes (normal cells). The reduction rates of BMP-4 gene expression compared to the control group for candidate substances 5 to 11 were 38.7%, 46.0%, 48.5%, 48.8%, 51.3%, 53.0%, and 84.7%, respectively. Among these, candidate substance 11, a Sanguinea pig extract, suppressed BMP-4 gene expression levels by more than 80% compared to the control group, suggesting that it has the strongest BMP-4 inhibitory effect among candidate substances 5 to 11 and is the most useful BMP-4 inhibitor.

[0079] (3) Inhibitory effect of Sanguisorba officinalis extract on melanin production Next, we examined whether the burnet extract, which had the most effective BMP-4 inhibitory effect in (2) above, exhibits an inhibitory effect on melanin production in actual cells.

[0080] Mouse melanoma cells (B-16, DS Pharma) were harvested by trypsinization and then suspended in a medium containing 1% PSN. The cells were centrifuged (300 × g, 3 minutes, room temperature) to remove the supernatant, and resuspended in the above medium. Then, 5 × 10 cells were placed in each well of a 12-well plate. 4 cells / cm 2 The amount of medium in each well was 1 mL. The next day, the medium was completely removed, and 1.0 mL of medium containing the desired concentration of Sanguisorba officinalis extract was added, followed by incubation in a CO2 incubator (37°C).

[0081] The target concentrations were 1 ppm and 5 ppm, and a stock solution of 100x the target concentration of burnet extract dissolved in DMSO was added at a ratio of 10 μL per 1 mL of medium. The final concentration of 1% DMSO was 1%. A medium containing 1% DMSO but no burnet extract was used as a control. Three groups (n=4 each) were tested: groups with 1 ppm and 5 ppm burnet extract concentrations, and a control group with no concentration.

[0082] Three days after seeding, the supernatant was removed from each well, washed twice with PBS, and the viable cell count was determined using WST reagent (Cell Counting Kit, Dojindo Laboratories). After measuring the viable cell count, each well was washed twice with PBS, and melanin dissolving solution (1 mol / L NaOH, 10% DMSO, aqueous solution) was added and completely dissolved by sonication at 50°C for 20 minutes.

[0083] After the dissolution, 200 μL of the melanin solution from each well was transferred to a 96-well plate, and the absorbance at 400 nm was measured at room temperature. The absorbance obtained from the measurement was corrected for the number of viable cells, and the final melanin amount was determined. The measurement results are shown in Figure 3. The vertical axis of the figure shows the relative amount of melanin produced, with the amount of melanin produced in the control group set at 100%. The leftmost bar shows the amount of melanin produced in the control group (concentration 0), the center bar shows the group with a 1 ppm burnet extract concentration, and the rightmost bar shows the group with a 5 ppm concentration. Note that the bar in each group represents the average value of four samples, and the error bars represent the standard deviation.

[0084] As shown in Figure 3, the results of the measurements showed that the 5 ppm and 1 ppm burnet extract groups produced approximately 76% and 82% of the melanin levels of the control group, respectively. Next, a homogeneity of variance test was performed between the control and experimental groups using an F-test. After confirming homogeneity of variance, a Student's t-test was performed. In Figure 3, an "*" indicates a significance level of greater than 1% and less than 5% in the Student's t-test. Therefore, from the results of Figure 3, it can be concluded that exposure to 5 ppm and 1 ppm burnet extract significantly reduced melanin production by melanocytes.

[0085] (4) Inhibitory effect of nano-sized Sanguisorba officinalis composition on melanin production (4-1) Preparation of nano-sized burnet composition Hereinafter, examples of cosmetics containing a nano-sized dispersion of Sanguisorba officinalis extract as a BMP-4 inhibitor as a skin whitening agent will be described. In this disclosure, "nanonized" is defined as particles with a median diameter of less than 1 μm.

[0086] (4-1-1) Preparation of nano-sized and non-nanofibrate compositions [Nanocomposition 1] (Adjustment of oil phase) The following ingredients were heated to 80°C and stirred for 1 hour while maintaining the temperature at 80°C to obtain an oil phase. γ-oryzanol: 0.5% by mass Sanguisorba officinalis extract: 1.0% by mass Hydrogenated polyisobutene: 12.5% by mass Lecithin (emulsifier): 1.0% by mass

[0087] (Preparation of aqueous phase) The following ingredients were heated to 80°C and stirred for 1 hour while maintaining the temperature at 80°C to obtain an aqueous phase. Polyglyceryl-10 oleate (emulsifier): 6.7% by mass Sucrose stearate (emulsifier): 3.3% by mass Glycerin (polyhydric alcohol) 40.0% by mass Water 35.0% by mass

[0088] While maintaining the temperature at 80°C, 15% by mass of the resulting oil phase was added to 85% by mass of the aqueous phase, and ultrasonic dispersion was performed for 1 minute using an ultrasonic homogenizer (US-600AT, Nippon Seiki Seisakusho). The resulting dispersion was subjected to high-pressure emulsification dispersion at a pressure of 245 MPa using a Starburst Mini (Sugino Machine), repeated three times in total. After high-pressure emulsification dispersion, the mixture was filtered using a microfilter with an average pore size of 1 μm to prepare nano-sized composition 1 as an emulsion composition cosmetic. The content of Sanguisorba officinalis extract in nano-sized composition 1 was 1.0% by mass.

[0089] [Nanocomposition 2] Nano-sized composition 2 as an emulsion composition cosmetic was prepared using the same process as nano-sized composition 1, except that the hydrogenated polyisobutene in nano-sized composition 1 was replaced with the same amount of squalane.

[0090] [Nanocomposition 3] Nano-composition 3, a cosmetic emulsion composition, was prepared using the same process as nano-composition 1, except that the hydrogenated polyisobutene in nano-composition 1 was replaced with the same amount of isotridecyl isononanoate.

[0091] [Nanocomposition 4] Nano-sized composition 4, a cosmetic emulsion composition, was prepared using the same process as nano-sized composition 1, except that the hydrogenated polyisobutene in nano-sized composition 1 was replaced with the same amount of tri(caprylic / caprylic acid)glyceryl.

[0092] [Non-nanoized composition] The oil phase composition was added to the aqueous phase composition prepared in the same manner as nano-sized composition 1, and then stirred for 1 minute using a propeller-type mixer (As One) to prepare a non-nanofiberized composition as a cosmetic.

[0093] (4-1-2) Evaluation of nanoparticles The nanosized compositions 1 to 4 and the non-nanized composition were evaluated by the following methods.

[0094] [Evaluation of particle size] The obtained cosmetic was diluted with Milli-Q water to a concentration of 1% by mass, and the average particle size (median size (d50)) of the dispersed particles was measured using a particle size analyzer (FPAR-1000, Otsuka Electronics Co., Ltd.).

[0095] [Evaluation of stability over time] Immediately after preparation of the obtained cosmetics, and immediately after preparation, after 3 days in a 50°C environment, each cosmetic was diluted 100 times with water and the turbidity was measured using a spectrophotometer (V-630, JASCO Corporation). The turbidity 3 days after preparation was evaluated on a 4-point scale of A, B, C, and D, from best to worst, according to the following evaluation criteria. A: The turbidity was 0.025 or less. B: The turbidity was greater than 0.025 and less than 0.2. C: The turbidity was greater than 0.2 and less than 0.5. D: Turbidity exceeds 0.5.

[0096] The evaluation results of the median diameter and stability over time for nanosized compositions 1 to 4 and the non-nanized composition are shown in Table 1 below.

[0097] [Table 1]

[0098] From Table 1 above, it can be seen that while the median diameter of the non-nanonized composition was 5.6 μm, the median diameter of the cosmetics of nanoized compositions 1 to 4 was 51 nm for nanoized composition 1, the smallest, and 115 nm for nanoized composition 4, the largest, indicating that all were nanoized. The stability of the cosmetics over time was evaluated as good, with nanoized composition 1 and nanoized composition 2 receiving an A rating. However, as the average particle size increased, the evaluation of stability over time worsened, with nanoized composition 3 receiving a B rating and nanoized composition 4 receiving a C rating. The non-nanonized composition with an average particle size of 5.6 μm, which was not nanoized, received the poorest rating, D rating. It is believed that particles with each median diameter in nanoized compositions 1 to 4 function as oil droplets containing burnet extract and as BMP-4 inhibitors.

[0099] (4-2) Evaluation of the effect of nano-sized Sanguisorba officinalis extract on melanin production Using a skin model containing melanocytes, we investigated the whitening effect of nano-sized burnet extract, which was found to have an inhibitory effect on BMP-4 gene expression levels. Burnet extract contains large amounts of tannins and saponins and is a hydrophobic material that is poorly soluble in water. Nano-sizing improves its penetration into the skin, and a higher whitening effect can be expected compared to non-nano-sized formulations. In this study, nano-sized composition 1, which had the smallest average particle size and the highest stability rating over time in (4-1) above, was used as the experimental group, and its whitening effect was evaluated in comparison to a control group using a non-nano-sized composition.

[0100] Immediately after the melanocyte-containing skin model (MEL-300B, Kurabo) arrived at the laboratory, the culture cup was transferred to a netwell and pre-incubated for one day using maintenance medium. After pre-incubation, the cosmetics of nano-sized example 1 and non-nano-sized example were applied, and the maintenance medium was changed once every three days before applying the cosmetics, and the model was cultured for one week.

[0101] After one week of culture, the skin model culture cups were removed and washed with PBS, and the number of viable cells was detected using the MTT reagent described in (1). After measuring the number of viable cells, the products of the reaction with the MTT reagent were completely removed from each skin model using isopropanol, and the melanin dissolving solution described in (1) was added and completely dissolved by heating at 80°C for 1 hour.

[0102] After dissolution, 200 μL of the melanin solution from each well was transferred to a 96-well plate, and the absorbance at 400 nm was measured at room temperature. The amount of melanin was calculated from the absorbance obtained from the measurement. The final amount of melanin was calculated by correcting the value based on the number of viable cells.

[0103] The results are shown in Figure 4. The vertical axis of Figure 4 shows the amount of melanin produced when the amount of melanin produced in the non-nano-sized composition is taken as 100%. The bar graph for "Non-nano-sized component" shows the control group, and the bar graph for "Nano-sized component" shows the experimental group. The bar graph for each group represents the average value of three samples, and the error bars represent the standard deviation. Furthermore, a homogeneity of variance test was performed using an F-test for each of the non-nano-sized composition and nano-sized composition 1, and after confirming homogeneity of variance, a Student's t-test was performed. In Figure 4, an "*" indicates that the risk rate in the above t-test is greater than 1% and less than 5%.

[0104] The results in Figure 4 show that application of a cosmetic containing nanoized burnet extract as a BMP-4 inhibitor as a whitening agent reduced melanin production by 28% compared to non-nanoized cases. This indicates that nanoizing the burnet extract improves its skin permeability, thereby enhancing its melanin production inhibitory effect.

[0105] (5) Manufacturing method of cosmetics A specific example of a method for producing a cosmetic product will be described below.

[0106] (5-1) Whitening agents Candidate substances 1 to 11, which are candidate substances for skin whitening agents as described in (2), were subjected to the screening method described in (2). Of candidate substances 5 to 11, which were confirmed to reduce the expression level of BMP-4, a melanocyte differentiation-inducing factor, candidate substance 11, which had the most effective BMP-4 inhibitory activity, was selected as the skin whitening agent. This burnet extract was nano-sized using the method described in (4-1-1) to prepare a nano-sized composition. Of the nano-sized compositions listed in (4-1-1), nano-sized composition 1 was used as the skin whitening agent.

[0107] (5-2) Astaxanthin-containing emulsion composition The astaxanthin-containing emulsion compositions that are components of the following Cosmetics 1 to 6 and 9 were prepared as follows.

[0108] [Aqueous phase composition A] The following ingredients were dissolved for 1 hour while heating at 70°C to obtain aqueous phase composition A. Sucrose stearate (HLB=16): 33.0g Decaglyceryl monooleate (HLB=12): 67.0g Glycerin: 450.0g Pure water: 300.0g

[0109] [Oil Phase Composition A] The following ingredients were dissolved for 1 hour while heating at 70°C to obtain an oil phase composition A. Krill extract: 15.0g Mixed tocopherols (Riken Vitamin, Riken E Oil 800): 32.0g Medium-chain fatty acid glyceride (Kao, Coconard MT): 93.0g Lecithin (Riken Vitamin, Resion P, soybean-derived): 10.0g

[0110] The aqueous phase composition A obtained above was stirred (10,000 rpm) with a homogenizer (model name: HP93, manufactured by SMT Co., Ltd.) while maintaining the temperature at 70°C, and the oil phase composition A was added to the aqueous phase composition A to obtain a preliminary emulsion.

[0111] The resulting pre-emulsified product was then cooled to approximately 40°C and subjected to high-pressure emulsification at a pressure of 200 MPa using an Ultimizer HJP-25005 (manufactured by Sugino Machine Co., Ltd.). The resulting mixture was then filtered through a microfilter with an average pore size of 1 μm to prepare an astaxanthin-containing emulsion composition (astaxanthin content: 0.3% by mass).

[0112] The obtained astaxanthin emulsion composition was diluted to 1% by mass with Milli-Q water, and the particle size of the dispersed particles was measured using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), which was found to be 58 nm.

[0113] Furthermore, an equivalent emulsion composition can be obtained by replacing the krill extract with product name: Astax-ST (Marine Daio Co., Ltd.) or product name: ASTOTS-S (Haematococcus algae extract, Fujifilm Corporation).

[0114] (5-3) Preparation of lycopene-containing emulsion composition The lycopene-containing emulsion compositions, which are components of the following cosmetics 1, 2 and 9, were prepared as follows.

[0115] [Aqueous phase composition B] The following components were weighed into a container and heated and mixed in a thermostatic bath at 70°C while stirring. After confirming that the components were well mixed, the temperature was maintained at 70°C to obtain an aqueous phase composition B. Decaglyceryl-10 oleate (HLB=12.0, Nikko Chemicals, Decaglyn 1-OV): 8.0g Sucrose stearate (Ryoto Sugar Ester S-1670, manufactured by Mitsubishi Chemical Foods): 2.0 g Glycerin: 45.0g Purified water: up to 100g remaining

[0116] [Oil phase composition B] The following components were weighed into a container and heated and mixed on a hot plate at 150°C for 5 minutes while stirring. After confirming that the components were well mixed, an oil phase composition B was obtained. Tomato oleoresin (Lyc-O-Mato 15% (containing 15% lycopene by mass), manufactured by Sunbright): 1.14g Lecithin (Resion P (soybean-derived), manufactured by Riken Vitamin): 1.0g Medium-chain fatty acid glyceride (Coconard MT, manufactured by Kao): 12.8g

[0117] The aqueous phase composition B obtained above was added to the oil phase composition B, and the mixture was stirred and mixed, followed by dispersing for a predetermined time using an ultrasonic homogenizer to obtain a crude dispersion. The crude dispersion was then further emulsified at a high pressure of 200 MPa using an ultra-high pressure emulsifier (Ultimizer, manufactured by Sugino Machine Co., Ltd.) to prepare a lycopene-containing composition (lycopene content: 0.17% by mass).

[0118] The obtained lycopene-containing emulsion composition was diluted to 1% by mass with Milli-Q water, and the particle size of the dispersed particles was measured using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), which was found to be 52 nm.

[0119] (5-4) Cosmetic Preparation The whitening agent obtained in (5-1) was added to each of the cosmetic ingredients listed below to produce the following cosmetics 1 to 9.

[0120] (5-4-1) Cosmetics 1 (lotion) As cosmetic preparation 1, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients having the following composition, and a lotion was prepared in a conventional manner (total amount 100% by mass). Arbutin: 2.0% by mass Dipotassium glycyrrhetinate: 1.0% by mass Dipropylene glycol: 4.0% by mass Polyoxyethylene methyl glucoside: 1.0% by mass 1,3-butylene glycol: 4.0% by mass Polyethylene glycol: 1.0% by mass Ethanol: 2.0% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Phenoxyethanol: 0.3% by mass Glycerin mono-2-ethylhexyl ether: 0.2% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20, manufactured by Nikko Chemicals): 0.03% by mass N-acetyl-L-hydroxyproline: 1.0% by mass Water-soluble collagen: 1.0% by mass Hydrolyzed collagen: 1.0% by mass Seaweed extract (1): 1.0% by mass Astaxanthin-containing emulsion composition: 0.2% by mass Lycopene-containing emulsion composition: 0.1% by mass Magnesium ascorbyl phosphate: 1.0% by mass Sodium hyaluronate: 0.2% by mass Citric acid: 1.0% by mass Sodium citrate: appropriate amount Water: remaining amount

[0121] (5-4-2) Cosmetics 2 (Serum) As cosmetic preparation 2, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients having the following composition, and a beauty serum was prepared in a conventional manner (total amount 100% by mass). Magnesium ascorbyl phosphate: 2.0% by mass Dipotassium glycyrrhetinate: 1.0% by mass Dipropylene glycol: 4.0% by mass Glycerin: 5.0% by mass Diglycerin: 2.0% by mass 1,2-pentanediol: 2.0% by mass Phenoxyethanol: 0.5% by mass Methyl parahydroxybenzoate: 0.1% by mass Alkaline Negatives Latex B-16 Polymer: 0.05% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.05% by mass Citric acid: 0.7% by mass Sodium citrate: appropriate amount N-acetyl-L-hydroxyproline: 1.0% by mass Water-soluble collagen: 1.0% by mass Hydrolyzed collagen: 1.0% by mass Yeast extract (1): 1.0% by mass Astaxanthin-containing emulsion composition: 0.2% by mass Lycopene-containing emulsion composition: 0.2% by mass Water: remaining amount

[0122] (5-4-3) Cosmetic 3 (Cream) Cosmetic 3 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition, to prepare a cream in a conventional manner (total amount 100% by mass). Arbutin: 2.0% by mass Dipotassium glycyrrhetinate: 1.0% by mass Magnesium ascorbyl phosphate: 0.1% by mass 1,2-pentanediol: 3.0% by mass Dipropylene glycol: 7.0% by mass Concentrated glycerin: 5.0% by mass Polyethylene glycol 6000 (molecular weight: 6000): 1.0% by mass Sodium hyaluronate: 0.5% by mass Trimethylglycine: 0.5% by mass 1,3-butylene glycol: 3.0% by mass Xanthan gum: 0.5% by mass Acrylic acid / alkyl methacrylate copolymer: 0.7% by mass Squalane: 0.5% by mass Shea butter: 1.0% by mass White beeswax: 1.0% by mass Behenyl alcohol: 1.0% by mass Glyceryl monostearate: 2.0% by mass Polyoxyethylene glyceryl isostearate: 1.0% by mass Tocopherol: 0.5% by mass Astaxanthin: 0.2% by mass Water-soluble collagen: 1.0% by mass Hydrolyzed collagen solution (fish-derived): 1.0% by mass N-acetyl-L-hydroxyproline: 1.0% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.1% by mass Sucrose fatty acid ester: 0.1% by mass Polyglyceryl monooleate: 0.1% by mass Citric acid: 0.7% by mass Sodium citrate: appropriate amount Phenoxyethanol: 0.3% by mass Purified water: remaining amount

[0123] (5-4-4) Cosmetics 4 (Sunscreen) Cosmetic 4 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition, to prepare a sunscreen agent in a conventional manner (total amount 100% by mass). Cyclopentasiloxane: 20.0% by mass Dimethicone mass%: 10.0 mass% Titanium oxide: 5.0% by mass t-Butyl methoxybenzoylmethane: 1.0% by mass HXMT-100ZA (manufactured by Teika, average primary particle size 15 nm): 6.0% by mass Aluminum hydroxide: 1.0% by mass Isostearic acid: 0.5% by mass Sorbitan sesquioleate: 1.0% by mass Dipotassium glycyrrhetinate: 0.5% by mass Magnesium ascorbyl phosphate: 0.1% by mass Krill extract: 0.5% by mass Water-soluble collagen: 1.0% by mass Citric acid: 0.7% by mass Sodium citrate: appropriate amount Tocopherol: 0.5% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.1% by mass Fragrance: Trace amount Methyl parahydroxybenzoate: 0.15% by mass Purified water: remaining amount

[0124] (5-4-5) Cosmetics 5 (lotion) [Oil phase composition C] An oil phase composition C was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition. Haematococcus algae extract: 0.2% by mass Astaxanthin-containing emulsion composition: 0.4% by mass Squalane: 8.0% by mass Jojoba oil: 7.0% by mass Cetyl alcohol mass%: 1.5 mass%

[0125] [Aqueous phase composition C] Additionally, the cosmetic ingredients having the following composition were mixed to prepare an aqueous phase composition C. Glycerin monostearate: 2.0% by mass Polyoxyethylene cetyl ether: 3.0% by mass Polyoxyethylene isorbitan monooleate: 2.0% by mass 1,3-butylene glycol: 1.0% by mass Glycerin: 2.0% by mass Sucrose stearate: 0.1% by mass Polyglyceryl-10 oleate: 0.1% by mass Polyglyceryl-2 stearate: 0.1% by mass Phenoxyethanol: 0.2% by mass Collagen: 1.0% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20, manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.05% by mass Arbutin: 0.5% by mass Dipotassium glycyrrhetinate: 0.5% by mass Citric acid: 1.0% by mass Sodium citrate: appropriate amount Fragrance: Trace amount Purified water: remaining amount

[0126] The above oil phase composition C and aqueous phase composition C were each heated to 70°C and stirred while emulsifying, to prepare an emulsion as cosmetic preparation 5 (total amount 100% by mass).

[0127] (5-4-6) Cosmetic 6 (jelly-like serum) Cosmetic 6 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition, and a jelly-like beauty serum was prepared in a conventional manner (total amount 100% by mass). Haematococcus algae extract: 0.1% by mass Astaxanthin-containing emulsion composition: 0.2% by mass Ceramide III and VI mixture: 1.0% by mass Hydrolyzed collagen: 1.0% by mass Acetylhydroxyproline: 1.0% by mass Ethylhexylglycerin: 0.1% by mass Oleic acid: 0.5% by mass 1,3-butylene glycol: 1.0% by mass Glycerin: 2.0% by mass Sucrose: 0.1% by mass Polyglyceryl-10 oleate: 0.1% by mass Polyglyceryl-2 stearate: 0.1% by mass Phenoxyethanol 0.2% by mass Collagen: 1.0% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.1% by mass Citric acid: 1.0% by mass Sodium citrate: appropriate amount (PEG-240 / Decyltetradeceth-20 / HDI) copolymer: 0.3% by mass Damask rose flower oil: trace amount Fragrance: Trace amount Purified water: remaining amount

[0128] (5-4-7) Cosmetic 7 (wet foundation) 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients of the composition below to prepare a slurry, which was then filled into a specified container and dried to produce a solid powder cosmetic (wet foundation) as cosmetic 7 (total amount 100% by mass). Talc (OTS-2 TALK JA-46R, manufactured by Daito Kasei): 18% by mass Titanium oxide (OTS-2 TiO2 CR-5, manufactured by Daito Kasei): 9.0% by mass Iron oxide yellow (OTS-2 YELLOW LLXLO, manufactured by Daito Kasei): 2.3% by mass Iron oxide red (OTS-2 RED R-516L, manufactured by Daito Kasei): 0.15% by mass Iron oxide black (OTS-2 BLACK BL-100, manufactured by Daito Kasei): 0.3% by mass Pearl pigment (gold) (Rona Flare Balance Gold, manufactured by Merck): 13% by mass Pearl pigment (red) (Transprismar Red, manufactured by Merck): 7.0% by mass Composite powder pigment (HNB RED7, manufactured by Daito Kasei Kogyo): 1.0% by mass Dimethicone trimethylsiloxysilicate (DC593, manufactured by Toray Dow Corning): 3.0% by mass Dimethicone (SH200C-20cs, manufactured by Toray Dow Corning): 7.0% by mass Phenoxyethanol: 0.5% by mass Sericite (OTS-2 SERICITE FSE, manufactured by Daito Kasei Co., Ltd.): remaining amount

[0129] (5-4-8) Cosmetics 8 (Liquid Foundation) Cosmetic 8 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition, to prepare a liquid foundation (W / O emulsion) in a conventional manner (total amount 100% by mass). Dipotassium glycyrrhizinate: 0.2% by mass Specific red composite pigment (HNB RED7, manufactured by Daito Kasei): 0.5% by mass Extender pigment (OTS-2 SERICITE PSE and OTS-2 TALK JA-46R (both manufactured by Daito Kasei) mixed in a ratio of 7:3): 15.0% by mass Colorant pigment (OTS-2 TiO2 CR-50, OTS-2 YELLOW LLXLO, OTS-2 RED R-516L, and OTS-2 BLACK BL-100 (all manufactured by Daito Kasei) mixed in a ratio of 78:19:1:2): 2.0% by mass Pearl pigment (a mixture of Naflare Balance Gold and Lance Prisma Red (both manufactured by MERCK) in a ratio of 7:3): 3.0% by mass Cyclomethicone: 25.0% by mass Dimethicone polyol: 5.0% by mass Lauryl PEG-9 polydimethylsiloxyethyl dimethicone: 3.0% by mass PEG-9 polydimethylsiloxyethyl dimethicone: 1.2% by mass Squalane: 0.1% by mass Sorbitan sesquiisostearate: 1.0% by mass Disteardimonium hectorite: 0.8% by mass Ethylhexyl methoxycinnamate: 2.5% by mass Haematococcus pluvialis oil: 0.1% by mass Tocopherol: 0.1% by mass Damask rose flower oil: trace amount Fragrance: Appropriate amount Phenoxyethanol: 0.3% by mass Glycerin: 10.0% by mass Dipropylene glycol: 4.0% by mass 1,3-butylene glycol: 3.0% by mass Water-soluble collagen: 0.1% by mass Royal jelly extract: 0.1% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.1% by mass Calcium chloride: 1.0% by mass Citric acid: 1.0% by mass Sodium citrate: appropriate amount Ion exchange water: remaining amount

[0130] (5-4-9) Cosmetics 9 (Facial Cleanser) Cosmetic 9 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic ingredients having the following composition, to prepare a facial cleanser in a conventional manner (total amount 100% by mass). Potassium myristate: 2.0% by mass Potassium palmitate: 0.5% by mass Potassium stearate: 0.5% by mass (Lauramide / Myristamide) DEA: 1.0% by mass Sodium cocoyl glycinate: 10.0% by mass Sodium lauroampho: 13.0% by mass PEG-32: 3.0% by mass Butylene glycol: 15.0% by mass Glycerin: 10.0% by mass Sorbitol: 5.0% by mass Potassium hydroxide: appropriate amount Glyceryl stearate: 1.5% by mass Haematococcus pluvialis oil: 0.05% by mass Astaxanthin-containing emulsion composition: 0.05% by mass Lycopene-containing emulsion composition: 0.1% by mass Water-soluble collagen: 1.0% by mass Tocopherol: 0.5% by mass Citric acid: 1.0% by mass Sodium citrate: appropriate amount Shea butter: 1.0% by mass Polyquaternium-7: 0.5% by mass Polyquaternium-39: 0.5% by mass Sodium lauroyl glutamate: 1.0% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20, manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Phenoxyethanol: 0.5% by mass Damask rose flower oil: trace amount Fragrance: Trace amount

[0131] (5-4-10) Cosmetics 10 (Serum) As cosmetic preparation 10, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients having the following composition, and a beauty serum was prepared in a conventional manner (total amount 100% by mass). Magnesium ascorbyl phosphate: 2.0% by mass Dipotassium glycyrrhetinate: 1.0% by mass Dipropylene glycol: 4.0% by mass Sanguisorba officinalis extract (product name: Jiyu Extract Powder, Maruzen Pharmaceutical Co., Ltd.): 0.001% by mass Glycerin: 5.0% by mass Diglycerin: 2.0% by mass 1,2-pentanediol: 2.0% by mass Phenoxyethanol: 0.5% by mass Methyl parahydroxybenzoate: 0.1% by mass Alkaline Negatives Latex B-16 Polymer: 0.05% by mass Oryzanol: 0.01% by mass Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals): 0.03% by mass Polyoxyethylene hydrogenated castor oil (60E.O.): 0.2% by mass Centella asiatica extract: 0.01% by mass Lecithin: 0.05% by mass Citric acid: 0.7% by mass Sodium citrate: appropriate amount N-acetyl-L-hydroxyproline: 1.0% by mass Water-soluble collagen: 1.0% by mass Hydrolyzed collagen: 1.0% by mass Yeast extract (1): 1.0% by mass Astaxanthin-containing emulsion composition: 0.2% by mass Lycopene-containing emulsion composition: 0.2% by mass Nicotinamide: 5.0% by mass Water: remaining amount

Claims

[Claim 1] A method for screening for a skin whitening agent, the method using as an index a decrease in the expression level of a melanocyte differentiation induction promoter when cells are exposed to a candidate substance for a skin whitening agent, compared to the expression level of the melanocyte differentiation induction promoter in a control group in which the cells are not exposed to the candidate substance for a skin whitening agent, the melanocyte differentiation promoter is bone morphogenetic protein 4, the cells are human normal cells, The method for screening a skin whitening agent, wherein the normal human cells are epidermal keratinocytes or fibroblasts.

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