JP7594051B2 - Bone morphogenetic protein 4 inhibitor - Google Patents

Description

The present invention relates to a skin whitening agent obtained by a method for screening a skin whitening agent .

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, bright skin. For example, the following Patent Document 1 discloses cosmetics containing a specific cysteine derivative or a salt of a cysteine derivative and a specific whitening agent, and discloses a number of plant extracts as the specific whitening agent.

Melanin, the black pigment in skin, is produced by pigment-producing cells known as melanocytes. The following Patent Document 2 discloses a method for promoting induction of melanocyte differentiation for the purpose of treating diseases such as vitiligo vulgaris, hereditary symmetrical pigmentary dyschromatosis, and albinism, which are caused by abnormal suppression of 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 cells and a component that induces terminal differentiation into keratinocytes.

The following Patent Document 3 discloses a method for screening for differentiation inducers of pluripotent stem cells, and describes many 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 below) and in promoting hair growth (Patent Document 5 below). Furthermore, the following Patent Document 6 reports that BMP-4 reduces melanin production in melanocytes.

International Publication No. 2013/081147 JP 2015-146803 A Special Publication No. 2005-500847 JP 2009-226069 A Special table 2008-534607 publication Special Publication No. 2006-508026

Skin pigmentation is caused by the differentiation and proliferation of melanocytes from stem cells, which then leads to increased melanin production by the proliferated melanocytes. Therefore, substances that can inhibit the differentiation and proliferation of melanocytes are thought to be useful as skin whitening agents.

The objective of the present disclosure is to screen for substances that are useful as skin whitening agents by utilizing a mechanism involved in the inhibition of melanocyte differentiation and proliferation, to provide a BMP-4 inhibitor as a skin whitening agent whose usefulness has been confirmed through screening, and to provide a cosmetic product containing a skin whitening agent whose usefulness has been confirmed through screening, and a method for producing the cosmetic product.

Specific means for solving the above problems include the following embodiments.
[1] A method for screening whitening agents, which uses as an indicator a reduction in the expression level of a melanocyte differentiation-inducing factor when cells are exposed to a candidate whitening agent, relative 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.

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

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

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

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

[6] A bone morphogenetic protein 4 inhibitor comprising at least one selected from the group consisting of Sanguisorba officinalis extract, Aloe vera leaf extract, fermented soy milk broth, Moss bean seed extract, Horse chestnut extract, Licorice extract, and Angelica keiskei leaf/stem extract.

[7] The 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.

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

[9] A cosmetic preparation 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 promoter by any one of the screening methods for whitening agents described in [1] to [5].

[10] A method for producing a cosmetic, comprising the steps of: determining, as a whitening agent, a candidate whitening agent for which a reduced expression level of a melanocyte differentiation-inducing factor has been confirmed by any one of the screening methods for whitening agents according to [1] to [5]; and adding the whitening agent to a cosmetic ingredient.

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

Graph showing the melanin production effect of BMP-4. Graph showing the results of screening for candidate substances for skin whitening agents. Graph showing the effect of the presence or absence of Sanguisorba officinalis extract on melanin production. Graph showing the effect of nano-sizing of Sanguisorba officinalis extract on melanin production.

(1) Screening method for whitening agents The screening method for whitening agents of the first embodiment uses as an indicator a reduction in the expression level of a melanocyte differentiation-inducing factor when cells are exposed to a candidate whitening agent, relative 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.

In this embodiment, when a substance is determined to be a candidate for a whitening agent, the expression levels of the melanocyte differentiation-inducing factor are verified 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 lower than that in the control group is used as an index for screening as a whitening agent. Here, a "whitening agent candidate substance" is a candidate substance that can become a whitening agent, and refers to a test substance for screening. Also, a "whitening agent" refers to a substance that has been confirmed to have the function of lowering the expression level of the melanocyte differentiation-inducing factor through screening, among the candidate substances for whitening agents.

A melanocyte differentiation-inducing promoter is a protein that promotes the differentiation of pluripotent stem cells (e.g., induced pluripotent stem cells (iPS cells)) or melanocyte stem cells into melanocytes. Examples of proteins that function as melanocyte differentiation-inducing promoters include BMP-2, BMP-4, BMP-7, stem cell factor (SCF), endothelin 1, endothelin 3, and Wnt3a.

Here, the expression level of the melanocyte differentiation induction promoting factor may be confirmed by detecting the melanocyte differentiation induction promoting factor itself as a produced protein, or may be confirmed by detecting the expression of the corresponding gene when the melanocyte differentiation induction promoting factor is produced. In other words, the expression level of the melanocyte differentiation induction promoting factor may be either the gene expression level or the protein expression level, or both.

Methods for confirming gene expression levels include, for example, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), a method for detecting gene amount using an RNA probe (microarray method), a method for sequencing an RNA sequence using a next-generation sequencer and detecting the expression amount, and a method for detecting the expression amount using nucleic acid hybridization (in situ hybridization method).

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 protein detection using proteome analysis.

The decrease in the expression level of the melanocyte differentiation induction promoting factor in the experimental group may be judged by whether the expression level in the experimental group is even slightly lower than that in the control group, and preferably by whether the decrease rate of the expression level in the experimental group relative to that of the control group (i.e., the rate calculated by "(A-X)/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 decrease 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 rate for judging statistical significance may be 5% or less.

According to this embodiment, candidate substances for skin whitening agents that may be useful as skin 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 skin whitening agents.

In the second embodiment of the method for screening skin whitening agents, the melanocyte differentiation-inducing factor in the first embodiment is bone morphogenetic protein 4.

Of the factors that can serve as indicators for melanocyte differentiation-inducing promoting factors in the screening method for skin whitening agents according to the first embodiment, a decrease in the gene expression level of bone morphogenetic protein 4 (BMP-4) can be used as an indicator to clearly screen for candidate skin whitening agents.

The screening method for skin whitening agents of the third embodiment is the same as that of the first or second embodiment, except that the cells are normal human cells.

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

The fifth embodiment of the method for screening skin whitening agents is the third embodiment, in which the normal human cells are fibroblasts.

Melanocytes that produce melanin differentiate from melanocyte stem cells. It is presumed that the differentiation of melanocyte stem cells into melanocytes involves melanocyte differentiation-promoting factors secreted from cells present in the skin surrounding melanocyte stem cells (e.g., human normal cells such as epidermal keratinocytes, fibroblasts, Langerhans cells, nerve cells, T cells, Merkel cells, and melanocytes, hereinafter referred to as "surrounding cells"). Therefore, if the secretion of melanocyte differentiation-promoting factors from surrounding cells can be suppressed, it is believed that the differentiation of melanocyte stem cells into melanocytes can also be suppressed, and as a result, melanin production by melanocytes can also be suppressed. Therefore, if a candidate whitening agent is exposed to human normal cells, particularly epidermal keratinocytes or fibroblasts, more preferably epidermal keratinocytes, and the expression level of melanocyte differentiation-promoting factors, such as BMP-4, in the exposed cells is reduced, this means that the candidate whitening agent exposed to the cells has the effect of suppressing melanocyte differentiation-promoting factors. It is believed that the whitening agent candidate substance exposed to the cells inhibits the differentiation of melanocyte stem cells into melanocytes through the effect of inhibiting the melanocyte differentiation induction promoting factor, and as a result, inhibits melanin production. That is, in the third to fifth embodiments, the whitening agent candidate substance is exposed to human normal cells, particularly epidermal keratinocytes or fibroblasts, more preferably epidermal keratinocytes, and the expression level of the melanocyte differentiation induction promoting factor is observed, which is considered to be screening for whitening effects. The reason why epidermal keratinocytes are considered to be more preferable as human normal cells is presumably because epidermal keratinocytes are located near melanocytes and therefore are more likely to affect melanocytes.

(2) Bone morphogenetic protein 4 (BMP-4) inhibitor The BMP-4 inhibitor of the sixth embodiment includes at least one selected from the group consisting of Sanguisorba officinalis 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.

When epidermal keratinocytes as normal human cells were exposed to various candidate substances for whitening agents, it was determined that each of the above substances has an effect as a BMP-4 inhibitor, since the extract of Sanguinea japonica, the extract of Aloe vera leaf, the fermented soy milk, the extract of Moss Bean seed, the extract of Horse Chestnut, the extract of Licorice Root, and the extract of Angelica keiskei leaf/stem significantly suppressed the gene expression level of BMP-4. In this way, it is expected that melanin production can be suppressed by reducing the gene expression level of BMP-4, and a whitening effect can be expected through the suppression of melanin production. It should be noted that the above-mentioned Patent Document 6 reports that BMP-4 reduces the production of melanin in melanocytes, whereas the sixth embodiment suppresses melanin production by reducing the expression level of BMP-4 with the BMP-4 inhibitor. Here, the BMP-4 inhibitor may be each of the above substances themselves, or may be a composition in which each of the above substances is dissolved in an appropriate solvent. The composition may also be a composition containing particles of any size finely divided by using each of the above substances alone or in combination of two or more together with an appropriate solvent or with any additive. Furthermore, the composition may be an emulsified or dispersed composition containing each of the above substances.

Burnet extract is an extract extracted from the roots and rhizomes of 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 juice of the leaves of Aloe vera (Aloe vera), and is available, for example, as "Aloe Extract Vera Miyakojima" (Ichimaru Falcos Co., Ltd.). Fermented soy milk liquid is a liquid obtained by fermenting and filtering soy milk, and is available, for example, as a product of 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 roots 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, and is available, for example, as "Angelica keiskei Extract B (Maruzen Pharmaceutical Co., Ltd.)." However, the above acquisition methods are not limited.

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

Hereinafter, the BMP-4 inhibitor of the seventh embodiment will be referred to as a nanoized sanguisorba composition. The nanoized sanguisorba composition is a sanguisorba composition that contains a sanguisorba extract, and the median diameter of the particles containing the sanguisorba extract is less than 1 μm.

Here, the median diameter is defined as the diameter at which the larger and smaller particles are equal if the powder is divided into two parts based on the 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.).

The nanoized Sanguinea Pig 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.

The particles contained in the nanoized sanguisorba 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. In addition to the sanguisorba extract, the particles contained in the nanoized sanguisorba composition may contain oily substances such as hydrogenated polyisobutene, squalane, isotridecyl isononanoate, and tri(capryl-caprylic acid)glyceryl.

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

The amount of oil phase in the nanoized Sanguisorba officinalis composition is preferably 1% by mass to 20% by mass, and 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. Also, an oil phase amount of 20% by mass or less is advantageous in terms of dispersing the Sanguisorba officinalis extract in the BMP-4 inhibitor.

The BMP-4 inhibitor disclosed herein can be added to, for example, cosmetics, quasi-drugs, pharmaceuticals, etc.

(3) Cosmetic and Manufacturing Method Thereof The cosmetic of the eighth embodiment contains the BMP-4 inhibitor of the sixth embodiment or any of the above embodiments as a skin whitening agent.

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 inhibits melanin production resulting from a decrease in the suppressed expression level of BMP-4. Therefore, by incorporating a BMP-4 inhibitor in a cosmetic product, a cosmetic product having a skin whitening effect can be provided.

The cosmetic of the ninth embodiment contains, as a whitening agent, a candidate substance for a whitening agent that has been confirmed to reduce the gene expression level of a melanocyte differentiation induction promoter by any one of the screening methods for whitening agents of the first to fifth embodiments.

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

The method for producing the cosmetic of the present disclosure is not particularly limited as long as it includes a step of adding a candidate whitening agent that has been screened by the screening method for whitening agents of the present disclosure to the cosmetic as a whitening agent. The cosmetic ingredients will be described later. The cosmetic of the present disclosure can be obtained, for example, by mixing a specific amount of a thickener, a specific amount of a moisturizing ingredient, a specific amount of a surfactant, water, and other ingredients as necessary to prepare the cosmetic ingredients, and further adding a specific amount of a BMP-4 inhibitor as a whitening agent, according to a known production method.

In particular, the cosmetic of the present disclosure is suitably manufactured by the cosmetic manufacturing method of the tenth embodiment. The cosmetic manufacturing method of the tenth embodiment includes a step of determining, as a whitening agent, a candidate whitening agent substance that has been confirmed to have a reduced expression level of a melanocyte differentiation induction promoter by any one of the screening methods for whitening agents of the first to fifth embodiments (hereinafter, the candidate substance determination step), and a step of adding the whitening agent to the cosmetic ingredients (hereinafter, the addition step).

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

In the addition step, the candidate substance for the whitening agent is added to the cosmetic component. The addition can be carried out by mixing the candidate substance for the whitening agent with the cosmetic component. The mixing can be carried out by a known method such as an emulsion dispersion method.
In the case of using the emulsion dispersion method 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.

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 components will be described later.
Examples of the emulsifier 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).

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 the emulsifier include lecithin, glycerin fatty acid ester, saponin, and sucrose fatty acid ester.
The organic solvent is preferably a water-soluble solvent, such as ethanol, polyhydric alcohols (butylene glycol, dipropylene glycol, glycerin, etc.).

The emulsification can be carried out in the 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 device US-600AT manufactured by Nippon Seiki Seisakusho Co., Ltd.).

The cosmetic product of the present disclosure may be in any form, such as a lotion, emulsion, cream, gel, wax, facial cleansing cream, or facial cleansing foam.

By using the method for screening whitening agents disclosed herein, it is possible to select candidate whitening agents useful as whitening agents using as an indicator the phenomenon that leads to a whitening effect, namely, a decrease in the gene expression level of melanocyte differentiation induction promoting factors (particularly BMP-4). Furthermore, by incorporating the candidate whitening agent selected by the method for screening whitening agents disclosed herein into a cosmetic product, it is possible to provide a cosmetic product having a whitening effect.

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

The cosmetic composition of the present disclosure may contain, as a cosmetic ingredient, an additive agent that is normally used in cosmetics. Examples of additive agents include functional ingredients (such as γ-oryzanol, hydrogenated polyisobutene, etc.) that exhibit useful beauty effects (such as moisturizing effects, skin conditioning effects, etc.) when used in cosmetics. Other additive agents include, for example, polyoxyethylene polyoxypropylene decyl tetradecyl ether (cosmetic ingredient name: PPG-6 decyl tetradeceth-20), solubilizers such as polyoxyethylene hydrogenated castor oil, surfactants other than specific surfactants, astaxanthin, other carotenoids other than astaxanthin (such as 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 removers, antimicrobial agents, anti-inflammatory agents, minerals, etc.

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. The melanin production inhibitory effect of Sanguisorba officinalis extract, which was the most effective BMP-4 inhibitor among the candidate substances, was examined. Furthermore, the melanin production inhibitory effect of nano-sizing Sanguisorba officinalis 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.

(1) Evaluation of the melanin production effect of BMP-4 Proteins that function as melanocyte differentiation-inducing promoters include, for example, BMP-2, BMP-4, BMP-7, SCF, endothelin 1, endothelin 3, Wnt3a, etc. Of these proteins, the melanin production effect of BMP-4 in inducing differentiation from iPS cells to melanocytes was examined.

In other words, 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.

iPS cells (Cellular Dynamics International) were seeded on a 6-well plate, and when they reached confluence, they were switched to a neural crest cell induction medium prepared according to the 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). The iPS cells were maintained in mTeSR1 medium (STEMCELL Technology) and cultured 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 induction medium containing Neurobasal Medium (Gibco) to which N2 and B27 supplement factors (Gibco) were added at the recommended concentrations, and then the medium was supplemented with 1% by mass 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.

Microscopic observation confirmed the appearance of melanocytes around the colonies around the 12th day. By the 21st day after induction, the emerged melanocytes were observed to further synthesize melanin. From the above, it was confirmed that this induction system can be used to reproduce the differentiation of melanocytes from stem cells and the synthesis of melanin. Based on these results, the effect of the presence or absence of BMP-4 on the final melanin production in this induction system was examined. As a control group, a differentiation induction medium of the same composition, except that BMP-4 was not added, was used and cultured under the same conditions.

Using the above differentiation induction system, the number of viable cells was measured 21 days 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.

After the above 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 live cells to determine the final amount of melanin. The results of the measurement are shown in Figure 1. The vertical axis of Figure 1 indicates the relative amount of melanin produced when the amount of melanin produced in the control group is set to 1. The bar graph with "(-)" indicates the control group in which a differentiation-inducing medium without BMP-4 was used, and the bar graph with "(+)" indicates the experimental group in which a differentiation-inducing medium with a BMP-4 concentration of 10 ng/mL was used. The bar graph in each group represents the average value of three samples, and the error bars represent the standard deviation.

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 between the control group and the experimental group using an F test, and after confirming that the variances were homogeneous, a Student's t-test was performed. In Figure 1, "**" indicates that the risk rate in the Student's t-test was 1% or less. In other words, from the results in Figure 1, it can be concluded that the presence of BMP-4 significantly increased the amount of melanin produced by melanocytes.

The results of the measurements showed that BMP-4 promotes increased melanin production during the differentiation process. Therefore, by suppressing the gene expression level of BMP-4 in the system in which 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 versus the experimental group in Figure 1.

(2) Screening of candidate substances for skin whitening agents Next, screening of candidate substances for skin whitening agents was carried out using the decrease in the gene expression level of BMP-4 as an index. Specifically, the following candidate substances 1 to 11, which are generally considered to have skin whitening effects, were screened.
Candidate substance 1: Loquat leaf extract (product name: Loquat Leaf Extract CA, Maruzen Pharmaceutical Co., Ltd.)
Candidate substance 2: Clove extract (product name: Clove Extract-J, Maruzen Pharmaceutical Co., Ltd.)
Candidate substance 3: Acacia extract (product 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 Ex Vera Miyakojima, Ichimaru Pharcos Co., Ltd.)
Candidate substance 6: Fermented soy milk liquid (Technoble Co., Ltd.)
Candidate substance 7: Moss bean seed extract (product 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 (product 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: Sanguinea oleracea extract (product name: Jiu Extract Powder, Maruzen Pharmaceutical Co., Ltd.)

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

Each of the above candidate substances was dissolved or suspended in a solution of DMSO and ultrapure water obtained from Milli-Q (Merck Millipore) mixed at a volume ratio of 1:1 to give a candidate substance solution with a solid content of 1 mg/mL.

Human fetal primary epidermal 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 min, room temperature), the supernatant was removed and the cells were resuspended in the above medium, and seeded in a 12-well plate at 1.5×10 ⁵ cells/cm ² per well. 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 and mixed well, and the cells were allowed to stand in a CO ₂ incubator (37° C.). The final concentration of each candidate substance was 10 ppm.

In addition, a control group was also prepared in which nHEKs were cultured under the same conditions except that none of the candidate substances were added. Three samples were prepared for each candidate substance group and the control group, and the following measurements were performed.

One day after the addition of the candidate substance, the supernatant was removed from each well and washed twice with phosphate-buffered saline (PBS), and total RNA was extracted using an RNA extraction reagent (RNeasy mini Kit, QUIAGEN) according to the protocol. After measuring the RNA concentration using an absorbance meter, 30 μg/μL was used as the RNA template and the Ct value of BMP-4 was measured using an RT-PCR system (Mx3000P, Agilent Technologies), and the amount of BMP-4 mRNA was measured using the relative quantification method (ΔΔCt method) using RT-PCR (PrimeScript One Step RT-PCR Kit, Takara Bio) based on the measured Ct value.

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

As a housekeeping gene in the 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'

The expression level of the BMP-4 gene quantified for each candidate substance (three samples were measured for each candidate substance, and the average value and standard deviation of the three samples are shown) is shown in the graph in Figure 2 as a relative value, with the expression level of the BMP-4 gene in the control group not exposed to the candidate substance set at 1. The vertical axis of the graph represents the relative expression level of the BMP-4 gene, and the numbers on the horizontal axis represent the number of each candidate substance. Each bar graph represents the average value of three samples for each candidate substance, and the standard deviation is also shown in the error bars. However, the bar graph labeled "none" on the left side represents the average value for the control group, and the standard deviation is also shown in the error bars. In addition, a homogeneity of variance test was performed using an F test for each of the control group and the groups exposed to each candidate substance, and after verifying that the variances of the two groups could not be said to be unequal, a Student's t-test was performed. In Figure 2, "*" indicates that the risk rate in the above t-test is greater than 1% and less than or equal to 5%, "**" indicates that the risk rate is greater than 0.5% and less than or equal to 1%, and "***" indicates that the risk rate is less than or equal to 0.1%.

From the results in Figure 2, the substances that significantly reduced the BMP-4 gene expression level compared to the control group were 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 keiskei leaf/stem extract, and candidate substance 11, Sanguisorba officinalis extract. Therefore, candidate substances 5 to 11 are useful as BMP-4 inhibitors that reduce the BMP-4 gene expression level in epidermal keratinocytes as normal cells. The reduction rates of BMP-4 gene expression level 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 the gene expression level of BMP-4 by more than 80% compared to the control group, suggesting that it has the most excellent BMP-4 inhibitory effect among candidate substances 5 to 11 and is the most useful BMP-4 inhibitor.

(3) Inhibitory effect of Sanguisorba officinalis extract on melanin production Next, it was examined whether the Sanguisorba officinalis extract, which had the most excellent BMP-4 inhibitory effect in (2) above, exhibits an inhibitory effect on melanin production in actual cells.

Mouse melanoma cells (B-16, DS Pharma) were collected by trypsinization and then suspended in a medium containing 1% PSN. After centrifugation (300×g, 3 min, room temperature) to remove the supernatant and resuspending in the above medium, the cells were seeded in a 12-well plate at 5×10 ⁴ cells/cm ² per well. The amount of medium in each well was 1 mL. The next day, all the medium was removed, and 1.0 mL of medium containing the desired concentration of Sanguisorba officinalis extract was added, followed by standing in a CO ₂ incubator (37° C.).

The target concentrations were 1 ppm and 5 ppm, and a stock solution of Sanguisorba officinalis extract at a concentration 100 times that of each target concentration dissolved in DMSO was added at a ratio of 10 μL per 1 mL of medium. The final concentration of 1% DMSO was 1%. As a control group, a medium containing 1% DMSO that did not contain Sanguisorba officinalis extract was used. The test was performed using a total of three groups (n=4 each): groups with Sanguisorba officinalis extract concentrations of 1 ppm and 5 ppm, and a control group with a concentration of 0.

Three days after seeding, the supernatant was removed from each well, and the wells were washed twice with PBS, and the number of viable cells was detected using a WST reagent (Cell Counting Kit, Dojindo Laboratories). After measuring the number of viable cells, each well was washed twice with PBS, and a melanin dissolving solution (1 mol/L NaOH, 10% DMSO, aqueous solution) was added and the cells were completely dissolved by ultrasonic treatment at 50°C for 20 minutes.

After the above 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 live cells to determine the final amount of melanin. The results of the measurement are shown in Figure 3. The vertical axis of the figure shows the relative amount of melanin produced when the amount of melanin produced in the control group is set to 100%. The leftmost bar graph shows the amount of melanin produced in the control group (concentration 0), the center bar graph shows the group with a 1 ppm concentration of Sanguisorba officinalis extract, and the rightmost bar graph shows the group with a 5 ppm concentration. The bar graphs in each group are the average value of four samples, and the error bars show the standard deviation.

As a result of the measurements, as shown in Figure 3, it was found that the 5 ppm and 1 ppm groups produced approximately 76% and 82% of the amount of melanin compared to the control group. Next, a homogeneity of variance test was performed between the control and experimental groups using an F-test, and after confirming homogeneity of variance, a Student's t-test was performed. In Figure 3, "*" indicates that the risk rate in the Student's t-test was greater than 1% and less than 5%. In other words, it can be concluded from the results in Figure 3 that exposure to 5 ppm and 1 ppm of Sanguinea Pig extract significantly reduced the amount of melanin produced by melanocytes.

(4) Melanin production inhibitory effect of nanoized Sanguisorba officinalis composition (4-1) Preparation of nanoized Sanguisorba officinalis composition Hereinafter, examples of cosmetics containing a nanoized dispersion of Sanguisorba officinalis extract as a BMP-4 inhibitor as a skin whitening agent will be described. Hereinafter, "nanonized" in the present disclosure is defined as a particle having a median diameter of less than 1 μm.

(4-1-1) Preparation of nanoized composition and non-nanized composition [Nanized composition 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

(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

15% by mass of the obtained oil phase was kept at 80°C and 85% by mass of the water phase was added, followed by ultrasonic dispersion for 1 minute using an ultrasonic homogenizer (US-600AT, Nippon Seiki Seisakusho). The obtained dispersion was subjected to high-pressure emulsification dispersion at a pressure of 245 MPa using a Starburst Mini (Sugino Machine) 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 Sanguinea gracilis extract in nano-sized composition 1 was 1.0% by mass.

[Nanized composition 2]
Nanoized composition 2, an emulsion composition cosmetic, was prepared using the same process as nanoized composition 1, except that the hydrogenated polyisobutene in nanoized composition 1 was replaced with the same amount of squalane.

[Nanized composition 3]
Nanosized composition 3 as an emulsion composition cosmetic was prepared in the same manner as nanosized composition 1, except that the hydrogenated polyisobutene of nanosized composition 1 was replaced with the same amount of isotridecyl isononanoate.

[Nanized composition 4]
Nanoized composition 4, an emulsion composition cosmetic, was prepared using the same process as nanoized composition 1, except that the hydrogenated polyisobutene in nanoized composition 1 was replaced with the same amount of tri(capryl-caprylic acid)glyceryl.

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

(4-1-2) Evaluation of Nanoparticles Nanoparticle compositions 1 to 4 and the non-nanoparticle composition were evaluated by the following methods.

[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.).

[Evaluation of temporal stability]
Immediately after preparation of the obtained cosmetics, and immediately after preparation and 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 four-level 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 equal to or less than 0.2.
C: The turbidity was greater than 0.2 and not more than 0.5.
D: Turbidity exceeded 0.5.

The evaluation results of the median diameter and temporal stability of nano-sized compositions 1 to 4 and the non-nanonized composition are shown in Table 1 below.

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, and that all of them were nanoized. The stability over time of the cosmetics was evaluated as good, A, for nanoized composition 1 and nanoized composition 2. However, as the average particle size increased, the evaluation of stability over time worsened, with B for nanoized composition 3 and C for nanoized composition 4. The non-nanonized composition with an average particle size of 5.6 μm was evaluated as the worst, D. It is believed that the particles with each median diameter in nanoized compositions 1 to 4 function as oil droplets containing Sanguinea oleifera extract and function as BMP-4 inhibitors.

(4-2) Evaluation of the effect of nano-sizing of Sanguisorba officinalis extract on melanin production Using a skin model containing melanocytes, the whitening effect of nano-sizing Sanguisorba officinalis extract, which was found to have an inhibitory effect on the expression level of the BMP-4 gene, was examined. Sanguisorba officinalis extract contains a lot of tannins and saponins and is a hydrophobic material that is difficult to dissolve in water, so nano-sizing improves its penetration into the skin and is expected to have a higher whitening effect than a non-nanoized formulation. In this study, nano-sized composition 1, which had the smallest average particle size and the highest stability over time evaluation in (4-1) above, was used as the experimental group, and the whitening effect was evaluated in comparison with the control group using a non-nanoized composition.

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-nanofiberized 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.

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 mentioned in (1). After measuring the number of viable cells, the products reacted with the MTT reagent were completely removed from each skin model using isopropanol, and the melanin dissolving solution mentioned in (1) was added and completely dissolved by heating at 80°C for one hour.

After the above 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.

The results are shown in Figure 4. The vertical axis in Figure 4 shows the amount of melanin produced when the amount of melanin produced in the non-nanoized 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 shows the average value of three samples, and the error bars show the standard deviation. Furthermore, a homogeneity of variance test was performed using an F-test for each of the non-nanoized composition and nanoized composition 1, and after confirming homogeneity of variance, a Student's t-test was performed. In Figure 4, "*" indicates that the risk rate in the above t-test is greater than 1% and less than 5%.

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

(5) Manufacturing Method of Cosmetics Specific examples of manufacturing methods of cosmetics are described below.

(5-1) Skin Whitening Agent Candidate substances 1 to 11, which are candidate substances for skin whitening agents described in (2), were subjected to the screening method described in (2). Among candidate substances 5 to 11, which were confirmed to reduce the expression level of BMP-4 as a melanocyte differentiation-inducing factor, candidate substance 11, which had the most excellent BMP-4 inhibitory effect, was selected as the skin whitening agent. This Sanguisorba officinalis extract was nanoized by the method described in (4-1-1) to prepare a nanoized composition. Of the nanoized compositions listed in (4-1-1), nanoized composition 1 was used as the skin whitening agent.

(5-2) Astaxanthin-Containing Emulsion Composition The astaxanthin-containing emulsion composition, which is a component of Cosmetics 1 to 6 and 9 below, was prepared as follows.

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

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

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

Then, the obtained preliminary emulsion was cooled to about 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.). After that, it was 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).

The resulting 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.

The same emulsion composition can be obtained by replacing the krill extract with Astax-ST (Marine Daio Co., Ltd.) or ASTOTS-S (Haematococcus algae extract, Fujifilm Corporation).

(5-3) Preparation of Lycopene-Containing Emulsion Composition The lycopene-containing emulsion composition, which is a component of Cosmetics 1, 2 and 9 below, was prepared as follows.

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

[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 with stirring. After confirming that they were well mixed, an oil phase composition B was obtained.
Tomato oleoresin (Lyc-O-Mato 15% (containing 15% by mass of lycopene), manufactured by Sunbright): 1.14 g
Lecithin (Resion P (derived from soybeans), manufactured by Riken Vitamin): 1.0g
Medium-chain fatty acid glyceride (Coconard MT, manufactured by Kao): 12.8g

The aqueous phase composition B obtained above was added to the oil phase composition B, stirred and mixed, and dispersed for a specified time using an ultrasonic homogenizer to obtain a crude dispersion. The crude dispersion obtained was then further emulsified at 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).

The resulting 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 52 nm.

(5-4) Preparation of Cosmetics The whitening agent obtained in (5-1) was added to each of the cosmetic ingredients listed below to prepare the following cosmetics 1 to 9.

(5-4-1) Cosmetic 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 to prepare a lotion 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 (60 E.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 weight
Citric acid: 1.0% by mass
Sodium citrate: appropriate amount Water: remaining amount

(5-4-2) Cosmetic 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 to prepare a beauty essence 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 paraoxybenzoate: 0.1% by mass
Alkaline Negative Latus 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 (60 E.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

(5-4-3) Cosmetic 3 (cream)
As cosmetic preparation 3, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients having the composition shown below, and a cream was prepared 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
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 weight
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 (60 E.O.): 0.2% by mass
Centella asiatica extract: 0.01% by mass
Lecithin: 0.1% by weight
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

(5-4-4) Cosmetic 4 (Sunscreen)
As cosmetic preparation 4, 0.1% by mass of the whitening agent obtained in (5-1) was added 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 (60 E.O.): 0.2% by mass
Centella asiatica extract: 0.01% by mass
Lecithin: 0.1% by weight
Fragrance: Trace amount Methyl paraoxybenzoate: 0.15% by mass
Purified water: remaining amount

(5-4-5) Cosmetic 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 components 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%

[Aqueous Phase Composition C]
In addition, 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 weight
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 (60 E.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

The above oil phase composition C and water phase composition C were each heated to 70°C and emulsified while stirring to prepare an emulsion as cosmetic preparation 5 (total amount 100% by mass).

(5-4-6) Cosmetic 6 (jelly-like serum)
As cosmetic preparation 6, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic components having the following composition to prepare a jelly-like beauty essence 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 weight
1,3-butylene glycol: 1.0% by mass
Glycerin: 2.0% by mass
Sucrose: 0.1% by weight
Polyglyceryl-10 oleate: 0.1% by mass
Polyglyceryl-2 stearate: 0.1% by mass
Phenoxyethanol 0.2% by weight
Collagen: 1.0% by mass
Oryzanol: 0.01% by mass
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemical Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60 E.O.): 0.2% by mass
Centella asiatica extract: 0.01% by mass
Lecithin: 0.1% by weight
Citric acid: 1.0% by mass
Sodium citrate: appropriate amount (PEG-240/Decyltetradeceth-20/HDI) copolymer: 0.3% by mass
Damask rose oil: trace amount Fragrance: trace amount Purified water: balance

(5-4-7) Cosmetic 7 (wet foundation)
0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic components of the composition shown below to prepare a slurry, which was then filled into a specified container and dried to prepare 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 Dow Corning Toray): 3.0% by mass
Dimethicone (SH200C-20cs, manufactured by Dow Corning Toray): 7.0% by mass
Phenoxyethanol: 0.5% by mass
Sericite (OTS-2 SERICITE FSE, manufactured by Daito Kasei Co., Ltd.): remaining amount

(5-4-8) Cosmetic 8 (Liquid Foundation)
Cosmetic 8 was prepared by adding 0.1% by mass of the whitening agent obtained in (5-1) to the cosmetic components 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
Color 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 Nafrea 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 weight
Damask rose flower oil: trace amount Fragrance: appropriate amount Phenoxyethanol: 0.3% by weight
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 (60 E.O.): 0.2% by mass
Centella asiatica extract: 0.01% by mass
Lecithin: 0.1% by weight
Calcium chloride: 1.0% by mass
Citric acid: 1.0% by mass
Sodium citrate: appropriate amount Ion exchange water: remaining amount

(5-4-9) Cosmetic 9 (Facial cleanser)
As cosmetic preparation 9, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic ingredients having the following composition to prepare a face wash 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 glycine: 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 weight
Citric acid: 1.0% by mass
Sodium citrate: appropriate amount Shea butter: 1.0% by weight
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 (60 E.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

(5-4-10) Cosmetic 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 composition shown below to prepare a beauty essence 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
Sanguinea oleracea extract (product name: Jiu 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 paraoxybenzoate: 0.1% by mass
Alkaline Negative Latus 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 (60 E.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

Claims (4)

Bone morphogenetic protein 4 inhibitor containing Sanguisorba officinalis extract. The bone morphogenetic protein 4 inhibitor according to claim 1, which contains particles containing the Sanguisorba officinalis extract, and the particles have a median diameter of less than 1 μm. The bone morphogenetic protein 4 inhibitor according to claim 2, wherein the particles containing the Sanguisorba officinalis extract are an oil-in-water emulsion. The bone morphogenetic protein 4 inhibitor according to claim 2 or 3, wherein the median diameter of the particles containing the Sanguisorba officinalis extract is 10 nm or more and less than 200 nm.