JP2021171036A - Whitening agent screening method, bone-forming protein 4 inhibitor, cosmetics, and method for manufacturing cosmetics - Google Patents

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

The present invention relates to a method for screening a whitening agent, and a whitening agent obtained by the method for screening a whitening agent and a cosmetic using the whitening agent.

Conventionally, whitening cosmetics containing various whitening active ingredients such as placenta, ellagic acid, kojic acid, tranexamic acid, cetyl tranexamic acid, and linoleic acid have been provided in order to prevent skin pigmentation and maintain white and bright skin. ing. 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 a large number of plant extracts are disclosed as the specific whitening agent.

Melanin, the black pigment of the skin, is produced by pigment-producing cells called melanocytes. The following Patent Document 2 describes melanocytes for the purpose of treating diseases such as vitiligo vulgaris, hereditary contralateral dyschromia and leukoderma caused by abnormal suppression of melanin production or deficiency of melanocytes. A method for promoting the induction of differentiation of melanocytes is disclosed. Specifically, a method including culturing human pluripotent stem cells in the presence of a component that induces epidermal cells and a component that induces final differentiation into keratinocytes is disclosed.

The following Patent Document 3 discloses a screening method for a pluripotent stem cell differentiation-inducing agent, and as a stem cell differentiation-inducing agent, so-called TGF-β (transforming growth factor-β, transforming growth factor β) ) A large number of factors belonging to the superfamily are described, and BMP-4 (Bone Morphogenetic Protein-4, bone morphogenetic protein 4) is described as one of the many factors. It has been reported that BMP-4 is involved in the differentiation of stem cells into retinal pigment epithelium (Patent Document 4 below) and in promoting hair growth (Patent Document 5 below). Further, Patent Document 6 below reports that BMP-4 reduces the production of melanin in melanocytes.

International Publication No. 2013/081147 Japanese Unexamined Patent Publication No. 2015-146803 Special Table 2005-500847 JP-A-2009-226069 Japanese Patent Application Laid-Open No. 2008-534607 Special Table 2006-5008026 Gazette

Skin pigmentation results from the differentiation and proliferation of melanocytes from stem cells, and the increased production of melanin by the proliferated melanocytes. Therefore, a substance capable of suppressing the differentiation and proliferation of melanocytes is considered to be useful as a whitening agent.

The present disclosure discloses a substance that is useful as a whitening agent by utilizing a mechanism involved in suppressing the differentiation and proliferation of melanocytes, and a BMP-4 inhibitor as a whitening agent whose usefulness has been confirmed by screening. An object of the present invention is to provide a cosmetic containing a whitening agent whose usefulness has been confirmed by screening, and a method for producing the same.

Specific means for solving the above problems include the following embodiments.
[1] The decrease in the expression level of the melanocyte differentiation-inducing factor when the cells are exposed to the whitening agent candidate substance is used as an index for the decrease in the expression level of the melanocyte differentiation-inducing factor in the control group in which the cells are not exposed to the whitening agent candidate substance. , Whitening agent screening method.

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

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

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

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

[6] Bone-forming protein containing at least one selected from the group consisting of crackle mulberry extract, aloe vera leaf extract, soymilk fermented liquid, moth bean seed extract, horse chestnut extract, horse chestnut extract and ashitaba leaf / stem extract. 4 Inhibitor.

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

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

[9] A cosmetic containing as a whitening agent a candidate substance for which a decrease in the gene expression level of a melanocyte differentiation-inducing factor has been confirmed by the screening method for a whitening agent according to any one of [1] to [5].

[10] A step of determining a whitening agent candidate substance whose expression level of a melanocyte differentiation-inducing factor has been confirmed to decrease by any of the whitening agent screening methods from [1] to [5] as a whitening agent, and whitening. A method for producing a cosmetic product, which comprises a step of adding an agent to a cosmetic ingredient.

According to the technique of the present disclosure, a substance useful as a whitening agent can be screened by utilizing a mechanism involved in suppressing the differentiation and proliferation of melanocytes. Further, it is possible to provide a cosmetic containing a BMP-4 inhibitor as a whitening agent whose usefulness has been confirmed by screening, and a whitening agent whose usefulness has been confirmed by screening, and a method for producing the same.

The graph which shows the melanin production effect of BMP-4. The graph which shows the screening result of the whitening agent candidate substance. The graph which shows the influence on the melanin production by the presence or absence of the Sanguisorba extract. The graph which shows the influence on the melanin production by the nano-izing of the Sanguisorba extract.

(1) Whitening agent screening method The whitening agent screening method of the first embodiment does not expose the cells to the expression level of the melanocyte differentiation-inducing factor when the cells are exposed to the whitening agent candidate substance. The decrease in the expression level of the melanocyte differentiation-inducing factor in the control group is used as an index.

In the present embodiment, when a certain substance is used as a candidate substance for a whitening agent, the expression level of the melanocyte differentiation-inducing factor is verified in the experimental group in which the substance is exposed to cells and the control group in which the cells are not exposed to the substance. do. Then, whether or not the expression level of the experimental group is lower than the expression level of the control group is used as an index for screening as a whitening agent. Here, the "whitening agent candidate substance" is a candidate substance that can be a whitening agent, and refers to a test substance for screening. In addition, the “whitening agent” refers to a substance among the whitening agent candidate substances whose function of lowering the expression level of the melanocyte differentiation-inducing factor has been confirmed by screening.

The melanosite differentiation-inducing factor is a protein that promotes the induction of differentiation of pluripotent stem cells (for example, induced pluripotent stem cells) or melanosite stem cells into melanocytes. Examples of proteins that function as melanocyte differentiation-inducing factors 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-inducing factor may be confirmed by detecting the melanocyte differentiation-inducing factor itself as the produced protein, or when the melanocyte differentiation-inducing factor is produced. It may be confirmed by detecting the expression of the corresponding gene of. That is, the expression level of the melanocyte differentiation-inducing factor may be either or both of the gene expression level and the protein expression level.

Examples of methods for confirming the gene expression level include PCR (polymerase chain reaction) method, RT-PCR (reverse transcription PCR) method, gene amount detection method using an RNA probe (microarray method), and RNA using a next-generation sequencer. A method of sequencing the sequence and detecting the expression level, a method of detecting the expression level using nucleic acid hybridization (in situ hybridization method), and the like can be adopted.

Methods for confirming the protein expression level include a method for semi-quantitatively detecting a protein using Western blotting, a method for quantitatively detecting a protein using the ELISA method, and fluorescence in an image of an immunostained specimen. A method of measuring the staining intensity, a method of detecting a protein using proteome analysis, and the like can be adopted.

The decrease in the expression level of the melanosite differentiation-inducing factor in the experimental group may be judged by the fact that the expression level in the experimental group is even slightly lower than the expression level in the control group, and the expression level in the experimental group is preferable. The rate of decrease with respect to the expression level of the control group (that is, the rate obtained by "(AX) / A" when the expression level of the control group is A and the expression level of the experimental group is X) is preferably 5% or more. May be determined by 10% or more, more preferably 20% or more, still more preferably 50% or more. Furthermore, it is desirable that the difference between the expression level of the experimental group and the expression level of the control group is statistically significant regardless of the value of the reduction rate. The risk factor when determining that it is statistically significant can be 5% or less.

According to the present embodiment, a whitening agent candidate substance that may be useful as a whitening agent by using a decrease in the gene expression level of a melanocyte differentiation-inducing factor as an index as a mechanism involved in suppressing the differentiation and proliferation of melanocytes. The substance obtained by the screening can be used as a whitening agent.

In the method for screening the whitening agent of the second embodiment, the melanocyte differentiation-inducing promoting factor is the bone-forming protein 4 in the first embodiment.

Among the factors that can be used as an index in the screening method for a whitening agent according to the first embodiment as a melanocyte differentiation-inducing factor, a whitening agent candidate can be obtained by using a decrease in the gene expression level of bone morphogenetic protein 4 (BMP-4) as an index. The substance can be clearly screened.

In the method for screening a whitening agent according to the third embodiment, in the first embodiment or the second embodiment, the cells are normal human cells.

In the method for screening the whitening agent of the fourth embodiment, in the third embodiment, human normal cells are epidermal keratinocytes.

In the method for screening the whitening agent of the fifth embodiment, in the third embodiment, human normal cells are fibroblasts.

Melanocytes that produce melanin differentiate from melanocyte stem cells. For the differentiation of melanocyte stem cells into melanocytes, cells existing around the melanocyte stem cells in the skin (for example, epidermal keratinized cells, fibroblasts, Langerhans cells, nerve cells, T cells, Merkel cells, etc. as human normal cells, And melanocytes, etc., hereinafter referred to as "peripheral cells"), it is presumed that melanocyte differentiation promoting factors are involved. Therefore, if the secretion of melanocyte differentiation-inducing factor can be suppressed from peripheral cells, 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, exposure of a whitening agent candidate to normal human cells, particularly epidermal keratinized cells or fibroblasts, more preferably epidermal keratinized cells, results in melanocyte differentiation-inducing factors in the exposed cells, such as BMP. The decrease in the expression level of -4 means that the whitening agent candidate substance exposed to the cells has an effect of suppressing the keratinocyte differentiation-inducing factor. Then, it is considered that the whitening agent candidate substance exposed to the cells suppresses the differentiation of melanocyte stem cells into melanocytes through the effect of suppressing the melanocyte differentiation-inducing factor, and as a result, the melanin production is suppressed. That is, in the third to fifth embodiments, a whitening agent candidate substance is exposed to human normal cells, particularly epidermal keratinized cells or fibroblasts, more preferably epidermal keratinized cells, and a melanocyte differentiation promoting factor. Observing the expression level of is regarded as a screening of whitening effect. It is presumed that the reason why epidermal keratinized cells are more preferable as human normal cells is that epidermal keratinocytes are located around melanocytes and therefore easily affect melanocytes.

(2) Bone morphogenetic protein 4 (BMP-4) inhibitor The BMP-4 inhibitor of the sixth embodiment is a crackle extract, aloe vera leaf extract, soymilk fermented liquid, moss bean seed extract, citrus extract, and citrus fruit. Includes at least one of the group consisting of root extract and Ashitaba leaf / stem extract.

When various whitening agent candidate substances are exposed to epidermal keratinized cells as normal human cells, crackle extract, aloe vera leaf extract, soymilk fermented liquid, moss bean seed extract, citrus extract, citrus root extract And the Ashitaba leaf / stem extract remarkably suppressed the gene expression level of BMP-4, so that each of the above substances was judged to have an effect as a BMP-4 inhibitor. As described above, it can be expected that melanin production is suppressed by lowering the gene expression level of BMP-4, and a whitening effect can be expected through suppression of melanin production. In the above-mentioned Patent Document 6, it is reported that BMP-4 reduces the production of melanin in melanocytes, whereas in the sixth embodiment, the expression level of BMP-4 is lowered by the BMP-4 inhibitor. By letting it suppress melanin production. Here, the BMP-4 inhibitor may be the above-mentioned substances themselves, or may be a composition in which each of the above-mentioned substances is dissolved in an appropriate solvent. In addition, each of the above substances may be used alone or in combination of two or more, and the composition may contain particles finely divided to an arbitrary size together with an appropriate solvent or an arbitrary additive. Further, the composition may be an emulsified / dispersed composition containing each of the above-mentioned substances.

The burnet extract is an extract extracted from the roots and rhizomes of burnet (Jiyo, English name: great burnet, scientific name: Sanguisorba officinalis), and is available as, for example, "Jiyu extract powder (Maruzen Pharmaceuticals Co., Ltd.)". be. The aloe vera leaf extract is an extract extracted from the juice of the leaves of aloe vera (scientific name: Aloe vera), and is available as, for example, "Aloe vera Miyakojima (Ichimaru Falcos Co., Ltd.)". The soymilk fermented liquid is a liquid obtained by fermenting soymilk and filtering it, and is available as a product of Technoble Co., Ltd., for example. The moth bean seed extract is an extract extracted from the seeds of moth bean (scientific name: Vigna aconitifolia), and is available as, for example, "VIT-A-LIKE PW LS 9898 (Yamakawa Trading Co., Ltd.)". The horse chestnut extract is an extract extracted from the seeds of horse chestnut (scientific name: Aesculus hippocastanum), and is available as a product of Sabinsa Japan Corporation, for example. The licorice root extract is an extract extracted from the root of licorice (scientific name: Glycyrrhiza glabra), and is available as, for example, "licorice extract (Maruzen Pharmaceuticals Co., Ltd.)". Ashitaba leaf / stem extract is an extract extracted from one or both of the leaves and stems of Ashitaba (scientific name: Angelica keiskei), and is obtained as, for example, "Ashitaba extract B (Maruzen Pharmaceuticals Co., Ltd.)". It is possible. The method of obtaining the above is not limited to the above.

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

Hereinafter, the BMP-4 inhibitor of the 7th embodiment will be referred to as a nano-sized Sanguisorba composition. The nano-sized Sanguisorba composition is a Sanguisorba composition containing a Sanguisorba extract, and the diameter of the particles containing the Sanguisorba extract is less than 1 μm in median diameter.

Here, the median diameter is defined as a diameter in which the large side and the small side have equal amounts, assuming that the powder is divided into two by the particle size. As an example of measuring the median diameter, it is obtained by applying the principle of dynamic light scattering. For example, the median diameter (d50) of the dispersed particles can be measured using a particle size analyzer (FPAR-1000, Otsuka Electronics Co., Ltd.).

The nano-sized Sanguisorba composition of the seventh embodiment can improve the effect of suppressing melanin production as compared with a dispersion containing particles having a median diameter of 1 μm or more. The median diameter of the particles 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 diameter of the particles may be 10 nm.

The particles contained in the nano-sized Sanguisorba composition used in the seventh embodiment may be produced by any step such as an emulsification step or a dispersion step as long as the particles have a median diameter of less than 1 μm. In addition to the Sanguisorba extract, the particles contained in the nano-sized Sanguisorba composition can contain, for example, an oily substance such as hydrogenated polyisobutene, squalane, isotridecyl isononanoate, and tri (caprylic caprylic acid) glyceryl.

The nano-sized crackle composition of the seventh embodiment can be produced as oil droplets in an oil-in-water emulsion by suspending the crackle extract in an oil phase and then emulsifying and dispersing it together with the aqueous phase.

The amount of the oil phase of the nano-sized Sanguisorba composition is preferably 1% by mass to 20% by mass, more preferably 5% by mass to 17.5% by mass. When the amount of the oil phase is 1% by mass or more, it is advantageous in that the BMP-4 inhibitory effect is exhibited. Further, when the amount of the oil phase is 20% by mass or less, it is advantageous in that the Sanguisorba extract is dispersed in the BMP-4 inhibitor.

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

(3) Cosmetics and Method for Producing The Cosmetics The cosmetics of the eighth embodiment contain the BMP-4 inhibitor of the sixth embodiment or the sixth embodiment as a whitening agent.

When the BMP-4 inhibitor of the 6th or 7th embodiment is exposed to normal human cells, particularly epidermal keratinized cells or fibroblasts, the expression level of BMP-4 is lowered as compared with the unexposed control group. Therefore, the BMP-4 inhibitor of the 6th embodiment or the 7th embodiment is useful as a whitening agent that suppresses melanin production caused by a decrease in the expression level of BMP-4 inhibition. Therefore, by incorporating a BMP-4 inhibitor in a cosmetic, it is possible to provide a cosmetic having a whitening effect.

The cosmetic of the ninth embodiment whitens a whitening agent candidate substance in which a decrease in the gene expression level of a melanocyte differentiation-inducing factor has been confirmed by the screening method for the whitening agent according to any one of the first to fifth embodiments. Contains as an agent.

The content of the whitening agent candidate substance (for example, crackle extract) in the cosmetic is preferably 0.0001% by mass to 0.05% by mass, more preferably 0.001% by mass to 0.025% by mass. When the content of the whitening agent candidate substance is 0.0001% by mass or more, it is advantageous in that the gene expression level of the melanocyte differentiation-inducing factor is lowered. Further, when it is 0.05% by mass or less, it is advantageous in that the whitening agent candidate substance is dispersed in the cosmetic.

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

Above all, the cosmetics of the present disclosure are suitably produced by the method for producing cosmetics according to the tenth embodiment. The method for producing the cosmetic of the tenth embodiment is a whitening agent candidate substance in which a decrease in the expression level of the melanocyte differentiation-inducing factor was confirmed by the screening method for the whitening agent according to any one of the first to fifth embodiments. Is included as a whitening agent (hereinafter, candidate substance determination step) and a step of adding the whitening agent to the cosmetic component (hereinafter, addition step).

In the candidate substance determination step, a whitening agent candidate substance in which a decrease in the expression level of the melanocyte differentiation-inducing factor is confirmed by the screening method for any of the whitening agents described in the first to fifth embodiments described above is determined. .. The details of the first to fifth embodiments are as described above, and thus the description thereof will be omitted here.

In the addition step, a whitening agent candidate substance is added to the cosmetic ingredient. The addition can be performed by mixing the whitening agent candidate substance and the cosmetic ingredient. The mixing can be carried out by a known method such as an emulsion dispersion method.
When the emulsification and dispersion method is used and the whitening candidate substance is an oil-soluble component, for example, an oil phase containing at least a whitening agent candidate substance and an aqueous phase are mixed and emulsified and dispersed to form an oil-in-water type or oil. Obtained as a medium-water emulsion.

The oil phase may contain a whitening agent candidate substance, an emulsifier if necessary, other additives, an organic solvent and the like. The whitening agent candidate substances are as described above, and other cosmetic ingredients will be described later.
Examples of the emulsifier include lecithin, glycerin fatty acid ester (for example, higher fatty acid ester such as polyglyceryl oleate), saponin, sucrose fatty acid ester (for example, higher fatty acid ester such as sucrose stearate) and the like.

The aqueous phase may contain water, emulsifiers and organic solvents and, if desired, other cosmetic ingredients. The whitening agent candidate substances are as described above, and other cosmetic ingredients will be described later.
Examples of the emulsifier include lecithin, glycerin fatty acid ester, saponin, sucrose fatty acid ester and the like.
As the organic solvent, a solvent soluble in water is preferable, and examples thereof include ethanol and polyhydric alcohols (butylene glycol, dipropylene glycol, glycerin, etc.).

Emulsification can be carried out in a temperature range of 20 ° C to 90 ° C.
Emulsification can be performed using a stirring device such as an ultra-high speed homogenizer or an ultrasonic homogenizer (for example, an ultrasonic homogenizer device US-600AT manufactured by Nissei Tokyo Office Co., Ltd.).

The resulting cosmetic form of the present disclosure may be any of lotions, emulsions, creams, gels, waxes, facial cleansing creams, facial cleansing foams and the like.

By the whitening agent screening method of the present disclosure, a whitening agent candidate substance useful as a whitening agent is selected by using a phenomenon leading to a whitening effect of a decrease in the gene expression level of a melanocyte differentiation-inducing factor (particularly BMP-4) as an index. be able to. In addition, by incorporating a whitening agent candidate substance selected by the whitening agent screening method of the present disclosure into a cosmetic, a cosmetic having a whitening effect can be provided.

Examples of the use of the cosmetics of the present disclosure include the use of cosmetics (for example, skin care cosmetics such as lotions and serums, especially skin care cosmetics for whitening). Examples of the cosmetics include skin care cosmetics (lotion, milky lotion, beauty essence, etc.), body cosmetics (body lotion, etc.), scalp cosmetics, and the like. However, the use of the cosmetics of the present disclosure is not limited to the above.

The cosmetics of the present disclosure may appropriately contain an additive compound usually used in cosmetics as a cosmetic ingredient. Examples of the additive compound include functional ingredients (for example, γ-oryzanol, hydrogenated polyisobutene, etc.) that exhibit useful beauty effects (for example, moisturizing effect, skin conditioning effect, etc.) when used in cosmetics. .. Examples of other additive additives include polyoxyethylene polyoxypropylene decyltetradecyl ether (cosmetic ingredient labeling name: PPG-6 decyltetradeceth-20), solubilizers such as polyoxyethylene hydrogenated castor oil, and specific surfactants. Surfactants other than activators, astaxanthin, other carotenoids other than astaxanthin (eg lycopene, carotene), preservatives such as phenoxyethanol, ethylhexyl glycerin, colorants, thickeners, pH adjusters such as sodium hydroxide, hydrochloric acid, etc. Examples thereof include buffers, fragrances, antibacterial agents, ultraviolet absorbers, active oxygen removers, antimicrobial agents, antiinflammatory agents, minerals and the like.

Hereinafter, according to Examples, the melanin production effect of BMP-4 was evaluated, a whitening agent candidate substance was screened based on the evaluation result, and Sanguisorba officinalis extract which exhibited the most excellent effect as a BMP-4 inhibitor among the candidate substances. The melanin production inhibitory effect was verified for the substance, and the melanin production inhibitory effect by nanonizing the Sanguisorba extract was further verified. The following examples show an example of an embodiment, and the present disclosure is not limited to the examples.

(1) Evaluation of melanin-producing effect of BMP-4 Examples of proteins that function as melanocyte differentiation-inducing factors include BMP-2, BMP-4, BMP-7, SCF, endothelin 1, endothelin 3, and Wnt3a. .. Among these proteins, BMP-4 was examined for its melanin-producing effect in inducing differentiation of iPS cells into melanocytes.

That is, a melanocyte differentiation-inducing system was prepared from iPS cells, and the effect of BMP-4 in the process of melanocyte differentiation-inducing was examined.

IPS cells (Cellular Dynamics International) were seeded on a 6-well plate, and when they became confluent, they were prepared according to the method described in publicly known literature (Cell Reports 3, 1140-1152, April 25, 2013). The neural crest cell-inducing medium was switched to obtain melanocyte stem cells (SOX10 and c-kit positive cells). For the maintenance of iPS cells, mTeSR1 medium (manufactured by STEMCELL Technology) was used, and the cells were cultured at _{37 ° C. in a 5% by volume CO 2 incubator.} The melanocyte stem cells obtained by the above method are further cultured in a confluent state, and a medium containing the cultured melanocyte stem cells is added to Neurobasal Medium (Gibco) at recommended concentrations of N2 and B27 additive factor (Gibco), and then 1% by mass. 100 ng / mL SCF, 100 ng / mL Wnt3a, 250 ng / mL EDN3 (Endothelin-3), 25 ng / mL BMP-4, 3 μM CHIR99021, 500 μM dbcAMP (dibutylyl circlic adenocine monophosphate) were added to the medium supplemented with GlutaMax (Gibco). Stem cells were induced to differentiate into melanocytes by culturing in a differentiation-inducing medium.

Microscopic observation confirmed the appearance of melanocytes around the colony around the 12th day. By the 21st day after induction, it was observed that the melanocytes that appeared further synthesized melanin. From the above, it was confirmed that all of melanocyte differentiation and melanin synthesis can be reproduced from stem cells by using this induction system. Based on the above results, the effect of the presence or absence of BMP-4 on the final melanin production in this induction system was verified. As a control group, the cells were cultured under the same conditions using a differentiation-inducing medium having the same composition except that BMP-4 was not added.

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

After the above lysis, 200 μL of the melanin lysate after melanin dissolution in each well was transferred to a 96-well plate, the absorbance at 400 nm was measured at room temperature, and the absorbance obtained from the measurement was corrected by the number of living cells to obtain the final value. The amount of melanin was adjusted. The measurement results are shown in FIG. The vertical axis of FIG. 1 shows the relative melanin production amount when the melanin production amount in the control group is 1. The bar graph of "(-)" uses a control group using a differentiation-inducing medium to which BMP-4 is not added, and the bar graph of "(+)" uses a differentiation-inducing medium with a BMP-4 concentration of 10 ng / mL. The experimental group was shown. The bar graph in each group represents the average value of the three samples, and the error bar represents the standard deviation.

As a result of the measurement, as shown in FIG. 1, the experimental group showed a melanin production amount more than twice that of the control group. Next, the control group and the experimental group were subjected to a homoscedasticity test by F-test, and after confirming that they were homoscedastic, a Student's t-test was performed. In FIG. 1, "**" indicates that the risk rate in Student's t-test is 1% or less. That is, from the results of FIG. 1, it can be concluded that the presence of BMP-4 significantly increased the amount of melanin produced by melanocytes.

From the measurement results, it was found that BMP-4 promotes an increase in melanin production during the process of differentiation. Therefore, in a system in which melanocytes produce melanin, suppressing the gene expression level of BMP-4 reduces the amount of BMP-4 in the system. As a result, as shown in the results of the control group with respect to the experimental group in FIG. 1, melanin It was suggested that production could be suppressed.

(2) Screening of whitening agent candidate substances Next, screening of whitening agent candidate substances was performed using the decrease in the gene expression level of BMP-4 as an index. Specifically, among the substances that are said to have a whitening effect on the street, the following candidate substances 1 to 11 were screened.
Candidate substance 1: Loquat leaf extract (trade name: loquat leaf extract CA, Maruzen Pharmaceuticals Co., Ltd.)
Candidate substance 2: Clove extract (trade name: Clove extract-J, Maruzen Pharmaceuticals Co., Ltd.)
Candidate substance 3: Asenyaku extract (trade name: Asenyaku extract, Maruzen Pharmaceuticals Co., Ltd.)
Candidate substance 4: Arnica flower extract (trade name: Arnica extract BG, Maruzen Pharmaceuticals Co., Ltd.)
Candidate substance 5: Aloe vera leaf extract (trade name: Aloe extract vera Miyakojima, Ichimaru Falcos Co., Ltd.)
Candidate substance 6: Soymilk fermented liquid (Technoble Co., Ltd.)
Candidate substance 7: Moth 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 Pharmaceuticals Co., Ltd.)
Candidate substance 10: Angelica keiskei leaf / stem extract (trade name: Angelica keiskei extract BG, Maruzen Pharmaceuticals Co., Ltd.)
Candidate substance 11: Sanguisorba extract (trade name: Jiyu extract powder, Maruzen Pharmaceuticals Co., Ltd.)

Candidate substance 1 is an extract extracted from the leaves of Biwa (scientific name: Eriobotrya japonica), candidate substance 2 is an extract extracted from the flower buds of clove (scientific name: Syzygium aromaticum), and candidate substance 3 is gambir tree (scientific name: Gambir tree). It is an extract extracted from leaves and shoots of scientific name: Uncaria gambir), and candidate substance 4 is an extract extracted from flowers of Arnica (scientific name: Arnica montana).

Each of the above candidate substances is dissolved or suspended in a solution obtained by mixing DMSO and ultrapure water obtained by Milli-Q (Merck Millipore) at a volume ratio of 1: 1 so that the solid content of the candidate substance is 1 mg / mL. Was used as a candidate substance solution.

The first representative epidermal keratinized cells (nHEK, Thermo Fisher Scientific) derived from a human foetation were collected by trypsin treatment and then suspended in a medium containing 1% PSN (penicillin-streptomycin-neomycin) (EpiLife, Gibco). Centrifugation (300 g, 3 min, room temperature) were seeded so that the above after re-suspended in culture medium, 12-well plates 1.5 per well × ¹⁰ 5 cells / cm ² except for the supernatant. The amount of medium in each well was 1.0 mL. The day after sowing, 10 μL of each of the above candidate substance solutions was added, mixed well, and allowed to stand in a _{CO 2 incubator (37 ° C.).} The final concentration of each candidate substance was 10 ppm.

A control group in which nHEK was cultured under the same conditions was also prepared, except that none of the candidate substances was included. Three samples were prepared for each of the 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, washed twice with phosphate buffered saline (PBS), and total RNA was extracted according to the protocol using an RNA extraction reagent (RNeasy mini Kit, QUIAGEN). .. After measuring the RNA concentration with an absorptiometer, use 30 μg / μL as an RNA template, measure the Ct value of BMP-4 with an RT-PCR system (Mx3000P, Agitant Technology), and RT-PCR based on the measured Ct value. The amount of BMP-4 mRNA was measured by a relative quantification method (ΔΔCt method) using (PrimeScript One Step RT-PCR Kit, Takara Bio).

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

In addition, as a housekeeping gene in relative quantification by RT-PCR, the base sequence of the forward primer for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is set to the following base sequence 3, and the reverse primer The base sequence was the following base sequence 4.
Nucleotide sequence 3: 5'-GCACCGTCAAGGCTGAGAAC-3'
Nucleotide sequence 4: 5'-TGGTGAAGACGCCAGTGGA-3'

The expression level of the BMP-4 gene quantified for each candidate substance (measure 3 samples for each candidate substance and show the average value and standard deviation of the 3 samples) was measured for the BMP-4 gene in the control group not exposed to the candidate substance. The relative value with the expression level of 1 as 1 is shown in the graph of FIG. The vertical axis of the graph represents the relative expression level of the BMP-4 gene, and the horizontal axis represents the number of each candidate substance. Each bar graph shows the average value of 3 samples for each candidate substance, and the error bar also shows the standard deviation. However, the bar graph of "none" at the left end shows the average value of the control group, and the error bar also shows the standard deviation. In addition, a homoscedasticity test was performed on each of the control group and the group exposed to each candidate substance by the F test, and after verifying that the dispersions of the two were not equal, the Student's t-test was performed. Was carried out. In FIG. 2, "*" indicates that the risk rate in the above t-test is more than 1% and 5% or less, and "**" is also that the risk rate is more than 0.5% and 1% or less. , And "***" also indicates that the risk rate is 0.1% or less.

From the results shown in FIG. 2, the gene expression level of BMP-4 was significantly reduced as compared with the control group by extracting the aloe vera leaf extract of the candidate substance 5, the soymilk fermented solution of the candidate substance 6, and the moss bean seed extraction of the candidate substance 7. It was a substance, an extract of Tochinoki of candidate substance 8, an extract of Kanzo of candidate substance 9, an extract of Ashitaba leaf / stem of candidate substance 10, and an extract of crackle of candidate substance 11. Therefore, candidate substances 5 to 11 are useful as BMP-4 inhibitors that lower the BMP-4 gene expression level in epidermal keratinized cells as normal cells. The reduction rates of BMP-4 gene expression levels of Candidate Substances 5 to 11 with respect to the control group were 38.7%, 46.0%, 48.5%, 48.8%, 51.3%, respectively. It was 53.0% and 84.7%. Among them, the Sanguisorba extract of Candidate Substance 11 has the best BMP-4 inhibitory effect among Candidate Substances 5 to 11 because it suppressed the gene expression level of BMP-4 by 80% or more compared to the control group. It was suggested that it is the most useful as a BMP-4 inhibitor.

(3) Melanin production inhibitory effect of Sanguisorba extract Next, it was verified whether or not the Sanguisorba extract, which had the best BMP-4 inhibitory effect in (2) above, exerts a melanin production inhibitory effect in actual cells. ..

Mouse melanoma cells (B-16, DS Pharma) were collected by trypsin treatment and then suspended in a medium containing 1% PSN. After centrifuging (300 × g, 3 minutes, room temperature) to remove the supernatant and resuspending in the above medium, seeds were seeded on 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, after removing all the medium, 1.0 mL of the medium containing the target concentration of Sanguisorba officinalis extract was added, and the medium was _{allowed to stand in a CO 2} incubator (37 ° C.).

The target concentrations were 1 ppm and 5 ppm, and a stock solution in which a crackle extract having a concentration 100 times the target concentration was dissolved in DMSO was added at a ratio of 10 μL to 1 mL of the medium. The final concentration of 1% DMSO was 1%. As a control group, a medium supplemented with 1% DMSO containing no Sanguisorba extract was used. The test was carried out using a total of 3 groups (n = 4 each) of the Sanguisorba extract concentration 1 ppm group and 5 ppm group and the control group having a concentration of 0.

Three days after seeding, the supernatant was removed from each well, washed twice with PBS, and the number of viable cells was detected by WST reagent (Cell Counting Kit, Dojin Chemical Laboratory). After measuring the number of viable cells, each well was washed twice with PBS, melanin lysate (1 mol / L NaOH, 10% DMSO, aqueous solution) was added, and the wells were completely dissolved by sonication at 50 ° C. for 20 minutes. I let you.

After the above lysis, 200 μL of the melanin lysate after melanin dissolution in each well was transferred to a 96-well plate, the absorbance at 400 nm was measured at room temperature, and the absorbance obtained from the measurement was corrected by the number of living cells to obtain the final value. The amount of melanin was adjusted. The measurement results are shown in FIG. The vertical axis of the figure shows the relative melanin production amount when the melanin production amount in the control group is 100%. The leftmost bar graph shows the amount of melanin produced in the control group (concentration 0), the center bar graph shows the crackle extract concentration 1 ppm group, and the rightmost bar graph shows the concentration 5 ppm group. The bar graph in each group is the average value of 4 samples, and the error bar is the standard deviation.

As a result of the measurement, as shown in FIG. 3, it was found that the Sanguisorba extract concentration 5 ppm group showed about 76% of the melanin production amount and the 1 ppm group showed 82% of the melanin production amount as compared with the control group. Next, a homoscedasticity test was performed between the control group and the experimental group by F-test, and after confirming that the homoscedasticity was equal, the Student's t-test was performed. In FIG. 3, "*" indicates that the risk rate in Student's t-test is more than 1% and 5% or less. That is, from the results of FIG. 3, it can be concluded that the amount of melanin produced by melanocytes was significantly reduced by exposure to 5 ppm and 1 ppm Sanguisorba extract.

(4) Melanin production inhibitory effect of nano-sized Sanguisorba composition (4-1) Adjustment of nano-sized Sanguisorba composition The following is a cosmetic containing a nano-sized dispersion of Sanguisorba extract as a BMP-4 inhibitor as a whitening agent. Examples will be described below. Here, in the present disclosure, "nano-ization" is defined as making the median diameter of the particles less than 1 μm.

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

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

While maintaining the obtained oil phase of 15% by mass at 80 ° C., an aqueous phase of 85% by mass was added, and ultrasonic dispersion was performed for 1 minute using an ultrasonic homogenizer (US-600AT, Nissei Tokyo Office). The obtained dispersion was subjected to high-pressure emulsification dispersion at a pressure of 245 MPa using a starburst mini (Sugino Machine) three times for convenience. After high-pressure emulsification dispersion, filtration was performed using a microfilter having an average pore size of 1 μm to prepare a nano-sized composition 1 as a cosmetic of the emulsified composition. The content of the Sanguisorba extract in the nano-sized composition 1 was 1.0% by mass.

[Nanoized composition 2]
The nano-sized composition 2 as a cosmetic of the emulsified composition was prepared in the same step as the nano-sized composition 1 except that the hydrogenated polyisobutene of the nano-sized composition 1 was replaced with the same amount of squalane.

[Nanoized composition 3]
The nano-sized composition 3 as a cosmetic of the emulsified composition was prepared in the same step as the nano-sized composition 1 except that the hydrogenated polyisobutene of the nano-sized composition 1 was replaced with the same amount of isotridecyl isononanoate.

[Nanoized composition 4]
The nano-sized composition 4 as a cosmetic of the emulsified composition is the same step as the nano-sized composition 1 except that the hydrogenated polyisobutene of the nano-sized composition 1 is replaced with the same amount of tri (caprylic caprylic acid) glyceryl. Prepared in.

[Non-nanoized composition]
After adding the oil phase composition to the aqueous phase composition prepared in the same manner as the nano-sized composition 1, the non-nano-sized composition as a cosmetic is prepared by stirring for 1 minute using a propeller type stirrer (AS ONE). bottom.

(4-1-2) Evaluation of Nano compound The nano-sized compositions 1 to 4 and the non-nano-sized 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 an average particle size (median size (d50)) of dispersed particles was used using a particle size analyzer (FPAR-1000, Otsuka Electronics Co., Ltd.). ) Was measured.

[Evaluation of stability over time]
Immediately after preparation of the obtained cosmetics and 3 days after preparation in an environment of 50 ° C., each cosmetic was diluted 100-fold with water and turbid with a spectrophotometer (V-630, JASCO Corporation). The degree was measured. The turbidity after 3 days of adjustment was evaluated on a scale of A, B, C and D from the one with the best evaluation according to the following evaluation criteria.
A: The turbidity was 0.025 or less.
B: The turbidity was more than 0.025 and 0.2 or less.
C: The turbidity was more than 0.2 and 0.5 or less.
D: The turbidity exceeded 0.5.

Table 1 below shows the evaluation results of the median diameter and the stability over time in the nano-sized compositions 1 to 4 and the non-nano-sized compositions.

From Table 1 above, the median diameter of the non-nano-sized composition was 5.6 μm, whereas the median diameter of the cosmetics of the nano-sized compositions 1 to 4 was 51 nm, which was the largest for the nano-sized composition 1. It was found that the nano-sized composition 4 was also nano-sized at 115 nm. The stability over time of the cosmetics was evaluated as A in the nano-sized composition 1 and the nano-sized composition 2. However, as the average particle size increased, the evaluation of stability over time became worse, with B for the nano-sized composition 3 and C for the nano-sized composition 4. The average particle size was 5.6 μm, which was the worst evaluation of D in the non-nano-sized composition which was not nano-sized. The particles having each median diameter in the nano-sized compositions 1 to nano-sized composition 4 are considered to function as BMP-4 inhibitors as oil droplets containing the Sanguisorba extract.

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

Immediately after the melanocyte-containing skin model (MEL-300B, Kurabo Industries Ltd.) arrived in the laboratory, the culture cups were transferred to netwells and pre-incubated with maintenance medium for 1 day. After the pre-incubation, the cosmetics of the nano-sized examples 1 and the non-nano-sized examples were applied, the maintenance medium was changed once every 3 days, the cosmetics were applied, and the cells were cultured for 1 week.

After culturing for 1 week, the skin model culture cup was taken out and washed with PBS, and then the number of viable cells was detected by the MTT reagent mentioned in the section (1). After measuring the number of viable cells, the product reacted with the MTT reagent with isopropanol for each skin model was completely removed, the melanin lysate mentioned in the section (1) was added, and heat treatment was performed at 80 ° C. for 1 hour. Completely dissolved.

After the above dissolution, 200 μL of the melanin solution after melanin dissolution in each well was transferred to a 96-well plate, the absorbance at 400 nm was measured at room temperature, and the amount of melanin was calculated from the absorbance obtained from the measurement. For the final amount of melanin, a value corrected by the above-mentioned number of living cells was adopted.

The results are shown in FIG. The vertical axis of FIG. 4 shows the amount of melanin produced when the amount of melanin produced in the non-nanoized composition is 100%. The "Non-nano-sized component" bar graph shows the control group, and the "Nano-sized component" bar graph shows the experimental group. The bar graph in each group represents the mean value of the three samples, and the error bar represents the standard deviation. Further, each of the non-nanoized composition and the nanonized composition 1 was subjected to a homoscedasticity test by F-test, and after confirming that the non-nanoized composition was homoscedastic, the Student's t-test was carried out. In FIG. 4, “*” indicates that the risk rate in the above t-test is more than 1% and 5% or less.

From the results shown in FIG. 4, the amount of melanin produced was reduced by 28% as compared with the non-nano-sized example by applying a cosmetic containing the nano-sized Sanguisorba extract as a BMP-4 inhibitor as a whitening agent. Therefore, it was shown that the nanonization of the Sanguisorba extract enhances the skin permeability and enhances the melanin production inhibitory effect.

(5) Manufacturing Method of Cosmetics A specific example of a manufacturing method of cosmetics will be described below.

(5-1) Whitening agent Candidate substances 1 to 11 as whitening agent candidate substances described in (2) were subjected to the screening method described in (2). Then, among the candidate substances 5 to 11 in which the decrease in the expression level of BMP-4 as a melanocyte differentiation-inducing factor was confirmed, the Sanguisorba extract of the candidate substance 11 having the most excellent BMP-4 inhibitory action was whitened. Determined as an agent. This Sanguisorba extract was nano-sized by the method described in (4-1-1) to prepare a nano-sized composition. Among the nano-sized compositions listed in (4-1-1), the nano-sized composition 1 was used as a whitening agent.

(5-2) Astaxanthin-containing emulsified composition The astaxanthin-containing emulsified composition, which is a component of the following cosmetics 1 to 6 and 9, 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 stearic acid ester (HLB = 16): 33.0 g
Decaglyceryl monooleate (HLB = 12): 67.0 g
Glycerin: 450.0 g
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 (made by RIKEN Vitamin, RIKEN E oil 800): 32.0 g
Medium-chain fatty acid glyceride (Kao, Coconard MT): 93.0 g
Lecithin (made by RIKEN Vitamin, derived from Resion P, soybean): 10.0g

The aqueous phase composition A obtained above is kept at 70 ° C. and stirred with a homogenizer (model name: HP93, manufactured by SMT Co., Ltd.) (10000 rpm), and the oil phase composition A is added to the aqueous phase composition A. To obtain a preliminary emulsion.

Subsequently, the obtained preliminary emulsion was cooled to about 40 ° C., and high-pressure emulsification was performed at a pressure of 200 MPa using an altimaizer HJP-25005 (manufactured by Sugino Machine Limited). Then, the astaxanthin-containing emulsified composition (astaxanthin content: 0.3% by mass) was prepared by filtering with a microfilter having an average pore size of 1 μm.

The obtained astaxanthin emulsified 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.) and found to be 58 nm. ..

An emulsified composition equivalent even if the above krill extract is replaced with a product name: Astax-ST (manufactured by Marine Daio Co., Ltd.) or a product name: ASTOTS-S (haematococcus algae extract, manufactured by FUJIFILM Corporation). Can be obtained.

(5-3) Preparation of Lycopene-Containing Emulsified Composition The lycopene-containing emulsified composition, which is a component of the following cosmetics 1, 2 and 9, was prepared as follows.

[Aqueous phase composition B]
The following components were weighed in a container, heated and mixed in a constant temperature bath at 70 ° C. with stirring, confirmed to be well mixed, and held at 70 ° C. to obtain an aqueous phase composition B.
Decaglyceryl oleate-10 (HLB = 12.0, manufactured by Nikko Chemicals, Decaglin 1-OV): 8.0 g
Sucrose stearic acid ester (Ryoto sugar ester S-1670, manufactured by Mitsubishi Chemical Foods): 2.0 g
Glycerin: 45.0 g
Purified water: Remaining amount up to 100 g

[Oil phase composition B]
The following components were weighed in a container and heated and mixed for 5 minutes while stirring on a hot plate at 150 ° C., and it was confirmed that they were well mixed to obtain an oil phase composition B.
Tomato oleoresin (Lyc-O-Mato 15% (containing 15% by mass of lycopene), manufactured by Sunbright): 1.14 g
Lecithin (Resion P (derived from soybeans), made by RIKEN Vitamin): 1.0 g
Medium-chain fatty acid glyceride (Coconade MT, manufactured by Kao): 12.8 g

The aqueous phase composition B obtained above was added to the oil phase composition B, stirred and mixed, and dispersed for a predetermined time using an ultrasonic homogenizer to obtain a crude dispersion liquid. Then, the obtained crude dispersion was further emulsified at 200 MPa using an ultra-high pressure emulsifier (Ultimizer, manufactured by Sugino Machine Limited) to form a lycopene-containing composition (lycopene content: 0.17% by mass). Was prepared.

The obtained lycopene-containing emulsified 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.) and found to be 52 nm. rice field.

(5-4) Adjustment of Cosmetics The whitening agent obtained in (5-1) was added to each of the following cosmetic components to produce the following cosmetics 1 to 9.

(5-4-1) Cosmetics 1 (Toner)
As cosmetic 1, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic component having the following composition to prepare a lotion by a conventional method (total amount 100% by mass).
Arbutin: 2.0% by mass
Dipotassium glycyrrhetinate: 1.0% by mass
Dipropylene glycol: 4.0% by mass
Polyoxyethylene methylglucoside: 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 (60EO): 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 emulsified composition: 0.2% by mass
Lycopene-containing emulsified composition: 0.1% by mass
Phosphate-L-ascorbyl magnesium: 1.0% by mass
Sodium hyaluronate: 0.2% by mass
Citric acid: 1.0% by mass
Sodium citrate: Appropriate amount Water: Remaining amount

(5-4-2) Cosmetics 2 (essence)
As cosmetics 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 by a conventional method (total amount 100% by mass).
Phosphate-L-ascorbyl magnesium: 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 Negesuteras B-16 Polymer: 0.05% by mass
Oryzanol: 0.01% by mass
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60EO): 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 emulsified composition: 0.2% by mass
Lycopene-containing emulsified composition: 0.2% by mass
Water: remaining amount

(5-4-3) Cosmetics 3 (cream)
As cosmetic 3, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic component having the following composition to prepare a cream by a conventional method (total amount 100% by mass).
Arbutin: 2.0% by mass
Dipotassium glycyrrhetinate: 1.0% by mass
Phosphate-L-ascorbyl magnesium: 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 methacrylic acid copolymer: 0.7% by mass
Squalene: 0.5% by mass
Shea butter: 1.0% by mass
Sarashi 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 (derived from fish): 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 Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60EO): 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

(5-4-4) Cosmetics 4 (sunscreen)
As the 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 by a conventional method (total amount 100% by mass).
Cyclopentasiloxane: 20.0% by mass
Dimethicone mass%: 10.0 mass%
Titanium oxide: 5.0% by mass
t-Butylmethoxybenzoylmethane: 1.0% by mass
HXMT-100ZA (manufactured by TAYCA, 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
Phosphate-L-ascorbyl magnesium: 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 Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60EO): 0.2% by mass
Centella asiatica extract: 0.01% by mass
Lecithin: 0.1% by mass
Fragrance: Trace amount Methyl paraoxybenzoate: 0.15% by mass
Purified water: remaining amount

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

[Aqueous phase composition C]
Further, the cosmetic components having the following compositions were mixed to prepare the aqueous phase composition C.
Glycerin monostearate: 2.0% by mass
Polyoxyethylene cetyl ether: 3.0% by mass
Polyoxyethylene sodium orbitan monoolate: 2.0% by mass
1,3-butylene glycol: 1.0% by mass
Glycerin: 2.0% by mass
Sucrose stearate: 0.1% by mass
Polyglyceryl oleate-10: 0.1% by mass
Polyglyceryl stearate-2: 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 (60EO): 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 aqueous phase composition C were each heated to 70 ° C. and stirred while emulsifying to prepare a milky lotion as cosmetic 5 (total amount 100% by mass).

(5-4-6) Cosmetic 6 (Jerry-like serum)
As the cosmetic 6, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic component having the following composition to prepare a jelly-like beauty essence by a conventional method (total amount 100% by mass).
Haematococcus algae extract: 0.1% by mass
Astaxanthin-containing emulsified composition: 0.2% by mass
Ceramide III, VI mixture: 1.0% by mass
Hydrolyzed collagen: 1.0% by mass
Acetyl hydroxyproline: 1.0% by mass
Ethylhexyl glycerin: 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 oleate-10: 0.1% by mass
Polyglyceryl stearate-2: 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 Chemical Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60EO): 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

(5-4-7) Cosmetics 7 (wet foundation)
0.1% by mass of the whitening agent obtained in (5-1) is added to the cosmetic component having the following composition to prepare a slurry, filled in a predetermined container, and dried to solidify the cosmetic component 7. A powdered cosmetic (wet foundation) was prepared (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) (Lonaflare Balance Gold, made by Merck): 13% by mass
Pearl Pigment (Red) (Transprismer 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

(5-4-8) Cosmetics 8 (Liquid Foundation)
As the cosmetic product 8, 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 liquid foundation (W / O emulsion) by a conventional method (total amount). 100% by mass).
Dipotassium glycyrrhizinate: 0.2% by mass
Specified red composite pigment (HNB RED7, manufactured by Daito Kasei): 0.5% by mass
Constituent pigment (OTS-2 SERICITE PSE and OTS-2 TALK JA-46R (both manufactured by Daito Kasei) mixed at a ratio of 7: 3): 15.0% by mass
Color material pigments (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) at a ratio of 78: 19: 1: 2) (Mixed in): 2.0% by mass
Pearl pigment (a mixture of Naflare Balance Gold and Lance Prisma-Red (both made by MERCK) at 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 weight
PEG-9 polydimethylsiloxyethyl dimethicone: 1.2% by weight
Squalene: 0.1% by mass
Sorbitan sesquiisostearate: 1.0% by mass
Disteardimonium hectorite: 0.8% by mass
Ethylhexyl methoxycinnamate: 2.5% by mass
Hematococcus spruviaris 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 (60EO): 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-exchanged water: Remaining amount

(5-4-9) Cosmetic 9 (face wash)
As the cosmetic 9, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic component having the following composition to prepare a face wash by a conventional method (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 cocoylglycine: 10.0% by mass
Lauroamphosodium: 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
Hematococcus spruviaris oil: 0.05% by mass
Astaxanthin-containing emulsified composition: 0.05% by mass
Lycopene-containing emulsified 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 (60EO): 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) Cosmetics 10 (essence)
As the cosmetic 10, 0.1% by mass of the whitening agent obtained in (5-1) was added to the cosmetic component having the following composition to prepare a beauty essence by a conventional method (total amount 100% by mass).
Phosphate-L-ascorbyl magnesium: 2.0% by mass
Dipotassium glycyrrhetinate: 1.0% by mass
Dipropylene glycol: 4.0% by mass
Sanguisorba extract (trade name: Jiyu extract powder, Maruzen Pharmaceuticals 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 Negesuteras B-16 Polymer: 0.05% by mass
Oryzanol: 0.01% by mass
Polyoxyethylene phytosterol (NIKKOL BPS-20: manufactured by Nikko Chemicals Co., Ltd.): 0.03% by mass
Polyoxyethylene hydrogenated castor oil (60EO): 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 emulsified composition: 0.2% by mass
Lycopene-containing emulsified composition: 0.2% by mass
Nicotinamide: 5.0% by mass
Water: remaining amount

Claims (10)

Whitening using a decrease in the expression level of the melanocyte differentiation-inducing factor when the cells are exposed to the whitening agent candidate substance as an index with respect to the expression level of the melanocyte differentiation-inducing factor in the control group in which the cells are not exposed to the whitening agent candidate substance. Drug screening method. The method for screening a whitening agent according to claim 1, wherein the melanocyte differentiation-inducing factor is bone-forming protein 4. The method for screening a whitening agent according to claim 1 or 2, wherein the cells are normal human cells. The method for screening a whitening agent according to claim 3, wherein the normal human cells are epidermal keratinocytes. The method for screening a whitening agent according to claim 3, wherein the human normal cells are fibroblasts. Bone-forming protein containing at least one selected from the group consisting of crackle mulberry extract, aloe vera leaf extract, fermented soymilk, moth bean seed extract, horse chestnut extract, horse chestnut root extract, and ashitaba leaf / stem extract. Inhibitor. The bone-forming protein 4 inhibitor according to claim 6, which contains particles containing the Sanguisorba extract, and the particles have a median diameter of less than 1 μm. A cosmetic containing the bone-forming protein 4 inhibitor according to claim 6 or 7 as a whitening agent. A cosmetic containing a whitening agent candidate substance as a whitening agent, for which a decrease in the expression level of the melanocyte differentiation-inducing factor has been confirmed by the whitening agent screening method according to any one of claims 1 to 5. A step of determining as a whitening agent a candidate substance for which a decrease in the expression level of the melanocyte differentiation-inducing factor has been confirmed by the screening method for a whitening agent according to any one of claims 1 to 5. ,
Step of adding the whitening agent to cosmetic ingredients,
Manufacturing method of cosmetics including.

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