Method for maintaining skin color homeostasis, topical agent for maintaining skin color homeostasis, and screening method

Abstract

To provide a novel method for maintaining skin color homeostasis, a novel topical agent for maintaining skin color homeostasis, and a novel screening method for an active ingredient thereof.SOLUTION: The present invention provides a method for maintaining skin color homeostasis that promotes sFRP1 expression in skin cells to inhibit melanocyte activity in the skin cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for maintaining skin color constancy, an external preparation for maintaining skin color constancy, and a method for screening an active ingredient thereof.

Background Art

[0002] Melanin is a brown to black pigment produced enzymatically from tyrosine, which is a kind of amino acid. In humans, melanin is said to be one of the factors that determine skin color and plays an important role in protecting the body from the adverse effects of ultraviolet rays. However, excessive production of melanin causes uneven distribution of melanin in the skin, leading to cosmetic skin problems such as freckles, chloasma, and skin discoloration.

[0003] As the melanin production mechanism, the following processes (a) to (d) have been elucidated. (a) Melanocyte activating factors are produced from keratinocytes by ultraviolet rays or stress, and these factors stimulate melanocytes present in the basal layer of the skin. (b) In activated melanocytes, tyrosinase, which is a melanin-producing enzyme, is excessively produced, and melanin is produced in the intracellular organelle melanosome. (c) This melanosome moves to the end of the dendritic protrusion of the melanocyte and is transferred to keratinocytes (keratinized cells). (d) The melanosome is transported into these cells and accumulated, ultimately changing the skin color to black.

[0004] Conventionally, as countermeasures against freckles, chloasma, and skin discoloration, many studies have been conducted to search for substances that suppress melanin production by suppressing factors that promote melanin production, substances that have an action of promoting melanosome degradation, and the like.

[0005] For example, Patent Document 1 describes luteolin and / or its glycoside, and an endothelin-1 production inhibitor containing an extract of Kudamono-tokeiso as an active ingredient, as substances that suppress melanin production by suppressing endothelin-1, which is a factor that promotes melanin production. In addition, for example, Patent Document 2 describes a lysosome activity promoter containing an adlay extract or a birch extract as an active ingredient, which improves lysosome activity in keratinocytes and promotes the degradation of melanosomes.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, despite the above conventional approaches, it has still not been possible to sufficiently eliminate the problems of skin color changes including freckles, liver spots, and uneven skin tone. Therefore, further elucidation of the mechanism of skin color change and further exploration of substances that suppress skin color change have been demanded.

[0008] In view of the above background, an object of the present invention is to provide a novel method for maintaining skin color constancy, an external preparation for maintaining skin color constancy, and a screening method for an active ingredient thereof.

Means for Solving the Problems

[0009] As a result of intensive studies to solve the above-described problems, the present inventors focused on the WNT signal transduction known to be involved in melanin synthesis in melanocytes, and found that the above problems could be solved by promoting the expression of sFRP1 (Secreted frizzled-related protein 1), which is a control factor that suppresses WNT signal transduction, in skin cells, leading to the present invention.

[0010] The present invention is as follows. [1] A method for maintaining skin color constancy, which comprises suppressing the activity of melanocytes in the skin cells by promoting the expression of sFRP1 in the skin cells. [2] An external preparation for maintaining skin color constancy for use in the method for maintaining skin color constancy according to [1], which comprises promoting the expression of sFRP1 in skin cells. [3] The external preparation for maintaining skin color constancy according to [2], which contains at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, licorice extract, and zingiber extract as an active ingredient. [4] A screening method for the active ingredient of the external preparation for maintaining skin color constancy according to [2], which comprises selecting the active ingredient using the expression level of sFRP1 in cultured skin cells to which a candidate substance has been added as an index. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a novel method for maintaining skin color constancy, an external preparation for maintaining skin color constancy, and a screening method for the active ingredient thereof. [Brief Description of the Drawings]

[0012]

Figure 1

Figure 2

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Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0013] Hereinafter, the mode for carrying out the present invention (hereinafter referred to as "the present embodiment") will be described in detail. The following present embodiment is an exemplification for explaining the present invention and is not intended to limit the present invention to the following contents.

[0014] In this specification, the structure of the skin, which is the object that brings about the skin color constancy maintaining effect, will be described as follows. First, the skin has, in this order from the outermost layer side, the epidermis, the dermis, and the subcutaneous tissue. Further, the epidermis has, in this order from the outermost layer side, the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. And the cells constituting the epidermis are called "epidermal cells", but epidermal cells generally often indicate keratinocytes. Also, in this specification, the term "skin cells" means the cells constituting the skin, and specifically includes the above - mentioned epidermal cells, fibroblasts, melanocytes, immune cells, etc. Skin cells may be those existing in vivo or cultured skin cells (those taken out from a living body and cultured, or those obtained by inducing differentiation from embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, etc.).

[0015] In the search and research for substances that suppress skin color changes, the inventors focused on the WNT signaling pathway (WNT / β-catenin pathway), which is important for cell growth and differentiation and is known to be involved in melanin synthesis in melanocytes. When the WNT ligand protein binds to the Frizzled receptor on the cell membrane of melanocytes, it is known that the melanocytes are activated (the expression of melanin-producing enzymes increases) and melanin production increases. When the inventors investigated and examined the expression levels and effects on melanocyte function of various regulatory factors involved in the WNT signaling pathway, they newly discovered that the expression level of sFRP1, a regulatory factor that suppresses WNT signaling, was decreased in the pigmented areas (pigmented sites) of human skin compared to the non-pigmented areas (healthy sites). Figure 1 shows the results of measuring the expression levels of sFRP1 mRNA in the healthy and pigmented sites of the skin obtained from volunteers by gene expression analysis.

[0016] sFRP1 is known to inhibit the binding of the WNT ligand protein to the Frizzled receptor by directly binding to the WNT ligand protein, thereby suppressing WNT signaling. Therefore, based on the above new finding that the expression level of sFRP1 is low in the pigmented sites, the inventors considered that in the pigmented sites, due to the low expression level of sFRP1, melanocytes are excessively activated by WNT signaling, resulting in increased melanin production and the appearance of pigmentation. They found the possibility that suppressing the excessive activation of melanocytes and suppressing melanin production by promoting the expression of sFRP1. Actually, when analyzing the effect of sFRP1 on the function of melanocytes, as shown in Figure 2, it was confirmed that the addition of sFRP1 significantly suppressed the cell proliferation of melanocytes. Figure 2 shows the change in the number of cells when the sFRP1-added group, to which 1 μg of the recombinant protein of sFRP1 (manufactured by Sigma-Aldrich) was added per 1 mL of the medium, and the control group without the addition of the recombinant protein of sFRP1 were each cultured for 6 days (T-test *** p<0.001). Furthermore, when measuring the activity of tyrosinase in the above sFRP1-added group and control group, as shown in Figure 3, it was confirmed that the activity significantly decreased in the sFRP1-added group compared to the control group (T-test *** p<0.001). From the above, it became clear that sFRP1 negatively controls the function of melanocytes and has the function of suppressing skin pigmentation.

[0017] Conventional techniques for suppressing melanin production (so-called whitening techniques) were approaches that suppressed the overproduction of melanin by suppressing factors that promote melanin production. In contrast, the present invention is an entirely new approach of enhancing a factor (sFRP1) that suppresses melanin production, and it enhances the function of not synthesizing melanin among the functions of regulating the amount of melanin originally present in the skin.

[0018] [Method for maintaining skin color constancy] The method for maintaining skin color constancy of the present embodiment is characterized by suppressing the activity of melanocytes in the skin cells by promoting the expression of sFRP1 in the skin cells. According to the method for maintaining skin color constancy of the present embodiment, since melanin production can be suppressed, the accumulation of melanin in the skin cells can be suppressed, and the change in skin color to black can be suppressed (skin color darkening suppression effect). Also, the occurrence of color unevenness due to local change in skin color to black (appearance of freckles, age spots, etc.) can be suppressed (skin color unevenness suppression effect), and the original skin color can be maintained (skin color constancy maintenance effect). Note that the method for maintaining skin color constancy in this embodiment does not include a method implemented for the purpose of treating human skin.

[0019] [External preparation for maintaining skin color constancy] The external preparation for maintaining skin color constancy in this embodiment is a drug used in the method for maintaining skin color constancy in the above-described embodiment, and is characterized by promoting the expression of sFRP1 in skin cells. According to the external preparation for maintaining skin color constancy in this embodiment, by promoting the expression of sFRP1, excessive activation of melanocytes can be suppressed, and melanin production can be suppressed. Therefore, the accumulation of melanin in skin cells can be suppressed, and the change in skin color to black can be suppressed (skin color darkening suppression effect). In addition, the local change in skin color to black (formation of freckles, chloasma, etc.) and the occurrence of color unevenness can be suppressed (skin color unevenness suppression effect), and the original skin color can be maintained (skin color constancy maintenance effect). That is, the external preparation for maintaining skin color constancy in this embodiment can be said to be an external preparation for suppressing skin color darkening and an external preparation for suppressing skin color unevenness. The external preparation for maintaining skin color constancy in this embodiment can be suitably used as cosmetics, pharmaceuticals, quasi-drugs, and the like.

[0020] The active ingredient of the external preparation for maintaining skin color constancy is not particularly limited as long as it can promote the expression of sFRP1 in skin cells. Examples include ascorbic acid, ascorbic acid derivatives (vitamin C derivatives), zingiber extract, licorice extract, and the like. Among them, ascorbic acid derivatives are preferred. These active ingredients may be used alone or in combination of two or more.

[0021] Ascorbic acid derivatives mean those obtained by chemically modifying and / or substituting a part of ascorbic acid. More specifically, ascorbic acid derivatives include metal salts of ascorbic acid (such as magnesium, sodium, potassium, calcium), organic salts (such as ammonium, amine), and sugar esters. Among them, L-ascorbyl magnesium phosphate (VC-PMG) and ascorbic acid 2-glucoside (AA-2G) are preferred. The ascorbic acid derivative may be a commercially available product or may be synthesized by a conventionally known method.

[0022] The calamus extract is an extract of Acorus calamus (scientific name: "Acorus calamus") of the genus Acorus in the family Acoraceae. Examples of the raw material for the calamus extract include the leaves, roots, stems, branches, flowers, stamens, pistils, fruits, seeds, cultured cells, callus, protoplasts, transformed plant cells, transformed plants, etc. of calamus, and the whole plant may be used. The raw material may be any of the living organism itself, frozen product, dried product, freeze-dried product, roasted product, etc. The method for producing the calamus extract is not particularly limited and can be produced according to a conventional method. Among them, it is preferable to use an aqueous solvent such as water or ethanol as the extraction solvent. The extract may be, for example, a concentrated or dried extraction solution. The form of the extract is not particularly limited and may be any of liquid, paste, gel, solid, powder, etc.

[0023] The mugwort extract is an extract of the leaves or shoot tips of Artemisia princeps Pampanini (scientific name: "Artemisia princeps Pampanini") or Artemisia montana Pampanini (scientific name: "Artemisia montana Pampanini") of the genus Artemisia in the family Asteraceae. The leaves or shoot tips of Artemisia princeps Pampanini or Artemisia montana Pampanini, which are the raw materials for the mugwort extract, may be any of the living organism itself, frozen product, dried product, freeze-dried product, roasted product, etc. The method for producing the mugwort extract is not particularly limited and can be produced according to a conventional method. Among them, it is preferable to use an aqueous solvent such as water or ethanol as the extraction solvent. The extract may be, for example, a concentrated or dried extraction solution. The form of the extract is not particularly limited and may be any of liquid, paste, gel, solid, powder, etc.

[0024] The external preparation for maintaining skin color constancy of this embodiment may contain water, ethanol, glycerin, glycol, etc. as a solvent for the active ingredient.

[0025] The content of the active ingredient in the external preparation for maintaining skin color constancy is preferably 0.00005% by mass or more, more preferably 0.0005% by mass or more, and even more preferably 0.005% by mass or more, in terms of mass% on a dry solid basis. When the content of the active ingredient in the external preparation for maintaining skin color constancy is at least the above lower limit, the effect of maintaining skin color constancy tends to be more significantly exhibited. Also, the upper limit of the content of the active ingredient in the external preparation for maintaining skin color constancy is not particularly limited. For example, the mass% on a dry solid basis may be 10% by mass or less, may be 5% by mass or less, and may be 1% by mass or less.

[0026] Unless it inhibits the effects of the present invention, the external preparation for maintaining skin color constancy of the present embodiment may, in addition to the active ingredient and the solvent, contain aqueous components (such as hydroxyproline), oily components, plant extracts, animal extracts, powders, surfactants, oils, alcohols, pH adjusters, preservatives, antioxidants, thickeners, pigments, and fragrances, etc., which are commonly used in cosmetics, pharmaceuticals, and quasi-drugs, as additive components as needed and can be used by blending. The content of the additive components in the external preparation for maintaining skin color constancy is not particularly limited unless it inhibits the effects of the present invention. For example, in terms of the mass-to-volume ratio concentration on a dry solid basis, it may be 0 to 99.99995% by mass, may be 90 to 99.9995% by mass, may be 95 to 99.995% by mass, and may be 99 to 99.995% by mass.

[0027] The dosage forms of the external preparation for maintaining skin color constancy of the present embodiment, as well as cosmetics, pharmaceuticals, and quasi-drugs using the external preparation for maintaining skin color constancy, are not particularly limited and may be various dosage forms such as lotion, emulsion, cream, pack, powder, spray, ointment, dispersion, and cleansing agent.

[0028] [Screening method] The screening method of the present embodiment is a method for screening the active ingredient of the external preparation for maintaining skin color constancy of the above-described present embodiment, and is characterized in that the active ingredient is selected using the expression level of sFRP1 in cultured skin cells to which a candidate substance is added as an index. According to the screening method of this embodiment, the external preparation for maintaining skin color constancy of the above-described this embodiment can be efficiently selected. The screening method of this embodiment preferably includes, for example, an addition step of adding a candidate substance to cultured skin cells, a measurement step of measuring the expression level of sFRP1 in the cultured skin cells to which the candidate substance has been added, and a selection step of selecting a candidate substance useful as an active ingredient using the expression level of sFRP1 obtained in the measurement step as an index.

[0029] [Addition step] In the addition step, a candidate substance for the active ingredient of the external preparation for maintaining skin color constancy is added to the cultured skin cells. The cultured skin cells to which the candidate substance is added may be a two-dimensional culture or a three-dimensional culture. The method for adding the candidate substance to the cultured skin cells is not particularly limited. For example, it may be added to the medium. From the viewpoint of enhancing the screening accuracy, it is preferable to add the candidate substance to the cultured skin cells while varying the concentration. The culture conditions such as the culture temperature are not particularly limited, and the cells may be cultured under the environment usually set when culturing skin cells.

[0030] [Measurement step] In the measurement step, the expression level of sFRP1 in the cultured skin cells to which the candidate substance has been added is measured. More specifically, the expression level of sFRP1 mRNA in the cultured skin cells cultured by adding a candidate substance at a predetermined concentration and the expression level of sFRP1 mRNA in the cultured skin cells (control group) cultured without adding the candidate substance are measured. The mRNA expression level of sFRP1 in the cultured skin cells can be measured by the method described in the examples below.

[0031] By adding the candidate substance to the cultured skin cells while varying the concentration of the candidate substance and measuring the expression level of sFRP1 at each concentration, the skin color constancy maintaining effect at each concentration can be evaluated. Then, based on the obtained evaluation, the preferable range of the effective concentration when applied as an active ingredient can be determined.

[0032] [Selection Step] In the selection step, a candidate substance useful as an active ingredient is selected using the expression level of sFRP1 obtained in the measurement step as an index. More specifically, the mRNA expression level of sFRP1 in cultured skin cells to which no candidate substance has been added is compared with the mRNA expression level of sFRP1 in cultured skin cells to which a candidate substance has been added, and a candidate substance useful as an active ingredient of the external preparation for maintaining skin color constancy is selected. When the mRNA expression level M1 of sFRP1 in cultured skin cells to which a candidate substance has been added is increased compared to the mRNA expression level M0 of sFRP1 in cultured skin cells to which no candidate substance has been added, that is, when M1 is more than 100% when M0 is set to 100%, it is determined that the candidate substance is useful as an active ingredient of the external preparation for maintaining skin color constancy and is selected.

Example

[0033] Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples.

[0034] Using cultured skin cells, screening was performed to select a component useful as an active ingredient of the external preparation for maintaining skin color constancy.

[0035] (Example 1) As a candidate substance for an active ingredient of the external preparation for maintaining skin color constancy, L-ascorbyl magnesium phosphate (VC-PMG) was used, and the expression level of sFRP1 in cultured skin cells to which VC-PMG was added was measured. Specifically, a medium ("HuMedia-KG2" manufactured by Kurabo Industries Ltd.) was added to a 12-well culture plate, and normal human epidermal keratinocytes (keratinocytes manufactured by Kurabo Industries Ltd.) were seeded and cultured in an environment of 37°C and 5% by volume CO2. After culturing for 24 hours, VC-PMG (manufactured by Fujifilm Wako Pure Chemical Corporation) was added so that the concentrations in the medium were 0.5 mM and 5 mM, respectively, and cultured in an environment of 37°C and 5% by volume CO2. After 24-hour culture, the cells were washed with phosphate buffer and collected. Total RNA was extracted and purified from the collected cells using the RNeasy Kit (manufactured by QIAGEN). The mRNA expression level of sFRP1 was measured from the purified total RNA using quantitative reverse transcription PCR (Bio-Rad's "Real-time PCR Detection System").

[0036] (Example 2) The mRNA expression level of sFRP1 was measured in the same manner as in Example 1, except that ascorbic acid 2-glucoside (AA-2G, manufactured by FUJIFILM Wako Pure Chemical Corporation) was used instead of VC-PMG as the candidate substance.

[0037] (Example 3) The mRNA expression level of sFRP1 was measured in the same manner as in Example 1, except that zingiber extract (a 1.6 w / v% dilution solution with 40 v / v% ethanol) was added as the candidate substance at concentrations of 0.01% and 0.1% in the medium.

[0038] (Example 4) The mRNA expression level of sFRP1 was measured in the same manner as in Example 1, except that licorice extract (a 0.8 w / v% dilution solution with 50 v / v% ethanol) was added as the candidate substance at concentrations of 0.1% and 0.5% in the medium.

[0039] (Comparative Example 1) The mRNA expression level of sFRP1 was measured in the same manner as in Example 1, except that niacinamide was used instead of VC-PMG as the candidate substance.

[0040] Figures 4 to 8 are graphs showing the measurement results of the mRNA expression levels of sFRP1 in Examples 1 to 4 and Comparative Example 1. That is, Figures 4 to 8 are graphs showing the effects of VC-PMG, AA-2G, Coptis japonica extract, Artemisia princeps extract, and niacinamide on the mRNA expression of sFRP1. In all of the graphs, the vertical axis represents the relative mRNA expression level (%) when the mRNA expression level of the control group (cultured without adding the candidate substance, indicated as a concentration of "0 mM" or "0%") is set to 100%, and the horizontal axis represents the concentration (mM or %) of each candidate substance. The error bars indicate the standard error (SE value).

[0041] As shown in Figure 4, in Example 1, in the group with 5 mM of VC-PMG added, a 45% increase in the mRNA expression level of sFRP1 was observed based on the control group, showing an excellent expression promoting effect (Dunnett's test * p < 0.05). Also, as shown in Figure 5, in the group with 5 mM of AA-2G added in Example 2, a 41% increase in the mRNA expression level of sFRP1 was observed based on the control group, showing a particularly excellent expression promoting effect (Dunnett's test # p = 0.058). In the groups with the addition of ascorbic acid derivatives (VC-PMG, AA-2G), overall, the mRNA expression level of sFRP1 increased compared to the control group without their addition. Therefore, it was found that by adding ascorbic acid derivatives, the production of sFRP1 in epidermal cells can be promoted, and ascorbic acid derivatives are useful as active ingredients of external preparations for maintaining skin color constancy. Also, from this result, when an external preparation for maintaining skin homeostasis containing ascorbic acid derivatives as an active ingredient is applied to skin cells, it can be seen that the produced sFRP1 suppresses the excessive activation of melanocytes, and as a result, can contribute to maintaining skin color constancy.

[0042] In addition, for Example 3, as shown in Fig. 6, in the groups supplemented with 0.01% and 0.1% of Canna extracts, increases in the mRNA expression levels of sFRP1 by 45% and 50% respectively were observed based on the control group, both showing excellent expression promoting effects. Also in Example 4, as shown in Fig. 7, in the groups supplemented with 0.1% and 0.5% of Galium extracts, increases in the mRNA expression levels of sFRP1 by 72% and 48% respectively were observed based on the control group, both showing excellent expression promoting effects (Dunnett's test p = 0.09). In general, in the groups supplemented with Canna extracts and Galium extracts, the mRNA expression level of sFRP1 increased compared to the control group without these supplements. Therefore, it was found that by adding Canna extracts and / or Galium extracts, the production of sFRP1 in epidermal cells can be promoted, and Canna extracts and Galium extracts are useful as active ingredients of an external preparation for maintaining skin color constancy. Also, from these results, it can be seen that when an external preparation for maintaining skin constancy containing Canna extracts and / or Galium extracts as active ingredients is applied to skin cells, the produced sFRP1 can suppress the excessive activation of melanocytes, and as a result, can contribute to maintaining skin color constancy.

[0043] On the other hand, in Comparative Example 1, the mRNA expression levels were 86% in the group supplemented with 0.5 mM of niacinamide and 75% in the group supplemented with 5 mM of niacinamide. Considering that the addition concentration on the high concentration side is the same 5 mM as in Examples 1 and 2 and the standard error of the measured values, it can be said that the presence or absence of niacinamide addition had no significant effect on the increase or decrease in the mRNA expression level of sFRP1. Therefore, it can be seen that depending on niacinamide, the effect of maintaining skin color constancy based on promoting the production of sFRP1 cannot be obtained.

[0044] Hereinafter, formulation examples using the external preparation for maintaining skin color constancy of the present invention (active ingredients: VC-PMG, AA-2G, Canna extracts, Galium extracts) are shown. These formulation examples are merely illustrative of the present invention, and the present invention is not limited to these formulation examples.

[0045] [Formulation Example 1: Lotion] (Ingredients) (Unit: %) 1. Glycerin 5.0 2. 1,3 - Butylene Glycol 5.0 3. Lactic Acid 0.05 4. Sodium Lactate 0.1 5. Polyoxyethylene (20 moles) Sorbitan Monooleate 1.2 6. Ethanol 8.0 7. Indole - 2 - Carboxylic Acid 0.01 8. Methyl Para - hydroxybenzoate 0.1 9. Perfume 0.05 10. Purified Water Balance 11. VC - PMG 0.1 12. Birch Sap 0.1 13. Extract of Prunus Mume Sieb. Et Zucc. 0.1 14. Hydrolyzed Collagen 0.1 15. Niacinamide 4.0 16. Tranexamic Acid 2.0

[0046] (Manufacturing Method) A: Ingredients 5 - 9 were mixed and dissolved. B: Ingredients 1 - 4 and 10 - 16 were mixed and dissolved. C: A was added to B and mixed to obtain a lotion.

[0047] [Formulation Example 2: Emulsified Lotion] (Ingredients) (Unit: %) 1. Hydrogenated Phosphatidylcholine from Soybean 0.5 2. Cetostearyl Alcohol 0.1 3. Polyoxyethylene (10 moles) Cholesterol Ether 0.2 4. dl - α - Tocopherol Acetate 0.1 5. Squalane 0.1 6. Hydroxyethyl Cellulose 0.03 7. Purified Water Balance 8. Stearyl Glycyrrhetinate 0.25 9. Disodium Hydrogen Phosphate 0.1 10. Sodium Dihydrogen Phosphate 0.1 11. Glycerin 3.0 12. Dipropylene glycol 2.0 13. Ethanol 7.0 14. Fragrance 0.1 15. VC-PMG 2.0 16. Birch sap 0.1 17. Houttuynia cordata extract 0.1 18. Hydrolyzed collagen 0.1 19. Canna extract (1.6 w / v% diluted solution with 40 v / v% ethanol) 0.05 20. Tranexamic acid 1.5

[0048] (Manufacturing method) A: Components 1 to 5 were heated to 75 °C and uniformly mixed and dissolved. B: Components 6 and 7 were heated to 75 °C and uniformly mixed and dissolved. C: B was added to A and emulsified at 75 °C. D: C was cooled to room temperature and components 8 to 20 were added to obtain an emulsion-type lotion.

[0049] [Formulation Example 3: Oil-in-water emulsion] (Components) (Unit: %) 1. 1,3-Butylene glycol 5.0 2. Dibutylene glycol 5.0 3. Purified water balance 4. 6-Phenyl-indole-2-carboxylic acid 0.001 5. Sodium hydroxide 2% solution 0.2 6. N-Myristoyl-L-glutamic acid 0.2 7. Polyoxyethylene (10 moles) hydrogenated castor oil (Note 1) 0.2 8. Phytosteryl macadamia nut oil fatty acid 1.0 9. Dimethylpolysiloxane (Note 2) 3.0 10. N-Stearoyl phytosphingosine 0.1 11. Tocopherol acetate 0.01 12. Stearyl alcohol 0.5 13. 1,2 - Pentanediol 0.1 14. Carboxyvinyl polymer (Note 3) 0.15 15. (Sodium acrylate / sodium acryloyldimethyltaurine) copolymer (Note 4) 0.1 16. Ethanol 5.0 17. Fragrance 0.1 18. VC - PMG 5.0 19. Extract of Ezo - kogitake 0.1 20. Extract of Hoonoki 0.1 21. Extract of Tabebuia impetiginosa bark 0.1 22. Niacinamide 2.0 23. Tranexamic acid 2.0 (Note 1) HCO - 10 (manufactured by Nippon Surfactant Industry Co., Ltd.) (Note 2) Silicon KF - 96A(6CS) (manufactured by Shin - Etsu Chemical Co., Ltd.) (Note 3) CARBOPOL980 (manufactured by LUBRIZOL ADVANNCED MATERIALS) (Note 4) SIMULGEL EG (manufactured by SEPIC)

[0050] (Manufacturing method) A: Components 1 - 5 were uniformly dissolved and mixed at 70°C. B: Components 6 - 14 were uniformly dissolved and mixed at 80°C. C: B was added to A and emulsified at 70°C. D: Components 15 - 23 were added to and mixed with C, and then cooled to 40°C to obtain an oil - in - water type emulsion.

[0051] [Formulation Example 4: Oil - in - water type emulsifying cream] (Components) (unit: %) 1. 1,3 - Butylene glycol 12.0 2. Glycerin 5.0 3. Purified water balance 4. Citric acid 0.1 5. Sodium ascorbate 0.5 6. Sodium metasilicate 0.05 7. Hydrogenated lecithin 0.5 8. Hydrogenated Lecithin (Note 5) 0.5 9. Phytosteryl Macadamia Nut Oil Fatty Acid 1.5 10. Glyceryl Tri(caprylyl / caprate) 2.0 11. Ceramide 2 0.1 12. Astaxanthin 0.1 13. Tocopherol 0.01 14. Cetearyl Alcohol 2.0 15. Behenyl Alcohol 1.0 16. Glyceryl Monostearate 1.0 17. Methyl Paraben 0.05 18. Carbomer 0.15 19. Xanthan Gum 0.1 20. Sodium Hydroxide 0.004 21. Fragrance 0.05 22. Galium Aparine Extract (0.8 w / v% Diluted Solution with 50 v / v% Ethanol) 0.01 23. Saururus Chinensis Extract 0.1 24. Phyllostachys Nigra Extract 0.1 25. Tabebuia Impetiginosa Bark Extract 0.1 26. Niacinamide 1.0 27. Tranexamic Acid 2.5 (Note 5) LP70H (manufactured by Nippon Fine Chemical Co., Ltd.)

[0052] (Manufacturing Method) A: Components 1 to 6 were uniformly dissolved and mixed at 70°C. B: Components 7 to 16 were uniformly dissolved and mixed at 80°C. C: B was added to A and emulsified at 70°C. D: After adding and mixing Components 17 to 27 to C, it was cooled to 40°C to obtain an oil-in-water type emulsified cream.

[0053] [Formulation Example 5: Non-woven Fabric Impregnated Type Packing Material] (Components) (Unit: %) 1. Polyoxyethylene (10 moles) Phytosterol 1.0 2. Stearyl alcohol 0.2 3. Cetyl alcohol 0.2 4. Hydrogenated soybean phospholipid (Note 6) 1.0 5. Propylene glycol 10.0 6. Diglycerin 8.0 7. Liquid paraffin 1.0 8. Meadowfoam oil 0.5 9. Coenzyme Q10 (Note 7) 0.02 10. Purified water 1.0 11. Ethanol 3.0 12. Purified water remainder 13. Iris extract (1.6 w / v% diluted solution with 40 v / v% ethanol) 0.5 14. Saffron extract 0.1 15. Sodium chondroitin sulfate 0.05 16. Pancratium maritimum extract 0.1 17. Niacinamide 0.5 18. Tranexamic acid 0.1 (Note 6) Nikkol Resinol S-10 (manufactured by Nikko Chemicals Co., Ltd.) (Note 7) Kaneka Coenzyme Q10 (manufactured by Kaneka Corporation)

[0054] (Manufacturing method) A: Components 1 to 6 were dissolved and mixed at 80°C. B: Components 7 to 9 were heated to 80°C, added to A, and then component 10 heated to 75°C was added and emulsified. C: B was stirred and cooled, components 11 to 18 were added and mixed to obtain an oil-in-water type emulsion. This was impregnated into a non-woven fabric to obtain a non-woven fabric impregnated type pack material.

[0055] [Formulation Example 6: Sunscreen] (Components) (unit: %) 1. Stearic acid 1.0 2. Polyoxyethylene (20 moles) sorbitan monooleate 0.5 3. Sorbitan sesquioleate 0.5 4. Behenyl alcohol 0.5 Glyceryl 5.2 - ethylhexanoate 2.0 6. 2 - Ethylhexyl paramethoxycinnamate 10.0 7. Hexyl diethylaminohydroxybenzoyl benzoate 2.0 8. Dimethicone diethylbenzylmalonate 1.0 9. 2,4 - Bis - [{4 - (2 - ethylhexyl oxy)-2 - hydroxy}-phenyl]-6 - (4 - methoxyphenyl)-1,3,5 - triazine (Note 8) 1.0 10. Octyl triazone 0.5 11. Dipropylene glycol 10.0 12. Triethanolamine 1.0 13. Purified water balance 14. Glycerin 5.0 15. 1,3 - Butylene glycol 5.0 16. Methylene bis - benzotriazolyl tetramethylbutylphenol (Note 9) 1.0 17. Acrylic acid / methacrylic acid alkyl ester copolymer (Note 10) 0.2 18. Purified water 5.0 19. Canna extract (1.6 w / v% diluted solution with 40 v / v% ethanol) 0.2 20. Iris extract (0.8 w / v% diluted solution with 50 v / v% ethanol) 0.1 21. Sodium chondroitin sulfate 0.05 22. Pancratium maritimum extract 0.4 23. Niacinamide 1.5 24. Tranexamic acid 0.5 25. Citric acid 0.1 26. Disodium succinate 0.1 27. Ethanol 10.0 28. Phenoxyethanol 0.1 29. Fragrance 0.1 (Note 8) Tinosorb S (manufactured by BASF) (Note 9) Tinosorb M (manufactured by BASF) (Note 10) CARBOPOL 1382 (manufactured by Lubrizol Advanced Materials)

[0056] (Manufacturing method) A: Components 1 to 10 were uniformly dissolved at 80°C. B: Components 11 to 18 were uniformly dissolved at 80°C. C: A was added to B and emulsified. D: C was stirred and cooled, and Components 19 to 29 were added to obtain a sunscreen.

[0057] [Formulation Example 7: Ointment] (Component) (unit: %) 1. Stearyl alcohol 18.0 2. Beeswax 20.0 3. Polyoxyethylene (20) monooleate 0.25 4. Glyceryl monostearate 0.3 5. Vaseline 40.0 6. Purified water balance 7. Glycerin 10.0 8. AA-2G 0.1 9. Neem leaf extract 0.5 10. Polyglutamic acid solution 0.1 11. Buchu extract 0.1 12. Niacinamide 2 13. Tranexamic acid 3.5

[0058] (Manufacturing method) A: 1 to 5 were uniformly mixed at 70°C. B: 6 to 8 were heated to 70°C. C: B was added to A and emulsified. D: C was cooled and 9 to 13 were added to obtain an ointment.

[0059] [Formulation Example 8: Foundation] (Component) (unit: %) 1. Polyoxyethylene methylsiloxane·polyoxypropylene oleylmethylsiloxane·dimethylsiloxane copolymer (Note 11) 2.0 2. PEG-9 Polydimethylsiloxyethyl Dimethicone (Note 12) 1.0 3. Dimethylpolysiloxane 5.0 4. Dimethylpolysiloxane Treated Red Iron Oxide 1.0 5. Dimethylpolysiloxane Treated Yellow Iron Oxide 1.5 6. Dimethylpolysiloxane Treated Black Iron Oxide 0.5 7. Dimethylpolysiloxane Treated Titanium Dioxide (Average Particle Size 400 nm) 10.0 8. Dimethylpolysiloxane Treated Talc (Plate-Like, Average Particle Size 12 μm) 5.0 9. 2-Ethylhexyl p-Methoxycinnamate 5.0 10. Stearyl Glycyrrhetinate 0.5 11. Sorbitan Sesquioleate 0.5 12. Purified Water Balance 13. 1,3-Butylene Glycol 15.0 14. AA-2G 1.0 15. Neem Leaf Extract 0.1 16. Polyglutamic Acid Solution 0.1 17. Buchu Extract 0.1 18. Niacinamide 0.1 19. Gaiyou Extract (0.8 w / v% Diluted Solution with 50 v / v% Ethanol) 0.2 20. Sodium Citrate 0.1 21. Sodium Dihydrogen Phosphate 0.1 22. Phenylbenzimidazole Sulfonic Acid 0.3 23. Triethanolamine 1.7 24. Caprylyl Glycol 0.1 25. Fragrance 0.1 (Note 11) ABIL EM-90 (Manufactured by EVONIK GOLDSCHMIDT GMBH) (Note 12) KF-6028P (Manufactured by Shin-Etsu Chemical Co., Ltd.)

[0060] (Manufacturing Method) A: Components 1 to 3 were uniformly mixed. B: Components 4 to 11 were uniformly dispersed using a roller. C: B was added to A and uniformly mixed. D: Components 12 to 25 were mixed and dissolved. E: D was added to C and emulsified to obtain a foundation.

[0061] [Formulation Example 9: Oil-in-Water Base] (Component) (Unit: %) 1. 1,3-Butylene Glycol 7.0 2. Dipropylene Glycol 5.0 3. Triethanolamine 0.4 4. Purified Water Balance 5. Sodium Lactate 0.04 6. Hydrogenated Lecithin 0.4 7. Stearic Acid 1.0 8. Stearic Acid PEG-55 0.3 9. Sorbitan Sesquisisostearate 0.1 10. Cetyl 2-Ethylhexanoate 2.0 11. Cetostearyl Alcohol 0.3 12. Behenyl Alcohol 0.5 13. Phytosteryl Oleate 0.1 14. Talc 0.4 15. Hydrous Silica 0.1 16. Mica 1.3 17. Titanium Oxide 6.0 18. Iron Oxide 0.1 19. Acrylic Acid / Alkyl Methacrylate Copolymer 0.3 20. Xanthan Gum 0.1 21. Sodium Hydroxide 0.008 22. Glycine 0.01 23. Arginine 0.01 24. Niacinamide 6.0 25. Tranexamic Acid 0.1 26. VC-PMG 2.5 27. Theanine 0.05 28. Quince Seed Extract 0.1 29. Dioscorea pantaiica root extract 0.1 30. Sodium adenosine triphosphate 0.05

[0062] (Manufacturing method) A: Components 1 to 5 were uniformly dissolved and mixed at 70°C. B: Components 6 to 13 were uniformly dissolved and mixed at 80°C. C: B was added to A and emulsified at 70°C. D: After adding and mixing components 14 to 25 to C, it was cooled to 40°C, and then components 26 to 30 were added and mixed to obtain an oil-in-water type base.

Industrial applicability

[0063] According to the present invention, it is possible to provide a novel method for maintaining skin color constancy, an external preparation for maintaining skin color constancy, and a screening method for its active ingredients.

Claims

1. A method for maintaining skin color homeostasis, comprising promoting expression of sFRP1 in skin cells, thereby suppressing melanocyte activity in the skin cells.

2. 2. An external preparation for maintaining skin color homeostasis for use in the method for maintaining skin color homeostasis according to claim 1, characterized in that the external preparation for maintaining skin color homeostasis promotes expression of sFRP1 in skin cells.

3. 3. The skin color homeostasis maintaining external preparation according to claim 2, comprising as an active ingredient at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, Gaiyo extract, and Calamus extract.

4. A method for screening an effective ingredient of the skin color homeostasis maintaining external preparation according to claim 2, characterized in that the effective ingredient is selected using as an indicator the expression level of sFRP1 in cultured skin cells to which a candidate substance has been added.

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