Composition for promoting melanin synthesis and composition for inhibiting tyrosinase protein degradation

Abstract

To provide compositions capable of promoting melanin synthesis enzyme by suppressing degradation of tyrosinase protein, a melanin producing enzyme.SOLUTION: The invention provides a composition for promoting melanin synthesis or a composition for suppressing tyrosinase protein degradation comprising a triterpene as the effective ingredient, where, in particular, the triterpene is pentacyclic triterpene, further the pentacyclic triterpene is oleanolic acid or a derivative thereof.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a melanin synthesis promoting composition that promotes melanin synthesis, and a tyrosinase protein degradation inhibiting composition that inhibits the degradation of tyrosinase protein. [Background technology]

[0002] Melanin is a pigment produced by melanocytes in the skin and hair bulbs, and determines the color of skin and hair.

[0003] Melanin is known to be a cause of age spots and sunburn, and when produced in excess in the skin, it can cause pigmentation such as age spots and freckles.

[0004] Therefore, in the past, active research and development has been conducted into compositions that inhibit melanin synthesis in order to prevent and treat pigmentation such as age spots and freckles (see, for example, Patent Document 1).

[0005] On the other hand, it is known that when melanin is not produced properly in the skin or hair bulbs due to a decrease or absence of melanin synthesis ability in melanocytes, it can cause vitiligo and gray hair.

[0006] However, the main treatments for vitiligo are steroids, immunosuppressants, and surgical ultraviolet irradiation therapy, and the establishment of a simpler treatment method for vitiligo is desired.

[0007] Furthermore, the main method for treating gray hair is to dye it, and there is a need to establish an effective method for preventing gray hair and restoring color. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-97945 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the present inventors have conducted extensive research aimed at preventing and treating gray hair and vitiligo, and as a result have arrived at the present invention, which relates to a composition that promotes melanin synthesis. [Means for solving the problem]

[0010] A first aspect of the present disclosure is a melanin synthesis promoting composition containing a triterpene as an active ingredient, which promotes melanin synthesis.A second aspect of the present disclosure is a tyrosinase proteolysis inhibiting composition, which inhibits the degradation of tyrosinase protein.

[0011] The melanin synthesis promoting composition is characterized in that the triterpene is a pentacyclic triterpene, and further characterized in that the pentacyclic triterpene is oleanolic acid or a derivative thereof. The tyrosinase protein degradation inhibiting composition is characterized in that the triterpene is a pentacyclic triterpene, and further characterized in that the pentacyclic triterpene is oleanolic acid or a derivative thereof.

[0012] A third aspect of the present disclosure is a topical preparation containing the melanin synthesis-promoting composition, more specifically a topical preparation for gray hair or a topical preparation for vitiligo.A fourth aspect of the present disclosure is a topical preparation containing the tyrosinase proteolysis-inhibiting composition, more specifically a topical preparation for gray hair or a topical preparation for vitiligo. [Effects of the Invention]

[0013] According to the present disclosure, the degradation of tyrosinase protein can be inhibited to promote melanin synthesis, and this can be applied to the prevention and treatment of symptoms caused by a decrease or lack of melanin synthesis ability, such as gray hair and vitiligo. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1 is a graph showing the experimental results of melanin production, which will be explained in the Examples section. [Figure 2] FIG. 2 is a Western blot showing the results of a protein expression experiment described in the Examples section. [Figure 3] FIG. 3 is a graph showing the experimental results of tyrosinase gene expression ability, which will be explained in the Examples section. [Figure 4] FIG. 4 is a Western blot showing the results of the protein degradation experiments described in the Examples section. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail. The following embodiments do not limit the content of the present invention described in the claims, but are examples to facilitate understanding of the present invention. Furthermore, not all of the components constituting the embodiments are necessarily essential as means for solving the problems of the present invention. An embodiment in which some of the components constituting the embodiments are omitted can also be a means for solving the problems of the present invention.

[0016] The terms used in the following description are for the purpose of embodiment only and are not intended to limit the scope of the present disclosure, i.e., as set forth in the claims, specification, and drawings. Elements described in the specification and claims are intended to include the plural unless the context clearly dictates otherwise.

[0017] The terms "and" and "or" can include any and all combinations of one or more of the listed elements before and after it. By way of example, "A or B" can mean "A, B, or both A and B." "A," "B," and "both A and B" all each satisfy "A or B."

[0018] The terms "includes," "including," "having," and / or "comprising" used in this disclosure mean that features, operations, elements, or steps are present, but do not exclude the presence or addition of one or more other features, operations, elements, steps, and / or groups thereof not listed in the specification and claims.

[0019] All embodiments and optional embodiments included in this disclosure may be combined with each other to form new embodiments. In this case, the new embodiments may include those formed by combining embodiments omitting some of the components of the embodiments exemplified in this disclosure. Furthermore, all technical features and optional technical features included in this disclosure may be combined with each other to form new technical features.

[0020] The present invention relates to a composition that prevents or improves gray hair or vitiligo (hereinafter sometimes simply referred to as "gray hair, etc.") and a topical preparation containing the composition. In particular, the present invention relates to a composition that can be used to prevent or treat symptoms such as gray hair caused by a decrease or lack of melanin synthesis ability, and a topical preparation containing the composition.

[0021] In the present disclosure, "prevention" refers to the action of preventing hair or skin whitening, and a "preventive agent" refers to a substance that has this action on hair or skin. "Treatment" refers to the action of reducing hair or skin whitening or inhibiting the progression of whitening, and an "improvement agent" refers to a substance that has this action on hair or skin. "Preventive agent" and "improvement agent" are terms used for convenience to facilitate understanding of the present invention and may be expressed alternatively by other terms (e.g., cosmetics). The term "topical agent" used in the present disclosure is a term that encompasses the "preventive agent" and "improvement agent" described herein. Furthermore, "topical agent" includes topical agents according to the intended use, such as the topical agent for hair and topical agent for gray hair according to the present invention.

[0022] In accordance with an embodiment of the present invention, a preventive or ameliorating agent for gray hair, etc., contains a triterpene as an active ingredient from the viewpoint of promoting melanin synthesis and inhibiting tyrosinase proteolysis. In particular, the triterpene is preferably a pentacyclic triterpene, and among pentacyclic triterpenes, oleanolic acid or a derivative thereof is particularly preferred. Oleanolic acid, which belongs to the triterpenes containing pentacyclic triterpenes, is inexpensive, is effective at relatively low concentrations (several tens of μM) in cultured cell experiments, shows no cytotoxicity at these concentrations, has been confirmed to be safe in animals, and has advantages such as being contained in natural products used as herbal medicines and for food. In particular, oleanolic acid is a type of pentacyclic triterpene compound produced by plants and can be extracted from natural products (plant roots and peels).

[0023] The concentration of triterpene (pentacyclic triterpene or a derivative thereof, oleanolic acid) in the agent for preventing or improving gray hair and the like is not particularly limited, but from the viewpoint of preventing or improving gray hair and the like, for example, 0.011 to 0.023% by mass is preferred.

[0024] Examples of agents for preventing or improving gray hair include soaps (solid soaps, liquid soaps), ointments, liniments, lotions, and packs. Agents for preventing or improving gray hair may also include cosmetics. Cosmetics may include, but are not limited to, hair cosmetics (shampoos, rinses, treatments, hair dyes such as gray hair dyes and hair colors, hair growth agents, hair care products), skin care products (lotions, creams, emulsions, serums, etc.), makeup cosmetics (base emulsions, liquid foundations, etc.), and sunscreen cosmetics. Cosmetics may include pharmaceuticals, quasi-drugs, and cosmetics. Examples of quasi-drugs include, but are not limited to, medicated soaps, medicated shampoos, medicated rinses, medicated cosmetics, medicated lotions, medicated packs, medicated gray hair dyes, medicated hair colors, medicated color treatments, and medicated hair growth agents.

[0025] The dosage form of the agent for preventing or improving gray hair etc. may include, but is not limited to, liquid, emulsion, cream, gel, paste, mousse, sheet, and ointment.

[0026] The agent for preventing or improving gray hair, etc. may contain triterpenes and other ingredients. The ingredients other than triterpenes can be selected depending on the intended use and function, and specifically, additives such as carriers such as water, alcohol, and oils and fats, excipients, emulsifiers, surfactants, buffers, diluents, solubilizers, preservatives, stabilizers, antioxidants, gelling agents, powders, water-soluble polymers, UV protection agents, inclusion compounds, fragrances, salts, pH adjusters, animal-derived extracts, microbial-derived extracts, plant-derived extracts, astringents, blood circulation promoters, whitening agents, moisturizers, anti-inflammatory agents, active oxygen scavengers, cell activators, chelating agents, keratolytic agents, enzymes, hormones, and vitamins can be appropriately blended.

[0027] Examples of methods for producing an agent for preventing or improving gray hair and the like include, but are not limited to, a method in which triterpenes and other ingredients depending on the intended use and function are added to water and mixed. [Example]

[0028] Examples are described below. The examples described below are merely illustrative and are intended to aid in understanding the present application and are not intended to limit the present application. References to "Examples" in this disclosure mean that specific features, compositions, properties, etc. described in connection with an Example may be included in at least one Example in this disclosure. Unless specific techniques or conditions are specified in the Examples, the techniques or conditions are carried out in accordance with the techniques or conditions described in the relevant technical literature or product instructions. Materials, reagents, or equipment used without a specified manufacturer are all common, commercially available products. Furthermore, ingredients derived from natural products are commercially available products or extracted from natural products. Unless otherwise specified, the content of each ingredient is based on mass.

[0029] The following experiment was carried out using oleanolic acid, which belongs to the triterpene group containing five ring-containing triterpenes.

[0030] <Measurement of melanin production>

[0031] First, we measured melanin production using oleanolic acid. For comparison, we also used a control (DMSO: dimethyl sulfoxide), 2-BP (2-bromopalmitate), which inhibits protein palmitoylation (palmitic acid modification), and α-melanocyte-stimulating hormone (α-MSH) at concentrations of 50 μM oleanolic acid, 5 μM 2-BP, and 1 μM α-MSH.

[0032] Here, B16 melanoma cells (a mouse melanoma cell line) were cultured in the presence of the compounds for 24 to 48 hours, and the amount of melanin produced was measured.

[0033] The results are shown in Figure 1. In Figure 1, the amount of melanin produced in the control is set at 100%, and the values ​​are shown as a percentage relative to this.

[0034] As shown in Figure 1, oleanolic acid produced 124% more melanin after 24 hours of culture and 193% more after 48 hours of culture compared to the control.

[0035] In contrast, the effects of 2-BP and α-MSH were 110% and 148% after 24 hours of culture, and 122% and 200% after 48 hours of culture, respectively, compared to the control.

[0036] Therefore, it can be seen that oleanolic acid exhibits the same amount of melanin production as α-MSH, which is known as melanocyte-stimulating hormone, and has the same melanin production-promoting effect.

[0037] Furthermore, oleanolic acid produces more melanin than 2-BP, which is known as a compound that inhibits the degradation of tyrosinase, and is therefore found to have a strong melanin production promoting effect.

[0038] <Measurement of protein expression ability>

[0039] Next, we measured the protein expression potential of oleanolic acid. Similar to the measurement of melanin production, we used oleanolic acid, DMSO, 2-BP, and α-MSH as controls for comparison. Here, oleanolic acid was used at 50 μM, 2-BP at 5 μM, and α-MSH at 1 μM.

[0040] Here, B16 melanoma cells were cultured in the presence of the compounds for 24 to 48 hours, and the protein expression levels of tyrosinase, a melanin-producing enzyme, and Tyrp-1 (Tyrosinase-related protein 1), one of the enzymes involved in melanin synthesis, were measured using Western blotting.

[0041] The results are shown in Figure 2. In Figure 2, β-actin, a cytoskeletal protein, was used as an internal standard.

[0042] As shown in FIG. 2, oleanolic acid promotes the protein expression of tyrosinase more than 2-BP, and it is clear that oleanolic acid has a stronger effect of promoting the expression of tyrosinase protein than 2-BP.

[0043] Furthermore, oleanolic acid has the same effect as α-MSH in promoting tyrosinase protein expression, but unlike α-MSH, oleanolic acid does not promote Tyrp-1 protein expression, whereas α-MSH does.

[0044] Therefore, it is clear that oleanolic acid specifically promotes the protein expression of tyrosinase.

[0045] <Measurement of melanin-producing enzyme tyrosinase gene expression>

[0046] To clarify the difference between oleanolic acid and α-MSH, we measured the gene expression of the melanin-producing enzyme tyrosinase. For comparison, we used oleanolic acid, DMSO, and α-MSH as controls. The concentrations of oleanolic acid and α-MSH were 50 μM and 1 μM, respectively.

[0047] Here, B16 melanoma cells were cultured in the presence of the compounds for 24 to 48 hours, and the mRNA expression levels of tyrosinase and Tyrp-1 were measured by RT-PCR (Real Time-Polymerase chain reaction).

[0048] The results are shown in Figure 3. In Figure 3, β-actin, a cytoskeletal protein, was used as an internal standard. The expression level of the control tyrosinase gene was set at 100%, and the results are shown as a percentage.

[0049] As shown in FIG. 3, it can be seen that α-MSH promotes the expression of the tyrosinase gene, whereas oleanolic acid does not promote the expression of the tyrosinase gene.

[0050] That is, as shown in FIG. 2, oleanolic acid has the same effect of promoting tyrosinase protein expression as α-MSH, but its mechanism is different from that of α-MSH.

[0051] <Protein degradation measurement>

[0052] Next, to clarify the mechanism of the melanin production-promoting effect of oleanolic acid, we measured its proteolytic activity. For comparison, we used oleanolic acid, DMSO, and 2-BP as controls. The concentrations of oleanolic acid and 2-BP were 50 μM and 5 μM, respectively.

[0053] Here, B16 melanoma cells were cultured in the presence of the compounds for 24 hours, and then further cultured in the presence of the protein synthesis inhibitor cycloheximide (CHX) for 4 to 8 hours, and the amount of proteolysis of tyrosinase and Tyrp-1 was measured by Western blotting.

[0054] The results are shown in Figure 4. In Figure 4, β-actin was used as an internal standard.

[0055] As shown in Figure 4, in the control (DMSO), degradation of tyrosinase protein and Tyrp-1 protein was confirmed in the presence of the protein synthesis inhibitor cycloheximide CHX, whereas oleanolic acid and 2-BP inhibited the degradation of tyrosinase protein but did not inhibit the degradation of Tyrp-1 protein.

[0056] Furthermore, it was found that oleanolic acid inhibited the degradation of tyrosinase protein more strongly than 2-BP.

[0057] Therefore, it is clear that oleanolic acid specifically inhibits the degradation of tyrosinase protein, a melanin-producing enzyme.

[0058] <Consideration>

[0059] From the above, it can be seen that oleanolic acid has the same melanin production-promoting effect as α-MSH, which is known as a melanocyte-stimulating hormone, and has the same tyrosinase protein expression-promoting effect as α-MSH. However, unlike α-MSH, oleanolic acid does not promote the expression of Tyrp-1 protein or the tyrosinase gene.

[0060] Furthermore, it was found that oleanolic acid has a stronger melanin production promoting effect than 2-BP, which is known as a compound that inhibits tyrosinase degradation, and has a stronger effect of promoting tyrosinase protein expression than 2-BP. Furthermore, it was found that oleanolic acid inhibits tyrosinase protein degradation more strongly than 2-BP, but does not inhibit Tyrp-1 protein degradation as 2-BP does.

[0061] Oleanolic acid has the specificity of inhibiting the degradation of tyrosinase protein rather than promoting the expression of the tyrosinase gene, which increases the expression level of tyrosinase and promotes melanin production.

[0062] Furthermore, since oleanolic acid has a different mechanism of action from existing α-MSH and the like, synergistic effects can be expected when these are used in combination.

[0063] In the above explanation, experimental data on oleanolic acid was presented, but this is not limited to oleanolic acid; similar actions and effects are also observed for derivatives of oleanolic acid and triterpenes, including the pentacyclic triterpene to which oleanolic acid belongs.

[0064] As explained above, compositions containing triterpenes, including oleanolic acid and its derivatives, including pentacyclic triterpenes, as active ingredients can inhibit the degradation of tyrosinase protein and promote melanin synthesis, and are useful as melanin synthesis-promoting compositions or tyrosinase protein degradation-inhibiting compounds, and can be applied to the prevention and treatment of symptoms caused by a decrease or lack of melanin synthesis ability, such as gray hair and vitiligo. Specifically, they can be used as topical preparations for various purposes, such as topical preparations for gray hair and vitiligo, which contain these as ingredients.

[0065] Furthermore, triterpenes, including oleanolic acid and its derivatives, including pentacyclic triterpenes, may be natural products with high content ratios, and these can provide highly safe compositions for promoting melanin synthesis and compositions for inhibiting tyrosinase proteolysis. Specifically, these can be used as topical agents for various purposes, such as topical agents for gray hair and topical agents for vitiligo, containing these as ingredients.

Claims

1. A melanin synthesis promoting composition characterized by having oleanolic acid or a derivative thereof as an active ingredient and promoting melanin synthesis.

2. A composition for inhibiting the degradation of tyrosinase protein, which contains oleanolic acid or a derivative thereof as an active ingredient and inhibits the degradation of tyrosinase protein.

3. An external preparation for gray hair, comprising the melanin synthesis promoting composition according to claim 1.

4. An external preparation for vitiligo containing the melanin synthesis promoting composition according to claim 1.

5. An external preparation for gray hair, comprising the composition for inhibiting tyrosinase proteolysis according to claim 2.

6. An external preparation for vitiligo containing the tyrosinase proteolysis inhibitor composition according to claim 2.

7. An external preparation for gray hair according to claim 3 or claim 5, which is either a soap, an ointment, a liniment, a lotion, a pack, or a cosmetic.

8. An external preparation for vitiligo as described in claim 4 or claim 6, which is either a soap, ointment, liniment, lotion, pack, or cosmetic.

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