Whitening cosmetic composition that acts via olfactory receptors

Abstract

The purpose of the present invention is to develop a cosmetic based on a new whitening action mechanism. Melanocytes are known to express olfactory receptors, and it was discovered that melanogenesis is suppressed when a scent component capable of stimulating the expressed olfactory receptors is applied to the melanocytes. A whitening cosmetic composition that acts via activation of olfactory receptors is provided according to these findings.

Description

Whitening cosmetic composition acting via olfactory receptors

[0001] The present invention relates to a cosmetic composition for whitening that acts via olfactory receptors expressed in melanocytes. It also relates to a method for screening whitening agents using activation of olfactory receptors expressed in melanocytes as an indicator. It also relates to the use of ingredients selected by such a screening method.

[0002] Olfactory receptors are G protein-coupled proteins expressed primarily in olfactory cells. Humans have 396 functional olfactory receptor genes. While receptors typically function by binding to specific ligands, olfactory receptors can be activated by multiple molecules by binding to specific structures of odor molecules. This allows organisms to detect a variety of odors. It is known that olfactory receptors are not necessarily found exclusively in olfactory cells. Sperm express olfactory receptors called MOR23 (mouse) or hOR17-4 (human), which are thought to be involved in chemotaxis. Furthermore, it has been reported that the olfactory receptor MOR23 in mouse muscle is involved in muscle fiber repair (Non-Patent Document 1: Dev Cell., 17, 649-661, (2009)). The kidney has been reported to express multiple olfactory receptors and to respond to various stimuli (Non-Patent Document 2: Proc Natl Acad Sci USA., 10, 2059-2064, (2009); Non-Patent Document 3: Proc Natl Acad Sci USA., 110, 4410-4415, (2013)). In addition, it has been reported that a receptor called OR2AT4 expressed by keratinocytes in the skin is involved in wound healing (Non-Patent Document 4: J Invest Dermatol., 134, 2823-2832, (2014)).

[0003] Meanwhile, nerol is a monoterpene found in the essential oils of lemongrass and hops. It has a rose-like scent and is used as a fragrance ingredient. Nerol has been shown to have tyrosinase inhibitory activity, and its inclusion in topical skin preparations is expected to have a whitening effect (Patent Document 1: Japanese Patent No. 4148862).

[0004] Patent No. 4148862

[0005] Dev Cell., 17, 649-661, (2009)Proc Natl Acad Sci USA., 10, 2059-2064, (2009)Proc Natl Acad Sci USA., 110, 4410-4415, (2013)J Invest Dermatol., 134, 2823-2832, (2014)

[0006] The aim is to develop cosmetics based on a new whitening mechanism.

[0007] While conducting research on melanocytes for skin whitening research, the present inventors discovered that melanocytes express olfactory receptors. Based on this finding, they applied fragrance components capable of activating the expressed olfactory receptors to melanocytes, and surprisingly found that the expression of tyrosinase, which is involved in melanin production, was reduced, thereby suppressing melanin production, leading to the present invention. The present invention therefore relates to the following: [1-1] A cosmetic composition for skin whitening containing nerol or (-)-carvone. [1-2] A method for perfume application using nerol or (-)-carvone, which induces skin whitening. [1-3] A method for skin whitening using nerol or (-)-carvone, which induces skin whitening via the fragrance. [1-4] Use of nerol or (-)-carvone for producing a cosmetic composition for skin whitening. [2] The cosmetic composition, perfume application method, skin whitening method, or use according to any one of Items 1-1 to 1-4, comprising 0.000075 to 0.015% nerol or (-)-carvone. [3] The cosmetic composition, perfume application method, skin whitening method, or use according to Items 1-1 to 1-4 or 2, further comprising adding a cosmetic excipient. [4] A cosmetic composition for skin whitening, comprising a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes. [4-2] A perfume application method using a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, which induces skin whitening. [4-3] A skin whitening method using a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, which induces skin whitening via perfume application. [4-4] Use of a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes in a cosmetic composition for whitening. [5] A cosmetic composition for inhibiting tyrosinase expression, comprising nerol or (-)-carvone. [5-2] A method for perfume application using a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, wherein tyrosinase expression is inhibited. [5-3] A method for whitening using a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, wherein tyrosinase expression is inhibited via perfume application.[5-4] Use of a ligand for olfactory receptor OR2W3 or OR2L13 expressed in melanocytes in a cosmetic composition for inhibiting tyrosinase expression. [6] The cosmetic composition, perfuming method, whitening method, or use according to any one of Items 5-1 to 5-4, comprising 0.000075 to 0.015% of nerol or (-)-carvone. [7] The cosmetic composition, perfuming method, whitening method, or use according to Items 5-1 to 5-4 or 6, further comprising a cosmetic excipient. [8] A whitening composition mediated by activation of olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, comprising a ligand for the olfactory receptor. [9] The whitening composition according to Item 8, wherein the ligand is nerol or (-)-carvone.

[10] A whitening composition comprising (-)-carvone.

[11] A method for screening for skin whitening agents using activation of olfactory receptor OR2W3 or OR2L13 expressed in melanocytes as an index.

[12] The method according to item 11, comprising: a step of allowing a candidate drug to act on olfactory receptor-expressing cells; a step of measuring the activation of olfactory receptors in the olfactory receptor-expressing cells; and a step of selecting a candidate drug that activates the olfactory receptor as a skin whitening agent.

[13] The method according to item 12, wherein the olfactory receptor-expressing cells are selected from the group consisting of melanocytes.

[14] A method for determining sensitivity to skin whitening treatment using expression of olfactory receptors in melanocytes as an index.

[15] The method according to item 14, wherein the sensitivity to skin whitening treatment is sensitivity to a ligand for the olfactory receptor.

[16] The method according to item 15, wherein the ligand is nerol or (-)-carvone.

[0008] It is possible to suppress melanin production through activation of olfactory receptors expressed in melanocytes.

[0009] Figure 1 shows that the olfactory receptors OR2L13 and OR2W3 are expressed specifically in melanocytes. Figure 2 shows that nerol, a ligand for the olfactory receptor OR2W3, suppresses tyrosinase expression in melanocytes without affecting the expression of MITF, TRP1, or TRP2 (Figure 2A). It also shows that the addition of nerol reduced melanin content in melanocyte cell cultures (Figure 2B). Figure 3 shows that (-)-carvone, a ligand for the olfactory receptor OR2L13, suppresses tyrosinase expression in melanocytes without affecting the expression of MITF, TRP1, or TRP2.

[0010] The present invention relates to a cosmetic composition for whitening, and also to a method for screening whitening agents using activation of olfactory receptors expressed in melanocytes as an indicator.

[0011] Olfactory receptors expressed in melanocytes include OR2W3 and OR2L13. These olfactory receptors have been found to be expressed in melanocytes (Figure 1). Furthermore, activation of these olfactory receptors in cultured melanocytes has been found to suppress tyrosinase expression (Figures 2 and 3). Tyrosinase is an enzyme that catalyzes the biosynthesis of dopaquinone from tyrosine and is a target enzyme in the melanin biosynthetic pathway. It is known that inhibiting tyrosinase can suppress melanin production and further exert a whitening effect. Therefore, any component that can activate OR2W3 or OR2L13 can act on melanocytes and exert a whitening effect. Such components that can activate OR2W3 or OR2L13 may be components already identified in the technical field or may be unidentified components. For example, a cosmetic composition for whitening may contain a ligand for OR2W3 or OR2L13.

[0012] As a component that activates the olfactory receptor OR2W3, a compound having the following structural formula: An example of such an ingredient is nerol, which has the formula: NEROL. Nerol is a monoterpene found in lemongrass and hops, has a rose-like scent, and is used as a fragrance ingredient. In the cosmetic composition of the present invention, nerol can typically be blended at 0.000075 to 0.015%. From the viewpoint of achieving a physiological effect on the skin via olfactory receptors, nerol is preferably blended at 0.00075% or more, more preferably at 0.0075% or more, and even more preferably at 0.01% or more. In terms of fragrance strength, nerol is preferably blended at 0.15% or less, more preferably at 0.00075% or more, and even more preferably at 0.0075% or more.

[0013] In the examples of the present specification, it was shown that the addition of nerol reduced the expression level of tyrosinase in melanocytes (Figure 2A) and further reduced the amount of melanin production (Figure 2B). On the other hand, nerol does not affect the expression of MITF, a regulatory factor that controls the expression of tyrosinase, or TRP1 and TRP2, which are involved in melanin synthesis, indicating that it suppresses tyrosinase expression in an MITF-independent manner, and that the reduction in tyrosinase expression is involved in the suppression of melanin production (Figure 2A). Therefore, nerol can be said to be a tyrosinase inhibitor, more specifically, an MITF-independent tyrosinase inhibitor. Because MITF is also involved in the onset and pathology of diseases such as melanoma, MITF-independent action is particularly important in developing a skin whitening agent. Prior art has shown that nerol inhibits melanin production in cultured melanocytes at 0.05% V / V. Based on these experimental results, it has been described that nerol is incorporated into topical skin preparations at a concentration of 0.0001 to 0.5% by weight (Patent Document 1). The melanin production inhibitory effect of nerol in Patent Document 1 is based on its inhibitory effect on tyrosinase activity, and its inhibitory effect on apple surface browning has also been investigated. In contrast, the present invention focuses on its action via olfactory receptors, thereby suppressing tyrosinase expression itself. Inhibition of melanin production by suppressing tyrosinase expression can have a sustained effect, unlike transient inhibition of tyrosinase activity. Furthermore, to inhibit tyrosinase activity, nerol must act on a significant proportion of tyrosinase present in cells, requiring a high concentration, whereas its action via olfactory receptors present on the cell surface can be effective at much lower concentrations. The present invention is characterized by being incorporated into a cosmetic composition for whitening, and is not intended to inhibit tyrosinase activity by direct application to the skin, but rather to have the volatilized nerol inhibit tyrosinase expression via olfactory receptors.

[0014] As a component that activates the olfactory receptor OR2L13, a compound having the following structural formula: An example of a suitable olfactory active ingredient is (-)-carvone, which has the formula (I). (-)-Carvone is a monoterpene found in caraway and spearmint, and is used as a fragrance due to its aromatic properties. In the examples, it was shown that the addition of (-)-carvone reduces the expression level of tyrosinase in melanocytes. In the cosmetic composition of the present invention, (-)-carvone can typically be incorporated at 0.000075 to 0.015%. From the perspective of achieving physiological effects on the skin via olfactory receptors, (-)-carvone is preferably incorporated at 0.00075% or more, more preferably at 0.0075% or more, and even more preferably at 0.01% or more. In terms of fragrance strength, (-)-carvone is preferably incorporated at 0.15% or less, more preferably at 0.00075% or more, and even more preferably at 0.0075% or more.

[0015] Ligands for olfactory receptors expressed in melanocytes, including nerol and (-)-carvone, can act as gases on melanocytes through activation of olfactory receptors. From the perspective of acting as gases on olfactory receptors, the cosmetic composition can be set to a preferred concentration to be achieved in the air, and as an example, it is preferable to achieve a concentration of 7.5 ppm to 150 ppm in the air, particularly in the air surrounding the subject.

[0016] The cosmetic composition of the present invention refers to a composition intended for fragrance application. Accordingly, a whitening cosmetic composition refers to a composition that exerts a whitening effect through activation of olfactory receptors by volatile components. Examples of cosmetic compositions include perfumes, antiperspirant sprays, hair care products (shampoos, rinses, treatments, hair styling products), soaps, body washes, detergents (laundry detergents, fabric softeners, finishing agents), air sprays, fabric sprays, deodorizers and air fresheners for homes, cars, toilets, and baths, incense, room fragrances, aroma diffusers, and the like. Laundry detergents, fabric softeners, and fabric sprays have the advantage of imparting fragrance directly to clothing and allowing it to act at close range to the skin for an extended period of time. Ingredients contained in cosmetic compositions, such as nerol or (-)-carvone, can exert a whitening effect by acting on olfactory receptors expressed in melanocytes. These ingredients may be formulated alone or in combination with other active ingredients, or may be combined with ingredients that act via olfactory receptors. The cosmetic composition may be in the form of a water-soluble or oil-soluble liquid, a paste or gel, a powder, a capsule, or a tablet. Various dosage forms can be produced by known methods. In particular, a volatile component can be encapsulated in a polymer resin or penetrated into the pores of a carrier such as silica, enabling a long-lasting fragrance.

[0017] In addition to fragrance components, cosmetic compositions may contain other active ingredients and may further contain various additives. The active ingredient may be a fragrance component that exerts a physiological effect, or may be an antibacterial agent, etc. Examples of additives include water; organic solvents such as alcohol, glycerin, and propylene glycol; excipients; oils and fats; emulsifiers; thickeners; preservatives; and colorants. These additives can be appropriately selected from those commonly used in cosmetic compositions. The cosmetic composition preferably contains, as an excipient, dextrin, etc., which is suitable for formulating monoterpenes such as nerol or (-)-carvone into cosmetics. The inclusion of dextrin provides sustained release, thereby extending the duration of action.

[0018] Cosmetic compositions typically act via the sense of smell through the fragrance components contained in the composition, and can produce various physiological effects such as allure, relaxation, improved concentration, and refreshment. However, the present invention provides a novel mechanism by which a cosmetic composition induces physiological effects by directly acting on human cells, rather than through the sense of smell. Although melanocytes in the epidermis, in particular, are separated from the outside world by the stratum corneum, it is known that skin gases and the like diffuse through the stratum corneum. Highly volatile, lipid-soluble, low-molecular-weight fragrance components can pass through the stratum corneum and act on epidermal melanocytes. While cosmetic compositions, such as perfumes, are sometimes applied to the skin, they can be clearly distinguished from topical skin compositions in that they act on melanocytes via the gases of their volatile components.

[0019] Because nerol or (-)-carvone has the effect of reducing the expression level of tyrosinase, the present invention may also relate to a tyrosinase expression inhibitor containing nerol or (-)-carvone. Furthermore, the cosmetic composition of the present invention may also relate to a cosmetic composition for inhibiting tyrosinase expression. In the present invention, the cosmetic composition may contain synthetic nerol or (-)-carvone, may contain it as a natural purified product purified from a plant, or may contain it as part of an extract extracted from a plant. For example, because nerol is found in lemongrass and hops, the cosmetic composition of the present invention may contain extracts of these. Furthermore, because (-)-carvone is found in caraway, spearmint, rosemary, and thyme, the cosmetic composition of the present invention may contain extracts of these.

[0020] In another aspect, the present invention may relate to a skin whitening composition containing (-)-carvone. Such a skin whitening composition exhibits a skin whitening effect when applied. The skin whitening composition may be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, or may be incorporated into foods, such as nutritional supplements. The skin whitening composition may be administered orally or parenterally, for example, transdermally. When administered transdermally, it can be formulated into a topical skin preparation. The topical skin preparation is not particularly limited as long as it is applicable to the skin, and any formulation may be used, for example, a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a topical skin preparation, bases and excipients commonly used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used. When incorporated into cosmetics, the compounds can be incorporated into facial or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. The use of cosmetics, pharmaceuticals, and quasi-drugs containing the components of the present invention can exert a whitening effect. More preferably, the whitening effect may be exerted via activation of olfactory receptors. In the present invention, (-)-carvone can also be used as a melanin formation inhibitor and a tyrosinase expression inhibitor.

[0021] The concentrations of the whitening composition, melanogenesis inhibitor, and tyrosinase expression inhibitor of the present invention can be selected as desired to achieve the desired effect. When formulated as a topical skin preparation, (-)-carvone can be formulated at 0.000075 to 0.015%. To fully achieve the desired effect, it can be formulated at preferably 0.00075% or more, and more preferably 0.0075% or more. To avoid a strong odor, it can be formulated at preferably 0.15% or less, and more preferably 0.075% or less.

[0022] Yet another aspect of the present invention relates to a method for screening for skin whitening agents using olfactory receptor activation as an index. More specifically, the method includes: a step of applying a candidate component to the olfactory receptor-expressing cells; a step of measuring the activation of the olfactory receptor in the olfactory receptor-expressing cells; and a step of selecting a candidate component that activates the olfactory receptor as a skin whitening agent. Examples of olfactory receptors include OR2W3 and OR2L13. Cells expressing OR2W3 or OR2L13 may be melanocytes, cells known to express such receptors, or cells in which expression has been induced by gene transfer. The olfactory receptor-expressing cells may be cultured cells, organ-cultured tissues, or three-dimensional cultures. Since olfactory receptors are G protein-coupled receptors, activated olfactory receptors activate the G protein GOLF, which activates adenylate cyclase and increases cAMP. Furthermore, the action of cAMP opens ion channels, allowing sodium and potassium ions to flow in. As an example, calcium imaging can be used to measure calcium influx when a candidate component is applied, thereby determining the activation of olfactory receptors.

[0023] The whitening agents screened in this way can inhibit tyrosinase expression and thus suppress melanin production through activation of olfactory receptors expressed in melanocytes. Therefore, the whitening agents screened by the screening method of the present invention can also be called tyrosinase expression inhibitors or melanin production inhibitors, and can exert their effects via activation of olfactory receptors.

[0024] The candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0025] A further aspect of the present invention relates to a method for determining susceptibility to whitening treatment using the expression of olfactory receptors in melanocytes as an indicator. This method includes a step of measuring the expression level of olfactory receptors in melanocytes of a skin sample obtained from a subject. Subjects who express high levels of olfactory receptors can be determined to be highly susceptible to the application of an olfactory receptor ligand. The olfactory receptor ligand is nerol or (-)-carvone. For subjects who are highly susceptible to whitening treatment with the application of an olfactory receptor ligand, the use of a fragrance or cosmetic product containing nerol or (-)-carvone can be suggested.

[0026] All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention.

[0027] Example 1: Presence of olfactory receptors in melanocytes Normal human epidermal melanocytes were seeded and cultured until subconfluent. The cells were harvested and subjected to Western blotting using anti-OR2L13 antibody (Thermo Fisher Scientific) and anti-OR2W3 antibody (Thermo Fisher Scientific) to measure the presence or absence of these proteins.

[0028] Example 2: Effect of Nerol on melanocytes. Melanocytes were cultured at 5.0 x 10 4 The cells were seeded in a 6-well plate at 1 mL / well and incubated in DermaLife BM medium at 37°C in 5% CO ２ Nerol (Tokyo Chemical Industry Co., Ltd.) was added at concentrations of 0 μM, 50 μM, 250 μM, 500 μM, and 1000 μM, and the cells were cultured in DermaLife BM medium at 37°C and 5% CO for 48 hours. ２The cells were cultured for 24 hours under ambient conditions. After culture, the cells were harvested, washed with PBS, and separated by SDS-PAGE. The gel was transferred to a PVDF membrane. Western blotting was performed using anti-tyrosinase antibody (Cell Signaling Technology), anti-MITF antibody (Cell Signaling Technology), anti-TRP1 antibody (Abcam), anti-TRP2 antibody (Abcam), and anti-β-actin antibody (Santa Cruz) as primary antibodies. AP-labeled antibody (Thermo Fisher Scientific) was used as the secondary antibody. The results are shown in Figure 2.

[0029] Example 3: Effect of (-)-carvone on melanocytes. Melanocytes were cultured at 5.0 x 10 4 The cells were seeded in a 6-well plate at 1 mL / well and incubated in DermaLife BM medium at 37°C in 5% CO ２ (-)-Carvone (Tokyo Chemical Industry Co., Ltd.) was added at concentrations of 0 μM, 50 μM, 250 μM, 500 μM, and 1000 μM, and the cells were cultured in DermaLife BM medium at 37°C and 5% CO for 48 hours. ２ The cells were cultured for 24 hours under ambient conditions. After incubation, the cells were harvested, washed with PBS, and separated by SDS-PAGE. The gel was transferred to a PVDF membrane. Western blotting was performed using anti-tyrosinase antibody (Cell Signaling Technology), anti-MITF antibody (Cell Signaling Technology), anti-TRP1 antibody (Abcam), anti-TRP2 antibody (Abcam), and anti-β-actin antibody (Santa Cruz) as primary antibodies. AP-labeled antibody (Thermo Fisher Scientific) was used as the secondary antibody. The results are shown in Figure 3.

Claims

1. A cosmetic composition for whitening containing nerol or (-)-carvone.

2. The cosmetic composition according to claim 1, comprising 0.000075 to 0.015% of nerol or (-)-carvone.

3. The cosmetic composition of claim 1, further comprising a cosmetic excipient.

4. A cosmetic composition for whitening, comprising a ligand for the olfactory receptor OR2W3 or OR2L13 expressed in melanocytes.

5. A cosmetic composition for inhibiting tyrosinase expression, comprising nerol or (-)-carvone.

6. The cosmetic composition according to claim 5, comprising 0.000075 to 0.015% of nerol or (-)-carvone.

7. The cosmetic composition of claim 5, further comprising a cosmetic excipient.

8. A composition for whitening that activates the olfactory receptor OR2W3 or OR2L13 expressed in melanocytes, the composition comprising a ligand for the olfactory receptor.

9. The whitening composition according to claim 8, wherein the ligand is nerol or (-)-carvone.

10. A skin whitening composition containing (-) carvone.

11. A method for screening skin whitening agents using activation of olfactory receptors OR2W3 or OR2L13 expressed in melanocytes as an indicator.

12. The method according to claim 11, comprising the steps of: applying a candidate drug to an olfactory receptor-expressing cell; measuring activation of the olfactory receptor in the olfactory receptor-expressing cell; and selecting a candidate drug that brings about activation of the olfactory receptor as a whitening agent.

13. The method of claim 12, wherein the olfactory receptor expressing cells are selected from the group consisting of melanocytes.

14. A method for determining sensitivity to skin whitening treatment using the expression of olfactory receptors in melanocytes as an indicator.

15. The method according to claim 14, wherein the sensitivity to whitening treatment is sensitivity to a ligand for an olfactory receptor.

16. The method of claim 15, wherein the ligand is nerol or (-)-carvone.

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