Melanin phagocytosis inhibitors

Abstract

The present invention provides a melanin phagocytosis inhibitor and a topical skin whitening agent that can produce a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation. [Solution] A melanin phagocytosis inhibitor containing a plant extract of the Commelinaceae family, for suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

Description

Technical Field

[0001] The present invention relates to a melanophage inhibitor and a skin external preparation for whitening. More specifically, the present invention relates to a melanophage inhibitor and a skin external preparation for whitening that can suppress the phagocytosis of melanin by keratinocytes caused by heat stimulation, thereby achieving a whitening effect.

Background Art

[0002] Conventionally, it has been reported that extracts of plants of the Commelinaceae family such as Commelina communis have been found to have an anti-aging effect and the like (for example, see Patent Document 1).

[0003] By the way, it has been reported that pigment spots are formed in epidermal cells due to various factors. For example, it is known that the formation of pigment spots in epidermal cells is carried out by the production and transport of melanosomes and melanin. Specifically, it is common for melanosomes taken up by keratinocytes to be uniformly distributed within the cells. However, in some cases, melanosomes may locally aggregate, which can cause the formation of pigment spots (for example, freckles or stains).

[0004] It is considered that in addition to genetic factors, ultraviolet exposure, hormones, etc. are involved in the formation of pigment spots.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, controlling only the factors related to chromatophoresis formation mentioned above was not sufficient to suppress chromatophoresis formation, suggesting that factors other than those mentioned above may also be involved in chromatophoresis formation. However, no such new factors had been previously known.

[0007] This invention has been made in view of the above-mentioned conventional problems, and aims to provide a melanin phagocytosis inhibitor and a topical skin whitening agent that can produce a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation, by focusing on thermal stimulation as a new factor related to pigment spot formation. [Means for solving the problem]

[0008] The present invention, which solves the above problems, mainly comprises the following configuration.

[0009] (1) Contains plant extracts from the Commelinaceae family, A melanin phagocytosis inhibitor used to suppress the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

[0010] With this composition, the melanin phagocytosis inhibitor contains an extract from a Commelinaceae plant. Such a melanin phagocytosis inhibitor exhibits an action that suppresses melanin phagocytosis by keratinocytes caused by thermal stimulation, which was previously unknown, and may produce a whitening effect.

[0011] (2) The melanin phagocytosis inhibitor according to (1), wherein the Commelinaceae plant extract is Commelina communis extract.

[0012] With this composition, a melanin phagocytosis inhibitor containing the extract of *Campanula punctata* may exert a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

[0013] (3) The melanin phagocytosis inhibitor according to (1) or (2), wherein the extract of the large-flowered daisy contains a flavonoid.

[0014] With this composition, the melanin phagocytosis inhibitors containing flavonoids in the extract of *Campanula punctata* exhibit an effect of suppressing melanin phagocytosis by keratinocytes caused by thermal stimulation, and may produce a whitening effect.

[0015] (4) The thermal stimulation is a thermal stimulation by applying a temperature exceeding 37°C, as described in any of (1) to (3).

[0016] With this configuration, when the skin is exposed to sunlight and subjected to heat exceeding body temperature, the melanin phagocytosis inhibitor exerts an effect that suppresses the phagocytosis of melanin by keratinocytes caused by thermal stimulation, thereby producing a skin whitening effect.

[0017] (5) A topical skin whitening agent comprising a melanin phagocytosis inhibitor as described in any of (1) to (4).

[0018] With this configuration, a topical skin whitening agent can exert a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

[0019] (6) A cosmetic, as described in (5), for topical use on the skin for whitening.

[0020] With this composition, a topical whitening skin preparation, which is a cosmetic product, can exert a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

[0021] (7) A topical whitening agent for the skin as described in (5), which is a pharmaceutical composition.

[0022] With this composition, the topical whitening agent, which is a pharmaceutical composition, can exert a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation. [Effects of the Invention]

[0023] According to the present invention, there can be provided a melanophage inhibitor and a skin external preparation for whitening, which can exhibit a whitening effect by suppressing the phagocytosis of melanin by keratinocytes caused by heat stimulation.

Brief Description of the Drawings

[0024] [Figure 1] FIG. 1 is a graph showing the relationship between the infrared irradiation amount and the skin temperature. [Figure 2] FIG. 2 is a micrograph for explaining the enhanced phagocytosis action of pseudo-melanosomes by heat stimulation. [Figure 3] FIG. 3 is a graph for explaining the enhanced phagocytosis action of pseudo-melanosomes by heat stimulation. [Figure 4] FIG. 4 is a micrograph for confirming the phagocytosis inhibitory effect by luteolin. [Figure 5] FIG. 5 is a graph for confirming the phagocytosis inhibitory effect by luteolin. [Figure 6] FIG. 6 is a micrograph for confirming the phagocytosis inhibitory effect by the extract of Anaphalis margaritacea (AA). [Figure 7] FIG. 7 is a graph for confirming the phagocytosis inhibitory effect by the extract of Anaphalis margaritacea (AA). [Figure 8] FIG. 8 is a micrograph for confirming the phagocytosis inhibitory effect by the combined use of luteolin and the extract of Anaphalis margaritacea (AA). [Figure 9] FIG. 9 is a graph for confirming the phagocytosis inhibitory effect by the combined use of luteolin and the extract of Anaphalis margaritacea (AA).

Embodiments for Carrying Out the Invention

[0025] <Melanophage Inhibitor> One embodiment of the melanin phagocytosis inhibitor contains an extract from a Commelinaceae plant. The melanin phagocytosis inhibitor is used to suppress the phagocytosis of melanin by keratinocytes caused by thermal stimulation. Each of these will be described below.

[0026] (Regarding the melanin phagocytic effect caused by thermal stimulation) First, we will explain the mechanism by which keratinocytes phagocytose melanin in response to "thermal stimulation," as discovered by the inventors.

[0027] Figure 1 is a graph showing the relationship between infrared radiation dose and skin temperature. Infrared radiation accounts for more than 50% of sunlight and has a radiation intensity equivalent to eight times that of total ultraviolet radiation. Therefore, the thermal effect of infrared radiation reaches even deeper than the dermis. As shown in Figure 1, continuous exposure to infrared radiation can cause skin temperature to rise above 37°C.

[0028] Furthermore, even heat insufficient to cause burns can, if exposed for a long period, cause a reticular, transient red rash. This is also known as thermal erythema, and there are known clinical cases in which repeated exposure to heat over a long period leads to erythema and hyperpigmentation, causing atrophy and thinning of the skin and underlying tissues. Based on this phenomenon, the inventors of this invention hypothesized that, in addition to previously known genetic factors and exposure to ultraviolet light, thermal stimulation (for example, exposure to infrared light) is one of the causes of pigmentation.

[0029] To verify this, we investigated whether thermal stimulation enhances the phagocytosis of melanosomes. Figure 2 is a micrograph illustrating the enhanced phagocytosis of pseudomelanosomes by thermal stimulation. Figure 3 is a graph illustrating the enhanced phagocytosis of pseudomelanosomes by thermal stimulation. The conditions for the phagocytosis test of pseudomelanosomes (fluorescent beads) were as follows.

[0030] (Conditions for phagocytosis testing of pseudo-melanosomes (fluorescent beads)) Cell type: Normal human epidermal keratinocytes (epidermal cells) Culture conditions: 5% CO2, 37°C, thermal stimulation at 43°C Normal human epidermal cells (Kurabo Industries Ltd.) were seeded in 96-well plates and cultured for 24 hours at 37°C in a 5% CO2 environment using Humedia-KG2. The sample was diluted with Humedia-KB2 (Kurabo Industries Ltd.), added to the cells, and cultured for 30 minutes at 37°C in a 5% CO2 environment to create two groups: one cultured at a constant 37°C and the other exposed to 43°C for 1 hour. The Humedia-KG2 containing fluorescent beads (0.2 μm) that mimic melanosomes was replaced, and culture was continued for 4 hours. The phagocytic activity of the mimic melanosomes was evaluated by imaging with a fluorescence microscope and detection with a microplate reader (Ex / Em=580 / 605 nm).

[0031] As shown in Figure 2, the fluorescent beads (0.2 μm) that act as pseudomelanosomes are observed as red spots. Compared to the cell group exposed to 37°C, the cell group exposed to 43°C showed increased uptake of fluorescent beads into the cells and stronger aggregation near the nucleus. In Figure 3, the fluorescence levels were shown by detection using a microplate reader (Ex / Em = 580 / 605 nm). Compared to the cell group exposed to 37°C, the cell group exposed to 43°C showed an increase in the amount of fluorescence taken up by the cells. From this, it was confirmed that the thermal stimulation at 43°C enhanced the phagocytosis of melanosomes. The melanin phagocytosis inhibitor of this embodiment is characterized by suppressing the phagocytosis of melanin caused by such thermal stimulation.

[0032] (Tractaceae plant extract) Returning to the description of this embodiment, the Commelinaceae plants are not particularly limited. Commelinaceae plants include the genus Commelina (such as Commelina communis, Commelina communis var. hortensis, Commelina benghalensis, and Commelina cyanea), the genus Tradescantia (such as Tradescantia virginiana, Tradescantia spathacea, and Tradescantia zebrina), the genus Rhoeo (such as Rhoeo discolor), the genus Zebrina (such as Zebrina pendula), the genus Setcreasea (such as Setcreasea purpurea), and Cleomena (Cochliostema). These include genera such as Cochliostema (Cleomena odoratissimum), Tinantia (Tinantia erecta), Cyanotis (Cyanotis somaliensis), Siderasis (Siderasis fuscata), and Tripogandra (Tripogandra multiflora).

[0033] Among these, the Commelinaceae plant is preferably of the genus Commelina, and more preferably Commelina communis. As a result, the melanin phagocytosis inhibitor containing Commelina communis extract exhibits an effect of suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation, and can produce a whitening effect.

[0034] The concentration of the Haworthia ovata extract is not particularly limited. For example, the concentration of the Haworthia ovata extract is preferably 0.06 mg / mL or higher, more preferably 0.13 mg / mL or higher, and even more preferably 0.25 mg / mL or higher. Furthermore, the concentration of the Haworthia ovata extract is preferably 10 mg / mL or lower, more preferably 1 mg / mL or lower, and even more preferably 0.5 mg / mL or lower. By having the concentration of the Haworthia ovata extract within the above range, the melanin phagocytosis inhibitor of this embodiment has an excellent effect in suppressing melanin phagocytosis by keratinocytes caused by thermal stimulation, and can produce an excellent whitening effect.

[0035] The melanin phagocytosis inhibitor of this embodiment preferably contains flavonoids found in the extract of *Campanula punctata*, among other things.

[0036] In other words, the large-leaved daisy contains several flavonoids, mainly in its leaves and flowers. For example, these flavonoids include apigenin, kaempferol, luteolin, and quercetin. The melanin phagocytosis inhibitor of this embodiment contains these flavonoids found in the large-leaved daisy extract, and thus exhibits an effect of suppressing melanin phagocytosis by keratinocytes caused by thermal stimulation, thereby producing a skin whitening effect.

[0037] It is even more preferable that the flavonoid contains luteolin. This results in a more effective melanin phagocytosis inhibitor of this embodiment in suppressing melanin phagocytosis by keratinocytes caused by thermal stimulation, and thus a more superior whitening effect.

[0038] The concentration of luteolin is not particularly limited. For example, the concentration of luteolin is preferably 15 μM or more, more preferably 20 μM or more, and even more preferably 25 μM or more. Furthermore, the concentration of luteolin is preferably 200 μM or less, more preferably 100 μM or less, and even more preferably 50 μM or less. By having a luteolin concentration within the above range, the melanin phagocytosis inhibitor of this embodiment has an excellent effect in suppressing melanin phagocytosis by keratinocytes caused by thermal stimulation, and can produce an excellent whitening effect.

[0039] The melanin phagocytosis inhibitor of this embodiment is a melanin phagocytosis inhibitor that suppresses the phagocytosis of melanin by keratinocytes caused by thermal stimulation, and exhibits a particularly superior effect when the thermal stimulation is caused by applying a temperature exceeding 37°C.

[0040] The thermal stimulation is preferably a temperature exceeding 37°C, more preferably a temperature of 39°C or higher, and even more preferably a temperature of 41°C or higher. Furthermore, the thermal stimulation is preferably a temperature of 43°C or lower. As described above in relation to Figure 1, exposure to infrared rays can raise the skin temperature above 37°C, up to about 42°C. Also, as described above in relation to Figures 2 and 3, when a thermal stimulation of 43°C is applied, the phagocytic activity of melanosomes is enhanced. Therefore, the melanin phagocytosis inhibitor of this embodiment is excellent at suppressing this enhancement even when melanin phagocytosis by keratinocytes is enhanced by such thermal stimulation. As a result, the melanin phagocytosis inhibitor exhibits an effect that suppresses melanin phagocytosis by keratinocytes caused by thermal stimulation, which was not previously known, and can produce a whitening effect.

[0041] (Uses of melanin phagocytosis inhibitors (topical skin whitening agents)) The melanin phagocytosis inhibitor of this embodiment exhibits an effect of suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation, and can produce a whitening effect. Therefore, it is suitable as a topical skin whitening agent.

[0042] In other words, the topical skin preparation of this embodiment contains the melanin phagocytosis inhibitor described above. Therefore, the whitening topical skin preparation exhibits an effect of suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation, and can produce a whitening effect.

[0043] The concentration of the melanin phagocytosis inhibitor in a topical whitening agent is not particularly limited. For example, the concentration of the melanin phagocytosis inhibitor is preferably 0.0001% or more, and more preferably 0.001% or more, in the topical whitening agent. Furthermore, the concentration of the melanin phagocytosis inhibitor is preferably 1% or less, and more preferably 0.1% or less, in the topical whitening agent. By having a concentration of the melanin phagocytosis inhibitor within the above range, the topical whitening agent can exhibit excellent whitening effects.

[0044] The specific use of a topical skin whitening agent may be a cosmetic or a pharmaceutical composition. Thus, a topical skin whitening agent, whether a cosmetic or a pharmaceutical composition, can exert a whitening effect by inhibiting the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

[0045] The specific uses of cosmetic or pharmaceutical compositions are not particularly limited. For example, the uses of cosmetic or pharmaceutical compositions include basic cosmetics such as lotions, emulsions, oils, gels, creams, serums, sunscreens, packs, masks, hand creams, body lotions, and body creams; cleansing cosmetics such as facial cleansers, makeup removers, body washes, shampoos, conditioners, and treatments; makeup cosmetics such as makeup bases, foundations, face powders, liquid face powders, face powders, greasepaints, eyeshadow bases, eyeshadows, nose shadows, lip pencils, lipsticks, lip glosses, blushes, and various colors; and lip balms, soaps, and body washes.

[0046] When a topical whitening agent is a cosmetic or pharmaceutical composition, any conventionally known components may be added to it. Examples of such components include water-soluble polymers, oil-soluble polymers, waxes, alcohols, hydrocarbon oils, fatty acids, higher alcohols, fatty acid esters, silicone oils, surfactants, UV scattering agents, and other powder components, pharmaceuticals, and various solvents.

[0047] As described above, the whitening topical skin preparation of this embodiment exhibits an effect of suppressing the phagocytosis of melanin by keratinocytes caused by thermal stimulation, and can produce a whitening effect. [Examples]

[0048] The present invention will be described more specifically below with reference to examples. The present invention is not limited in any way to these examples. Unless otherwise specified, "%" means "mass%" and "parts" means "parts by mass".

[0049] <Confirmation of the phagocytic inhibitory effect of luteolin, Examples 1-3 and Comparative Example 1> The phagocytic inhibitory effect of luteolin was confirmed under the following conditions. The concentrations of luteolin used in each example and comparative example were as follows: Comparative Example 1 (0 μM), Example 1 (25 μM), Example 2 (50 μM), and Example 3 (100 μM). (conditions) Cell type: Normal human epidermal keratinocytes (epidermal cells) Culture conditions: 5% CO2, 37°C, thermal stimulation at 43°C Normal human epidermal cells (Kurabo Industries Ltd.) were seeded in 96-well plates and cultured for 24 hours at 37°C in a 5% CO2 environment using Humedia-KG2. Luteolin (Lut) was diluted with Humedia-KB2 (Kurabo Industries Ltd.), added to the cells, and cultured for 30 minutes at 37°C in a 5% CO2 environment to create two groups: one cultured at a constant 37°C and the other exposed to 43°C for 1 hour. The Humedia-KG2 containing fluorescent beads (0.2 μm) that mimic melanosomes was replaced, and culture was continued for 4 hours. The phagocytic activity of the mimic melanosomes was evaluated by imaging with a fluorescence microscope and detection with a microplate reader (Ex / Em=580 / 605 nm). The results are shown in Figures 4 and 5. Furthermore, Figures 4 and 5 show a concentration-dependent uptake of fluorescent beads and a decrease in fluorescence intensity (Ex / Em = 580 / 605 nm), indicating that phagocytosis of melanin by keratinocytes is suppressed. The same applies to the following points.

[0050] As shown in Figures 4 and 5, the melanin phagocytosis inhibitors of Examples 1 to 3, which contain the flavonoid (luteolin) found in the extract of the large-flowered honeysuckle, were able to suppress the increased melanosome phagocytosis when subjected to thermal stimulation, compared to the melanin phagocytosis inhibitor of Comparative Example 1, which does not contain luteolin.

[0051] To 1 g (dry weight) of cut, dried whole plants of C. communis var. hortensis, 10 mL of distilled water was added and the mixture was left to stand in a refrigerator (4°C) for 5 days, then filtered. To this, 0.1 mL of cellulase solution prepared to a concentration of 100 mg / mL was added, and the mixture was treated with enzymes at 45°C for 18 hours with stirring. 15 mL of distilled water and 25 mL of ethanol were added, and the mixture was left to stand in a refrigerator (4°C) for 5 days to extract C. communis var. hortensis extract (AA).

[0052] <Confirmation of the phagocytic inhibitory effect of *Campanula punctata* extract (AA), Examples 4-8 and Comparative Example 2> The phagocytic inhibitory effect of the *Campanula punctata* extract was confirmed under the following conditions. The concentrations of the *Campanula punctata* extract used in each example and comparative example were as follows: Comparative Example 2 (0 mg / mL), Example 4 (0.06 mg / mL), Example 5 (0.25 mg / mL), and Example 6 (1 mg / mL). (conditions) Cell type: Normal human epidermal keratinocytes (epidermal cells) Culture conditions: 5% CO2, 37°C, thermal stimulation at 43°C Normal human epidermal cells (Kurabo Industries Ltd.) were seeded in 96-well plates and cultured for 24 hours at 37°C and 5% CO2 using Humedia-KG2. A large-flowered daisy extract (AA) was diluted with Humedia-KB2 (Kurabo Industries Ltd.), added to the cells, and cultured for 30 minutes at 37°C and 5% CO2. Two groups were created: one cultured at a constant 37°C and the other exposed to 43°C for 1 hour. The Humedia-KG2 containing fluorescent beads (0.2 μm) that mimic melanosomes was replaced, and culture was continued for 4 hours. The phagocytic activity of the mimic melanosomes was evaluated by fluorescence microscopy and detection using a microplate reader (Ex / Em=580 / 605 nm). The results are shown in Figures 6 and 7.

[0053] As shown in Figures 6 and 7, the melanin phagocytosis inhibitors of Examples 4 to 6, which contained the extract of *Hymenophyllum sibiricum*, were able to suppress the increased melanosome phagocytosis in response to thermal stimulation, compared to the melanin phagocytosis inhibitor of Comparative Example 2, which did not contain the extract of *Hymenophyllum sibiricum*.

[0054] <Confirmation of phagocytic inhibitory effect by combining luteolin and *Crepis japonica* extract (AA), Examples 7-9 and Comparative Example 3> The phagocytic inhibitory effects of luteolin alone, Crescent Moonflower extract alone, and luteolin in combination with Crescent Moonflower extract were confirmed under the following conditions. The concentrations of luteolin and Crescent Moonflower extract used in each example and comparative example were as follows: Comparative Example 3 (0 mg / mL), Example 7 (luteolin: 13 μM), Example 8 (Crescent Moonflower extract: 50 μM), and Example 9 (luteolin: 13 μM + Crescent Moonflower extract: 50 μM). (conditions) Cell type: Normal human epidermal keratinocytes (epidermal cells) Culture conditions: 5% CO2, 37°C, thermal stimulation at 43°C Normal human epidermal cells (Kurabo Industries Ltd.) were seeded in 96-well plates and cultured for 24 hours at 37°C and 5% CO2 using Humedia-KG2. Luteolin alone, hibiscus extract alone, and luteolin and hibiscus extract diluted with Humedia-KB2 (Kurabo Industries Ltd.) were added to the cells and cultured for 30 minutes at 37°C and 5% CO2. Two groups were created: one cultured at a constant 37°C and the other exposed to 43°C for 1 hour. The Humedia-KG2 containing fluorescent beads (0.2 μm) that mimic melanosomes was replaced, and culture was continued for 4 hours. The phagocytic activity of the mimic melanosomes was evaluated by fluorescence microscopy and detection using a microplate reader (Ex / Em=580 / 605 nm). The results are shown in Figures 8 and 9.

[0055] As shown in Figures 8 and 9, the melanin phagocytosis inhibitor of Example 7 containing luteolin, the melanin phagocytosis inhibitor of Example 8 containing daisy flower extract, and the melanin phagocytosis inhibitor of Example 9 containing luteolin and daisy flower extract were able to suppress the increased melanosome phagocytosis upon thermal stimulation compared to the melanin phagocytosis inhibitor of Comparative Example 3, which contained none of these inhibitors. In particular, the melanin phagocytosis inhibitor of Example 9 containing luteolin and daisy flower extract showed a superior melanin phagocytosis inhibitory effect compared to the melanin phagocytosis inhibitors of Examples 7 and 8, which contained either of these inhibitors individually.

Claims

1. Contains plant extracts from the Commelinaceae family, A melanin phagocytosis inhibitor used to suppress the phagocytosis of melanin by keratinocytes caused by thermal stimulation.

2. The melanin phagocytosis inhibitor according to claim 1, wherein the aforementioned plant extract of the Commelinaceae family is a Commelinaceae extract.

3. The melanin phagocytosis inhibitor according to claim 1 or 2, wherein the aforementioned *Campanula punctata* extract contains flavonoids.

4. The melanin phagocytosis inhibitor according to claim 1 or 2, wherein the thermal stimulation is a thermal stimulation by applying a temperature exceeding 37°C.

5. A topical skin whitening agent comprising the melanin phagocytosis inhibitor according to claim 1 or 2.

6. A cosmetic, a topical whitening skin preparation according to claim 5.

7. A topical whitening agent for the skin according to claim 5, which is a pharmaceutical composition.

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