Actin reorganization inhibitor, phagocytosis inhibitor, stain formation inhibitor containing the inhibitor, and external skin preparation containing the inhibitor

Abstract

To provide actin reorganization inhibitors and phagocytosis inhibitors, which inhibit the actin reorganization and induction of phagocytosis in epidermal keratinocytes by contacting the epidermal keratinocytes with a fibroblast conditioned medium; to provide stain formation inhibitors containing the inhibitor; and to provide external skin preparations containing the inhibitor.SOLUTION: Provided is an inhibitor that inhibits the actin reorganization and induction of phagocytosis in epidermal keratinocytes by contacting the epidermal keratinocytes with a fibroblast conditioned medium, and contains a specific plant extract or a specific compound as an active ingredient. Also provided are a spot formation inhibitor composed of the inhibitor, and a skin external preparation containing the inhibitor.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel actin reorganization inhibitor, a phagocytosis inhibitor, a scar formation inhibitor containing the inhibitor, and a topical skin preparation containing the inhibitor.

Background Art

[0002] Pigmented spots (solar lentigines) that appear with age are an important issue in skin beauty, especially for women. UVB-induced hyperpigmentation (UVB melanosis) disappears within 2 weeks to several months after UVB exposure. On the other hand, solar lentigines occur on sun-exposed skin, especially the face, and do not disappear because there is a possibility of DNA damage to epidermal keratinocytes by repeated UVB irradiation of the lesional epidermis. In fact, it is known that an individual's UV exposure history is the cause of the appearance of solar lentigines (see, for example, Non-Patent Document 1). Therefore, prevention and improvement of solar lentigines are important issues for cosmetics, especially for Asian and Caucasian women.

[0003] As histological features of solar lentigines, destruction of the basement membrane and an increase in the thickness of the epidermis due to developed rete ridges have been reported (see, for example, Non-Patent Documents 2 and 3). Furthermore, immunohistochemical analysis using the pigment cell-specific marker MART-1 has revealed an increase in the number of pigment cells in solar lentigines (senile pigmented spots) (see, for example, Non-Patent Document 4). Also, it has been clarified that when using a tyrosinase antibody, the number of tyrosinase-positive pigment cells significantly doubles in the lesional epidermis of solar lentigines (see, for example, Non-Patent Document 5).

[0004] The mechanism underlying pigmentation spots initiated by UV irradiation can be broadly classified into enhanced melanogenesis in melanocytes and the transfer of melanosomes to adjacent epidermal keratinocytes. Regarding enhanced melanogenesis, it has been reported that various cytokines such as endothelin-1 and granulocyte-macrophage colony-stimulating factor produced by epidermal keratinocytes act on melanocytes to promote melanogenesis (see, for example, Non-Patent Documents 6 and 7). Disruption of the basement membrane is expected to promote crosstalk not only between melanocytes and epidermal keratinocytes but also between the dermis and epidermis. In fact, in some studies, the contribution of fibroblasts to the initiation and maintenance of solar lentigines has been emphasized (see, for example, Non-Patent Document 8).

[0005] On the other hand, regarding the transfer of melanosomes, it has been reported that activation of Toll-like receptor 3 in epidermal keratinocytes promotes phagocytosis of melanosomes via activation of the Rho family (see, for example, Non-Patent Document 9). Also, the action of other factors such as epidermal growth factor / epidermal growth factor receptor has been reported (see, for example, Non-Patent Document 10).

[0006] An action of suppressing freckle formation by inhibiting the transfer of melanosomes from melanocytes to epidermal keratinocytes is known. At this time, it has been reported that epidermal keratinocytes take up melanosomes by phagocytosis. Also, it is known that phagocytosis by the epidermal keratinocytes is suppressed by a plant extract obtained from a plant belonging to the genus Melissa of the Lamiaceae family containing an unsaturated fatty acid derivative (see, for example, Patent Document 1). However, the mechanism of action of inducing actin reorganization and phagocytosis of the epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts is completely unknown. Also, an actin reorganization inhibitor, a phagocytosis inhibitor, and a freckle formation inhibitor containing these inhibitors that suppresses the mechanism of action are completely unknown. Moreover, it is completely unknown whether the action of the inhibitor exists in a specific plant extract or a specific compound.

Prior Art Documents

Patent Document

[0007]

Patent Document 1

Non-Patent Document

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0009] Therefore, the present invention provides an actin rearrangement inhibitor and a phagocytosis inhibitor that inhibit the actin rearrangement and induction of phagocytosis of epidermal keratinocytes by contacting the epidermal keratinocytes with a fibroblast conditioned medium. The present invention also provides a freckle formation inhibitor containing the above inhibitor and a topical skin preparation containing the above inhibitor.

[0010] In view of such circumstances, the inventors have made intensive efforts to find a method for preventing or improving the occurrence of freckle formation after contacting epidermal keratinocytes with a fibroblast-conditioned medium. As a result, they have found that rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia cordata extract, parsley extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, goreshi extract, hop extract, red algae extract, somemiyoshino extract, photinia extract, kibanaoilandasenni extract, keratin, acenyaku extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, pithecellobium extract have an actin reorganization inhibitory effect and a phagocytosis inhibitory effect, which are factors directly related to the prevention or improvement of freckle formation, and have completed the invention. That is, the present invention is as follows.

Means for Solving the Problems

[0011] That is, the present invention relates to (1) An inhibitor that inhibits the reorganization of actin in epidermal keratinocytes by contacting the epidermal keratinocytes with a fibroblast-conditioned medium, the inhibitor containing, as an active ingredient, one or more selected from rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia cordata extract, parsley extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, goreshi extract, hop extract, red algae extract, somemiyoshino extract, photinia extract, kibanaoilandasenni extract, keratin, acenyaku extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, pithecellobium extract, characterized in that it is an actin reorganization inhibitor. (2) A freckle formation inhibitor characterized by containing the actin reorganization inhibitor described in (1) above. (3) An inhibitor that inhibits phagocytosis of epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts, which is selected from one or more of rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, parsley extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba sasine extract, hydrolyzed wheat protein, gorenji extract, hop extract, red algae extract, somemiyoshino extract, photinia extract, nasturtium officinale extract, keratin, acen yak extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, and pithecellobium extract. A phagocytosis inhibitor characterized by containing as an active ingredient. (4) A freckle formation inhibitor characterized by containing the phagocytosis inhibitor according to (3) above. (5) A topical skin preparation characterized by containing the freckle formation inhibitor according to (2) or (4) above. Regarding.

Effects of the Invention

[0012] Specific plant extracts and specific compounds that are the active ingredients of the present invention have an extremely excellent inhibitory effect on actin reorganization and phagocytosis induction of epidermal keratinocytes after contacting the epidermal keratinocytes with a conditioned medium of fibroblasts, and the inhibitory effect effectively acts on freckle formation, exhibiting an extremely excellent effect.

[0013] In addition, since it exhibits an extremely excellent inhibitory effect on actin reorganization and phagocytosis induction involved in freckle formation, it has the effect of being suitably used as a topical skin preparation such as a whitening agent that does not form freckles.

Brief Description of the Drawings

[0014]

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Embodiments for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described.

[0016] An inhibitor that inhibits actin reorganization and induction of phagocytosis of the present invention, a scab formation inhibitor comprising the inhibitor, and a topical skin preparation containing the inhibitor contain a specific plant extract or a specific compound as an active ingredient.

[0017] 〔Inhibition of actin reorganization〕 The present inventors have found that when epidermal keratinocytes are brought into contact with a conditioned medium from fibroblasts, actin reorganization in the epidermal keratinocytes is induced. Furthermore, the present inventors have found that the actin reorganization induced by contact with the conditioned medium from fibroblasts is correlated with the inhibition by an actin reorganization inhibitor and the expression of the phagocytosis inhibitory action by the actin reorganization inhibitor. The present invention is based on these findings.

[0018] The inhibition of actin reorganization in the present invention is characterized by inhibiting actin reorganization induced by bringing epidermal keratinocytes into contact with a conditioned medium from fibroblasts.

[0019] Examples of the above epidermal keratinocytes include any epidermal keratinocytes derived from humans or non-human animals. From the viewpoint of being able to more accurately evaluate a test substance in anticipation of application to human skin, the epidermal keratinocytes used in the present invention are preferably human epidermal keratinocytes. The human epidermal keratinocytes are not particularly limited to normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but it is more preferable to use normal human epidermal keratinocytes from the viewpoint of being able to more accurately capture the phenomenon of actin reorganization and evaluate it accurately.

[0020] Examples of the fibroblast include any fibroblast derived from a human or a non-human animal. From the viewpoint of being able to more accurately evaluate a test substance in anticipation of application to human skin, the fibroblast used in the present invention is preferably a human fibroblast. The human fibroblast is not particularly limited to a normal human fibroblast or an immortalized human fibroblast, but from the viewpoint of more accurately capturing the phenomenon of actin reorganization and being able to evaluate it accurately, it is more preferable to use a normal human fibroblast.

[0021] The acclimation medium for the fibroblast is, for example, replaced with fresh KB2 medium or DMEM (5% FBS) medium from the state where the fibroblasts have become confluent in a 58 cm 2 dish, cultured for 12 to 36 hours, and then the medium with the supernatant collected can be used. Note that the acclimation medium is not limited to the above-described preparation method. In the present invention, from the viewpoint of sufficiently inducing actin reorganization, it is preferable to use the medium with the supernatant collected after culturing for 18 to 30 hours.

[0022] The epidermal keratinocytes can be seeded, for example, at 500 to 5,000 cells / cm 2 in a 4-chamber slide. In the present invention, from the viewpoint of facilitating the observation of the induction of actin reorganization, it is preferable to seed at 2,000 to 4,000 cells / cm 2 .

[0023] The time for contacting the epidermal keratinocytes with the acclimation medium from the fibroblast is not particularly limited, but from the viewpoint of sufficiently inducing actin reorganization by contacting with the acclimation medium from the fibroblast, it is preferable to contact with the acclimation medium from the fibroblast for 2 to 24 hours, and more preferably for 4 to 8 hours.

[0024] As used herein, "induction of actin reorganization by contact with conditioned medium from fibroblasts" means that, in response to stimulation of epidermal keratinocytes by conditioned medium from the above fibroblasts, the induction of actin reorganization in the epidermal keratinocytes is more significantly recognized compared to the actin reorganization in epidermal keratinocytes not in contact with the conditioned medium from the above fibroblasts.

[0025] The induction of actin reorganization by contact with conditioned medium from the above fibroblasts can be confirmed, for example, by staining the actin polymerization of epidermal keratinocytes and observing it with a fluorescence microscope. The reagent that can stain actin polymerization is not particularly limited, and examples include rhodamine phalloidin, commercially available actin antibodies, and the like.

[0026] Since the present invention can accurately observe and evaluate the phenomenon in which actin reorganization is induced by contacting epidermal keratinocytes with conditioned medium from fibroblasts, it can accurately evaluate the presence or absence of an inhibitory effect on actin reorganization of a test substance. That is, it can be suitably used as a method for screening actin reorganization inhibitors.

[0027] 〔Phagocytosis inhibition〕 The present inventors have also found that when epidermal keratinocytes are contacted with conditioned medium from fibroblasts, not only the above-described actin reorganization but also phagocytosis is induced in the epidermal keratinocytes. Furthermore, the present inventors have found that phagocytosis induced by contact with conditioned medium from fibroblasts is inhibited by phagocytosis inhibitors and actin reorganization inhibitors, and there is a correlation with the expression of the inhibitory effect on pigmentation formation by phagocytosis inhibitors and actin reorganization inhibitors. The present invention is based on these findings.

[0028] The above phagocytosis refers to the phagocytic action, ingestion, etc. of epidermal keratinocytes. The phagocytosis inhibition in the present invention is characterized by inhibiting the phagocytosis induced by contacting epidermal keratinocytes with a conditioned medium from fibroblasts.

[0029] Examples of the above epidermal keratinocytes include any epidermal keratinocytes derived from humans or non-human animals. The epidermal keratinocytes used in the present invention are preferably human epidermal keratinocytes from the viewpoint of accurately evaluating the test substance in anticipation of application to human skin. The human epidermal keratinocytes are not particularly limited to normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but normal human epidermal keratinocytes are more preferably used from the viewpoint of more accurately capturing the phenomenon of phagocytosis and accurately evaluating it.

[0030] Examples of the above fibroblasts include any fibroblasts derived from humans or non-human animals. The fibroblasts used in the present invention are preferably human fibroblasts from the viewpoint of accurately evaluating the test substance in anticipation of application to human skin. The human fibroblasts are not particularly limited to normal human fibroblasts or immortalized human fibroblasts, but normal human fibroblasts are more preferably used from the viewpoint of more accurately capturing the phenomenon of phagocytosis and accurately evaluating it.

[0031] The conditioned medium of the above fibroblasts is, for example, replaced with fresh KB2 medium or DMEM (5% FBS) medium from the state where the fibroblasts have become confluent in a 58 cm 2 culture dish, cultured for 12 to 36 hours, and then the supernatant is collected and the resulting medium can be used. Note that the conditioned medium is not limited to the above-described preparation method. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to use the medium from which the supernatant has been collected after culturing for 18 to 30 hours.

[0032] The above epidermal keratinocytes are, for example, seeded at 50,000 to 130,000 cells / cm in a 96-well plate or a 24-well plate2 It can be seeded so as to be 70,000 to 110,000 cells / cm. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, 2 it is preferably seeded so as to be

[0033] The time for contacting the epidermal keratinocytes with the conditioned medium from the fibroblasts is not particularly limited. However, from the viewpoint of sufficiently inducing phagocytosis by contacting with the conditioned medium from the fibroblasts, it is preferable to contact the conditioned medium from the fibroblasts for 2 to 24 hours, and more preferably for 4 to 8 hours.

[0034] In this specification, "induction of phagocytosis by contact with the conditioned medium from fibroblasts" means that, in response to the stimulation of epidermal keratinocytes by the conditioned medium from the above fibroblasts, the amount of microparticles taken up in the epidermal keratinocytes is significantly more than the amount of microparticles taken up in epidermal keratinocytes not contacted with the conditioned medium from the fibroblasts. Here, "significant" means that the p-value of the t-test is less than 0.05, preferably less than 0.01.

[0035] Furthermore, in this specification, "phagocytosis inhibition" means that the cell viability is 90% or more (compared with when the cell viability of epidermal keratinocytes contacted with the conditioned medium is 100%), and the epidermal keratinocytes contacted with the conditioned medium containing the test substance have a fluorescence intensity of 50% or less compared with the epidermal keratinocytes contacted with the conditioned medium not containing the test substance.

[0036] Examples of the above microparticles include melanosomes, melanin, coloring substances, and the like.

[0037] For the above melanosomes, any pigment cells derived from humans or non-human animals may be used. In the present invention, it is preferable to use those prepared from a homogenate of pigment cells derived from humans.

[0038] The above melanin may be natural melanin or synthetic melanin. Also, commercially available synthetic melanin may be used. The amount of melanin used is preferably 1 μg or more and 100 μg or less in one well (0.35 cm 2 ) of a 96-well plate.

[0039] As the above coloring substance, fluorescent beads can be used. The fluorescent beads "FluoSpheres TM Carboxylate Modified Microspheres, 0.2 μm, Blue Fluorescence (365 / 415), 2% Solids" can be obtained from ThermoFisher Scientific (Waltham, Massachusetts, USA). The amount of fluorescent beads used is preferably 1 μg or more and 100 μg or less in one well (0.35 cm 2 ) of a 96-well plate.

[0040] Induction of phagocytosis by contact with the conditioned medium from the above fibroblasts can be evaluated, for example, by measuring fluorescent beads taken up by epidermal keratinocytes with a fluorescence plate reader.

[0041] Since the present invention can accurately observe and evaluate the phenomenon in which phagocytosis is induced by contacting epidermal keratinocytes with the conditioned medium from fibroblasts, it can accurately evaluate the presence or absence of an inhibitory effect of a test substance on phagocytosis. That is, it can be suitably used as a method for screening phagocytosis inhibitors.

[0042] As described above, the present invention can accurately observe and evaluate the actin reorganization and the phenomenon of phagocytosis induced by contacting epidermal keratinocytes with the conditioned medium from fibroblasts. That is, the present invention can accurately observe and evaluate the actin reorganization involved in the formation of freckles and the phenomenon of phagocytosis, and can accurately confirm and evaluate the presence or absence of an inhibitory effect of a test substance on actin reorganization and phagocytosis.

[0043] In addition, according to the present invention, an inhibitor that exhibits an extremely excellent inhibitory effect on actin reorganization and phagocytosis induction involved in the formation of spots is useful as a spot formation inhibitor, and it can be said that the inhibitor can be suitably used as a topical preparation for the skin such as a whitening agent that does not form spots.

[0044] 〔Test Substance〕 The inhibitor, inhibitor, and topical preparation of the present invention contain a specific plant extract or a specific compound as an active ingredient. In the present invention, among these specific plant extracts and specific compounds, from the viewpoint of exhibiting an extremely excellent inhibitory effect, rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, carrot extract, foxtail millet extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, gorenji extract, hop extract, red algae extract, yosino extract, photomomo extract, kibanaoilandasenni extract, keratin, asenyak extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, pithecellobium extract, etc. It is preferable to contain one or more selected extracts and compounds as active ingredients. In the present invention, a mixed raw material containing these extracts and compounds may also be used.

[0045] The "extract" in this specification includes any form such as an extract obtained by extracting various natural products and the like with various solvents as extraction raw materials, a diluted solution of the extract, a concentrated solution of the extract, a dried product obtained by drying the extract, and a purified product obtained by drying the extract.

[0046] The extracts and compounds used in the present invention are rooibos (scientific name: Aspalathus Linearis), aloe (scientific name: Aloe), skullcap (scientific name: Scutellaria Baicalensis), raspberry (scientific name: Rubus), kiwi (scientific name: Actinidia Chinensis), houttuynia cordata (scientific name: Houttuynia Cordata), ginseng (scientific name: Panax Ginseng), isodon japonicus (scientific name: Isodon Japonicus), loquat (scientific name: Eriobotrya Japonica), peach (scientific name: Prunus Persica), saxifraga stolonifera (scientific name: Saxifraga Stolonifera), Japanese pepper (scientific name: Zanthoxylum Piperitum), bamboo vinegar, ganoderma (scientific name: Ganodermataceae), wild ginger (scientific name: Asarum Sieboldii), hydrolyzed wheat protein, starfruit (scientific name: Averrhoa Carambola), hop (scientific name: Humulus Lupulus), red algae (scientific name: Rhodophyta), yoshino cherry (scientific name: Prunus Yedoensis), rose apple (scientific name: Syzygium Jambos), toothache plant (scientific name: Acmella Oleracea), keratin, gambeer (scientific name: Uncaria Gambir), cassia bark (Cinnamomum Cassia), oligopeptide-20, isomerized sugar, tree peony (scientific name: Paeonia Suffruticosa), lychee (scientific name: Litchi Chinensis), lotus (scientific name: Nelumbo Nucifera), eclipta prostrata (scientific name: Eclipta Prostrata), and pithecellobium (scientific name: Pithecellobium).

[0047] Rooibos (scientific name: Aspalathus Linearis) is cultivated in the Republic of South Africa and is readily available from this region.

[0048] Aloe (scientific name: Aloe) is cultivated in the Republic of South Africa and is readily available from this region.

[0049] Skullcap (Scutellaria baicalensis) is cultivated in Russia, China, Mongolia, and the Korean Peninsula, and is easily available from these regions.

[0050] Strawberry (Rubus) is commonly found in the cold to temperate zones of the Northern Hemisphere and is easily available from these regions.

[0051] Kiwi (Actinidia chinensis) is cultivated in Italy, China, New Zealand, and Japan, and is easily available from these regions.

[0052] Heartleaf Houttuynia (Houttuynia cordata) is cultivated in Japan and is easily available from this region.

[0053] Ginseng (Panax ginseng) is cultivated in China and the Korean Peninsula, and is easily available from these regions.

[0054] Japanese Isodon (Isodon japonicus) is cultivated in Japan and is easily available from this region.

[0055] Loquat (Eriobotrya japonica) is cultivated in China and Japan, and is easily available from these regions.

[0056] Peach (Prunus persica) is cultivated in China, the United States, Italy, and Japan, and is easily available from these regions.

[0057] Saxifrage (Saxifraga stolonifera) is cultivated in China and Japan, and is easily available from these regions.

[0058] Sansho (scientific name: Zanthoxylum Piperitum) is cultivated in the Korean Peninsula and Japan and is easily available from these regions.

[0059] Bamboo vinegar is a liquid obtained by cooling the smoke produced when burning bamboo charcoal and is easily available.

[0060] Reishi (scientific name: Ganodermataceae) is cultivated in China and Japan and is easily available from these regions.

[0061] Wild ginger (scientific name: Asarum Sieboldii) is cultivated in China, the Korean Peninsula, and Japan and is easily available from these regions.

[0062] Hydrolyzed wheat gluten is obtained by hydrolyzing wheat gluten with acid, enzymes, or other methods and is easily available.

[0063] Star fruit (scientific name: Averrhoa Carambola) is cultivated in China, Brazil, and Japan and is easily available from these regions.

[0064] Hops (scientific name: Humulus Lupulus) are cultivated in Germany, the United States, the Czech Republic, the United Kingdom, and Japan and are easily available from these regions.

[0065] Red algae (scientific name: Rhodophyta) mostly grow in coastal areas Rock and are easily available from these regions.

[0066] Somei Yoshino (scientific name: Prunus Yedoensis) is cultivated in Japan and is easily available from this region.

[0067] Java plum (scientific name: Syzygium Jambos) is cultivated in Japan and is easily available from this region.

[0068] Acmella oleracea is cultivated in Japan and is easily available in this region.

[0069] Keratin is a protein obtained from wool and is easily available.

[0070] Uncaria gambir is cultivated in Japan and is easily available in this region.

[0071] Cinnamomum cassia is cultivated in China, India, and Indonesia and is easily available from these regions.

[0072] Oligopeptide-20 is a synthetic peptide composed of 12 amino acids including alanine, arginine, cysteine, glutamic acid, leucine, lysine, methionine, proline, and tyrosine and is easily available.

[0073] Isomerized sugar is a carbohydrate complex made by rearranging a sugar mixture by base catalysis and is easily available.

[0074] Paeonia suffruticosa is cultivated in China and Japan and is easily available from these regions.

[0075] Litchi chinensis is cultivated in China and is easily available from this region.

[0076] Nelumbo nucifera is cultivated in India and Japan and is easily available from these regions.

[0077] Eclipta prostrata is cultivated in China, the Korean Peninsula, and Japan and is easily available from these regions.

[0078] Pithecellobium (scientific name: Pithecellobium) is a woody plant that grows into a tall tree of the genus Pithecellobium in the legume family, with about 200 species differentiated in the tropical region, and is easily available.

[0079] Examples of the parts that can be used as the above extraction raw materials include, for example, leaf parts, bark parts, trunk parts, stem parts, root parts, flower parts, fruit parts, pericarp parts, seed parts, whole herbs, or mixtures of these parts.

[0080] For example, when extracting the above raw materials, after drying, it can be directly or crushed using a crusher and then subjected to extraction with an extraction solvent. Drying can be carried out in the sun or using a commonly used dryer. Also, pretreatment such as degreasing can be performed with a non-polar solvent such as hexane and then used as an extraction raw material. By performing pretreatment such as degreasing, the extraction treatment of the above extraction raw materials with a polar solvent can be carried out efficiently.

[0081] As the extraction solvent, it is preferable to use a polar solvent. For example, water, hydrophilic organic solvents, etc. can be mentioned, and it is preferable to use these alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent.

[0082] Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc., and in addition, those obtained by performing various treatments on these are included. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, the water that can be used as the extraction solvent in the present invention also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer solution, phosphate buffered saline, etc.

[0083] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0084] The extraction treatment is not particularly limited as long as the soluble components contained in the extraction raw material can be eluted into the extraction solvent, and it can be carried out according to a conventional method. For example, the extraction raw material can be immersed in the extraction solvent, and the soluble components can be extracted at room temperature or under reflux heating, and then the extraction residue can be removed by filtration to obtain an extract. When the solvent is distilled off from the obtained extract, a paste-like concentrate can be obtained, and when this concentrate is further dried, a dried product can be obtained.

[0085] The extract obtained as described above has excellent actin reorganization inhibitory action and phagocytosis inhibitory action, and thus can be used as an active ingredient of an actin reorganization inhibitor, a phagocytosis inhibitor, a stain formation inhibitor comprising the inhibitor, and a topical skin preparation containing the inhibitor.

[0086] In the present invention, any one of rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, carrot extract, pearl barley extract, loquat extract, peach extract, pimpinella extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, gorenji extract, hop extract, red algae extract, yoshino cherry extract, photinia extract, kibanaoilandasenni extract, keratin, asenyak extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, pithecellobium extract may be used as the above active ingredient, or these may be mixed and used as the above active ingredient.

[0087] The sima formation inhibitor of the present invention may be any one of a rooibos extract, an aloe extract, a saffron extract, a strawberry extract, a kiwi extract, a houttuynia extract, a parsnip extract, a pearl millet extract, a loquat extract, a peach extract, a snowdrop extract, a sansho extract, a bamboo vinegar extract, a jujube extract, a usuba saishin extract, a hydrolyzed wheat gluten, a gorenji extract, a hop extract, a red algae extract, a somemiyoshino extract, a photinia extract, a kibanolandasenichi extract, keratin, an asenyak extract, a cinnamon bark extract, an oligopeptide-20, an isomerized sugar, a button extract, a lychee extract, a lotus extract, a brinjarige extract, a pithecellobium extract, or a formulation of a mixture thereof.

[0088] The actin reorganization inhibitor and the phagocytosis inhibitor of the present invention can be formulated into any dosage form such as powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier and any other auxiliary agent.

[0089] When formulating the actin reorganization inhibitor and the phagocytosis inhibitor of the present invention, the content of any one of a rooibos extract, an aloe extract, a saffron extract, a strawberry extract, a kiwi extract, a houttuynia extract, a parsnip extract, a pearl millet extract, a loquat extract, a peach extract, a snowdrop extract, a sansho extract, a bamboo vinegar extract, a jujube extract, a usuba saishin extract, a hydrolyzed wheat gluten, a gorenji extract, a hop extract, a red algae extract, a somemiyoshino extract, a photinia extract, a kibanolandasenichi extract, keratin, an asenyak extract, a cinnamon bark extract, an oligopeptide-20, an isomerized sugar, a button extract, a lychee extract, a lotus extract, a brinjarige extract, a pithecellobium extract, or a mixture thereof is not particularly limited and can be appropriately set according to the purpose.

[0090] In addition, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention, if necessary, may contain other natural extracts having an actin reorganization inhibitory action and a phagocytosis inhibitory action, such as rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, carrot extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, collenchyma extract, hop extract, red algae extract, yoshino cherry extract, photinia extract, kibanaoilandasenni extract, keratin, asen yak extract, cassia bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjal extract, pithecellobium extract, and can be used as an active ingredient in combination with any one of these or a mixture thereof.

[0091] The actin reorganization inhibitor and phagocytosis inhibitor of the present invention can prevent, treat or improve skin spots through the actin reorganization inhibitory action and phagocytosis inhibitory action of rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, carrot extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, collenchyma extract, hop extract, red algae extract, yoshino cherry extract, photinia extract, kibanaoilandasenni extract, keratin, asen yak extract, cassia bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjal extract, pithecellobium extract.

[0092] In addition, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention can be used as an active ingredient of a pharmaceutical or quasi-drug for preventing and treating diseases caused by abnormal production of melanin. However, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention can be used for all applications that are meaningfully capable of exerting an actin reorganization inhibitory action and a phagocytosis inhibitory action, in addition to these applications.

[0093] In addition, since the actin reorganization inhibitor and phagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory action and phagocytosis inhibitory action, they are suitable for being formulated in, for example, external skin preparations. In this case, any one of rooibos extract, aloe extract, saffron extract, strawberry extract, kiwi extract, houttuynia extract, carrot extract, pearl barley extract, loquat extract, peach extract, snowdrop extract, sansho extract, bamboo vinegar extract, jujube extract, usuba saishin extract, hydrolyzed wheat gluten, gorenji extract, hop extract, red algae extract, yosino extract, photinia extract, kibanaoilandasenni extract, keratin, asen yak extract, cinnamon bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, lotus extract, brinjaraj extract, pithecellobium extract, or a mixture thereof may be formulated.

[0094] When the above plant extract is formulated and used in an external skin preparation, the content is not particularly limited as long as the desired effect can be exhibited, but it is preferably formulated in an amount of 0.00000001 to 5% by mass as an extraction raw material in the total amount of the external preparation, more preferably 0.0000001 to 3% by mass, still more preferably 0.000001 to 2% by mass, and particularly preferably 0.00001 to 1% by mass.

[0095] Here, the external skin preparation is not limited to its classification and widely includes skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally. Specifically, for example, external skin preparations such as ointments, creams, emulsions, beauty liquids, lotions, packs, foundations, lip creams, and bath agents can be mentioned.

[0096] In addition, since the actin reorganization inhibitor and phagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory activity and phagocytosis inhibitory activity, they can also be suitably used as reagents for research related to the mechanism of melanin production.

[0097] Next, a method for screening a test substance for evaluating the inhibitory effect and suppression effect of the present invention will be described.

[0098] As a specific example of the method for screening a test substance, there is a method for screening a test substance for evaluating the inhibitory effect of spot formation, which comprises: (A) contacting epidermal keratinocytes with fine particles and a test substance, and contacting the epidermal keratinocytes with a conditioned medium from fibroblasts before, simultaneously with, or after this contact; (B) measuring the amount of fine particles taken up by the epidermal keratinocytes from the fine particle uptake activity of the epidermal keratinocytes obtained in the step (A); and (C) examining the inhibition of phagocytosis of the epidermal keratinocytes based on the amount of fine particles taken up by the epidermal keratinocytes measured in the step (B), and evaluating the inhibitory effect of spot formation of the test substance.

[0099] According to the above screening method, by contacting epidermal keratinocytes with fine particles and a test substance, and contacting the epidermal keratinocytes with a conditioned medium from fibroblasts before, simultaneously with, or after this contact, it is possible to quickly and simply evaluate whether the test substance inhibits phagocytosis of epidermal keratinocytes. In addition, based on the presence or absence of inhibition of phagocytosis of epidermal keratinocytes and the degree of the above inhibition, it is possible to quickly and simply evaluate the inhibitory effect of spot formation of the test substance.

[0100] In the above step (A), epidermal keratinocytes are contacted with fine particles and a test substance, and the epidermal keratinocytes are contacted with a conditioned medium from fibroblasts before, simultaneously with, or after this contact.

[0101] Examples of the epidermal keratinocytes include any epidermal keratinocytes derived from humans or non-human animals. From the viewpoint of accurately evaluating a test substance in anticipation of application to human skin, the epidermal keratinocytes used in the screening method are preferably human epidermal keratinocytes. The human epidermal keratinocytes are not particularly limited whether they are normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but from the viewpoint of more accurately capturing the phenomenon of phagocytosis and accurately evaluating, it is more preferable to use normal human epidermal keratinocytes.

[0102] Examples of the fibroblasts include any fibroblasts derived from humans or non-human animals. From the viewpoint of accurately evaluating a test substance in anticipation of application to human skin, the fibroblasts used in the screening method are preferably human fibroblasts. The human fibroblasts are not particularly limited whether they are normal human fibroblasts or immortalized human fibroblasts, but from the viewpoint of more accurately capturing the phenomenon of phagocytosis and accurately evaluating, it is more preferable to use normal human fibroblasts.

[0103] Examples of the fine particles include melanosomes, melanin, coloring substances, and the like.

[0104] Any pigment cells derived from humans or non-human animals may be used for the melanosomes. In the present invention, those prepared from a homogenate of human-derived pigment cells are preferably used.

[0105] The melanin may be natural melanin or synthetic melanin. The amount of melanin used is preferably 1 μg or more and 100 μg or less in one well (0.35 cm 2 ) of a 96-well plate.

[0106] Fluorescent beads can be used as the coloring substance. The fluorescent beads "FluoSpheres" TM"Carboxylate-modified microspheres, 0.2 μm, blue fluorescence (365 / 415), 2% solids" can be obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The amount of fluorescent beads used is preferably 1 μg or more and 100 μg or less in 1 well (0.35 cm 2 ) in a 96-well plate.

[0107] When bringing the above epidermal keratinocytes into contact with the test substance, the type of test substance used is not particularly limited, and examples include low-molecular compounds, plant extracts, extracts of natural products such as microorganisms, proteins, peptides, nucleic acids, synthetic polymer compounds, and the like. Since the amount of the test substance varies depending on the type of the test substance and the like, it cannot be determined unconditionally.

[0108] The acclimation medium for the above fibroblasts is, for example, a medium obtained by replacing the medium with fresh KB2 medium or DMEM (5% FBS) medium from the state where the fibroblasts have become confluent in a 58 cm 2 dish, culturing for 12 to 36 hours, and then collecting the supernatant. Note that the acclimation medium is not limited to the above-described preparation method. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to use the medium from which the supernatant has been collected after culturing for 18 to 30 hours.

[0109] The above epidermal keratinocytes can be seeded, for example, at a density of 50,000 to 130,000 cells / cm 2 in a 96-well plate or a 24-well plate. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to seed at a density of 70,000 to 110,000 cells / cm 2 .

[0110] The time for contacting the above epidermal keratinocytes with the conditioned medium from the above fibroblasts is not particularly limited. However, from the viewpoint of sufficiently inducing phagocytosis by contacting with the conditioned medium from fibroblasts, it is preferable to contact the conditioned medium from fibroblasts for 2 to 24 hours, and more preferably for 4 to 8 hours.

[0111] The timing for contacting the above epidermal keratinocytes with the conditioned medium from the above fibroblasts may be any of before, simultaneously with, or after the contact of the microparticles of epidermal keratinocytes and the test substance. In the present invention, from the viewpoint of enhancing the accuracy of the screening method, it is preferable to contact the microparticles and the test substance during the contact with the conditioned medium from fibroblasts. That is, the timing for contacting the epidermal keratinocytes with the conditioned medium from fibroblasts is preferably simultaneous with the contact of the microparticles of epidermal keratinocytes and the test substance. In this case, it can be carried out by mixing the microparticles and the test substance in the conditioned medium from fibroblasts and contacting them with the epidermal keratinocytes. Further, from the viewpoint of further enhancing the accuracy of the screening method, it is preferable to contact for 2 to 24 hours, and more preferably for 4 to 8 hours. From the viewpoint of suppressing the burden on cell growth and survival, it is preferably 24 hours or less.

[0112] Next, in step (B), the uptake amount of the microparticles of epidermal keratinocytes is measured from the microparticle uptake activity of the epidermal keratinocytes obtained in the above step (A).

[0113] Examples of the method for measuring the uptake amount of the microparticles of the above epidermal keratinocytes include a method of measuring fluorescent beads incorporated into epidermal keratinocytes with a fluorescence plate reader.

[0114] Next, in step (C), based on the uptake amount of the microparticles of epidermal keratinocytes measured in the above step (B), the inhibition of phagocytosis of epidermal keratinocytes is examined, and the inhibitory effect of the above test substance on stain formation is evaluated.

[0115] Whether the test substance inhibits phagocytosis can be examined, for example, by comparing the fluorescence intensity of phagocytosis measured in step (B) above with the fluorescence intensity of phagocytosis without contacting the test substance.

[0116] In step (C), when the fluorescence intensity of phagocytosis measured in step (B) above is significantly less than, for example, the fluorescence intensity of phagocytosis without contacting the test substance, it can be determined that the test substance inhibits phagocytosis.

[0117] Thus, when it is determined that the test substance inhibits phagocytosis, it can be evaluated that the test substance is a substance having a freckle formation inhibitory effect. Also, the greater the difference between the fluorescence intensity of phagocytosis measured in step (B) above and the fluorescence intensity of phagocytosis of the cell group not contacted with the test substance, the more it can be evaluated that the test substance has a stronger freckle formation inhibitory effect.

[0118] As described above, according to the screening method, based on the presence or absence of inhibition of phagocytosis and the degree of inhibition, the freckle formation inhibitory effect of the test substance can be easily evaluated. Therefore, screening for freckle formation inhibitors useful for freckle formation inhibitors and the like, and evaluation of the freckle formation inhibitory effect of freckle formation inhibitors and the like can be carried out quickly and simply.

Example

[0119] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to such examples.

[0120] (Production Example 1) Normal human epidermal keratinocytes (NHEK) 58cm 2 Using a petri dish (manufactured by IWAKI), normal human epidermal keratinocytes (manufactured by Kurashiki Boseki) were seeded in KG2 medium (manufactured by Kurashiki Boseki) and cultured under conditions of 37°C and 5% CO 2 2.

[0121] (Production Example 2) Normal human fibroblasts (NHDF) 58 cm 2 Using a petri dish (manufactured by IWAKI), normal human fibroblasts (manufactured by Kurashiki Boseki) were seeded in DMEM medium (manufactured by Nissui Pharmaceutical) containing 10% FBS, and cultured at 37 °C and 5% CO 2 under the conditions.

[0122] (Production Example 3) Preparation of conditioned medium from normal human fibroblasts From the state where the normal human fibroblasts cultured in Production Example 2 became confluent, the medium was replaced with fresh KB2 medium (manufactured by Kurashiki Boseki). After culturing for 24 hours, the supernatant was collected and used as the conditioned medium from normal human fibroblasts.

[0123] (Production Example 4) Preparation of conditioned medium containing synthetic melanin The conditioned medium containing synthetic melanin was prepared by adjusting the concentration of synthetic melanin to 100 μg / mL in the conditioned medium of Production Example 3. Note that synthetic melanin was prepared using the raw material name "Melanin" (manufactured by MERCK).

[0124] (Production Example 5) Preparation of conditioned medium containing fluorescent beads The conditioned medium containing fluorescent beads was prepared by adding 500 times the amount of the conditioned medium of Production Example 3 to 1 part by mass of fluorescent beads. Note that the fluorescent beads were TM "FluoSpheres carboxylate-modified microspheres, 0.2 μm, blue fluorescence (365 / 415), 2% solids" (manufactured by ThermoFisher Scientific) was used.

[0125] (Test Example 1) Verification of phagocytosis of synthetic melanin by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0126] The normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate (manufactured by IWAKI) at 90,000 cells / cm 2 and cultured at 37 °C and 5% CO 2It was cultured for 24 hours under the conditions. Then, it was replaced with the conditioned medium prepared in Production Example 4, and further cultured for 6 hours at 37°C under 5% CO 2 conditions. Next, the conditioned medium was removed, and after washing with PBS(-), the cells were visually observed under an optical microscope with a magnification of 100 (manufactured by Olympus). The results are shown in Fig. 1.

[0127] Note that as a control, normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate (manufactured by IWAKI) at a density of 90,000 cells / cm 2 and cultured for 24 hours at 37°C under 5% CO 2 conditions. Then, the medium was replaced with a medium prepared by adjusting the concentration of synthetic melanin to 100 μg / mL in KB2 medium, and further cultured for 6 hours at 37°C under 5% CO 2 conditions, and visually observed.

[0128] As is clear from Fig. 1, in the cells in which the conditioned medium from normal human fibroblasts was brought into contact with normal human epidermal keratinocytes, a clear difference was observed in the degree of presence or absence of synthetic melanin compared to the non-contact control. From this, it can also be seen that phagocytosis is significantly promoted by bringing the conditioned medium from normal human fibroblasts into contact with normal human epidermal keratinocytes.

[0129] (Test Example 2) Verification of phagocytosis of fluorescent beads by bringing the conditioned medium from normal human fibroblasts into contact with normal human epidermal keratinocytes (observation by fluorescence microscope)

[0130] Normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate at a density of 90,000 cells / cm 2 and cultured for 24 hours at 37°C under 5% CO 2 conditions. Then, it was replaced with the conditioned medium prepared in Production Example 5, and further cultured for 6 hours at 37°C under 5% CO 2 conditions. Next, the conditioned medium was removed, and after washing with PBS(-), the cells were visually observed under a fluorescence microscope (product name: IX71, manufactured by OLYMPUS). The results are shown in Fig. 2.

[0131] Note: As for the control, normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate (manufactured by IWAKI) at a density of 90,000 cells / cm 2 so as to achieve a density of 90,000 cells / cm 2 and cultured at 37°C under 5% CO 2 for 24 hours. After that, the medium was replaced with a medium prepared by adding 500 times the amount of KB2 medium to 1 part by mass of fluorescent beads, and further cultured at 37°C under 5% CO

[0132] As is clear from Figure 2, it was confirmed that in cells in which the conditioning medium from normal human fibroblasts was brought into contact with normal human epidermal keratinocytes, the fluorescence intensity incorporated into the cells was higher than that of the non-contact control. From this, it can also be seen that phagocytosis is significantly promoted by bringing the conditioning medium from normal human fibroblasts into contact with normal human epidermal keratinocytes.

[0133] (Test Example 3) Verification of phagocytosis of fluorescent beads by bringing the conditioning medium from normal human fibroblasts into contact with normal human epidermal keratinocytes (measurement using a fluorescence microplate reader)

[0134] Normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate at a density of 90,000 cells / cm 2 so as to achieve a density of 90,000 cells / cm 2 and cultured at 37°C under 5% CO 2 for 24 hours. Then, the medium was replaced with the conditioning medium prepared in Production Example 5, and further cultured at 37°C under 5% CO

[0135] Note: As for the control, normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate (manufactured by IWAKI) at a density of 90,000 cells / cm 2 so as to achieve a density of 90,000 cells / cm 2After culturing for 24 hours under the conditions, the medium was replaced with the prepared medium by adding 500 times the amount of KB2 medium to 1 part by mass of fluorescent beads, and further cultured at 37°C and 5% CO 2 The fluorescence intensity of the cells cultured for 6 hours under the conditions was measured.

[0136] As is clear from Fig. 3, it was confirmed that the fluorescence intensity was higher in the cells in which the conditioned medium from normal human fibroblasts was brought into contact with normal human epidermal keratinocytes than in the non-contact control. From this, it can also be seen that phagocytosis is significantly promoted by bringing the conditioned medium from normal human fibroblasts into contact with normal human epidermal keratinocytes.

[0137] (Test Example 4) Verification of actin reorganization by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0138] Normal human epidermal keratinocytes were seeded on a 4-chamber slide (manufactured by BD Falcon) at 2,500 cells / cm 2 and cultured at 37°C and 5% CO 2 for 24 hours. Then, the medium was replaced with the conditioned medium prepared in Production Example 3, and further cultured at 37°C and 5% CO 2 for 2 to 6 hours. Next, the conditioned medium was removed and the cells were washed with PBS(-). Then, the cells were fixed with 4% formaldehyde, treated with 0.5% Triton-100, and blocked with 1% BSA. 200 μL of 165 nM rhodamine phalloidin (manufactured by FUJIFILM Wako Pure Chemical Corporation) and 20 μM Hoechst 33258 (manufactured by Takara Bio Inc.) dissolved in PBS(-) were added to the cells, stained for 30 minutes, mounted with Entellan New (manufactured by MERCK), and the cells were visually observed and evaluated with a fluorescence microscope (product name: IX71, manufactured by OLYMPUS, rhodamine phalloidin Ex: 550 nm / Em: 580 nm, Hoechst 33258 Ex: 360 nm / Em: 420 nm). The results are shown in Fig. 4. In the figure, the scale bar indicates 50 μm.

[0139] Note that the control was prepared by seeding normal human epidermal keratinocytes on a 4-chamber slide (manufactured by BD Falcon) at 2,500 cells / cm2 Seed so as to obtain [a certain density], and culture for 24 hours under the conditions of 37°C and 5% CO 2 After culturing for 24 hours under the above conditions, without replacing with the conditioned medium prepared in Production Example 3, the cells were visually observed and evaluated in the same manner as above.

[0140] As is clear from FIG. 4, in the cells in which the conditioned medium from normal human fibroblasts was brought into contact with normal human epidermal keratinocytes, it was confirmed that F-actin (filamentous actin) was stained with rhodamine phalloidin in the vicinity of the cells as compared to the non-contact control. From this also, it can be seen that by bringing the conditioned medium from normal human fibroblasts into contact with normal human epidermal keratinocytes, F-actin polymerization is increased (actin reorganization).

[0141] (Test Examples 5, 6) Verification of the inhibitory effect of actin reorganization inhibitors on phagocytosis

[0142] As the test substance in Test Example 5, BAPTA-AM (manufactured by Fujifilm Wako Pure Chemical Corporation), which is a chelating agent, was used (concentration: 5.0 μM, 7.5 μM). As the test substance in Test Example 6, Cytochalasin D (manufactured by Fujifilm Wako Pure Chemical Corporation), which is an actin inhibitor, was used (concentration: 0.3 μM, 0.6 μM).

[0143] Normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate at 90,000 cells / cm 2 Seed so as to obtain [a certain density], and culture for 24 hours under the conditions of 37°C and 5% CO 2 After culturing for 24 hours under the above conditions, replace with the conditioned medium prepared in Production Example 5 containing the test substances in Test Examples 5 and 6, and further culture for 6 hours under the conditions of 37°C and 5% CO 2 Then, remove the conditioned medium, wash with PBS(-), and measure the fluorescence intensity with a fluorescence plate reader (manufactured by Thermo Fisher Scientific) (Ex: 365 nm, Em: 415 nm). The results are shown in FIGS. 5 and 6. "***" in the graph indicates that the p-value of the t-test is less than 0.001. Also, "*" indicates that the p-value of the t-test is less than 0.05.

[0144] In addition, a substance not containing the test substance was used as a control in the same test method as in Test Examples 5 and 6, and the fluorescence intensity was similarly measured with a fluorescence plate reader.

[0145] As is clear from FIGS. 5 and 6, it was confirmed that the cells contacted with the actin rearrangement inhibitor had a lower fluorescence intensity than the non-contacted control. From this, it was confirmed that phagocytosis can be inhibited by contacting with an actin rearrangement inhibitor.

[0146] (Test Example 7) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0147] As the test substances, rooibos extract (trade name: Asparagus linearis, manufactured by Release Kagaku Kogyo Co., Ltd., concentration: 1.0%) and aloe extract (trade name: Asparagus linearis, manufactured by Release Kagaku Kogyo Co., Ltd., concentration: 1.0%) were each used.

[0148] Normal human epidermal keratinocytes cultured in Production Example 1 were seeded in a 96-well plate at a density of 90,000 cells / cm 2 and cultured at 37°C and 5% CO 2 for 24 hours. Then, the conditioned medium prepared in Production Example 5 containing the plant extract as the test substance was replaced, and the cells were further cultured at 37°C and 5% CO 2 for 6 hours. Next, the conditioned medium was removed, and after washing with PBS(-), the fluorescence intensity was measured with a fluorescence plate reader (manufactured by Thermo Fisher Scientific) (Ex: 365 nm, Em: 415 nm). The results are shown in FIG. 7.

[0149] In addition, a substance not containing the test substance was used as a control in the same test method as in Test Example 7, and the fluorescence intensity was similarly measured with a fluorescence plate reader.

[0150] As is clear from Fig. 7, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0151] (Test Example 8) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0152] Using the same test method as in Test Example 7, saffron extract (trade name: Saffron Flow Extract SP, manufactured by Kojima Pharmaceutical Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 8. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 8 above.

[0153] As is clear from Fig. 8, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0154] (Test Example 9) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0155] Using the same test method as in Test Example 7, strawberry extract (trade name: Dermo Fruit Raspberry / N, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) and kiwi extract (trade name: Kiwi Extract Solution, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) were used as the test substances. The results are shown in Fig. 9. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 9 above.

[0156] As is clear from Fig. 9, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0157] (Test Example 10) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0158] Using the same test method as in Test Example 7, dokudami extract (trade name: Jyuyaku-ryu Extract, manufactured by Kotani Pharmaceutical Co., Ltd., concentration: 1.0%) and carrot extract (trade name: Ninjin-ryu Extract P, manufactured by Kotani Pharmaceutical Co., Ltd., concentration: 1.0%) were used as test substances. The results are shown in Fig. 10. Note that a substance not containing a test substance was used as a control in the same test method as in Test Example 10 above.

[0159] As is clear from Fig. 10, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non-contacted control. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0160] (Test Example 11) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0161] Using the same test method as in Test Example 7, hikiokoshi extract (trade name: Enmaisou Extract, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) and loquat extract (trade name: Loquat Extract BG-J, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) were used as test substances. The results are shown in Fig. 11. Note that a substance not containing a test substance was used as a control in the same test method as in Test Example 11 above.

[0162] As is clear from Fig. 11, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non-contacted control. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0163] (Test Example 12) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0164] Using the same test method as in Test Example 7, peach extract (product name: Peach Extract Solution, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) and saxifrage extract (product name: Saxifrage Extract Solution BG, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) were used as the test substances. The results are shown in Fig. 12. In addition, a substance not containing the test substance was used as a control in the same test method as in Test Example 12 above.

[0165] As is clear from Fig. 12, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0166] (Test Example 13) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0167] Using the same test method as in Test Example 7, sansho extract (product name: Falcorex Sansho, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 1.0%), bamboo vinegar extract (product name: T.P. Bamboo Extract, manufactured by Tachibana Bamboo Co., Ltd., concentration: 1.0%), and jujube extract (product name: Jujube Extract Solution K5, manufactured by Komachi Pharmaceutical Co., Ltd., concentration: 1.0%) were used as the test substances. The results are shown in Fig. 13. In addition, a substance not containing the test substance was used as a control in the same test method as in Test Example 13 above.

[0168] As is clear from Fig. 13, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0169] (Test Example 14) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0170] Using the same test method as in Test Example 7, an extract of Usbasaisin (trade name: Falcorex Cysin E, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 14. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 14 above.

[0171] As is clear from Fig. 14, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0172] (Test Example 15) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0173] Using the same test method as in Test Example 7, hydrolyzed wheat gluten protein (trade name: TRITISOL - LQ - (WD), manufactured by Krodal Japan Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 15. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 15 above.

[0174] As is clear from Fig. 15, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0175] (Test Example 16) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0176] Using the same test method as in Test Example 7, an extract of Gorenji (trade name: Starfruit Leaf Extract BG30, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 16. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 16 above.

[0177] As is clear from Fig. 16, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0178] (Test Example 17) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0179] Using the same test method as in Test Example 7, hop extract (trade name: Hop Extract, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 17. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 17 above.

[0180] As is clear from Fig. 17, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0181] (Test Example 18) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0182] Using the same test method as in Test Example 7, red algae extract (trade name: Beta - Helin, manufactured by Technoble Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Fig. 18. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 18 above.

[0183] As is clear from Fig. 18, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0184] (Test Example 19) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0185] Using the same test method as in Test Example 7, Yoshino cherry extract (trade name: Sakura Extract B, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 1.0%) and loquat extract (trade name: Loquat Leaf Extract BG-30, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) were used as test substances. The results are shown in Fig. 19. In addition, a substance not containing a test substance was used as a control using the same test method as in Test Example 19 above.

[0186] As is clear from Fig. 19, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non-contacted control. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0187] (Test Example 20) Inhibitory effect test of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0188] Using the same test method as in Test Example 7, nasturtium extract (trade name: GATULINE EXPRESSION, manufactured by Gattefosse, concentration: 1.0%) was used as a test substance. The results are shown in Fig. 20. In addition, a substance not containing a test substance was used as a control using the same test method as in Test Example 20 above.

[0189] As is clear from Fig. 20, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non-contacted control. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

[0190] (Test Example 21) Inhibitory effect test of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0191] Using the same test method as in Test Example 7, keratin (trade name: KERATEC (Keratec) IFP-HMW, manufactured by Keratec Ltd., concentration: 1.0%) was used as a test substance. The results are shown in Fig. 21. In addition, a substance not containing a test substance was used as a control using the same test method as in Test Example 21 above.

[0192] As is apparent from FIG. 21, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0193] (Test Example 22) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0194] Using the same test method as in Test Example 7, as test substances, Asenyaku extract (trade name: Asenyaku extract solution, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) and Cinnamon bark extract (trade name: Keihi extract solution W - LA, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1.0%) were used. The results are shown in FIG. 22. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 22 above.

[0195] As is apparent from FIG. 22, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0196] (Test Example 23) Test for the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0197] Using the same test method as in Test Example 7, as the test substance, oligopeptide - 20 (trade name: CG - IDP5 BG, manufactured by Caregen Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. 23. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 23 above.

[0198] As is apparent from FIG. 23, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0199] (Test Example 24) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0200] Using the same test method as in Test Example 7, isomerized sugar (trade name: PENTAVITIN, manufactured by DSM, concentration: 1.0%) was used as the test substance. The results are shown in Figure 24. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 24 above.

[0201] As is clear from Figure 24, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0202] (Test Example 25) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0203] Using the same test method as in Test Example 7, button extract (trade name: Button Extract - BG, manufactured by Yamada Pharmaceutical Research, concentration: 1.0%) was used as the test substance. The results are shown in Figure 25. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 25 above.

[0204] As is clear from Figure 25, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity compared to the non - contacted control. Thus, it was confirmed that phagocytosis can be inhibited by contacting with the test substance.

[0205] (Test Example 26) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0206] Using the same test method as in Test Example 7, lychee extract (trade name: Lychee Seed Extract - LC, manufactured by Oriza Oil & Fat Co., Ltd., concentration: 1.0%) was used as the test substance. The results are shown in Figure 26. Note that a substance not containing the test substance was used as a control in the same test method as in Test Example 26 above.

[0207] As is apparent from Fig. 26, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis could be inhibited by contacting the test substance.

[0208] (Test Example 27) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0209] Using the same test method as in Test Example 7, as test substances, lotus extract (1.0%), burdock extract (1.0%), and pithecellobium extract (trade name: Pithecellobium Extract, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) were used. The results are shown in Fig. 27. Note that a sample without the test substance was used as a control in the same test method as in Test Example 27 above.

[0210] As is apparent from Fig. 27, it was confirmed that the cells contacted with the test substance had a lower fluorescence intensity than the non - contacted control. Thus, it was confirmed that phagocytosis could be inhibited by contacting the test substance.

[0211] (Test Example 28) Test for the inhibitory effect on phagocytosis by contacting normal human epidermal keratinocytes with a conditioned medium from normal human fibroblasts

[0212] Using the same test method as in Test Example 7, as test substances, arbutin (manufactured by Nippon Seika Co., Ltd., concentrations: 50 μg / mL, 100 μg / mL), nicotinamide (manufactured by DSM, concentrations: 50 μg / mL, 100 μg / mL), kojic acid (manufactured by Tokyo Chemical Industry Co., Ltd., concentrations: 50 μg / mL, 100 μg / mL), and black tea extract (trade name: Black Tea Liquid, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 0.1%) were used. The results are shown in Fig. 28. Note that a sample without the test substance was used as a control in the same test method as in Test Example 28 above.

[0213] As is apparent from Fig. 28, it was confirmed that the fluorescence intensity of the cells contacted with arbutin, nicotinamide, and kojic acid did not change compared to the non - contacted control. That is, it was found that arbutin, nicotinamide, and kojic acid, which are known to have a whitening effect, have no effect on the induction of phagocytosis of epidermal keratinocytes after contacting the epidermal keratinocytes with the conditioned medium of fibroblasts.

[0214] (Test Examples 29, 30) Inhibitory effect test of phagocytosis by contacting normal human epidermal keratinocytes with the conditioned medium from normal human fibroblasts

[0215] Using the same test method as in Test Example 7, adlay extract (trade name: Yokukinin extract, manufactured by Koei Kogyo Co., Ltd., concentration: 1%), cimicifuga racemosa extract (trade name: Falcorex cimicifuga, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 1%), and alpinia zerumbet extract (trade name: Alpinia zerumbet extract BG, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1%) were used as test substances. The results are shown in Figs. 29 and 30. In addition, a substance not containing the test substance was used as a control in the same test method as in Test Examples 29 and 30 above.

[0216] As is apparent from Figs. 29 and 30, it was confirmed that the fluorescence intensity of the cells contacted with adlay extract, cimicifuga racemosa extract, and alpinia zerumbet extract did not change compared to the non - contacted control. That is, it was found that adlay extract, cimicifuga racemosa extract, and alpinia zerumbet extract, which are known to inhibit melanin production (tyrosinase inhibition), have no effect on the induction of phagocytosis of epidermal keratinocytes after contacting the epidermal keratinocytes with the conditioned medium of fibroblasts.

[0217] From the above results, it was confirmed that the specific plant extracts and specific compounds, which are the active ingredients of the present invention, have an extremely excellent inhibitory effect on actin reorganization and induction of phagocytosis of epidermal keratinocytes after contacting the epidermal keratinocytes with the conditioned medium of fibroblasts, and are useful as a stain - forming inhibitor. In addition, it has become possible to provide a stain - forming inhibitory effect by applying this to a skin external preparation.

[0218] On the other hand, arbutin, nicotinamide, kojic acid, coix seed extract, cimicifuga racemosa extract, and polygonum hydropiper extract, which are known for their whitening effects, showed no effect on the induction of phagocytosis of epidermal keratinocytes after contacting the epidermal keratinocytes with a fibroblast-conditioned medium.

Claims

1. An inhibitor that inhibits phagocytosis by epidermal keratinocytes, characterized in that it contains one or more active ingredients selected from the group consisting of Asarum extract, raspberry extract, kiwi extract, Japanese knotweed extract, lychee extract, red algae extract, Somei-Yoshino cherry extract, myrtle extract, cassia bark extract, peony extract, Japanese pepper extract, bamboo vinegar, aloe extract and saxifrage extract.

2. A pigmentation formation inhibitor used for inhibiting pigmentation formation involving phagocytosis by epidermal keratinocytes, characterized by comprising the phagocytosis inhibitor according to claim 1.

3. 10. An external skin preparation used to inhibit the formation of age spots associated with phagocytosis by epidermal keratinocytes, comprising the age spot formation inhibitor according to claim 2.

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