JP2022058065A - Actin reorganization inhibitor, phagocytosis inhibitor, stain formation inhibitor containing the inhibitor, and external skin preparation containing the inhibitor - Google Patents

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

The present invention relates to a novel actin rearrangement inhibitor, a phagocytosis inhibitor, a stain formation inhibitor containing the inhibitor, and a skin external preparation containing the inhibitor.

Pigmented spots (Nikko Kuroko) that appear with age are important issues, especially in the beauty of the skin for women. UVB-induced hyperpigmentation (UVB melanosis) disappears within 2 weeks to several months after exposure to UVB. On the other hand, Nikko Kuroko occurs on the skin exposed to sunlight, especially on the face, and does not disappear because there is a possibility of DNA damage of epidermal keratinocytes due to repeated UVB irradiation on the epidermis of the lesion. In fact, it is known that the history of UV exposure of an individual is responsible for the appearance of Nikko Kuroko (see, for example, Non-Patent Document 1). Therefore, prevention and improvement of Nikko Kuroko is an important issue for cosmetics, especially for Asian and Caucasian women.

Histological features of Nikko Kuroko have been reported to include destruction of the basement membrane and increased epidermal thickness due to developed reticular ridges (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 Nikko Kuroko (senile pigment spots) (see, for example, Non-Patent Document 4). ). In addition, it has been clarified that the use of tyrosinase antibody significantly increases tyrosinase-positive pigment cells in the lesion epidermis of Nikko kuroko by a factor of 2 (see, for example, Non-Patent Document 5).

The underlying mechanism of pigmentation spots initiated by UV irradiation is broadly divided into enhanced melanin formation in pigment cells and migration of melanosomes to adjacent epidermal keratinocytes. Regarding the enhancement of melanin formation, it has been reported that various cytokines such as endoserin-1 and granulocyte-macrionic colony stimulating factor produced from epidermal keratinocytes act on pigment cells to promote melanin formation ( For example, see Non-Patent Document 6 and Non-Patent Document 7). Destruction of the basement membrane is expected to promote crosstalk between the dermis and the epidermis as well as between the pigment cells and the epidermal keratinocytes. In fact, some studies have highlighted the contribution of fibroblasts to the initiation and maintenance of Nikko Kuroko (see, eg, Non-Patent Document 8).

On the other hand, regarding melanosome metastasis, it has been reported that activation of Toll-like receptors 3 in epidermal keratinized cells promotes melanosomal phagocytosis through activation of the Rho family (for example, non-). See Patent Document 9). The actions of other factors such as epidermal keratinocyte proliferation factor / epidermal keratinocyte proliferation factor receptor have also been reported (see, for example, Non-Patent Document 10).

It is known that it has an inhibitory effect on the formation of spots by inhibiting the migration of melanosomes from pigment cells to epidermal keratinocytes. At this time, it has been reported that epidermal keratinized cells take up melanosomes by phagocytosis (phagocytosis). Further, it is known that fagocytosis caused by the epidermal keratinized cells is suppressed by a plant extract obtained from a plant belonging to the genus Melissa of the Labiatae family containing an unsaturated fatty acid derivative (for example, Patent Document 1). reference). However, the mechanism of action of contacting epidermal keratinocytes with a fibroblast conditioned medium to induce actin rearrangement and fagocytosis of the epidermal keratinocytes is completely unknown. Further, an actin rearrangement inhibitor, a phagocytosis inhibitor, and a stain formation inhibitor containing these inhibitors, which suppress the mechanism of action, are not known at all. Furthermore, it is not known at all that a specific plant extract or a specific compound has the action of the inhibitor.

Japanese Unexamined Patent Publication No. 2011-68575

E. Holzle, Br J Dermatol. , 127 Suppl 41, 48-50 (1992) S. Iriyama, T.I. Ono, H. Aoki, S.A. Amano, J Dermatol Sci. , 64 (3), 223-228 (2011) H. Hattori, M. et al. Kawashima, Y.M. Ichikawa, G.M. Imokawa, J Invest Dermatol. , 122 (5), 1256-65 (2004) W. Choi, L. et al. Yin, C.I. Smuda, J.M. Batter, V.I. J. Hearing, L. Kolbe, Exp Dermatol. , 26 (3), 242-248 (2017) S. Kadono, I. Manaka, M. et al. Kawashima, T.W. Kobayashi, G.M. Imokawa, J Invest Dermatol. , 116 (4), 571-7 (2001) G. Imokawa, M.M. Miyagishi, Y.M. Yada, J Invest Dermatol. , 105 (1), 32-7 (1995) G. Imokawa, Y. Yada, M. et al. Kimura, N.M. Morisaki, Biochem J. et al. , 313 (2): 625-31 (1996) N. Chen, Y. Hu, W. H. Li, M. Eisinger, M. et al. Seiberg, C.I. B. Lin, Exp Dermatol. , 19 (10), 856-72 (2010) S. Koike, K.K. Yamasaki, T.M. Yamauchi, R.M. Shimada-Omori, K.K. Tsuchiyama, H. et al. Ando, S.A. Aiba, J Dermatol Sci. 96 (3), 168-177 (2019) G. Cardinali, S.A. Ceccarelli, D.I. Kovacs, N. et al. Aspite, L. et al. V. Lotti, M. et al. R. Torrisi, M. et al. Picardo, J. et al. Invest. Dermatol. 125,1190-1199 (2005)

Therefore, the present invention is an actin rearrangement inhibitor, phagocytosis inhibition, in which epidermal keratinocytes are brought into contact with an acclimatized medium of fibroblasts to inhibit actin rearrangement and induction of fagocytosis of the epidermal keratinocytes. To provide the agent. Further, the present invention is to provide a stain formation inhibitor containing the inhibitor and a skin external preparation containing the inhibitor.

In view of these circumstances, the present inventors have sought a method for preventing or ameliorating the occurrence of stain formation after contacting epidermal keratinized cells with an conditioned medium for fibroblasts, and made diligent efforts. As a result, Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Momo extract, Yukinoshita extract, Sansho extract , Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Factors directly linked to the prevention or improvement of stain formation in Asenyaku extract, Cassia bark extract, Oligopeptide-20, isomerized sugar, Button extract, Litchi extract, Hass extract, Bringalage extract, Pitetherobium extract We have found an inhibitory effect on actin rearrangement and an inhibitory effect on phagocytosis, and have completed the invention. That is, the present invention is as shown below.

That is, the present invention
(1) An inhibitor that inhibits the reorganization of the actin of the epidermal keratinized cells by contacting the epidermal keratinized cells with an acclimatized medium of fibroblasts, and is an inhibitor that inhibits the rearrangement of the actin of the epidermal keratinized cells. Matter, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Momo extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usubasaishin Extract, hydrolyzed wheat protein, gorenshi extract, hop extract, red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, keratin, Asenyaku extract, Cassia bark extract, oligopeptide- 20. Actin reorganization characterized by containing one or more selected from isomerized sugar, button extract, lychee extract, hass extract, blingaraj extract, and pitecerobium extract as an active ingredient. Inhibitor,
(2) A stain formation inhibitor, which comprises the actin reorganization inhibitor according to (1) above.
(3) An inhibitor that inhibits fagocytosis of epidermal keratinized cells by contacting the epidermal keratinized cells with an acclimatized medium of fibroblasts, and is an inhibitor that inhibits fagocytosis of the epidermal keratinized cells, such as Louis Boss extract, Aloe extract, Ogon extract, and strawberry. Extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, Heterogeneous A phagocytosis inhibitor characterized by containing one or more selected from sucrose, button extract, lychee extract, hass extract, blingaraj extract, and pitecerobium extract as an active ingredient.
(4) A stain formation inhibitor, which comprises the phagocytosis inhibitor according to (3) above.
(5) An external skin preparation, which comprises the stain formation inhibitor according to (2) or (4).
Regarding.

Specific plant extracts and specific compounds, which are the active ingredients of the present invention, are used to induce actin rearrangement and fagocytosis of epidermal keratinized cells after contacting the epidermal keratinized cells with an conditioned medium for fibroblasts. It has a remarkably excellent inhibitory effect on the cells, and exerts a remarkably excellent effect that the inhibitory effect effectively acts on the suppression of stain formation.

In addition, since it exerts a remarkably excellent inhibitory effect on actin rearrangement and phagocytosis induction involved in the formation of age spots, it can be suitably used as an external skin agent such as a whitening agent that does not cause age spots. It works.

(A) is a drawing-substituting photograph showing a cell group in which synthetic melanin was administered to normal human epidermal keratinocytes in Test Example 1 without contacting the conditioned medium from normal human fibroblasts. (B) is a drawing-substituting photograph showing a cell group in which synthetic melanin was administered by contacting normal human epidermal keratinocytes with an conditioned medium from normal human fibroblasts in Test Example 1. In the drawings of the present specification, the normal human epidermal keratinized cells are abbreviated as "NHEK". Incidentally, "NHEK" is an abbreviation for Normal Human Epidermal Keratinocytes. (A) is a drawing-substituting photograph showing a cell group in which fluorescent beads were administered to normal human epidermal keratinocytes in Test Example 2 without contacting the conditioned medium from normal human fibroblasts. (B) is a drawing-substituting photograph showing a cell group to which fluorescent beads were administered by contacting normal human epidermal keratinocytes with an conditioned medium from normal human fibroblasts in Test Example 2. In Test Example 3, it is a graph which shows the difference in fluorescence intensity depending on whether or not a normal human epidermal keratinized cell is brought into contact with an conditioned medium from a normal human fibroblast. (A) is a drawing substitute photograph in Test Example 4 in which normal human epidermal keratinocytes were stained without contacting the conditioned medium from normal human fibroblasts. (B) is a drawing-substituting photograph in which normal human epidermal keratinocytes were contacted with an conditioned medium from normal human fibroblasts for 2 hours and stained in Test Example 4. (C) is a drawing-substituting photograph in which normal human epidermal keratinocytes were contacted with an conditioned medium from normal human fibroblasts for 4 hours and stained in Test Example 4. (D) is a drawing-substituting photograph in which normal human epidermal keratinocytes were contacted with an conditioned medium from normal human fibroblasts for 6 hours and stained in Test Example 4. It is a graph which shows that BAPTA-AM (chelating agent) inhibited phagocytosis in Test Example 5. It is a graph which shows that Cytochalasin D (actin polymerization inhibitor) inhibited phagocytosis in Test Example 6. 6 is a graph showing that phagocytosis in rooibos extract or aloe extract was inhibited in Test Example 7. It is a graph which shows that the Scutellaria baicalensis extract inhibited phagocytosis in Test Example 8. It is a graph which shows that the phagocytosis in the raspberry extract or the kiwi extract was inhibited in Test Example 9. It is a graph which shows that the phagocytosis in the Houttuynia cordata extract or the Panax ginseng extract was inhibited in Test Example 10. It is a graph which shows that phagocytosis in the Hikiokoshi extract or the loquat extract was inhibited in Test Example 11. It is a graph which shows that phagocytosis in the peach extract or the Saxifraga stolon extract was inhibited in Test Example 12. It is a graph which shows that phagocytosis in the Japanese pepper extract, the bamboo vinegar extract, or the lychee extract was inhibited in Test Example 13. It is a graph which shows that the ginger extract inhibited phagocytosis in Test Example 14. It is a graph which shows that hydrolyzed wheat protein inhibited phagocytosis in Test Example 15. It is a graph which shows that the carambola extract inhibited phagocytosis in Test Example 16. It is a graph which shows that the hop extract inhibited phagocytosis in Test Example 17. It is a graph which shows that the red algae extract inhibited phagocytosis in Test Example 18. It is a graph which shows that phagocytosis in the Yoshino cherry tree extract or the rose-apple extract was inhibited in Test Example 19. It is a graph which shows that the Kibana Paracress extract inhibited phagocytosis in Test Example 20. It is a graph which shows that keratin inhibited phagocytosis in Test Example 21. It is a graph which shows that Phagocytosis in the cinnamon extract or the cassia bark extract was inhibited in Test Example 22. It is a graph which shows that oligopeptide-20 inhibited phagocytosis in Test Example 23. It is a graph which shows that the isomerized sugar inhibited phagocytosis in Test Example 24. It is a graph which shows that the button extract inhibited phagocytosis in Test Example 25. It is a graph which shows that the lychee extract inhibited phagocytosis in Test Example 26. It is a graph which shows that phagocytosis in the lotus extract, the Bringalage extract or the pitecerobium extract was inhibited in Test Example 27. It is a graph which shows that arbutin, nicotinamide or kojic acid could not suppress phagocytosis in Test Example 28. It is a graph which shows that the Coix seed extract could not inhibit phagocytosis in Test Example 29. It is a graph which shows that the rockweed extract or the shell ginger extract could not inhibit phagocytosis in Test Example 30.

Hereinafter, embodiments of the present invention will be described.

The inhibitor of the present invention that inhibits the induction of actin rearrangement and fagocytosis, the stain formation inhibitor composed of the inhibitor, and the external skin preparation containing the inhibitor can be used as a specific plant extract or a specific compound. It is contained as an active ingredient.

[Actin reorganization inhibition]
The present inventors have found that when epidermal keratinocytes are contacted with an acclimatized medium from fibroblasts, actin rearrangement in epidermal keratinocytes is induced. Furthermore, we found that actin reorganization induced by contact with conditioned medium from fibroblasts is inhibited by actin reorganization inhibitors and that actin reorganization inhibitors have an inhibitory effect on fagocytosis. We found that there was a correlation with expression. The present invention is based on these findings.

Inhibition of actin rearrangement in the present invention is characterized by inhibiting actin rearrangement induced by contacting epidermal keratinized cells with an conditioned medium from fibroblasts.

Examples of the epidermal keratinocytes include 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 being able to evaluate the test substance more accurately with a view to application to human skin. The human epidermal keratinocyte is not particularly limited to a normal human epidermal keratinocyte cell or an immortalized human epidermal keratinocyte cell, but the phenomenon of actin rearrangement can be more accurately grasped and evaluated accurately. From the viewpoint, it is more preferable to use normal human epidermal keratinocytes.

Examples of the 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 being able to evaluate the test substance more accurately with a view to application to human skin. The human fibroblast is not particularly limited to normal human fibroblast or immortalized human fibroblast, but from the viewpoint of more accurately grasping the phenomenon of actin rearrangement and being able to evaluate it accurately. It is more preferable to use normal human fibroblasts.

The conditioned medium for fibroblasts is, for example, replaced with fresh KB2 medium or DMEM (5% FBS) medium from a state in which fibroblasts are confluent in 58 cm ² petri dishes, and then cultured for 12 to 36 hours. , The culture medium collected from the supernatant can be used. The conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficiently inducing actin rearrangement, it is preferable to use a medium obtained by culturing for 18 to 30 hours and then recovering the supernatant.

The epidermal keratinocytes can be seeded, for example, on a 4-chamber slide at a rate of 500 to 5,000 cells / cm ² . In the present invention, from the viewpoint of facilitating the observation of the induction of actin rearrangement, it is preferable to sow so as to be 2,000 to 4,000 pieces / cm ² .

The time for contacting the epidermal keratinized cells with the acclimatized medium from the fibroblasts is not particularly limited, but from the viewpoint of sufficiently inducing actin rearrangement by contacting the acclimatized medium from the fibroblasts, the fibroblasts. The conditioned medium from the cells is preferably contacted for 2 to 24 hours, more preferably 4 to 8 hours.

As used herein, the term "induction of actin rearrangement by contact with an acclimatized medium from fibroblasts" refers to the epidermal keratinization in response to stimulation of the epidermal keratinized cells by the acclimatized medium from the fibroblasts. It means that the actin rearrangement in the cells is more prominently inducing the actin rearrangement than the actin rearrangement in the epidermal keratinized cells which are not in contact with the acclimatized medium from the fibroblasts.

The induction of actin rearrangement by contact with the acclimatized medium from the fibroblasts can be confirmed, for example, by staining the actin polymerization of epidermal keratinized cells and observing with a fluorescence microscope. The reagent capable of staining actin polymerization is not particularly limited, and examples thereof include rhodamine phalloidin and commercially available actin antibodies.

Since the present invention can accurately observe and evaluate the phenomenon that actin rearrangement is induced by contacting epidermal keratinized cells with an acclimatized medium from fibroblasts, the presence or absence of an inhibitory effect on actin rearrangement of the test substance. Can be accurately evaluated. That is, it can be suitably used as a screening method for actin rearrangement inhibitors.

[Inhibition of phagocytosis]
When the epidermal keratinocytes are contacted with an acclimatized medium from fibroblasts, the present inventors induce fagocytosis not only from the above-mentioned actin rearrangements but also from actin rearrangements in the epidermal keratinocytes. I also found that. Furthermore, we found that phagocytosis induced by contact with conditioned medium from fibroblasts is inhibited by phagocytosis inhibitors, actin reorganization inhibitors, and phagocytosis inhibitors, It was found that there is a correlation with the expression of the stain formation inhibitory effect of actin reorganization inhibitors. The present invention is based on these findings.

The above-mentioned phagocytosis means phagocytosis, phagocytosis, etc. of epidermal keratinocytes. Inhibition of phagocytosis in the present invention is characterized by inhibiting phagocytosis induced by contacting epidermal keratinized cells with an conditioned medium from fibroblasts.

Examples of the epidermal keratinocytes include 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 being able to evaluate the test substance more accurately with a view to application to human skin. The human epidermal keratinocyte is not particularly limited to a normal human epidermal keratinocyte or an immortalized human epidermal keratinocyte, but the phenomenon of fagocytosis can be more accurately grasped and evaluated accurately. From the viewpoint, it is more preferable to use normal human epidermal keratinocytes.

Examples of the 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 being able to evaluate the test substance more accurately with a view to application to human skin. The human fibroblast is not particularly limited to normal human fibroblast or immortalized human fibroblast, but from the viewpoint of more accurately grasping the phenomenon of phagocytosis and being able to evaluate it accurately. It is more preferable to use normal human fibroblasts.

The conditioned medium for fibroblasts is, for example, replaced with fresh KB2 medium or DMEM (5% FBS) medium from a state in which fibroblasts are confluent in 58 cm ² petri dishes, and then cultured for 12 to 36 hours. , The culture medium collected from the supernatant can be used. The conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to use a medium obtained by culturing for 18 to 30 hours and then collecting the supernatant.

The epidermal keratinized cells can be seeded, for example, in a 96-well plate or a 24-well plate at a rate of 50,000 to 130,000 cells / cm ² . In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to sow so as to be 70,000 to 110,000 seeds / cm ² .

The time for contacting the epidermal keratinized cells with the conditioned medium from the fibroblasts is not particularly limited, but from the viewpoint of sufficiently inducing phagocytosis by contacting the conditioned medium from the fibroblasts, the fibroblasts The conditioned medium from the cells is preferably contacted for 2 to 24 hours, more preferably 4 to 8 hours.

As used herein, "induction of phagocytosis by contact with an acclimatized medium from fibroblasts" refers to the epidermal keratinization in response to stimulation of epidermal keratinized cells by the acclimatized medium from the fibroblasts. It means that the amount of fine particles taken up by the cells is significantly larger than the amount of fine particles taken up by the epidermal keratinized cells that are not in contact with the acclimatized medium from the fibroblasts. In addition, "significant" means that the p-value of the t-test is less than 0.05, preferably less than 0.01.

Further, in the present specification, "inhibition of phagocytosis" means that the cell viability is 90% or more (compared to the case where the cell viability of the epidermal keratinized cells in contact with the conditioned medium is 100%). Moreover, it means that the epidermal keratinized cells contacted with the acclimatized medium containing the test substance have a fluorescence intensity of 50% or less as compared with the epidermal keratinized cells contacted with the acclimatized medium containing no test substance. ..

Examples of the fine particles include melanosomes, melanin, and color-developing substances.

As the melanosomes, any pigment cells derived from humans or non-human animals may be used. In the present invention, it is preferable to use one prepared from homogenates of human-derived pigment cells.

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

Fluorescent beads can be used as the coloring substance. Fluorescent beads "FluoroSpheres ^TM carboxylate modified microspheres, 0.2 μm, blue fluorescence (365/415), 2% solids" are available from Thermo Fisher Scientific (Waltham, Mass., 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.

The induction of phagocytosis by contact with the conditioned medium from the fibroblasts can be evaluated, for example, by measuring the fluorescent beads taken up by the epidermal keratinized cells with a fluorescent plate reader.

Since the present invention can accurately observe and evaluate the phenomenon that phagocytosis is induced by contacting epidermal keratinized cells with an acclimatized medium from fibroblasts, the presence or absence of phagocytosis-inhibiting action of the test substance is possible. Can be accurately evaluated. That is, it can be suitably used as a screening method for phagocytosis inhibitors.

As described above, the present invention can accurately observe and evaluate the actin rearrangement induced by contacting epidermal keratinized cells with a conditioned medium from fibroblasts, and the phenomenon of fagocytosis. That is, the present invention can accurately observe and evaluate the actin rearrangement involved in the formation of stains and the phenomenon of phagocytosis, and determine the presence or absence of the actin rearrangement of the test substance and the inhibitory effect on phagocytosis. It can be confirmed and evaluated accurately.

Further, according to the present invention, an inhibitor that exerts a remarkably excellent inhibitory effect on actin rearrangement and fagocytosis induction involved in the formation of spots is useful as a spot formation inhibitor, and the inhibitor. Can be suitably used as an external skin preparation such as a whitening agent that does not cause spots.

[Test substance]
The inhibitor, inhibitor, and external 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 exerting a remarkably excellent inhibitory effect, Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami Extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract , Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch sennichi extract, keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, isomerized sugar, button extract, lychee It is preferable to contain one or more extracts selected from an extract, a hass extract, a blingalage extract, and a pitecellobium extract, and a compound as an active ingredient. In the present invention, these extracts and a mixed raw material containing a compound may be used.

As used herein, the term "extract" refers to an extract obtained by extracting each natural product or the like with various solvents as an extraction raw material, a diluted solution of the extract, a concentrated solution of the extract, and drying of the extract. It includes any form such as a dried product obtained by drying the extract and a purified product obtained by drying the extract.

The extracts and compounds used in the present invention include Louis Boss (scientific name: Aspalathus Linearis), Aloe (scientific name: Aloe), Ogon (Koganebana) (scientific name: Scutellaria Bacillensis), Kistrawberry (scientific name: Rubus), Kiwi (scientific name: Actini). Chinniss), Dokudami (scientific name: Houttuynia Cordata), Otaneninjin (scientific name: Panax Ginseng), Hikiokoshi (scientific name: Isodon Japannicus), Biwa (scientific name: Eriobotria Pana) , Sansho (scientific name: Zanthoxylum Piperitum), bamboo vinegar, Reishi (scientific name: Gandormataceae), Usbasaicin (scientific name: Asalum Sieboldii), hydrolyzed wheat protein, Gorenshi (scientific name: AverrhoaHulabul) Red algae (scientific name: Rhodophyta), Somei Yoshino (scientific name: Prunus Yedouensis), Futomomo (scientific name: Sysygium Jambos), Kibana Holland Sennichi (scientific name: Acella Oleracea), keratin (Cinnamomum Cassia), oligopeptide-20, isomerized sugar, button (scientific name: Paeonia Sufruticosa), lyche (scientific name: Litchi Chinnensis), hass (scientific name: Nelumbo Nucifera), blingaraji (takasaburo) Pitecellovium (scientific name: Pitecellobium).

Rooibos (scientific name: Cape gorses Linearis) is cultivated in South Africa and is readily available from this region.

Aloe (scientific name: Aloe) is cultivated in South Africa and is readily available from this region.

Scutellaria baicalensis (scientific name: Scutellaria Baicalensis) is cultivated in Russia, China, Mongolia and the Korean Peninsula, and is easily available from these regions.

Rubus (scientific name: Rubus) is abundant in the boreal to temperate zones of the Northern Hemisphere. It is readily available from these areas.

Kiwi (scientific name: Actinidia chinensis) is cultivated in Italy, China, New Zealand and Japan and is readily available from these regions.

Houttuynia (scientific name: Houttuynia Cordata) is cultivated in Japan and is easily available from this area.

Panax ginseng (scientific name: Panax Ginseng) is cultivated in China and the Korean Peninsula, and is easily available from these areas.

Isodon japonicus (scientific name: Isodon Japanicus) is cultivated in Japan and is easily available from this area.

Loquat (scientific name: Eriobotrya Japanica) is cultivated in China and Japan, and is easily available from these regions.

Peach (scientific name: Plumus Persica) is cultivated in China, the United States, Italy and Japan, and is easily available from these regions.

Yukinoshita (scientific name: Saxifraga stolonifera) is cultivated in China and Japan, and is easily available from these regions.

Zanthoxylum (scientific name: Zanthoxylum Piperitum) is cultivated in the Korean Peninsula, Japan, and is easily available from these areas.

Bamboo vinegar is a liquid obtained by cooling the smoke produced when baking bamboo charcoal, and is easily available.

Litchi (Reishi) (scientific name: Ganodermataceae) is cultivated in China and Japan, and is easily available from these regions.

Wild ginger (scientific name: Asalum Sieboldi) is cultivated in China, the Korean Peninsula, and Japan, and is easily available from these regions.

Hydrolyzed wheat protein is obtained by hydrolyzing wheat protein by acid, enzyme or other methods and is easily available.

Carambola (scientific name: Averhoroa Carambola) is cultivated in China, Brazil and Japan and is readily available from these regions.

Hops (scientific name: Humulus Lupus) are cultivated in Germany, the United States, the Czech Republic, the United Kingdom, and Japan, and are readily available from these regions.

Red algae (scientific name: Rhodophyta) mostly grow on coastal reefs and are readily available from these areas.

Yoshino cherry tree (scientific name: Plumus Yedonsis) is cultivated in Japan and is easily available from this area.

Futomomo (scientific name: Syzygium Jambos) is cultivated in Japan and is easily available from this area.

Kibana Paracress (scientific name: Amella Olacea) is cultivated in Japan and is readily available from this region.

Keratin is a protein obtained from wool and is readily available.

Asenyaku (Gambir) (scientific name: Uncaria Gambir) is cultivated in Japan and is easily available from this area.

Cinnamomum Cassia is cultivated in China, India and Indonesia and is readily available from these regions.

Oligopeptide-20 is a synthetic peptide consisting of 12 amino acids composed of alanine, arginine, cysteine, glutamic acid, leucine, lysine, methionine, proline and tyrosine, and is readily available.

High fructose corn syrup is a carbohydrate complex formed by rearranging a saccharide mixture by base catalysis and is easily available.

Moutan peony (scientific name: Paeonia Sufruticosa) is cultivated in China and Japan and is easily available from these regions.

Lychee (scientific name: Litchi Chineseis) is cultivated in China and is readily available from this region.

Lotus (scientific name: Nelumbo Nucifera) is cultivated in India and Japan and is easily available from these regions.

False daisy (scientific name: Eclipta Prostrata) is cultivated in China, the Korean Peninsula, and Japan, and is readily available from these regions.

Pitecellobium (scientific name: Pitecellobium) is a tree-based plant that grows into a tree of the genus Kinkiju of the family Leguminosae, in which about 200 species are differentiated in the tropical zone, and is easily available.

Examples of the part that can be used as the extraction raw material include a leaf part, a bark part, a stem part, a stem part, a root part, a flower part, a fruit part, a pericarp part, a seed part, a whole plant, or a mixture of these parts. ..

For example, when the above raw material is extracted, it can be obtained by drying it, crushing it as it is or using a coarse crusher, and subjecting it to extraction with an extraction solvent. Drying may be carried out in the sun or by using a commonly used dryer. Further, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment using the polar solvent of the extraction raw material can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent and the like, and these are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferable.

Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmoregulation, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

Hydrophilic organic solvents that can be used as the extraction solvent 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; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be carried out according to a conventional method. For example, an extract can be obtained by immersing the extraction raw material in an extraction solvent, extracting the soluble component at room temperature or under reflux heating, and then filtering to remove the extraction residue. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

Since the extract obtained as described above has an excellent actin reorganization inhibitory action and a fagocytosis inhibitory action, it is a stain composed of an actin reorganization inhibitor, a fagocytosis inhibitor, and the inhibitor. It can be used as an active ingredient of a formation inhibitor and an external skin preparation containing the inhibitor.

In the present invention, Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Momo extract, Yukinoshita extract. , Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Holland Sennichi One of the following: extract, keratin, asenyaku extract, cassia bark extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, hass extract, bringarage extract, pitecerobium extract. May be used as the above-mentioned active ingredient, or these may be mixed and used as the above-mentioned active ingredient.

The stain formation inhibitor of the present invention includes Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita. Extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Holland One of Sennichi extract, keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, isomerized sugar, button extract, lychee extract, hass extract, blingaraj extract, and pitecerobium extract. Alternatively, it may be a formulation of a mixture thereof.

The actin reorganization inhibitor and the phagocytosis inhibitor of the present invention may be powdered, granular, tableted, liquid or the like according to a conventional method using a pharmaceutically acceptable carrier or any other auxiliary agent. It can be formulated into the dosage form of.

When the actin rearrangement inhibitor of the present invention and the fagocytosis inhibitor are formulated, Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi Extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract Things, Somei Yoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, isomerized sugar, Button extract, Litchi extract, Hass extract, Brin The content of any one of the garage extract and the pitetherobium extract, or a mixture thereof, is not particularly limited and may be appropriately set according to the intended purpose.

In addition, the actin reorganization inhibitor of the present invention and the fagocytosis inhibitor can be obtained by, if necessary, using other natural extracts having an actin rearrangement inhibitory action and a fagocytosis inhibitory action. Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark As an active ingredient, it is blended with any one of the extract, oligopeptide-20, isomerized sugar, button extract, lychee extract, hass extract, blingaraji extract, pitecellobium extract, or a mixture thereof. Can be used.

The actin rearrangement inhibitor and the fagocytosis inhibitor of the present invention include Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, and Biwa. Extract, peach extract, yukinoshita extract, sansho extract, bamboo vinegar extract, leishi extract, usbasaicin extract, hydrolyzed wheat protein, gorenshi extract, hop extract, red algae extract, someiyoshino extract Extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, isomerized sugar, Button extract, Litchi extract, Hass extract, Bringalage extract, The stain can be prevented, treated or ameliorated through the actin reorganization inhibitory action and the phagocytosis inhibitory action of the pitecerobium extract.

In addition, the actin reorganization inhibitor and the phagocytosis inhibitor of the present invention can be used as an active ingredient of a drug for preventing / treating a disease caused by abnormal production of melanin or a quasi-drug. However, the actin reorganization inhibitor and the phagocytosis inhibitor of the present invention should be used for all uses other than these uses that are significant in exerting the actin reorganization inhibitory action and the phagocytosis inhibitory action. Can be done.

In addition, the actin reorganization inhibitor and the fagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory action and fagocytosis inhibitory action, and are therefore suitable for blending with, for example, an external preparation for skin. .. In this case, Louis Boss extract, Aloe extract, Ogon extract, Kistrawberry extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Momo extract, Yukinoshita extract, San Show extract, bamboo vinegar extract, leishi extract, usbasaicin extract, hydrolyzed wheat protein, gorenshi extract, hop extract, red algae extract, someiyoshino extract, futomomo extract, kibana Dutch sennichi extract , Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, Iisomerized sugar, Button extract, Litchi extract, Hass extract, Bringalage extract, Pitecerobium extract, or any of these May be blended with the mixture of.

The content of the above plant extract when used in combination with an external preparation for skin is not particularly limited as long as it can exert a desired effect, but 0.00000001 to 5% by mass is blended as an extraction raw material in the total amount of the external preparation. It is preferably 0.0000001 to 3% by mass, more preferably 0.000001 to 2% by mass, and particularly preferably 0.00001 to 1% by mass.

Here, the external skin preparation is not limited in its classification, and includes a wide range of skin cosmetics, non-pharmaceutical products, pharmaceuticals, etc. used percutaneously, and specifically, for example, an ointment. Examples include external skin preparations such as creams, milky lotions, beauty liquids, lotions, packs, foundations, lip creams, and bathing agents.

Further, since the actin reorganization inhibitor and the fagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory action and fagocytosis inhibitory action, they can also be used as reagents for research related to the melanin production mechanism. It can be suitably used.

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

A specific example of the test substance screening method is a test substance screening method for evaluating the stain formation inhibitory effect of the test substance, wherein (A) the epidermal keratinized cells are brought into contact with the fine particles and the test substance, and the contact is made. From the step of contacting the epidermal keratinized cells with the acclimatized medium from the fibroblasts before, simultaneously, or after, (B) the fine particle uptake activity of the epidermal keratinized cells obtained in the above step (A), the epidermal keratinized cells Steps for measuring the uptake amount of fine particles, (C) Based on the uptake amount of fine particles of epidermal keratinized cells measured in the step (B), inhibition of fagocytosis of epidermal keratinized cells was investigated, and the above-mentioned test substance was used. The steps for evaluating the stain formation inhibitory effect can be exemplified.

According to the above screening method, the epidermal keratinocytes are brought into contact with the fine particles and the test substance, and the epidermal keratinocytes are brought into contact with the acclimatized medium from the fibroblasts before, simultaneously, or after the contact. Whether or not the test substance inhibits fagocytosis of epidermal keratinocytes can be rapidly and easily evaluated. Further, based on the presence or absence of inhibition of phagocytosis of epidermal keratinocytes and the degree of inhibition, the stain formation inhibitory action of the test substance can be quickly and easily evaluated.

In step (A) above, the epidermal keratinocytes are contacted with the fine particles and the test substance, and the epidermal keratinocytes are contacted with the acclimatized medium from the fibroblasts before, simultaneously, or after the contact.

Examples of the epidermal keratinocytes include epidermal keratinocytes derived from humans or non-human animals. The epidermal keratinocytes used in the above screening method are preferably human epidermal keratinocytes from the viewpoint of being able to evaluate the test substance more accurately with a view to application to human skin. The human epidermal keratinocyte is not particularly limited to a normal human epidermal keratinocyte or an immortalized human epidermal keratinocyte, but the phenomenon of fagocytosis can be more accurately grasped and evaluated accurately. From the viewpoint, it is more preferable to use normal human epidermal keratinocytes.

Examples of the fibroblasts include any fibroblasts derived from humans or non-human animals. The fibroblasts used in the above screening method are preferably human fibroblasts from the viewpoint of more accurately evaluating the test substance in view of application to human skin. The human fibroblast is not particularly limited to normal human fibroblast or immortalized human fibroblast, but from the viewpoint of more accurately grasping the phenomenon of phagocytosis and being able to evaluate it accurately. It is more preferable to use normal human fibroblasts.

Examples of the fine particles include melanosomes, melanin, and color-developing substances.

As the melanosomes, any pigment cells derived from humans or non-human animals may be used. In the present invention, it is preferable to use one prepared from homogenates of human-derived pigment cells.

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

Fluorescent beads can be used as the coloring substance. Fluorescent beads "FluoroSpheres ^TM carboxylate modified microspheres, 0.2 μm, blue fluorescence (365/415), 2% solids" are available from Thermo Fisher Scientific (Waltham, Mass., 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.

The type of the test substance used in contacting the epidermal keratinized cells with the test substance is not particularly limited, and for example, small molecule compounds, plant extracts, natural extract 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 unconditionally determined.

The conditioned medium for fibroblasts is, for example, replaced with fresh KB2 medium or DMEM (5% FBS) medium from a state in which fibroblasts are confluent in 58 cm ² petri dishes, and then cultured for 12 to 36 hours. , The culture medium collected from the supernatant can be used. The conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to use a medium obtained by culturing for 18 to 30 hours and then collecting the supernatant.

The epidermal keratinized cells can be seeded, for example, in a 96-well plate or a 24-well plate at a rate of 50,000 to 130,000 cells / cm ² . In the present invention, from the viewpoint of sufficiently inducing phagocytosis, it is preferable to sow so as to be 70,000 to 110,000 seeds / cm ² .

The time for contacting the epidermal keratinized cells with the conditioned medium from the fibroblasts is not particularly limited, but from the viewpoint of sufficiently inducing phagocytosis by contacting the conditioned medium from the fibroblasts, the fibroblasts The conditioned medium from the cells is preferably contacted for 2 to 24 hours, more preferably 4 to 8 hours.

The timing of contacting the epidermal keratinocytes with the acclimatized medium from the fibroblasts may be before, at the same time, or after the contact of the fine particles of the epidermal keratinocytes and the test substance. In the present invention, from the viewpoint of increasing the accuracy of the screening method, it is preferable to bring the fine particles and the test substance into contact with each other during the contact with the conditioned medium from the fibroblasts. That is, the timing of contacting the epidermal keratinocytes with the acclimatized medium from the fibroblasts is preferably performed at the same time as the contact of the fine particles of the epidermal keratinocytes and the test substance. In this case, it can be done by mixing fine particles and a test substance with a conditioned medium from fibroblasts and contacting them with epidermal keratinized cells. Further, from the viewpoint of further improving the accuracy of the screening method, contact is preferably performed for 2 hours to 24 hours, and more preferably contacted for 4 hours to 8 hours. From the viewpoint of suppressing the load on cell growth and survival, it is preferably 24 hours or less.

Next, in step (B), the amount of fine particles taken up by epidermal keratinocytes is measured from the fine particle uptake activity of epidermal keratinocytes obtained in step (A).

Examples of the method for measuring the amount of fine particles taken up by the epidermal keratinocytes include a method of measuring the fluorescent beads taken up by the epidermal keratinocytes with a fluorescent bullet reader.

Next, in step (C), based on the amount of fine particles taken up by epidermal keratinocytes measured in step (B), inhibition of fagocytosis of epidermal keratinocytes was investigated, and stain formation of the test substance was suppressed. Evaluate the effect.

Whether or not the test substance inhibits phagocytosis is determined by, for example, comparing the fluorescence intensity of phagocytosis measured in step (B) with the fluorescence intensity of phagocytosis not in contact with the test substance. It can be investigated by doing.

In step (C), if the fluorescence intensity of phagocytosis measured in step (B) is significantly lower than, for example, the fluorescence intensity of phagocytosis not in contact with the test substance, the test is performed. It can be determined that the substance inhibits phagocytosis.

As described above, when it is determined that the test substance inhibits phagocytosis, it can be evaluated that the test substance is a substance exhibiting a spot formation inhibitory action. Further, the larger the difference between the fluorescence intensity of phagocytosis measured in step (B) and the fluorescence intensity of phagocytosis of the cell group not in contact with the test substance, the more the test substance has an effect of suppressing the formation of stains. Can be evaluated as having.

As described above, according to the above screening method, the stain formation inhibitory effect of the test substance can be easily evaluated based on the presence or absence of inhibition of phagocytosis and the degree of inhibition. It is possible to perform screening of a stain formation inhibitor useful for the purpose and evaluation of a stain formation inhibitory action such as a stain formation inhibitor quickly and with a simple operation.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to such examples.

(Production Example 1) Normal human epidermal keratinocytes (NHEK)
Normal human epidermal keratinized cells (manufactured by Kurabo Industries Ltd.) were seeded in KG2 medium (manufactured by Kurabo Industries Ltd.) using a 58 cm ² petri dish (manufactured by Kurabo Industries Ltd.) and cultured under 37 ° C. and 5% CO ² conditions.

(Production Example 2) Normal human fibroblast (NHDF)
Using a 58 cm ² petri dish (manufactured by IWAKI), normal human fibroblasts (manufactured by Kurabo Industries Ltd.) were seeded in DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FBS, and the conditions were 37 ° C. and 5% CO ² . Cultured below.

(Production Example 3) Preparation of acclimated medium from normal human fibroblasts When the normal human fibroblasts cultured in Production Example 2 became confluent, they were replaced with fresh KB2 medium (manufactured by Kurashiki Spinning Co., Ltd.). After culturing for 24 hours, the supernatant was collected and used as an conditioned medium from normal human fibroblasts.

(Production Example 4) Preparation of Synthetic Melanin-Containing Acclimation Medium The synthetic melanin-containing acclimation medium was prepared so that the concentration of synthetic melanin in the acclimation medium of Production Example 3 was 100 μg / mL.
As the synthetic melanin, the raw material name "melanin" (manufactured by MERCK) was used.

(Production Example 5) Preparation of Adaptation Medium Containing Fluorescent Beads The acclimation medium containing fluorescent beads was prepared by adding 500 times the amount of the acclimation medium of Production Example 3 to 1 part by mass of fluorescent beads.
As the fluorescent beads, "FluoroSpheres ^TM carboxylate modified microsphere, 0.2 μm, blue fluorescence (365/415), 2% solid content" (manufactured by Thermo Fisher Scientific) was used.

(Test Example 1) Verification of phagocytosis of synthetic melanin by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts.

The normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate (manufactured by IWAKI) so as to be 90,000 cells / cm ² , and cultured at 37 ° C. for 24 hours under 5% CO ² conditions. bottom. Then, it was replaced with the conditioned medium prepared in Production Example 4, and further cultured at 37 ° C. under 5% CO ² conditions for 6 hours. Then, the acclimation medium was removed, washed with PBS (−), and the cells were visually observed with a 100-magnification optical microscope (manufactured by Olympus). The results are shown in FIG.

As a control, the normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate (manufactured by IWAKI) so as to be 90,000 cells / cm ² , and the conditions were 37 ° C. and 5% CO ² . After culturing for 24 hours underneath, synthetic melanin was replaced with a medium prepared to have a concentration of 100 μg / mL in KB2 medium, and further cultured at 37 ° C. and 5% CO ² conditions for 6 hours was visually observed.

As is clear from FIG. 1, there is a clear difference in the presence or absence of synthetic melanin in cells in which normal human epidermal keratinocytes are contacted with an conditioned medium from normal human fibroblasts, as compared with controls without contact. Admitted. From this, it can be seen that phagocytosis is remarkably promoted by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts.

(Test Example 2) Verification of phagocytosis of fluorescent beads by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts (observation with a keratinocyte microscope).

The normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate at 90,000 cells / cm ² , and cultured at 37 ° C. under 5% CO ² conditions for 24 hours. Then, it was replaced with the conditioned medium prepared in Production Example 5, and further cultured at 37 ° C. under 5% CO ² conditions for 6 hours. Then, the conditioned medium was removed, washed with PBS (−), and the cells were visually observed with a fluorescence microscope (product name: IX71, manufactured by OLYMPUS). The results are shown in FIG.

As a control, the normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate (manufactured by IWAKI) so as to be 90,000 cells / cm ² , and the conditions were 37 ° C. and 5% CO ² . After culturing for 24 hours underneath, 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 ² conditions for 6 hours was visually observed.

As is clear from FIG. 2, cells in which normal human epidermal keratinocytes are contacted with an conditioned medium from normal human fibroblasts have higher intracellular fluorescence intensity than controls without contact. I was able to confirm that. From this, it can be seen that phagocytosis is remarkably promoted by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts.

(Test Example 3) Verification of phagocytosis of fluorescent beads by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts (measured by a keratinocyte plate reader).

The normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate at 90,000 cells / cm ² , and cultured at 37 ° C. under 5% CO ² conditions for 24 hours. Then, it was replaced with the conditioned medium prepared in Production Example 5, and further cultured at 37 ° C. under 5% CO ² conditions for 6 hours. Then, the acclimation medium was removed, washed with PBS (−), and the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) with a fluorescent plate reader (manufactured by Thermo Fisher Scientific). The results are shown in FIG. “***” in the graph indicates that the p-value of the t-test is less than 0.001.

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

As is clear from FIG. 3, it was confirmed that the cells in which the normal human epidermal keratinized cells were contacted with the conditioned medium from the normal human fibroblasts had higher fluorescence intensity than the non-contacted controls. From this, it can be seen that phagocytosis is remarkably promoted by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts.

(Test Example 4) Verification of actin rearrangement by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts.

Normal human epidermal keratinized cells were seeded on a 4-chamber slide (manufactured by BD Falcon) at a rate of 2,500 cells / cm ² , and cultured at 37 ° C. under 5% CO ² conditions for 24 hours. Then, it was replaced with the conditioned medium prepared in Production Example 3, and further cultured at 37 ° C. under 5% CO ² conditions for 2 to 6 hours. The conditioned medium was then removed and washed with PBS (-). It was then fixed with 4% formaldehyde, treated with 0.5% Triton-100 and blocked with 1% BSA. Add 200 μL of 165 nM rhodamine phalloidin (manufactured by Fuji Film Wako Junyaku Co., Ltd.) and 20 μM Hoechst 33258 (manufactured by Takara Bio) dissolved in PBS (-) to cells, stain for 30 minutes, and stain with Enteran New (manufactured by MERCK). 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. In the figure, the scale bar indicates 50 μm.

As a control, normal human epidermal keratinized cells were seeded on a 4-chamber slide (manufactured by BD Falcon) at a rate of 2,500 cells / cm ² , and after culturing at 37 ° C. and 5% CO ² conditions for 24 hours. , The cells were visually observed and evaluated in the same manner as above without replacing with the conditioned medium prepared in Production Example 3.

As is clear from FIG. 4, in cells in which normal human epidermal keratinized cells were contacted with an conditioned medium from normal human fibroblasts, F-actin (F-actin) was used in the vicinity of the cells in the vicinity of the cells as compared with the non-contacted control. It was confirmed that fibrous actin) was stained. From this, it can be seen that F-actin polymerization is increased by contacting normal human epidermal keratinized cells with an conditioned medium from normal human fibroblasts (actin rearrangement).

(Test Examples 5 and 6) Verification of phagocytosis inhibitory effect by actin reorganization inhibitor

As the test substance of Test Example 5, BAPTA-AM (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), which is a chelating agent, was used (concentration: 5.0 μM, 7.5 μM).
Cytochalasin D (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), which is an actin inhibitor, was used as the test substance of Test Example 6 (concentration: 0.3 μM, 0.6 μM).

The normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate at 90,000 cells / cm ² , and cultured at 37 ° C. under 5% CO ² conditions for 24 hours. Then, it was replaced with the conditioned medium prepared in Production Example 5 containing the test substances of Test Examples 5 and 6, and further cultured at 37 ° C. under 5% CO ² conditions for 6 hours. Then, the acclimation medium was removed, washed with PBS (−), and the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) with a fluorescent plate reader (manufactured by Thermo Fisher Scientific). 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. Further, "*" indicates that the p-value of the t-test is less than 0.05.

In addition, the same test method as in Test Examples 5 and 6 above was used as a control, and the fluorescence intensity was measured with a fluorescence plate reader in the same manner.

As is clear from FIGS. 5 and 6, it was confirmed that the cells contacted with the actin reorganization inhibitor had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting with an actin rearrangement inhibitor.

(Test Example 7) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

As test substances, rooibos extract (trade name: Cape gorses linearis, manufactured by Release Kagaku Kogyo Co., Ltd., concentration: 1.0%), aloe extract (trade name: Asparasus linearis, manufactured by Release Kagaku Kogyo Co., Ltd., concentration: 1) .0%) were used respectively.

The normal human epidermal keratinized cells cultured in Production Example 1 were seeded on a 96-well plate at 90,000 cells / cm ² , and cultured at 37 ° C. under 5% CO ² conditions for 24 hours. Then, it was replaced with the conditioned medium prepared in Production Example 5 containing the above-mentioned test substance, plant extract, and further cultured at 37 ° C. under 5% CO ² conditions for 6 hours. Then, the acclimation medium was removed, washed with PBS (−), and the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) with a fluorescent plate reader (manufactured by Thermo Fisher Scientific). The results are shown in FIG.

In addition, the same test method as in Test Example 7 above, which did not contain the test substance, was used as a control, and the fluorescence intensity was measured with a fluorescent plate reader in the same manner.

As is clear from FIG. 7, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 8) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

In the same test method as in Test Example 7, Scutellaria baicalensis extract (trade name: Scutellaria baicalensis extract SP, manufactured by Koshiro Pharmaceutical Co., Ltd., concentration: 1.0%) was used as a test substance. The results are shown in FIG. The same test method as in Test Example 8 above, which did not contain the test substance, was used as a control.

As is clear from FIG. 8, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 9) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Using the same test method as in Test Example 7, raspberry extract (trade name: Dermo Fruit Raspberry / N, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) and kiwi extract (trade name: kiwi extract) were used as test substances. Liquid, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. In addition, the same test method as in Test Example 9 which did not contain the test substance was used as a control.

As is clear from FIG. 9, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 10) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

As test substances in the same test method as in Test Example 7, Houttuynia cordata extract (trade name: Juyaku style extract, manufactured by Koshiro Pharmaceutical Co., Ltd., concentration: 1.0%), Panax ginseng extract (trade name: Carrot style extract P, Koshiro Pharmaceutical Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. In addition, the same test method as in Test Example 10 which did not contain the test substance was used as a control.

As is clear from FIG. 10, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 11) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Hikiokoshi extract (trade name: Enmeisou extract, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%) and loquat extract (trade name: loquat extract BG-) are used as test substances in the same test method as in Test Example 7. J, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. The same test method as in Test Example 11 and not containing the test substance was used as a control.

As is clear from FIG. 11, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 12) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

As a test substance in the same test method as in Test Example 7, peach extract (trade name: peach extract, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%), Saxifraga extract (trade name: Saxifraga extract BG, Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. In addition, the same test method as in Test Example 12 which did not contain the test substance was used as a control.

As is clear from FIG. 12, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 13) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Using the same test method as in Test Example 7, Sansho extract (trade name: Falcolex Sansho, manufactured by Ichimaru Falcos, concentration: 1.0%) and bamboo vinegar extract (trade name: T.I. P bamboo extract, Tachibana Bamboo Co., Ltd., concentration: 1.0%) and Reishi extract (trade name: Reishi extract K5, manufactured by Koshiro Pharmaceutical Co., Ltd., concentration: 1.0%) were used. The results are shown in FIG. In addition, the same test method as in Test Example 13 which did not contain the test substance was used as a control.

As is clear from FIG. 13, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 14) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A ginger extract (trade name: Falcorex choy sum E, manufactured by Ichimaru Falcos, concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. In addition, the same test method as in Test Example 14 which did not contain the test substance was used as a control.

As is clear from FIG. 14, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 15) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Hydrolyzed wheat protein (trade name: TRITISOL-LQ- (WD), manufactured by Croda Japan, concentration: 1.0%) as a test substance in the same test method as in Test Example 7.
It was used. The results are shown in FIG. The same test method as in Test Example 15 that did not contain the test substance was used as a control.

As is clear from FIG. 15, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 16) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A carambola extract (trade name: star fruit leaf extract BG30, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. In addition, the same test method as in Test Example 16 which did not contain the test substance was used as a control.

As is clear from FIG. 16, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 17) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A hop extract (trade name: hop extract, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. In addition, the same test method as in Test Example 17 which did not contain the test substance was used as a control.

As is clear from FIG. 17, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted with the test substance. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 18) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A red algae extract (trade name: Betaherin, manufactured by Technoble Co., Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. The same test method as in Test Example 18 and not containing the test substance was used as a control.

As is clear from FIG. 18, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 19) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Using the same test method as in Test Example 7, Someiyoshino extract (trade name: Sakura Extract B, manufactured by Ichimaru Falcos, concentration: 1.0%) and rose-apple extract (trade name: Roseapple Leaf Extract BG) were used as test substances. -30, manufactured by Koei Kogyo Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. In addition, the same test method as in Test Example 19 which did not contain the test substance was used as a control.

As is clear from FIG. 19, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 20) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

In the same test method as in Test Example 7, Kibana Paracress extract (trade name: GATTULINE EXPRESSION, manufactured by GATTEFOSSE, concentration: 1.0%) was used as a test substance. The results are shown in FIG. In addition, the same test method as in Test Example 20 which did not contain the test substance was used as a control.

As is clear from FIG. 20, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 21) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Keratin (trade name: KERATEC IFP-HMW, manufactured by Keratec Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. In addition, the same test method as in Test Example 21 which did not contain the test substance was used as a control.

As is clear from FIG. 21, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 22) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

As test substances in the same test method as in Test Example 7, cinnamon extract (trade name: cinnamon extract, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1.0%), cassia bark extract (trade name: cinnamon extract W). -LA, manufactured by Maruzen Pharmaceuticals, concentration: 1.0%) was used. The results are shown in FIG. A test method similar to that of Test Example 22 and containing no test substance was used as a control.

As is clear from FIG. 22, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 23) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Oligopeptide-20 (trade name: CG-IDP5 BG, manufactured by Caregen, concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. The same test method as in Test Example 23, which did not contain the test substance, was used as a control.

As is clear from FIG. 23, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 24) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

High fructose corn syrup (trade name: PENTAVITIN, manufactured by DSM, concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. The same test method as in Test Example 24, which did not contain the test substance, was used as a control.

As is clear from FIG. 24, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 25) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A peony extract (trade name: peony extract-BG, manufactured by Yamada Yakuken Co., Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. The same test method as in Test Example 25, which did not contain the test substance, was used as a control.

As is clear from FIG. 25, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 26) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

A lychee extract (trade name: lychee seed extract-LC, manufactured by Oryza Yuka Co., Ltd., concentration: 1.0%) was used as a test substance in the same test method as in Test Example 7. The results are shown in FIG. The same test method as in Test Example 26, which did not contain the test substance, was used as a control.

As is clear from FIG. 26, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 27) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

In the same test method as in Test Example 7, as test substances, Hass extract (1.0%), Bringalage extract (1.0%), Pitecerobium extract (trade name: Pitecerobium extract, Koei) Kogyo Co., Ltd., concentration: 1.0%) was used. The results are shown in FIG. The same test method as in Test Example 27, which did not contain the test substance, was used as a control.

As is clear from FIG. 27, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the controls not contacted. From this, it was confirmed that phagocytosis can be inhibited by contacting the test substance.

(Test Example 28) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Albutin (manufactured by Nippon Seika Co., Ltd., concentration: 50 μg / mL, 100 μg / mL) and nicotinic acid amide (manufactured by DSM Co., Ltd., concentration: 50 μg / mL, 100 μg / mL) were used as test substances in the same test method as in Test Example 7. ), Kodiic acid (manufactured by Tokyo Kasei Kogyo Co., Ltd., concentration: 50 μg / mL, 100 μg / mL), and tea extract (trade name: tea liquid, manufactured by Ichimaru Falcos Co., Ltd., concentration: 0.1%) were used. The results are shown in FIG. The same test method as in Test Example 28, which did not contain the test substance, was used as a control.

As is clear from FIG. 28, it was confirmed that the cells contacted with arbutin, nicotinamide, and kojic acid did not change the fluorescence intensity as compared with the control not contacted. That is, albutin, nicotinic acid amide, and kojic acid, which are known to have a whitening effect, have no effect on the induction of fagocytosis of epidermal keratinocytes after contacting the epidermal keratinized cells with an conditioned medium for fibroblasts. Was found not to show.

(Test Examples 29 and 30) Phagocytosis inhibitory effect test by contacting normal human epidermal keratinized cells with conditioned medium from normal human fibroblasts

Adlay extract (trade name: Yokuinin extract, manufactured by Koei Kogyo Co., Ltd., concentration: 1%), Hibamata extract (trade name: Falcolex Hibamata, Ichimaru Falcos) as test substances using the same test method as in Test Example 7. Co., Ltd., concentration: 1%), Ghetto extract (trade name: Tsukimoha extract BG, manufactured by Maruzen Pharmaceuticals Co., Ltd., concentration: 1%) was used. The results are shown in FIGS. 29 and 30. The same test method as in Test Examples 29 and 30 and not containing the test substance was used as a control.

As is clear from FIGS. 29 and 30, it was confirmed that the cells contacted with the Coix seed extract, the rockweed extract, and the shell ginger extract had the same fluorescence intensity as those of the non-contacted control. That is, the honeybee extract, hibamata extract, and ghetto extract, which are known to suppress melanin production (tyrosinase inhibition), are the fago of the epidermal keratinized cells after contacting the epidermal keratinized cells with a fibroblast conditioned medium. It was found to have no effect on the induction of cytosis.

From the above results, the specific plant extract and the specific compound, which are the active ingredients of the present invention, are used to reorganize the epidermal keratinocytes into actin after contacting the epidermal keratinocytes with a fibroblast conditioned medium. , And it has a remarkably excellent inhibitory effect on the induction of fagocytosis, and it was confirmed that it is useful as a stain formation inhibitor. In addition, by applying this to an external skin preparation, it has become possible to provide a spot formation inhibitory effect.

On the other hand, albutin, nicotinic acid amide, kojic acid, honeybee extract, hibamata extract, and ghetto extract, which are known to have a whitening effect, are used after contacting epidermal keratinized cells with a fibroblast conditioned medium. It had no effect on the induction of fagocytosis in epidermal keratinized cells.

Claims (5)

It is an inhibitor that inhibits the reorganization of actin of the epidermal keratinized cells by contacting the epidermal keratinized cells with a fibroblast conditioned medium, and is an inhibitor that inhibits the rearrangement of the actin of the epidermal keratinized cells. Extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, Heterogeneous An actin reorganization inhibitor comprising one or more selected from sucrose, button extract, lychee extract, hass extract, blingaraj extract, and pitecerobium extract as an active ingredient. A stain formation inhibitor, which comprises the actin reorganization inhibitor according to claim 1. It is an inhibitor that inhibits the reorganization of the actin of the epidermal keratinized cells by contacting the epidermal keratinized cells with the conditioned medium of the fibroblasts. Extract, Kiwi extract, Dokudami extract, Otaneninjin extract, Hikiokoshi extract, Biwa extract, Peach extract, Yukinoshita extract, Sansho extract, Bamboo vinegar extract, Reishi extract, Usbasaicin extract, Hydrolyzed wheat protein, Gorenshi extract, Hop extract, Red algae extract, Someiyoshino extract, Futomomo extract, Kibana Dutch Sennichi extract, Keratin, Asenyaku extract, Cassia bark extract, Oligopeptide-20, Heterogeneous A phagocytosis inhibitor comprising one or more selected from sucrose, button extract, lychee extract, hass extract, blingaraj extract, and pitecerobium extract as an active ingredient. A stain formation inhibitor, which comprises the phagocytosis inhibitor according to claim 3. An external skin preparation containing the stain formation inhibitor according to claim 2 or 4.

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