Actin reorganization inhibitor, phagocytosis inhibitor, pigmentation formation inhibitor containing said inhibitor, and skin external preparation containing said 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 polyphenol 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, an agent for suppressing pigmentation formation containing said inhibitor, and an external skin preparation containing said inhibitor. [Background technology]

[0002] Pigmented spots (solar lentigines) that appear with age are an important challenge 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 on the face, and do not disappear due to possible DNA damage in epidermal keratinocytes caused by repeated UVB irradiation of the lesioned epidermis. In fact, it is known that an individual's history of UV exposure is responsible for the appearance of solar lentigines (see, for example, Non-Patent Document 1). Therefore, the prevention and improvement of solar lentigines is an important challenge for cosmetics, especially for Asian and Caucasian women.

[0003] Histological characteristics of solar lentigines include destruction of the basement membrane and increased epidermal thickness due to developed reticular ridges (see, for example, Non-Patent Document 2 and Non-Patent Document 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 lentigo) (see, for example, Non-Patent Document 4). Furthermore, the use of tyrosinase antibodies has revealed a significant two-fold increase in tyrosinase-positive pigment cells in the lesional epidermis of solar lentigines (see, for example, Non-Patent Document 5).

[0004] The mechanisms underlying pigmented spots initiated by UV irradiation can be broadly divided into enhanced melanogenesis in pigment cells and transfer of melanosomes to adjacent epidermal keratinocytes. Regarding enhanced melanogenesis, it has been reported that various cytokines, such as endothelin-1 and granulocyte-monocyte colony-stimulating factor, produced by epidermal keratinocytes, act on pigment cells to promote melanogenesis (see, for example, Non-Patent Documents 6 and 7). It is expected that the destruction of the basement membrane will promote crosstalk not only between pigment cells and epidermal keratinocytes, but also between the dermis and epidermis. Indeed, some studies have highlighted the contribution of fibroblasts to the initiation and maintenance of solar lentigines (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 through activation of the Rho family (see, for example, Non-Patent Document 9). In addition, the effects of other factors such as epidermal keratinocyte growth factor / epidermal keratinocyte growth factor receptor have also been reported (see, for example, Non-Patent Document 10).

[0006] It is known that the pigmentation inhibitory effect is due to the inhibition of the transfer of melanosomes from pigment cells to epidermal keratinocytes. In this case, it has been reported that epidermal keratinocytes take up melanosomes by phagocytosis. It is also known that the phagocytosis by the epidermal keratinocytes is inhibited by a plant extract obtained from a plant belonging to the genus Melissa of the family Lamiaceae, which contains unsaturated fatty acid derivatives (see, for example, Patent Document 1). However, the mechanism of action of inducing actin reorganization and phagocytosis of epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts is not known at all. Furthermore, nothing is known about actin reorganization inhibitors and phagocytosis inhibitors that inhibit the mechanism of action, or pigmentation inhibitors containing these inhibitors. Furthermore, it is not known at all that polyphenol compounds have the inhibitory effect. [Prior art documents] [Patent documents]

[0007]

Patent Document 1

Non - Patent Documents

[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

[0009] Therefore, the present invention aims to provide an actin reorganization inhibitor and a phagocytosis inhibitor that inhibit the induction of actin reorganization and phagocytosis in epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts.The present invention also aims to provide a pigmentation formation inhibitor containing the inhibitor, and an external skin preparation containing the inhibitor.

[0010] In view of these circumstances, the present inventors have sought a method for preventing or ameliorating the formation of age spots after contacting epidermal keratinocytes with a conditioned medium for fibroblasts, and as a result of extensive efforts, have discovered that polyphenol compounds such as gallic acid, epicatechin gallate, and epigallocatechin gallate have an inhibitory effect on actin reorganization and phagocytosis, which are factors directly linked to the prevention or amelioration of age spots, and have thus completed the present invention. That is, the present invention is as follows. [Means for solving the problem]

[0011] That is, the present invention provides (1) An inhibitor for inhibiting actin reorganization in epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts, the inhibitor comprising a polyphenol compound as an active ingredient; (2) The actin reorganization inhibitor according to (1), wherein the polyphenol compound is one or more selected from the group consisting of gallic acid and catechin derivatives. (3) The actin reorganization inhibitor according to (2), wherein the catechin derivative is epicatechin gallate and / or epigallocatechin gallate. (4) A pigmentation formation inhibitor comprising the actin reorganization inhibitor according to any one of (1) to (3). (5) A phagocytosis inhibitor which inhibits phagocytosis of epidermal keratinocytes by contacting the epidermal keratinocytes with a conditioned medium of fibroblasts, the phagocytosis inhibitor comprising a polyphenol compound as an active ingredient. (6) The phagocytosis inhibitor according to (5), wherein the polyphenol compound is one or more selected from the group consisting of gallic acid and catechin derivatives. (7) The phagocytosis inhibitor according to (6), wherein the catechin derivative is epicatechin gallate and / or epigallocatechin gallate. (8) A pigmentation formation inhibitor comprising the phagocytosis inhibitor according to any one of (5) to (7). (9) A skin external preparation comprising the pigmentation inhibitor according to (4) or (8). Regarding. Effect of the Invention

[0012] The polyphenol compound, which is an active ingredient of the present invention, has an exceptionally excellent inhibitory effect on the induction of actin reorganization and phagocytosis in epidermal keratinocytes after the epidermal keratinocytes are contacted with a conditioned medium for fibroblasts, and this inhibitory effect exerts an exceptionally excellent effect of effectively suppressing the formation of age spots.

[0013] In addition, since it exhibits a significantly superior inhibitory effect on actin reorganization and phagocytosis induction, which are involved in the formation of age spots, it can be advantageously used as an external skin preparation, such as a whitening agent, that does not cause age spots. [Brief description of the drawings]

[0014] [Figure 1] (a) is a drawing-substitute photograph showing a cell group in Test Example 1, in which synthetic melanin was administered to normal human epidermal keratinocytes without contacting them with a conditioned medium from normal human fibroblasts. (b) is a drawing-substitute photograph showing a cell group in Test Example 1, in which synthetic melanin was administered to normal human epidermal keratinocytes after contacting them with a conditioned medium from normal human fibroblasts. In the drawings of this specification, the normal human epidermal keratinocytes are abbreviated as "NHEK." Incidentally, "NHEK" is an abbreviation for Normal Human Epidermal Keratinocytes. [Diagram 2] Photograph (a) is a drawing-substitute photograph showing a cell group in Test Example 2, in which fluorescent beads were administered to normal human epidermal keratinocytes without contacting them with a conditioned medium from normal human fibroblasts. Photograph (b) is a drawing-substitute photograph showing a cell group in Test Example 2, in which fluorescent beads were administered to normal human epidermal keratinocytes after contacting them with a conditioned medium from normal human fibroblasts. [Diagram 3] 1 is a graph showing the difference in fluorescence intensity depending on whether or not normal human epidermal keratinocytes are contacted with a conditioned medium from normal human fibroblasts in Test Example 3. [Figure 4] (a) is a photograph in Test Example 4 in which normal human epidermal keratinocytes were stained without being contacted with a conditioned medium from normal human fibroblasts. (b) is a photograph in Test Example 4 in which normal human epidermal keratinocytes were stained by being contacted with a conditioned medium from normal human fibroblasts for 2 hours. (c) is a photograph in Test Example 4 in which normal human epidermal keratinocytes were stained by being contacted with a conditioned medium from normal human fibroblasts for 4 hours. (d) is a photograph in Test Example 4 in which normal human epidermal keratinocytes were stained by being contacted with a conditioned medium from normal human fibroblasts for 6 hours. [Diagram 5] 1 is a graph showing that BAPTA-AM (chelating agent) inhibited phagocytosis in Test Example 5. [Figure 6] 1 is a graph showing that Cytochalasin D (an actin polymerization inhibitor) inhibited phagocytosis in Test Example 6. [Figure 7] 1 is a graph showing that phagocytosis was inhibited at each concentration of gallic acid in Test Example 7. [Figure 8] 1 is a graph showing that epicatechin gallate or epigallocatechin gallate inhibited phagocytosis at various concentrations in Test Example 8. [Figure 9] 1 is a graph showing that arbutin, nicotinamide, or kojic acid could not inhibit phagocytosis in Test Example 9. [Figure 10] 1 is a graph showing that the Job's Tears extract was unable to inhibit phagocytosis in Test Example 10. [Figure 11] 1 is a graph showing that in Test Example 11, Fucus vesiculosus extract or Alpinia speciosa extract could not inhibit phagocytosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0016] The inhibitor of the present invention that inhibits the induction of actin reorganization and phagocytosis, the agent for suppressing the formation of spots comprising said inhibitor, and the external skin preparation containing said inhibitor contain a polyphenol compound as an active ingredient.

[0017] [Inhibition of actin reorganization] The present inventors have found that actin reorganization is induced in epidermal keratinocytes when the epidermal keratinocytes are contacted with a conditioned medium from fibroblasts. Furthermore, the present inventors have found that the actin reorganization induced by the contact of the conditioned medium from fibroblasts is inhibited by an actin reorganization inhibitor, and there is a correlation between this and the expression of the phagocytosis inhibitory effect of the actin reorganization inhibitor. The present invention is based on these findings.

[0018] In the present invention, inhibition of actin reorganization is characterized by inhibiting actin reorganization induced by contacting epidermal keratinocytes with a conditioned medium from fibroblasts.

[0019] The epidermal keratinocytes may be, for example, epidermal keratinocytes derived from either humans or non-human animals. In view of application to human skin and the ability to evaluate a test substance more accurately, the epidermal keratinocytes used in the present invention are preferably human epidermal keratinocytes. The human epidermal keratinocytes may be normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but are more preferably normal human epidermal keratinocytes in view of the ability to more accurately capture and evaluate the phenomenon of actin reorganization.

[0020] The above-mentioned fibroblasts may be, for example, fibroblasts derived from either human or non-human animals. In view of application to human skin, the fibroblasts used in the present invention are preferably human fibroblasts, from the viewpoint of being able to evaluate the test substance more accurately. The human fibroblasts are not particularly limited to normal human fibroblasts or immortalized human fibroblasts, but it is more preferable to use normal human fibroblasts, from the viewpoint of being able to more accurately capture and evaluate the phenomenon of actin reorganization.

[0021] The conditioned medium for the fibroblasts may be, for example, 58 cm 2When the cells become confluent in the petri dish, the medium is replaced with fresh KB2 medium or DMEM (5% FBS) medium, and the medium is cultured for 12 to 36 hours, after which the supernatant is recovered, and the medium can be used. Note that the conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficient induction of actin reorganization, it is preferable to use the medium from which the supernatant is recovered after culture for 18 to 30 hours.

[0022] The epidermal keratinocytes are cultured, for example, at 500 to 5,000 cells / cm in a four-chamber slide. 2 In the present invention, from the viewpoint of facilitating observation of the induction of actin reorganization, the cells can be seeded at a density of 2,000 to 4,000 cells / cm. 2 It is preferable to sow the seeds so that

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

[0024] As used herein, "induction of actin rearrangement by contact with conditioned medium from fibroblasts" means that in response to stimulation of epidermal keratinocytes with the conditioned medium from the fibroblasts, the induction of actin rearrangement in the epidermal keratinocytes is observed to be more pronounced than in epidermal keratinocytes that have not been contacted with the conditioned medium from the fibroblasts.

[0025] The induction of actin reorganization by contact with the conditioned medium from the fibroblasts can be confirmed, for example, by staining actin polymerization in epidermal keratinocytes and observing them under a fluorescent microscope. Reagents capable of staining actin polymerization are not particularly limited, but examples thereof include rhodamine phalloidin and commercially available actin antibodies.

[0026] The present invention allows the accurate observation and evaluation of the phenomenon in which actin reorganization is induced by contacting epidermal keratinocytes with a conditioned medium from fibroblasts, and therefore allows accurate evaluation of the presence or absence of an inhibitory effect on actin reorganization of a test substance, and thus can be suitably used as a method for screening actin reorganization inhibitors.

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

[0028] The above-mentioned phagocytosis means the phagocytosis, engulfment, etc. of epidermal keratinocytes. In the present invention, the inhibition of phagocytosis is characterized by inhibiting phagocytosis induced by contacting epidermal keratinocytes with a conditioned medium from fibroblasts.

[0029] The epidermal keratinocytes may be, for example, epidermal keratinocytes derived from either humans or non-human animals. In view of application to human skin and the ability to evaluate a test substance more accurately, the epidermal keratinocytes used in the present invention are preferably human epidermal keratinocytes. The human epidermal keratinocytes may be normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but are more preferably normal human epidermal keratinocytes in view of the ability to more accurately capture and evaluate the phenomenon of phagocytosis.

[0030] The above-mentioned fibroblasts may be, for example, fibroblasts derived from either human or non-human animals. In view of application to human skin, the fibroblasts used in the present invention are preferably human fibroblasts, from the viewpoint of being able to evaluate the test substance more accurately. The human fibroblasts may be normal human fibroblasts or immortalized human fibroblasts, but are more preferably normal human fibroblasts, from the viewpoint of being able to capture the phenomenon of phagocytosis more accurately and evaluate it more accurately.

[0031] The conditioned medium for the fibroblasts may be, for example, 58 cm 2 When the cells become confluent in the petri dish, the medium is replaced with fresh KB2 medium or DMEM (5% FBS) medium, and the medium is cultured for 12 to 36 hours, after which the supernatant is recovered, and the medium can be used. Note that the conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficient induction of phagocytosis, it is preferable to use the medium from which the supernatant is recovered after culture for 18 to 30 hours.

[0032] The epidermal keratinocytes are, for example, plated in a 96-well plate or a 24-well plate at a density of 50,000 to 130,000 cells / cm. 2 In the present invention, from the viewpoint of sufficient induction of phagocytosis, the cells can be seeded at a density of 70,000 to 110,000 cells / cm. 2 It is preferable to sow the seeds so that

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

[0034] As used herein, "induction of phagocytosis by contact with conditioned medium from fibroblasts" refers to the fact that the amount of microparticles taken up by epidermal keratinocytes in response to stimulation of the epidermal keratinocytes by the conditioned medium from the fibroblasts is significantly greater than the amount of microparticles taken up by epidermal keratinocytes not contacted with the conditioned medium from the fibroblasts. Note that "significant" refers to a p-value of t-test of less than 0.05, preferably less than 0.01.

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

[0036] Examples of the fine particles include melanosomes, melanin, and color-developing substances.

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

[0038] The melanin may be natural or synthetic. Alternatively, commercially available synthetic melanin may be used. The amount of melanin used is 1 well (0.35 cm) in a 96-well plate. 2 ), it is preferable that the amount is 1 μg or more and 100 μg or less.

[0039] The color-developing substance may be fluorescent beads. TM Carboxylate-modified microspheres, 0.2 μm, blue fluorescent (365 / 415), 2% solids, are available from ThermoFisher Scientific (Waltham, Massachusetts, USA). The amount of fluorescent beads used is 1 well (0.35 cm) in a 96-well plate.2 ), it is preferable that the amount is 1 μg or more and 100 μg or less.

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

[0041] The present invention allows the phenomenon of phagocytosis induction to be observed and evaluated with high accuracy by contacting epidermal keratinocytes with a conditioned medium from fibroblasts, and therefore allows accurate evaluation of the presence or absence of a test substance's inhibitory effect on phagocytosis, and thus can be suitably used as a method for screening phagocytosis inhibitors.

[0042] As described above, the present invention allows for accurate observation and evaluation of the actin reorganization and phagocytosis phenomena induced by contacting epidermal keratinocytes with a conditioned medium from fibroblasts. That is, the present invention allows for accurate observation and evaluation of the actin reorganization and phagocytosis phenomena involved in the formation of age spots, and allows for accurate confirmation and evaluation of the presence or absence of an inhibitory effect of a test substance on actin reorganization and phagocytosis.

[0043] Furthermore, according to the present invention, an inhibitor that exhibits a significantly superior inhibitory effect against actin reorganization and phagocytosis induction involved in the formation of age spots is useful as an inhibitor of age spot formation, and said inhibitor can be suitably used as an external skin preparation such as a whitening agent that does not cause age spots.

[0044] [Test substance] The inhibitor, suppressor, and topical agent of the present invention contain a polyphenol compound as an active ingredient.

[0045] Specific examples of polyphenol compounds used in the present invention include catechin derivatives, anthocyanins, flavones, flavanones, rutin, chlorogenic acid, ellagic acid, gallic acid, propyl gallate, lignans, curcumin, ferulic acid, chlorogenic acid, etc. In the present invention, mixed raw materials containing these polyphenol compounds or plant extracts containing these polyphenol compounds may also be used.

[0046] In the present invention, from the viewpoint of exerting an excellent inhibitory effect among the above-mentioned polyphenol compounds, it is preferable to contain, as an active ingredient, one or more polyphenol compounds selected from gallic acid and catechin derivatives.

[0047] The above-mentioned gallic acid (INCI name: Gallic Acid) is contained in many plants, for example, Chinese gallnut, gall nut, witch hazel, tea leaves, oak bark, etc. Examples of the above-mentioned catechin derivatives include epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, etc.

[0048] In the present invention, it is most preferable to contain, among the above-mentioned polyphenol compounds, one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate as active ingredients, in view of exhibiting a significantly superior inhibitory effect.

[0049] The polyphenol compound used in the present invention has excellent actin reorganization inhibitory effects and phagocytosis inhibitory effects, and therefore can be used as an active ingredient in an actin reorganization inhibitor, a phagocytosis inhibitor, a spot formation inhibitor consisting of said inhibitor, and an external skin preparation containing said inhibitor.

[0050] The pigmentation formation inhibitor of the present invention may be a formulation containing one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate, or a mixture thereof.

[0051] The actin reorganization inhibitors and phagocytosis inhibitors of the present invention can be formulated into any dosage form, such as powder, granules, tablets, liquid, etc., using phagocytosis inhibitors and phagocytosis inhibitors that are pharma- ceutically acceptable carriers and any other auxiliary agents, in accordance with conventional methods.

[0052] When the actin reorganization inhibitor and phagocytosis inhibitor of the present invention are formulated, the content of one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate is not particularly limited and can be set appropriately depending on the purpose.

[0053] In addition, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention can be used as an active ingredient by combining, if necessary, other natural extracts having actin reorganization inhibitory activity and phagocytosis inhibitory activity with one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate.

[0054] The actin reorganization inhibitor and phagocytosis inhibitor of the present invention can prevent, treat, or improve blemishes through the actin reorganization inhibitory effect and phagocytosis inhibitory effect of one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate.

[0055] Furthermore, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention can be used as an active ingredient of pharmaceuticals or quasi-drugs for the prevention and treatment of diseases caused by abnormal production of melanin. However, in addition to these uses, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention can be used for all uses in which it is meaningful to exert an inhibitory effect on actin reorganization and phagocytosis.

[0056] In addition, the actin reorganization inhibitor and phagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory activity and phagocytosis inhibitory activity, and are therefore suitable for, for example, incorporation into skin external preparations. In this case, one or more polyphenol compounds selected from gallic acid, epicatechin gallate, and epigallocatechin gallate, or a mixture thereof may be incorporated.

[0057] When the polyphenol compound is incorporated into a skin topical preparation, the content is not particularly limited as long as the desired effect can be exhibited; however, the content of the polyphenol compound in the total amount of the topical preparation is preferably 0.000000001 to 5 mass%, more preferably 0.00000001 to 3 mass%, even more preferably 0.000001 to 2 mass%, and particularly preferably 0.000001 to 1 mass%.

[0058] Here, topical skin preparations are not limited to specific classifications and include a wide range of skin cosmetics, quasi-drugs, and pharmaceuticals that are used percutaneously. Specific examples of topical skin preparations include ointments, creams, milky lotions, beauty serums, lotions, packs, foundations, lip balms, and bath additives.

[0059] Furthermore, since the actin reorganization inhibitor and phagocytosis inhibitor of the present invention have excellent actin reorganization inhibitory effects and phagocytosis inhibitory effects, they can be suitably used as reagents for research related to the melanin production mechanism.

[0060] Next, a method for screening a test substance for evaluating the inhibitory effect and suppressive effect of the present invention will be described.

[0061] A specific example of a screening method for a test substance is a screening method for a test substance for evaluating the effect of the test substance in inhibiting the formation of age spots, which includes the steps of: (A) contacting epidermal keratinocytes with microparticles and the 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 microparticles taken up by the epidermal keratinocytes from the microparticle uptake activity of the epidermal keratinocytes obtained in step (A); and (C) examining the inhibition of phagocytosis in epidermal keratinocytes based on the amount of microparticles taken up by the epidermal keratinocytes measured in step (B), and evaluating the effect of the test substance in inhibiting the formation of age spots.

[0062] According to the screening method, by contacting epidermal keratinocytes with microparticles and a test substance, and contacting the epidermal keratinocytes with a conditioned medium from fibroblasts before, simultaneously with, or after the contact, it is possible to quickly and simply evaluate whether the test substance inhibits phagocytosis of epidermal keratinocytes. Furthermore, based on the presence or absence of inhibition of phagocytosis of epidermal keratinocytes and the level of the inhibition, it is possible to quickly and simply evaluate the effect of the test substance in inhibiting the formation of spots.

[0063] In the above step (A), the epidermal keratinocytes are contacted with the microparticles and the test substance, and before, simultaneously with, or after this contact, the epidermal keratinocytes are contacted with a conditioned medium from fibroblasts.

[0064] The epidermal keratinocytes may be, for example, epidermal keratinocytes derived from either humans or non-human animals. In view of the application to human skin and the ability to evaluate the test substance more accurately, the epidermal keratinocytes used in the screening method are preferably human epidermal keratinocytes. The human epidermal keratinocytes may be normal human epidermal keratinocytes or immortalized human epidermal keratinocytes, but it is more preferable to use normal human epidermal keratinocytes in view of the ability to more accurately capture and evaluate the phenomenon of phagocytosis.

[0065] The above-mentioned fibroblasts may be, for example, fibroblasts derived from either human or non-human animals. In view of applying to human skin and evaluating the test substance more accurately, the fibroblasts used in the above-mentioned screening method are preferably human fibroblasts. The human fibroblasts may be normal human fibroblasts or immortalized human fibroblasts, but are more preferably normal human fibroblasts in view of capturing the phenomenon of phagocytosis more accurately and evaluating it more accurately.

[0066] Examples of the fine particles include melanosomes, melanin, and color-developing substances.

[0067] The melanosomes may be prepared from pigment cells derived from either humans or non-human animals. In the present invention, it is preferable to use melanosomes prepared from a homogenate of pigment cells derived from humans.

[0068] The melanin may be natural or synthetic. The amount of melanin used is determined by measuring the amount of melanin in one well (0.35 cm) of a 96-well plate. 2 ), it is preferable that the amount is 1 μg or more and 100 μg or less.

[0069] The color-developing substance may be fluorescent beads. TM Carboxylate-modified microspheres, 0.2 μm, blue fluorescent (365 / 415), 2% solids, are available from ThermoFisher Scientific (Waltham, Massachusetts, USA). The amount of fluorescent beads used is 1 well (0.35 cm) in a 96-well plate. 2 ), it is preferable that the amount is 1 μg or more and 100 μg or less.

[0070] The type of test substance used when contacting the epidermal keratinocytes with the test substance is not particularly limited, and examples thereof include low molecular weight compounds, plant extracts, extracts of natural products such as microorganisms, proteins, peptides, nucleic acids, synthetic polymer compounds, etc. The amount of the test substance varies depending on the type of test substance, and therefore cannot be determined in general.

[0071] The conditioned medium for the fibroblasts may be, for example, 58 cm 2 When the cells become confluent in the petri dish, the medium is replaced with fresh KB2 medium or DMEM (5% FBS) medium, and the medium is cultured for 12 to 36 hours, after which the supernatant is recovered, and the medium can be used. Note that the conditioned medium is not limited to the above-mentioned preparation method. In the present invention, from the viewpoint of sufficient induction of phagocytosis, it is preferable to use the medium from which the supernatant is recovered after culture for 18 to 30 hours.

[0072] The epidermal keratinocytes are, for example, plated in a 96-well plate or a 24-well plate at a density of 50,000 to 130,000 cells / cm. 2 In the present invention, from the viewpoint of sufficient induction of phagocytosis, the cells can be seeded at a density of 70,000 to 110,000 cells / cm. 2 It is preferable to sow the seeds so that

[0073] The time for which the epidermal keratinocytes are contacted with the conditioned medium from the fibroblasts is not particularly limited, but from the viewpoint of sufficiently inducing phagocytosis by contact with the conditioned medium from the fibroblasts, it is preferable to contact the epidermal keratinocytes with the conditioned medium from the fibroblasts for 2 to 24 hours, and more preferably for 4 to 8 hours.

[0074] The timing of contacting the epidermal keratinocytes with the conditioned medium from the fibroblasts may be any of before, at the same time, or after the contact of the epidermal keratinocytes with the microparticles and the test substance. In the present invention, from the viewpoint of improving the accuracy of the screening method, it is preferable to contact the microparticles and the test substance during contact with the conditioned medium from the fibroblasts. That is, it is preferable to contact the epidermal keratinocytes with the conditioned medium from the fibroblasts at the same time as the contact of the epidermal keratinocytes with the microparticles and the test substance. In this case, the microparticles and the test substance are mixed with the conditioned medium from the fibroblasts and contacted with the epidermal keratinocytes. In addition, from the viewpoint of further improving 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 load on the growth and survival of the cells, it is preferable to contact for 24 hours or less.

[0075] Next, in step (B), the amount of microparticles taken up by the epidermal keratinocytes is measured based on the microparticle uptake activity of the epidermal keratinocytes obtained in step (A) above.

[0076] The amount of microparticles taken up by epidermal keratinocytes can be measured, for example, by measuring the amount of fluorescent beads taken up by epidermal keratinocytes using a fluorescent plate reader.

[0077] Next, in step (C), inhibition of phagocytosis of epidermal keratinocytes is examined based on the amount of microparticles taken up by the epidermal keratinocytes measured in step (B) above, and the effect of the test substance in inhibiting the formation of spots is evaluated.

[0078] Whether or not the test substance inhibits phagocytosis can be determined, for example, by comparing the fluorescence intensity of phagocytosis measured in step (B) above with the fluorescence intensity of phagocytosis in the absence of contact with the test substance.

[0079] In step (C), if the fluorescence intensity of phagocytosis measured in step (B) is, for example, significantly less than the fluorescence intensity of phagocytosis in the absence of contact with the test substance, it can be determined that the test substance inhibits phagocytosis.

[0080] In this way, when it is determined that the test substance inhibits phagocytosis, the test substance can be evaluated as a substance that exhibits a pigmentation formation inhibitory effect. Furthermore, the greater the difference between the phagocytosis fluorescence intensity measured in step (B) above and the phagocytosis fluorescence intensity of a cell group not contacted with the test substance, the greater the pigmentation formation inhibitory effect of the test substance can be evaluated.

[0081] As described above, the above screening method makes it possible to easily evaluate the stain formation inhibitory effect of a test substance based on the presence or absence of inhibition of phagocytosis and the degree of inhibition, thereby making it possible to quickly and easily screen stain formation inhibitors that are useful as stain formation inhibitors and evaluate the stain formation inhibitory effect of stain formation inhibitors and the like. EXAMPLES

[0082] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to such examples.

[0083] (Production Example 1) Normal human epidermal keratinocytes (NHEK) 58cm 2 Normal human epidermal keratinocytes (manufactured by Kurashiki Boseki Co., Ltd.) were seeded on KG2 medium (manufactured by Kurashiki Boseki Co., Ltd.) using a petri dish (manufactured by IWAKI Co., Ltd.), and incubated at 37°C and 5% CO 2 The cells were cultured under the conditions.

[0084] (Production Example 2) Normal human fibroblasts (NHDF) 58cm 2 Normal human fibroblasts (Kuraray Industries, Inc.) were seeded in DMEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% FBS using a petri dish (IWAKI Co., Ltd.), and incubated at 37°C and 5% CO2 The cells were cultured under the conditions.

[0085] (Production Example 3) Preparation of conditioned medium from normal human fibroblasts When the normal human fibroblasts cultured in Production Example 2 reached confluence, the medium was replaced with fresh KB2 medium (Kuraray Industries, Inc.), and after culturing for 24 hours, the supernatant was recovered and used as the conditioned medium from the normal human fibroblasts.

[0086] (Production Example 4) Preparation of synthetic melanin-containing conditioned medium The synthetic melanin-containing conditioned medium was prepared by adding synthetic melanin to the conditioned medium of Production Example 3 to give a concentration of 100 μg / mL. The synthetic melanin used was "Melanin" (manufactured by MERCK).

[0087] (Production Example 5) Preparation of conditioned medium containing fluorescent beads The fluorescent bead-containing conditioned medium was prepared by adding 500 times the amount of the conditioned medium of Production Example 3 to 1 part by mass of fluorescent beads. The fluorescent beads are "FluoSpheres TM Carboxylate-modified microspheres, 0.2 μm, blue fluorescence (365 / 415), 2% solids (ThermoFisher Scientific) were used.

[0088] (Test Example 1) Verification of phagocytosis of synthetic melanin by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0089] The normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate (IWAKI) at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 The medium was then replaced with the conditioned medium prepared in Production Example 4 and incubated at 37°C, 5% CO 2 The cells were cultured for 6 hours under these conditions. The conditioned medium was then removed, and the cells were washed with PBS(-), after which they were visually observed under a 100x optical microscope (Olympus). The results are shown in Figure 1.

[0090] As a control, normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate (IWAKI) at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 After culturing for 24 hours under these conditions, the medium was replaced with KB2 medium containing synthetic melanin at a concentration of 100 μg / mL, and then incubated at 37°C, 5% CO 2 The cells were cultured under the above conditions for 6 hours and then visually observed.

[0091] As is clear from Figure 1, a clear difference was observed in the degree of synthetic melanin in normal human epidermal keratinocytes exposed to conditioned medium from normal human fibroblasts, compared to the control cells that were not exposed to the medium. This also shows that phagocytosis is significantly promoted by exposing normal human epidermal keratinocytes to conditioned medium from normal human fibroblasts.

[0092] (Test Example 2) Verifying phagocytosis of fluorescent beads by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts (observation by fluorescence microscope)

[0093] The normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 The medium was then replaced with the conditioned medium prepared in Production Example 5 and incubated at 37°C, 5% CO 2 The cells were cultured for 6 hours under these conditions. The conditioned medium was then removed, and the cells were washed with PBS(-), after which they were visually observed under a fluorescent microscope (product name: IX71, manufactured by OLYMPUS). The results are shown in Figure 2.

[0094] As a control, normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate (IWAKI) at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 After culturing for 24 hours under these conditions, the medium was replaced with a medium prepared by adding 500 times the amount of KB2 medium per part by mass of fluorescent beads, and then incubated at 37°C and 5% CO2 The cells were cultured under the above conditions for 6 hours and then visually observed.

[0095] As is clear from Figure 2, it was confirmed that the intensity of the fluorescence taken up into the cells was higher in the cells in which the conditioned medium from normal human fibroblasts was contacted with normal human epidermal keratinocytes than in the control where no contact was made. This also shows that phagocytosis is significantly promoted by contacting normal human epidermal keratinocytes with the conditioned medium from normal human fibroblasts.

[0096] (Test Example 3) Verifying phagocytosis of fluorescent beads by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts (measured with a fluorescent plate reader)

[0097] The normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 The medium was then replaced with the conditioned medium prepared in Production Example 5 and incubated at 37°C, 5% CO 2 The cells were cultured for 6 hours under these conditions. The conditioned medium was then removed, and the cells were washed with PBS(-), after which the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) using a fluorescent plate reader (Thermo Fisher Scientific). The results are shown in Figure 3. "***" in the graph indicates that the p-value of the t-test is less than 0.001.

[0098] As a control, normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate (IWAKI) at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 After culturing for 24 hours under these conditions, the medium was replaced with a medium prepared by adding 500 times the amount of KB2 medium per part by mass of fluorescent beads, and then incubated at 37°C and 5% CO 2 The fluorescence intensity was measured after incubation for 6 hours under the above conditions.

[0099] As is clear from Figure 3, it was confirmed that the fluorescence intensity was higher in normal human epidermal keratinocytes exposed to the conditioned medium from normal human fibroblasts than in the control, which was not exposed to the cells. This also shows that phagocytosis is significantly promoted by exposing normal human epidermal keratinocytes to the conditioned medium from normal human fibroblasts.

[0100] (Test Example 4) Verification of actin reorganization by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0101] Normal human epidermal keratinocytes were plated on a 4-chamber slide (BD Falcon) at 2,500 cells / cm. 2 The cells were seeded at 37°C and 5% CO 2 After that, the conditioned medium was replaced with the conditioned medium prepared in Production Example 3, and the medium was then incubated at 37°C and 5% CO 2 The cells were cultured for 2 to 6 hours under the above conditions. The conditioned medium was then removed and washed with PBS(-). The cells were then fixed with 4% formaldehyde, treated with 0.5% Triton-100, and blocked with 1% BSA. 200 μL of 165 nM rhodamine phalloidin (Fujifilm Wako Pure Chemical Industries, Ltd.) and 20 μM Hoechst 33258 (Takara Bio Inc.) dissolved in PBS(-) were added to the cells, stained for 30 minutes, and sealed in an Entelaneu (MERCK Co., Ltd.), and the cells were visually observed and evaluated under a fluorescent microscope (product name: IX71, OLYMPUS Co., Ltd., rhodamine phalloidin Ex: 550 nm / Em: 580 nm, Hoechst 33258 Ex: 360 nm / Em: 420 nm). The results are shown in Figure 4. In the figure, the scale bar indicates 50 μm.

[0102] As a control, normal human epidermal keratinocytes were cultured on a 4-chamber slide (BD Falcon) at 2,500 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 After culturing for 24 hours under these conditions, the conditioned medium was not replaced with the conditioned medium prepared in Production Example 3, and the cells were visually observed and evaluated in the same manner as above.

[0103] As is clear from Figure 4, in cells in which conditioned medium from normal human fibroblasts was contacted with normal human epidermal keratinocytes, F-actin (filamentous actin) was stained with rhodamine phalloidin near the cells, compared to the control cells that were not contacted. This also shows that F-actin polymerization is increased (actin reorganization) by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts.

[0104] (Test Examples 5 and 6) Verification of phagocytosis inhibitory effect by actin reorganization inhibitors

[0105] As a test substance in Test Example 5, a chelating agent, BAPTA-AM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), was used (concentrations: 5.0 μM, 7.5 μM). As a test substance in Test Example 6, an actin inhibitor, Cytochalasin D (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used (concentrations: 0.3 μM, 0.6 μM).

[0106] The normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 The medium was then replaced with the conditioned medium prepared in Production Example 5 containing the test substances of Test Examples 5 and 6, and then incubated at 37° C., 5% CO 2 The cells were cultured for 6 hours under these conditions. The conditioned medium was then removed, and the cells were washed with PBS(-), after which the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) using a fluorescent plate reader (Thermo Fisher Scientific). The results are shown in Figures 5 and 6. "***" in the graphs 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.

[0107] As a control, a test sample not containing the test substance was used in the same test methods as in Test Examples 5 and 6 above, and the fluorescence intensity was measured in the same manner using a fluorescent plate reader.

[0108] As is clear from Figures 5 and 6, it was confirmed that the cells exposed to the actin reorganization inhibitor had lower fluorescence intensity than the control cells that were not exposed to the actin reorganization inhibitor, confirming that phagocytosis can be inhibited by exposure to the actin reorganization inhibitor.

[0109] (Test Example 7) Test of the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0110] Gallic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., concentrations: 0.00125%, 0.0025%, 0.005%) was used as the test substance.

[0111] The normal human epidermal keratinocytes cultured in Production Example 1 were plated on a 96-well plate at 90,000 cells / cm 2 The cells were seeded at 37°C and 5% CO 2 The medium was then replaced with the conditioned medium prepared in Production Example 5 containing the plant extract, which was the test substance, and incubated at 37° C., 5% CO 2 The cells were cultured for 6 hours under the above conditions. The conditioned medium was then removed, and the cells were washed with PBS(-), after which the fluorescence intensity was measured (Ex: 365 nm, Em: 415 nm) using a fluorescent plate reader (Thermo Fisher Scientific). The results are shown in FIG.

[0112] As a control, a test sample not containing the test substance was used in the same test method as in Test Example 7 above, and the fluorescence intensity was measured in the same manner using a fluorescent plate reader.

[0113] As is clear from Figure 7, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the control cells that were not contacted with the test substance, confirming that phagocytosis can be inhibited by contacting the cells with the test substance.

[0114] (Test Example 8) Test of the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0115] The test substances used in the same test method as in Test Example 7 were epicatechin gallate (manufactured by Nagara Science Co., Ltd., concentrations: 0.0025% and 0.005%) and epigallocatechin gallate (manufactured by Nagara Science Co., Ltd., concentrations: 0.0025% and 0.005%). The results are shown in Figure 8. Note that a test substance not containing the test substance was used as a control in the same test method as in Test Example 8 above.

[0116] As is clear from Figure 8, it was confirmed that the cells contacted with the test substance had lower fluorescence intensity than the control cells that were not contacted with the test substance, confirming that phagocytosis can be inhibited by contacting the cells with the test substance.

[0117] (Test Example 9) Test of the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0118] The test substances used in the same test method as in Test Example 7 were arbutin (manufactured by Nippon Fine Chemicals, 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 (product name: Black Tea Liquid, manufactured by Ichimaru Pharcos, concentration: 0.1%). The results are shown in Figure 11. Note that a test substance not containing the test substance was used as a control in the same test method as in Test Example 9 above.

[0119] As is clear from Figure 9, it was confirmed that the fluorescence intensity was unchanged in the cells contacted with arbutin, nicotinamide, or kojic acid, compared to the control without contact. In other words, it was found that arbutin, nicotinamide, and kojic acid, which are known to have whitening effects, had no effect at all on the induction of phagocytosis in epidermal keratinocytes after contacting the epidermal keratinocytes with a conditioned medium for fibroblasts.

[0120] (Test Examples 10 and 11) Test of the inhibitory effect of phagocytosis by contacting normal human epidermal keratinocytes with conditioned medium from normal human fibroblasts

[0121] The same test method as in Test Example 7 was used, and the test substances used were Job's tears extract (product name: Coix seed extract, manufactured by Koei Kogyo Co., Ltd., concentration: 1%), Hibamata extract (product name: Falcorex Hibamata, manufactured by Ichimaru Pharcos Co., Ltd., concentration: 1%), and Alpinia speciosa extract (product name: Alpinia alpina leaf extract BG, manufactured by Maruzen Pharmaceutical Co., Ltd., concentration: 1%). The results are shown in Figures 10 and 11. Note that a substance not containing the test substance was used as a control, using the same test method as in Test Examples 10 and 11 above.

[0122] 10 and 11, it was confirmed that the fluorescence intensity did not change in the cells contacted with Job's tears extract, Fucus extract, or Alpinia speciosa extract, compared to the control that was not contacted. In other words, it was found that Job's tears extract, Fucus extract, and Alpinia speciosa extract, which are known to inhibit melanin production (inhibit tyrosinase), had no effect whatsoever on the induction of phagocytosis in epidermal keratinocytes after the epidermal keratinocytes were contacted with a conditioned medium for fibroblasts.

[0123] From the above results, it was confirmed that the polyphenol compound, which is the active ingredient of the present invention, has a significantly excellent inhibitory effect on the actin reorganization and phagocytosis induction of epidermal keratinocytes after contacting the epidermal keratinocytes with a conditioned medium of fibroblasts, and is useful as a pigmentation formation inhibitor. In addition, it is possible to provide a pigmentation formation inhibitor effect by applying this to a skin external preparation.

[0124] On the other hand, arbutin, nicotinamide, kojic acid, Job's tears extract, Fucus vesiculosus extract, and Alpinia speciosa extract, which are known to have whitening effects, showed no effect whatsoever on the induction of phagocytosis in epidermal keratinocytes after contacting the epidermal keratinocytes with a conditioned medium for fibroblasts.

Claims

1. An inhibitor that inhibits phagocytosis, wherein the phagocytosis is induced when conditioned medium from fibroblasts comes into contact with epidermal keratinocytes, and the phagocytosis inhibitor is characterized by containing a polyphenol compound as an active ingredient.

2. The phagocytosis inhibitor according to claim 1, wherein the polyphenol compound is one or more selected from the group consisting of gallic acid and catechin derivatives.

3. The phagocytosis inhibitor according to claim 2, wherein the catechin derivative is epicatechin gallate and / or epigallocatechin gallate.

4. A stain formation inhibitor for inhibiting stain formation involving phagocytosis of epidermal keratinocytes, wherein the phagocytosis is induced when conditioned medium from fibroblasts comes into contact with epidermal keratinocytes, and the stain formation inhibitor is characterized in that it contains a phagocytosis inhibitor described in any one of claims 1 to 3.

5. An external skin preparation for inhibiting the formation of age spots involving phagocytosis of epidermal keratinocytes, the phagocytosis being induced when epidermal keratinocytes come into contact with a conditioned medium from fibroblasts, and characterized in that the external skin preparation contains the age spot formation inhibitor described in claim 4.

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