JP6460426B2 - Screening method for skin tone improver - Google Patents

Description

  The present invention relates to a method for screening for a skin tone improving agent.

Pigmentation, blemishes, liver spots, senile pigment spots, etc. after sunburn in the skin are a state in which melanin production is remarkably enhanced by activation of melanocytes (pigment cells) present in the skin. Since the occurrence and worsening of troubles related to skin pigmentation hinders the beauty of the skin, research on ingredients having an action to prevent or improve these has been actively conducted, and various action mechanisms have been studied. A whitening ingredient based on the
For example, ascorbic acids, hydrogen peroxide, colloidal sulfur, glutathione, hydroquinone, catechol and the like (Non-Patent Document 1) are well known as whitening components. In recent years, various whitening components based on a new mechanism of action have been developed. Targets for suppressing melanin production include tyrosinase-related protein inhibitors (Patent Literature 1), endothelin action inhibitors (Patent Literature 2), proton pump inhibitors (Patent Literature 3), stem cell factor binding inhibitors (Patent Literature) 4) etc. Examples of the target for suppressing the migration of the produced melanin to the epidermis include a melanocyte dendrite elongation inhibitor (Patent Document 5), a melanin transfer or release inhibitor (Patent Document 6), and the like. In addition, an integrin production promoter (Patent Document 7) and the like have been reported as targets for promoting epidermal turnover.

By the way, melanin synthesized by melanocytes located in the basal layer, which is the lowest part of the epidermis in the skin, is delivered to adjacent keratinocytes (epidermal keratinocytes). In keratinocytes, the transferred melanin moves on the microtubules and gathers above the cell nucleus to form a melanin aggregate called a melanin cap (nuclear cap). Here, in keratinocytes, the role of carrying melanin to the periphery of the cell nucleus is responsible for the intracellular vesicle transport mechanism, and it is known that various transport-related factors are involved in this mechanism (non-patent literature). 2).
In relation to this, a method for improving the skin condition by regulating the expression of dynein, a protein that transports melanin in keratinocytes, has been disclosed (Patent Document 8).

JP 2010-195732 A JP2012-020950A JP 2011-178760 A JP 2008-031094 A Japanese Patent Laid-Open No. 2003-113027 JP 2008-143796 A JP 2006-045087 A International Publication No. 2012/091832 Pamphlet

Control of melanin pigment and development of whitening agent (Fragrance Journal, extra edition), No. 14, P.I. 118-126 (1995) The Journal of investigative dermatology, vol.121, No 4, Oct., 2003, 813-820

Although the whitening component based on the known mechanism of action as described above has a certain skin tone improvement effect, it is difficult to say that a sufficiently satisfactory effect is obtained. Further, in order to obtain a higher whitening effect, it is common that components based on different action mechanisms are contained in one composition, and therefore a whitening component based on a new action mechanism is also required.
Therefore, aiming at the development of whitening cosmetics that can still achieve higher effects, search for new ingredients effective in improving skin tone, and study of new mechanisms of action that can be targets for skin tone improvers. Has been made.
In view of such circumstances, an object of the present invention is to search for an effective component as a skin tone improving agent based on a new action mechanism, and to establish a new screening method therefor.

  As a result of intensive research to solve the above problems, the present inventor has found that cells are promoted to promote differentiation of keratinocytes incorporating melanin on the basis of a known fact that the upper layer of skin undergoing differentiation is light in color tone. We obtained new findings at the cellular level that melanin diffuses inside. In addition, new findings were found that melanin diffuses into cells by regulating the activity of intracellular vesicle transport-related factors in keratinocytes. And based on these knowledge, it discovered that the activity regulator of a differentiation related factor and / or an intracellular vesicle transport related factor can become a skin color improvement agent, and came to complete this invention.

That is, the present invention is as follows.
[1] A method of screening for a skin tone improving agent using the activity of differentiation-related factors and / or intracellular vesicle transport-related factors in cells as an index (hereinafter also referred to as “screening method of the present invention”).
[2] The activity of the differentiation-related factor is an expression level of a gene encoding a protein constituting the differentiation-related factor,
When the expression level of the gene in the cell to which the test substance is added is smaller or larger than the expression level of the gene in the cell to which the test substance is not added, the test substance has an effect of improving the skin tone. The method according to [1], wherein the method is determined to have.
[3] The activity of the intracellular vesicle transport-related factor is an expression level of a gene encoding a protein constituting the intracellular vesicle transport-related factor,
When the expression level of the gene in the cell to which the test substance is added is smaller or larger than the expression level of the gene in the cell to which the test substance is not added, the test substance has an effect of improving the skin tone. The method according to [1], wherein the method is determined to have.
[4] The differentiation-related factor is involucrin, keratin 1, kera
tin 5, keratin 10, keratin 14, filaggrin, lor
icrin, desmoglein 1, desmoglein 2, desmoglein 3, desmocolin 1, transglutaminase 1, and tra
The method according to [1] or [2], which is selected from nsglutaminase 3.
[5] The intracellular vesicle transport-related factor is syntaxin-2, syntaxin-3, syntaxin-4, annexin-1, annexin-2, VAMP-1, VAMP-2, VAMP-3, VAMP-4, VAMP. -5, VAMP-7, VAMP-8, Sec22b, Ykt6, SNAP-23, myosin V, myosin-10, m
The method according to [1] or [3], which is selected from yosin2a, uKHC, nKHC, kintin, KIFC3, Rab7, RILP, PLEKHA7, Nezha, 120catenin, α-tubulin, and β-tubulin.
[6] The method according to [5], wherein the intracellular vesicle transport-related factor is VAMP-1.
[7] The expression level of a gene encoding a protein constituting a differentiation-related factor in a cell to which the test substance is added and / or a protein encoding a protein constituting an intracellular vesicle transport-related factor is added to the test substance. When there is a variation of 10% or more with respect to the expression level of the gene in the cells that are not present, it is determined that the test substance has an action of improving the skin tone, [2] to [6] the method of.
[8] The method according to any one of [1] to [7], wherein the skin color tone improving action is an action of diffusing melanin into cells.
[9] A composition containing a substance determined to have an action of improving skin tone by the method according to any one of [1] to [8] (hereinafter also referred to as “the composition of the present invention”) .
[10] The composition according to [9], which is an external preparation for skin.
[11] The composition according to [9] or [10], which is a cosmetic (including quasi drugs).
[12] The composition according to any one of [9] to [11], which is for whitening.
[13] Production of a composition comprising a step of blending a composition with a substance determined to have an effect of improving skin tone by the method according to any one of [1] to [8] Method.

  According to the present invention, there is provided a screening method capable of searching for an effective component as a skin color tone improving agent based on a new mechanism of action.

A bright-field observation image showing the influence on the melanin distribution in cells by the presence or absence of differentiation induction. The graph showing the area ratio of the melanin assembly per unit cell number by the presence or absence of differentiation induction. The bright field observation image showing the influence on the melanin distribution in a cell by the presence or absence of inhibition of VAMP-1 mRNA expression. The graph showing the area ratio of the melanin assembly per unit cell number by the presence or absence of inhibition of VAMP-1 mRNA expression.

The screening method of the present invention is characterized by screening a skin tone improving agent using the activity of differentiation-related factors and / or intracellular vesicle transport-related factors in cells as an index.
The differentiation-related factor in the present invention refers to a protein that decreases or increases in expression with cell differentiation. The intracellular vesicle transport-related factor in the present invention refers to a protein involved in vesicle transport that transports melanin to the periphery of the cell nucleus in keratinocytes. The present inventor suppresses the formation of a melanin cap structure by promoting the differentiation of keratinocytes and / or regulating the activity of intracellular vesicle transport-related factors, and allows melanin to diffuse inside the cell. Focused on. Here, it is speculated that the promotion of differentiation of keratinocytes has some influence on the activity of intracellular vesicle transport-related factors. The inventor has also found that even in the case of the same amount of melanin in the cell, the aggregate is smaller in size and diffused to be brighter and lighter in color. Therefore, the differentiation promoting agent and / or the activity regulator of intracellular vesicle transport-related factor can be a skin tone improving agent.

  In a preferred embodiment of the present invention, the activity of the differentiation-related factor and / or intracellular vesicle transport-related factor is the expression level of a gene encoding a protein constituting the differentiation-related factor and / or intracellular vesicle transport-related factor. It is. Here, the gene expression level refers to either the transcription level of mRNA of the gene or the translation level of the protein encoded by the gene.

In a preferred embodiment of the screening method of the present invention, the expression level of a gene encoding a protein constituting a differentiation-related factor in a cell to which a test substance is added is compared with the expression level of the gene in a cell to which no test substance is added. The test substance is determined to have an action of promoting cell differentiation and improving skin tone. Here, the variation in the expression level includes a decrease in the expression level for a gene whose expression decreases with cell differentiation and an increase in the expression level for a gene whose expression increases with cell differentiation.
Here, differentiation-related factors are involucrin, keratin 1, and kerati.
n 5, keratin 10, keratin 14, filaggrin, loric
rin, desmoglein 1, desmoglein 2, desmoglein
3, desmocolin 1, transglutaminase 1, and transglutaminase 3 are preferable. Among these, keratin 5, keratin 14 and the like have the effect that the test substance improves the skin tone when the expression level of the gene is small when the test substance is added compared to when the test substance is not added. It is determined that Also, involucrin, keratin 1, keratin 10, filaggrin, loricrin, desmoglein 1, desmog
lein 2, desmoglein 3, desmocolin 1, transglu
Taminate 1, transglutaminase 3 and the like, when the test substance is added, when the expression level of the gene is larger than when the test substance is not added, the test substance has an action to improve the skin tone Determined.

In another preferred embodiment of the screening method of the present invention, the expression level of a gene encoding a protein constituting an intracellular vesicle transport-related factor in a cell to which a test substance is added is a cell to which no test substance is added. When the expression level of the gene fluctuates in comparison with the above, the test substance is determined to have an action of improving skin tone. Here, the fluctuation of the expression level is such that the expression level decreases for genes involved in the direction of promoting vesicle transport to the periphery of the cell nucleus, and the genes involved in the direction of suppressing vesicle transport to the periphery of the cell nucleus. This includes increasing the expression level.
Here, intracellular vesicle transport-related factors are syntaxin-2, syntaxin-3, syntaxin-4, annexin-1, annexin-2, VAMP-1, VAMP-2, VAMP-3, VAMP-4, VAMP-. 5, VAMP-7, VAMP-8, Sec22b, Ykt6, SNAP-23, myosin V, myosin-10, m
Preferred examples include yosin2a, uKHC, nKHC, kinectin, KIFC3, Rab7, RILP, PLEKHA7, Nezha, 120 catenin, α-tubulin, and β-tubulin. Among these, VAMP-1, VAMP-2, VAMP-3, VAMP-4, VAMP-5, VAMP-7, VAMP-8, Sec22b, Ykt6, KIFC3, Rab7, RILP, etc. were added with test substances. Sometimes, when the expression level of the gene is small compared to when the test substance is not added, the test substance is determined to have an action of improving the skin tone. In addition, uKHC, nKHC, kinectin and the like have the effect of improving the skin tone when the test substance is added and the gene expression level is larger than when the test substance is not added. It is determined to have.

Among the above-mentioned factors related to cell vesicle transport, VAMP which is a vesicle-binding membrane protein is more preferable, specifically, VAMP-1 / Synaptobrevin1, VAMP-2 / Synaptobrevin2, VAMP-3 / Cellubrevin, VAMP-4, VAMP. -5, VAMP-7 / Ti-VAMP, VAMP-8 / Endobrevin, Sec22b, Ykt6 and the like, and VAMP-1 is particularly preferable.
VAMP-1 has been reported to be involved in expression in human keratinocytes and vesicle trafficking in keratinocytes. The nucleotide sequence of human VAMP-1 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.

The expression level of a gene encoding a protein constituting a differentiation-related factor and / or an intracellular vesicle transport-related factor can be measured using any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer, and quantitative detection is performed. The gene sequences encoding the various factors described above are publicly disclosed, and those skilled in the art can appropriately design primers and use them for PCR. In particular, when screening is performed using the expression level of VAMP-1 as an index, a primer kit for detection is commercially available and can be suitably used.
Further, for example, the intracellular expression level of the protein may be quantitatively measured by a conventional method to obtain the gene expression level.

  The cell type used for screening is not particularly limited as long as it is a cell that can express a differentiation-related factor and / or an intracellular vesicle transport-related factor, but keratinocytes or fibroblasts are preferable, keratinocytes are more preferable, and human-derived keratinocytes are Further preferred. Cell culture conditions include normal culture conditions, for example, a commercially available KG2 medium, and other factors that do not interfere with the execution of the screening method of the present invention, specifically differentiation-related factors and / or intracellular vesicle transport-related factors. As long as the culture conditions do not interfere with the measurement of the expression level of the gene encoding the protein that constitutes the above, it can be applied without particular limitation.

The test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals or plants, or a mixture thereof.
The extracts derived from animals and plants mean not only animal or plant-derived extracts themselves, but also generic names of extract fractions, purified fractions, extracts or fractions, and solvent-removed products of purified products. Examples of plant-derived extracts include native or grown plants, extracts using products sold as herbal medicine ingredients, and commercially available extracts.
For the extraction operation, the whole plant part can be used, and other parts such as the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, the stem part, the flower ear, and the flower bud can be used. It is preferable to improve the extraction efficiency by cutting. Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. 1 type or 2 types or more selected from polar solvents, such as these, can be illustrated as a suitable thing. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. If so, there may be mentioned a method of immersing for several hours, cooling to room temperature, removing insoluble matters and / or solvents if desired, and fractionating and purifying by column chromatography or the like.

An example of the procedure in the screening method of the present invention is given below, but is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.
First, a test substance is added to cells and incubated for 24 hours. Thereafter, mRNA is extracted from the cells, and the expression level of the gene encoding the differentiation-related factor and / or intracellular vesicle transport-related factor as an index is RT-PCR using a primer that specifically detects the gene And measure quantitatively. As a control, the expression level of the gene is also measured in cells to which no test substance was added. When the expression level of the gene in the cell to which the test substance is added is smaller or larger than the expression level (control) in the cell to which the test substance is not added, preferably 10% or more with respect to the control, More preferably, when it fluctuates 15% or more, more preferably 20% or more, it is determined that the test substance has an action of improving skin tone. The determined substance can be a skin tone improving agent.

In the present invention, the “skin color tone” refers to the degree of lightness and darkness of the skin. “Improved color tone” means that the color tone is lighter and brighter than the untreated state.
The improvement in color tone can be confirmed by analyzing the bright-field image of the microscope at the cellular level, that the area measured as intracellular melanin aggregates is smaller than the control. . That is, when the size of the melanin aggregate in the cell is smaller than that of the control and is diffused into the cell in a fine particle state, and the melanin aggregate apparently decreases or disappears, the cell It can be said that the cells have improved color tone. In this case, the actual increase or decrease in the total amount of melanin in the cell is not questioned.
Further, at the skin tissue level, it can be confirmed that the color tone has been improved by measurement with a known colorimeter such as a color difference meter or a spectrocolorimeter, visual evaluation, or the like.

The substance determined to improve the skin tone by the screening method of the present invention (skin tone improver) can be contained in the composition by any preparation method.
As the composition, cosmetics, quasi-drugs, pharmaceuticals and the like can be suitably exemplified, and since they can be used on a daily basis, cosmetics and quasi-drugs are more preferable. The route of administration is not particularly limited, but in order to exert an effect of improving the skin tone, the transdermal administration is applied to the skin in consideration of the storage property on the skin, the efficiency of reaching the target site, etc. An external preparation is preferred.

The composition containing the skin tone improver of the present invention can be suitably used for whitening.
Conventional whitening agents and whitening skin external preparations have been directed to whitening the skin by suppressing the production of melanin by inhibiting tyrosinase synthesis.
On the other hand, the substance (skin color tone improving agent) determined to improve the skin tone by the screening of the present invention suppresses the formation of the melanin cap structure presumed to be caused by the differentiation state of the cells as described above. Thus, the melanin aggregate has a function of making fine particles and diffusing into the cells, so that it can be a whitening agent by a new mechanism.

The content (blending amount) of the skin color tone improving agent in the composition of the present invention is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more. Yes, usually 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass. If the content (blending amount) of the skin tone improver is too small, it may be difficult to obtain the desired effect, and if it is too much, the effect will reach its peak and the freedom of formulation of the composition may be impaired. It is.
Moreover, the kind of skin color tone improving agent contained in the composition is not limited to one, but may be two or more.

  In the production of the composition, optional components usually used in the preparation of cosmetics, quasi drugs, pharmaceuticals, and the like can be blended. For example, hydrocarbons such as squalane, petrolatum, microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow, coconut oil, stearic acid, oleic acid, retinoic acid Fatty acids such as oleyl alcohol, stearyl alcohol, higher alcohols such as octyldodecanol, anionic surfactants such as sulfosuccinate and sodium polyoxyethylene alkyl sulfate, amphoteric surfactants such as alkylbetaine salts, dialkylammonium salts Cationic surfactants such as, sorbitan fatty acid ester, fatty acid monoglyceride, these polyoxyethylene adducts, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester Nonionic surfactants, polyhydric alcohols such as polyethylene glycol, glycerin, 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, etc. Can be optionally blended.

Moreover, you may mix | blend the whitening component other than the skin color tone improving agent of this invention with the composition of this invention. For example, whitening ingredients such as ascorbic acids, hydrogen peroxide, colloidal sulfur, glutathione, hydroquinone, catechol, tyrosinase related protein inhibitors, endothelin action inhibitors, proton pump inhibitors, stem cell factor binding inhibitors, etc. Examples thereof include dendrite elongation inhibitors, melanin transport or release inhibitors, integrin production promoters, and the like.
Manufacture of a composition can be performed without difficulty by processing and mix | blending these components in accordance with a conventional method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.

<Reference example 1> Examination of influence on differentiation of melanin by promotion of differentiation In the following procedure, distribution of melanin in cells when differentiation was promoted under high calcium conditions was examined.
B16 mouse melanoma cells 5 × 10 ⁶ cells were collected by trypsin treatment and suspended in 3 mL of PBS. After centrifugation (1000 rpm, 2 minutes) and removal of the supernatant, cold homogenization buffer (0.25M sucrose, 10 mM Tris, 10 mM KCl)
2.5 mL was added to suspend the cells. The mixture was homogenized 20 times with a 2.5 mL syringe and a 25 G syringe on an ice bath, and then centrifuged (700 g, 10 minutes, 4 ° C.). The supernatant is recovered and 90% p
361.3 μL of ercoll and 800 μL of supernatant were added and suspended (final concentration 28% pe).
rcoll). Centrifugation (20000 g, 45 minutes, 4 ° C.) was performed, and the black layer that was visible near the bottom of the Eppendorf was collected to obtain isolated melanosomes.

Neonatal human normal keratinocytes were seeded in a 4-well chamber slide with KG2 medium at 1.0 × 10 ⁵ cells / well. Two hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01%. Twenty-four hours later, the melanosomes adhering to the cell membrane were removed by pipetting well with PBS, and then replaced with KG2 medium (250 μL / well) containing 1.0 mM calcium to induce differentiation. After 24 hours, keratinocytes were fixed with 4% paraformaldehyde, and then cell nuclei were stained with Hoechst 33342 (Dojindo Laboratories). After encapsulation, the nucleus and melanin distribution images are obtained by fluorescence observation and bright field observation with a fluorescence microscope, and the total area of the black region of the binarized melanin distribution image using image analysis software ImageJ (National Institutes of Health, USA) The average area and the number of cells for each particle were measured, and the average area of the melanin aggregate per unit cell number was calculated from these.

FIG. 1 shows an image of melanin distribution in keratinocytes taken in a bright field, and FIG. 2 shows an average area ratio of melanin aggregates per unit cell number calculated. By promoting differentiation, the average area of the melanin aggregate was suppressed to 62% of the control. When cells were lysed and the amount of intracellular melanin was quantified by measuring absorbance, it was also confirmed that the total amount of intracellular melanin did not change depending on whether differentiation was induced.
Therefore, melanin was not excreted extracellularly, but melanin aggregates were microparticulated and diffused into the cells, so that melanin apparently decreased or disappeared, resulting in an improvement in color tone at the cellular level.

<Example 1> Screening using keratin 10 mRNA expression level as an index Screening for skin tone improvers was performed using the activity of keratin 10 as a differentiation-related factor as an index according to the following procedure. The base sequence of human keratin 10 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 3 and 4, respectively.
Neonatal human normal keratinocytes were seeded in KG2 medium on 24-well plates and 4-well chamber slides at 1.0 × 10 ⁵ cells / well, respectively. Two hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01%. Twenty-four hours later, the melanosomes adhered to the cell membrane were removed by pipetting well with PBS, and then replaced with KG2 medium (250 μL / well). Furthermore, various plant extracts were added as test substances (2.5 μL / well). After 24 hours, the medium was removed, the plate was washed with PBS, RNeasyMiniKit (QIAGEN) RLT buffer was added to the keratinocytes of the 24-well plate, and mRNA was extracted.
RT-PCR was performed using tiTect Primer Assay (QIAGEN, Catalog No. QT00017045) to measure the amount of keratin 10 mRNA.
The mRNA expression level of keratin 10 was calculated by the comparative CT method using β-actin as an endogenous control, and keratin 10 mRN in keratinocytes to which no extract was added.
The relative expression when the expression level of A is “1” is shown. The keratinocytes on the 4-well chamber slide were fixed with 4% paraformaldehyde, and then the cell nucleus was fluorescently stained with Hoechst 33342 (Dojindo Laboratories). After encapsulation, images of cell nuclei and melanin distribution are obtained by fluorescence observation and bright field observation of a fluorescence microscope, and using image analysis software ImageJ, the total area of the black region of the binarized melanin distribution image, the average area for each particle and The number of cells was measured, and from these, the average area of melanin aggregates per unit cell number was calculated.

The results are shown in Table 1. The peony root extract, water-soluble proteoglycan, olive leaf extract, and ghetto leaf extract were found to have a keratin 10 expression enhancing action and a significant intracellular melanin diffusion action.

<Reference Example 2> Examination of influence on melanin distribution by inhibition of expression of intracellular vesicle transport-related factor The distribution of melanin in cells when siRNA was used to inhibit the expression of VAMP-1 in the following procedure was examined. .
B16 mouse melanoma cells 5 × 10 ⁶ cells were collected by trypsin treatment and suspended in 3 mL of PBS. After centrifugation (1000 rpm, 2 minutes) and removal of the supernatant, cold homogenization buffer (0.25M sucrose, 10 mM Tris, 10 mM KCl)
2.5 mL was added to suspend the cells. The mixture was homogenized 20 times with a 2.5 mL syringe and a 25 G syringe on an ice bath, and then centrifuged (700 g, 10 minutes, 4 ° C.). The supernatant is recovered and 90% p
361.3 μL of ercoll and 800 μL of supernatant were added and suspended (final concentration 28% pe).
rcoll). Centrifugation (20000 g, 45 minutes, 4 ° C.) was performed, and the black layer that was visible near the bottom of the Eppendorf was collected to obtain isolated melanosomes.

Neonatal human normal keratinocytes were seeded in KG2 medium on 24-well plates and 4-well chamber slides at 1.0 × 10 ⁵ cells / well, respectively. Two hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01%. Twenty-four hours later, the melanosomes adhering to the cell membrane were removed by pipetting well with PBS, and then replaced with antibiotic-free KG2 medium (250 μL / well). Separately, a transfection reagent (Opti-Mem 50 μL + Trans
IT-TKO (Mirus) 1 μL) was prepared, mixed well, and incubated for 10 minutes. To this transfection reagent, FlexiTube VAMP-1 siRNA (QIAGEN, catalog No. SI03151365) was added to a final concentration of 25 nM, mixed well, and incubated for 10 minutes. This siRNA-added transfection reagent was added to the keratinocytes (50 μL / well). After 24 hours, the medium was removed, the plate was washed with PBS, RNeasyMiniKit (QIAGEN) RLT buffer was added to the keratinocytes of the 24-well plate, mRNA was extracted, and J Invest Dermatol. 2001 Feb; 116 (2): 296-304. RT-PCR was performed using the custom DNA primers (SEQ ID NOs: 5 and 6, Life Technologies Japan Co., Ltd.) designed with reference to the above, and the amount of VAMP-1 mRNA was measured. The keratinocytes on the 4-well chamber slide were fixed with 4% paraformaldehyde, and then the cell nucleus was fluorescently stained with Hoechst 33342 (Dojindo Laboratories). After encapsulation, the nucleus and melanin distribution images are obtained by fluorescence observation and bright field observation with a fluorescence microscope, and the total area of the black region of the binarized melanin distribution image using image analysis software ImageJ (National Institutes of Health, USA) The average area and the number of cells for each particle were measured, and the average area of the melanin aggregate per unit cell number was calculated from these.

The amount of VAMP-1 mRNA in the keratinocytes to which siRNA was added was 38% of the control, and it was confirmed that the activity of VAMP-1 was inhibited in the cells. FIG. 3 shows an image of melanin distribution in keratinocytes taken in a bright field, and FIG. 4 shows an average area ratio of melanin aggregates per unit cell count. By inhibiting the expression of VAMP-1, the average area of the melanin aggregate was suppressed to 86% of the control. In addition, when the cell was melt | dissolved and the amount of intracellular melanin was quantified by the light absorbency measurement, it was also confirmed that the total amount of intracellular melanin does not change with the presence or absence of the inhibition of mRNA expression of VAMP-1.
Therefore, melanin was not excreted extracellularly, but melanin aggregates were microparticulated and diffused into the cells, so that melanin apparently decreased or disappeared, resulting in an improvement in color tone at the cellular level.

<Example 2> Screening using VAMP-1 mRNA expression level as an index Screening for skin tone improvers was performed using the activity of VAMP-1 which is an intracellular vesicle transport-related factor as an index according to the following procedure.
Neonatal human normal keratinocytes were seeded in KG2 medium on 24-well plates and 4-well chamber slides at 1.0 × 10 ⁵ cells / well, respectively. Two hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01%. Twenty-four hours later, the melanosomes adhered to the cell membrane were removed by pipetting well with PBS, and then replaced with KG2 medium (250 μL / well). Furthermore, various plant extracts were added as test substances (2.5 μL / well). After 24 hours, the medium was removed, washed with PBS, RNeasyMiniKit (QIAGEN) RLT buffer was added to the keratinocytes of the 24-well plate, mRNA was extracted, and custom DNA primers (SEQ ID NOs: 5 and 6, Life Technologies Japan) RT-PCR was used to measure the amount of VAMP-1 mRNA. The mRNA expression level of VAMP-1 was calculated by the comparative CT method using β-actin as an endogenous control, and the relative value when the expression level of VAMP-1 mRNA in the cornified cells not added with the extract was “1”. Indicated. The keratinocytes on the 4-well chamber slide were fixed with 4% paraformaldehyde, and then the cell nucleus was fluorescently stained with Hoechst 33342 (Dojindo Laboratories). After encapsulation, images of cell nuclei and melanin distribution are obtained by fluorescence observation and bright field observation of a fluorescence microscope, and using image analysis software ImageJ, the total area of the black region of the binarized melanin distribution image, the average area for each particle and The number of cells was measured, and from these, the average area of melanin aggregates per unit cell number was calculated.

  The results are shown in Table 2. Eight extracts of loquat leaf extract, polyglutamic acid, cocoon extract, aloe vera leaf extract, chlorella extract, bilberry leaf extract, hydrolyzed black bean extract and seagrass extract were found to have VAMP-1 expression inhibitory action and significant intracellular melanin diffusion action.

  The screening method of the present invention makes it possible to search for an effective component as a skin color tone improving agent based on a new mechanism of action, which is very useful industrially.

Claims (8)

A method of screening for a skin tone improving agent using the activity of VAMP-1 in cells as an index. The VAMP-1 activity is the expression of a gene encoding proteins that comprise the VAMP-1,
The expression level of the gene in cells supplemented with test substance, in comparison with not small as if the expression level of the gene in cells not adding the test substance, the test substance is an action of improving the tone of skin The method of claim 1, wherein the method is determined to have. When the expression level of the gene encoding the protein constituting VAMP-1 in the cell to which the test substance is added varies by 10% or more with respect to the expression level of the gene in the cell to which the test substance is not added, The method according to claim 2, wherein the test substance is determined to have an action of improving skin tone. Tone improving effect of the skin is a function of diffusing the melanin in the cells, A method according to any one of claims 1-3. A step of screening a skin tone improver by the method according to any one of claims 1 to 4 , and a step of blending a composition determined to have an action of improving the skin tone in the step with the composition. A method for designing a composition comprising the steps of: The design method according to claim 5 , wherein the composition is an external preparation for skin. The design method according to claim 5 or 6 , wherein the composition is a cosmetic (including a quasi-drug). The design method according to any one of claims 5 to 7 , wherein the composition is for whitening.

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