JP6583704B2 - Screening method for pigmentation improvers - Google Patents

Description

  The present invention relates to a method for screening for a pigmentation improving agent using the melanin excretion mechanism of keratinocytes. It also relates to a pigmentation improving agent based on a new mechanism of activation of the melanin excretion mechanism.

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.

  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). Keratinocytes divide and are gradually pushed up, and eventually turn into keratinocytes without cell nuclei and become exfoliated (turnover). Melanin also peels off with the cells following a turnover with a period of about 28 days. An integrin production promoter (Patent Document 7) and the like have been reported as targets for promoting this epidermal turnover.

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

Control of melanin pigment and development of whitening agent (Fragrance Journal, extra edition), No. 14, P.I. 118-126 (1995)

Although the whitening component based on the known mechanism of action as described above has a certain pigmentation improving 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, with the aim of developing whitening cosmetics that can still achieve higher effects, search for new ingredients effective in improving pigmentation and investigation of new mechanisms of action that can be targets for pigmentation-improving agents have been made. ing.
In view of such circumstances, an object of the present invention is to search for an effective component as a pigmentation-improving agent based on a new mechanism of action, and to establish a new screening method therefor.

  As a result of intensive studies to solve the above problems, the present inventor has found that keratinocytes incorporating melanin have reduced exocytosis activity. It was also found that the expression of exocytosis-related factors increased / decreased in keratinocytes with reduced exocytosis activity. Furthermore, it has also been found that melanin excretion of keratinocytes is activated by regulating the activity of exocytosis-related factors. And based on these knowledge, it discovered that the activity regulator of the exocytosis related factor could become a pigmentation improvement agent, and came to complete this invention.

That is, the present invention is as follows.
[1] A method for screening for a pigmentation-improving agent using the activity of an exocytosis-related factor in cells as an index (hereinafter also referred to as “the screening method of the present invention”).
[2] The activity of the exocytosis-related factor is an expression level of a gene encoding a protein constituting the exocytosis-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 is determined to have a pigmentation improving effect. The method according to [1].
[3] The exocytosis-related factor is selected from VAMP-1, syntaxin-2, syntaxin-3, syntaxin-4, SNAP-23, annexin-1, annexin-2, myosin V, myosin-10, and myosin-2a The method according to [1] or [2].
[4] The method according to [3], wherein the exocytosis-related factor is syntaxin-3 or SNAP-23.
[5] The expression level of the gene encoding the protein constituting the exocytosis factor in the cell to which the test substance is added varies by 10% or more relative to the expression level of the gene in the cell to which the test substance is not added. In some cases, the method according to any one of [2] to [4], wherein the test substance is determined to have a pigmentation-improving action.
[6] A composition containing a substance determined to have a pigmentation-improving action by the method according to any one of [1] to [5] (hereinafter also referred to as “the composition of the present invention”).
[7] The composition according to [6], which is an external preparation for skin.
[8] The composition according to [6] or [7], which is a cosmetic (however, including quasi drugs). [9] The composition according to any one of [6] to [8], which is for whitening.
[10] A method for producing a composition, comprising a step of blending a composition that is determined to have a pigmentation-improving action by the method according to any one of [1] to [5].

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

The graph showing the change of the mRNA expression level of an exocytosis related factor by melanin uptake. The graph showing the change of melanin output by exocytosis activation. The graph showing the change of the total area of the black area | region of the binarized melanin distribution image by exocytosis activation. The graph showing the change of the mRNA expression level of the exocytosis related factor by exocytosis activation. Graph showing changes in melanin excretion due to inhibition of expression of exocytosis-related factors. The melanin distribution image at the time of the plant extract addition which accelerates | stimulates the expression of an exocytosis related factor. The graph showing the change of the total area of the black area | region of the binarized melanin distribution image at the time of the plant extract addition which accelerates | stimulates the expression of an exocytosis related factor.

The screening method of the present invention is characterized by screening a pigmentation-improving agent using the activity of an exocytosis-related factor in a cell as an index.
The exocytosis-related factor in the present invention refers to a protein that exists in the cell membrane of keratinocytes and is involved in the exocytosis mechanism.
Exocytosis (exocytosis) is one of the secreted forms to the outside of cells. Substances such as proteins synthesized outside the cell are stored in the secretory granule and released outside the cell by exocytosis. Secretory granules move in the cytoplasm by the action of intracellular fibers and approach the cell membrane. The outer layer of secretory granule membrane and the inner layer of secretory granule membrane are fused with the outer layer of cell membrane, whereby the secretory granule lumen communicates with the extracellular space, and the granule contents migrate out of the cell. In the present specification, various proteins involved in this series of mechanisms are referred to as exocytosis-related factors.

  The inventor has found that keratinocytes incorporating melanin have reduced exocytosis activity, keratinocytes with reduced exocytosis activity have increased / decreased expression of exocytosis-related factors, and exocytosis. It was found that keratinocyte melanin excretion mechanism is activated by regulating the activity of tosis-related factors. When the melanin excretion mechanism of keratinocytes is activated, the melanin excretion is promoted to the outside of the cells, and the melanin stays in the keratinocytes. Therefore, an activity regulator of an exocytosis-related factor can be a pigmentation improving agent.

  In a preferred embodiment of the present invention, the activity of the exocytosis-related factor is an expression level of a gene encoding a protein constituting the exocytosis-related factor. 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 another preferred embodiment of the screening method of the present invention, the expression level of a gene encoding a protein constituting an exocytosis-related factor in a cell to which a test substance is added is expressed in the cell to which the test substance is not added. The test substance is determined to have a pigmentation-improving action when it varies compared to the gene expression level. Here, fluctuations in the expression level indicate that the expression level increases for genes involved in the direction of promoting exocytosis, and the expression level decreases for genes related to the direction of suppressing exocytosis. Including.
Here, exocytosis-related factors include VAMP-1, syntaxin-2, syntaxin-3, syntaxin-4, SNAP-23, annexin-1, and annexin-2, which are known as vesicle transport-related proteins, and motor proteins Preferred examples include myosin V, myosin-10, and myosin2a. Among these, VAMP-1, syntaxin-4, annexin-1, annexin-2, and myosin V are when the expression level of the gene is smaller when the test substance is added than when the test substance is not added. The test substance is determined to have a pigmentation improving agent action. Also, syntaxin-2, syntaxin-3, SNAP-23, myosin-10, and myosin2a are expressed when the expression level of the gene is larger when the test substance is added than when the test substance is not added. The test substance is determined to have a pigmentation improving agent action.
In the screening method of the present invention, the exocytosis-related factor using the activity as an index may be one kind or a combination of two or more kinds. From the viewpoint of the accuracy of screening, it is preferable to combine two or more exocytosis-related factors, for example, a combination of syntaxin-3 and SNAP-23 is more preferable. Syntaxin-3 and SNAP-23 are considered to function simultaneously to act on keratinocytes exocytosis, and it can be determined that substances that increase both of these expression levels have an excellent pigmentation-improving effect.

The expression level of the gene encoding the protein constituting the exocytosis-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.
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 type of cell used for screening is not particularly limited as long as it can express an exocytosis-related factor, but keratinocyte or fibroblast is preferred, keratinocyte is more preferred, and human-derived keratinocyte is more preferred. Cell culture conditions include normal culture conditions, for example, a commercially available KG2 medium, or a gene encoding a protein constituting an exocytosis-related factor, which does not interfere with the execution of the screening method of the present invention. Any culture conditions that do not interfere with the measurement of the expression level of 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, solvent removal products of purified products, Examples of plant-derived extracts include native or grown plants, extracts that are sold as herbal medicine raw materials, commercially available extracts, and the like.
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, ethers such as diethyl ether and tetrahydrofuran. One or two or more selected from polar solvents such as the above can be exemplified as suitable ones. 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 part used for extraction of a plant or the like or a dried product thereof. For example, after immersion for several hours and cooling to room temperature, the insoluble matter and / or solvent may be removed if desired, and fractional purification may be performed 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 a gene encoding an exocytosis-related factor as an index is quantitatively measured by RT-PCR using a primer that specifically detects the gene. . 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) of the gene in the cell to which the test substance is not added, preferably 10% or more with respect to the control More preferably, when the sample fluctuates by 15% or more, more preferably by 20% or more, the test substance is determined to have a pigmentation-improving action. The determined substance can be a pigmentation improving agent.

As used herein, “pigmentation” means that melanin stays in keratinocytes. “Improvement” of pigmentation means that keratinocytes that have already taken up melanin promote the discharge of melanin out of the cell. In addition, “improvement” of pigmentation includes “prevention” of pigmentation, and “prevention” prevents the decrease in the activity of the exocytosis mechanism in keratinocytes delivered from melanocytes. This refers to maintaining or enhancing the extracellular excretion effect. By improving or preventing pigmentation, the skin tone becomes lighter or brighter, and a whitening effect can be obtained.
The improvement in pigmentation means that at the cellular level, the total amount of intracellular melanin decreased and the area measured as intracellular melanin aggregates was smaller compared to the control. Can be confirmed by analyzing. Alternatively, it can be confirmed by measuring the amount of melanin excreted outside the cell and increasing it as compared to the control.
In addition, at the skin tissue level, it can be confirmed that pigmentation has been improved by measurement using a known colorimeter such as a color difference meter or spectrocolorimeter, visual evaluation, or the like.

A substance (pigmentation improving agent) determined to have a pigmentation improving action by the screening method of the present invention 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, however, in order to exert pigmentation-improving action, taking into account the retention in the skin, the efficiency of reaching the target site, etc. It is preferable to use an agent.

The composition containing the pigmentation-improving agent of the present invention can be suitably used for whitening applications.
Conventional whitening agents and skin whitening external preparations have led to skin whitening by suppressing the production of melanin by inhibiting tyrosinase synthesis.
On the other hand, a substance (pigmentation improving agent) determined to improve pigmentation by the screening of the present invention can be a whitening agent by a new mechanism.
Normally, melanin synthesized in melanocytes located in the basal layer, which is the lowest part of the epidermis in the skin, is delivered to adjacent keratinocytes. Keratinocytes divide and are gradually pushed up, and eventually turn into keratinocytes without cell nuclei and become exfoliated (turnover). As the melanin peels off with the cells following a turnover of about 28 days, it takes time for the produced melanin to disappear from the skin cells. However, since the pigmentation-improving agent of the present invention has an action of activating the melanin excretion mechanism of keratinocytes whose activity is reduced by melanin uptake as described above, the melanin is positively activated without waiting for turnover. It can be discharged outside. Also, if the turnover cycle is disturbed due to aging, excessive ultraviolet rays, excessive skin irritation, stress, etc., the metabolism will stagnate, melanin will also stay in the epidermis, pigmentation will progress and symptoms such as spots The pigmentation-improving agent of the present invention actively discharges melanin out of the cell, suppresses melanin retention,
It can prevent pigmentation from occurring.

The content (blending amount) of the pigmentation-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. The amount is usually 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass. If the content (blending amount) of the pigmentation-improving agent is too small, the desired effect may be difficult to obtain. If the content is too large, the effect may reach its peak, and the degree of freedom of composition formulation may be impaired. is there.
Moreover, the kind of pigmentation-improving agent contained in the composition may be not only one but also two or more kinds.

  In the production of the composition of the present invention, 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 pigmentation 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. Additive and synergistic effects can be expected by combining with the pigmentation-improving agent of the present invention, which has a different mechanism of action from conventional whitening components.
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 expression of exocytosis-related factor by melanin uptake The expression change of exocytosis-related factor in keratinocytes incorporating melanin was examined by the following procedure.
5 × 10 ⁶ B16 mouse melanoma cells were collected by trypsin treatment and suspended in 3 mL of PBS. After centrifugation (1000 rpm, 2 minutes), the supernatant was removed, and 2.5 mL of cold homogenization buffer (0.25 M sucrose, 10 mM Tris, 10 mM KCl) 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 was collected, and suspended by adding 361.3 μL of 90% (v / v) percoll and 800 μL of the supernatant (final concentration 28% (v / v) percoll). Centrifugation (20000 g, 45 minutes, 4 ° C.) was performed, and the black layer that was visible near the bottom of the Eppendorf tube was collected to obtain isolated melanosomes.
Neonatal human skin keratinocytes were seeded in a 24-well plate with KG2 medium so that the number of cells was 5.0 × 10 ⁴ cells / well. 24 hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01% (v / v). After 24 hours, the melanosomes adhering to the cell membrane were removed by pipetting well with PBS, and RNeasy MiniKit (QIAGEN) RLT buffer was added to the keratinocytes of the 24-well plate to extract mRNA. RT-PCR was performed using QuantTect Primer Assay, and the amounts of mRNA of vesicle transport-related proteins (syntaxin3 and SNAP-23) and motor proteins (myosin10, myosinV, myosin2a) expressed in keratinocytes were measured. The nucleotide sequences of human syntaxin 3 and SNAP-23 gene and the encoded amino acid sequences are shown in SEQ ID NOs: 1 to 4, respectively. The used primer is shown to sequence number 5-8. Using 18 srRNA as an endogenous control, the mRNA expression level of each gene was calculated by the comparative CT method, and the relative value when the gene expression level in keratinocytes not added with melanosomes was set to “1” was expressed as the gene expression level in melanosome-added keratinocytes did.
As shown in FIG. 1, it can be seen that the expression level of both syntaxin3 and SNAP-23 in keratinocytes was reduced by melanin uptake.

<Reference Example 2> Examination of melanin excretion when exocytosis is promoted In the following procedure, the presence or absence of melanin excretion during exocytosis in keratinocytes incorporating melanin was confirmed.
Isolated melanosomes were obtained in the same manner as in Reference Example 1.
Neonatal human skin keratinocytes were seeded in a 24-well plate with KG2 medium at 7.0 × 10 ⁴ cells / well. 24 hours after seeding, the medium was replaced with KG2 medium (500 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01% (v / v). After 24 hours, the melanosomes adhering to the cell membrane were removed by pipetting well with PBS, and phorbol 12-myristate 13-acetate (PMA), an exocytosis promoting reagent, was added to a final concentration of 100 nM, Exposure for 15 minutes activated exocytosis. As a control, PMA-free keratinocytes were also prepared in the same manner. Thereafter, the medium was replaced with KG2 medium and cultured for 24 hours, and then the culture supernatant was collected and stored at −80 ° C.
The medium stored at −80 ° C. was taken out, dissolved, and the amount of melanosomes in the medium was quantified by ELISA. The quantification method is shown below. First, 100 μL of the collected medium was added to ELISA plate (Sumitomo Bakelite, catalog No. MS-8596F), and reacted at 4 ° C. overnight. Thereafter, the medium was removed, and 300 μL of a 3% BSA-containing PBS solution was added to each well, followed by blocking at room temperature for 1 hour. The solution was removed, and 100 μL of a primary antibody: gp100 (abcam, catalog No. ab137078) diluted 1000-fold with PBS containing 3% BSA was added to each well and allowed to react at room temperature for 2 hours. Thereafter, the solution was removed and washed 3 times with a PBS solution containing 0.1% Tween20. Further, 100 μL of a secondary antibody: Anti-Rabbit IgG H & L (HRP) (abcam, catalog No. ab6721) diluted 1000-fold with PBS containing 3% BSA was added to each well and allowed to react at room temperature for 2 hours. Thereafter, the solution was removed and washed 3 times with a PBS solution containing 0.1% Tween20. 100 μL of TMB solution (Ceracare Life Sciences, catalog No. 52-00-01) was added, color was developed for 20 minutes, and 1N hydrochloric acid solution was added to stop the reaction. The absorbance at 450 nm was measured, and the relative value when the absorbance value in the keratinocytes not added with PMA was “1” was defined as the melanin excretion amount in the PMA-added keratinocytes.
As shown in FIG. 2, it can be seen that the amount of melanin excretion to the outside of the cells increased in the keratinocytes that promoted exocytosis.

<Reference Example 3> Examination of the influence of melanin uptake on the expression of exocytosis-related factors Preparation of keratinocytes that activated exocytosis by addition of PMA and control keratinocytes without addition of PMA as in Reference Example 2 did. After replacing with KG2 medium and culturing for 24 hours, the medium was removed, the cells were washed with PBS, fixed and encapsulated with 4% paraformaldehyde. A melanin distribution image was acquired with a confocal microscope, binarized using image analysis software ImageJ, and the total area of the black region in the same visual field area was measured.
As shown in FIG. 3, it can be seen that in keratinocytes that promoted exocytosis, the amount of melanin distribution was small and pigmentation was improved.

<Reference example 4> Examination of influence on expression of exocytosis-related factor at the time of melanin excretion In the following procedure, expression change of exocytosis-related factor expressed in keratinocyte at the time of melanin excretion was confirmed.
Neonatal human skin keratinocytes were seeded in a 24-well plate with KG2 medium so that the number of cells was 5.0 × 10 ⁴ cells / well. 24 hours after sowing, PMA was added to a final concentration of 100 nM, and exposure was performed for 15 minutes to activate exocytosis. As a control, PMA-free keratinocytes were also prepared in the same manner. After culturing for 24 hours, RLT buffer of RNeasyMiniKit (QIAGEN) was added, mRNA was extracted, RT-PCR was performed using QuantTect Primer Assay, and factors that decreased expression during melanin uptake (syntaxin3, SNAP-23) The amount of mRNA was measured. In addition, the primer shown to sequence number 5-8 was used. Using 18 srRNA as an endogenous control, the mRNA expression level of each gene was calculated by the comparative CT method, and the relative value when the gene expression level in keratinocytes not added with PMA was set to “1” was expressed as the gene expression level in PMA-added keratinocytes. did.
As shown in FIG. 4, it can be seen that the expression levels of syntaxin3 and SNAP-23 increased in keratinocytes that promoted exocytosis.

<Reference Example 5> Examination of influence on melanin excretion by inhibition of expression of exocytosis-related factor In the following procedure, expression of exocytosis-related factor (syntaxin3, SNAP-23) expressed in keratinocytes is expressed by siRNA. The effect on melanin excretion when inhibited by using was investigated.
Isolated melanosomes were obtained in the same manner as in Reference Example 1.
Neonatal human skin keratinocytes were seeded in a 24-well plate with KG2 medium so that the number of cells was 7.0 × 10 ⁴ cells / well. 24 hours after sowing, 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 + TransIT-TKO (Mirus) 1 μL) was prepared, mixed well, and incubated for 10 minutes. To this transfection reagent, Flexitube syntaxin3 siRNA (QIAGEN, catalog No. SI03035942) or SNAP-23 siRNA (QIAGEN, catalog No. SI000564448) was added to a final concentration of 50 nM, mixed well, and incubated for 10 minutes. did. This siRNA-added transfection reagent was added to the keratinocytes (50 μL / well). For comparison, keratinocytes without siRNA added transfection reagent were also prepared in the same manner. After 24 hours, the medium was removed, washed with PBS, PMA was added to a final concentration of 100 nM, and exocytosis was activated by exposure for 15 minutes, followed by further incubation for 24 hours. As a control, PMA-free keratinocytes were also prepared in the same manner. The culture supernatant was collected and stored at -80 ° C.
The medium stored at −80 ° C. was taken out and dissolved, and the amount of melanosomes in the medium was quantified in the same manner as in Reference Example 2. The absorbance at 450 nm was measured, and the relative value when the absorbance value in the keratinocytes not added with PMA was “1” was defined as the melanin excretion amount in each keratinocyte.
As shown in FIG. 5, in keratinocytes that inhibited the expression of exocytosis-related factors, it was found that melanin excretion to the outside of the cells was suppressed even when an exocytosis promoting reagent was used.

<Example 1> Screening using mRNA expression level of exocytosis-related factor as an indicator Screening for pigmentation improving agents using the activity of exocytosis-related factor syntaxin3 or SNAP-23 as an indicator was performed by the following procedure. .
Neonatal human skin keratinocytes were seeded in a 24-well plate with KG2 medium so that the number of cells was 5.0 × 10 ⁴ cells / well. 24 hours after sowing, the plant extract shown in Table 1 was added as a test substance (1 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, RT-PCR was performed using QuantiTect Primer Assay as an index, and syntaxin3 And each expression level of SNAP-23 was measured. In addition, the primer shown to sequence number 5-8 was used. The relative expression when each mRNA expression level of syntaxin3 and SNAP-23 was calculated by comparative CT method using 18srRNA as an endogenous control, and each mRNA expression level of syntaxin3 and SNAP-23 in the keratinocytes to which no extract was added was “1”. The value was defined as the gene expression level in the extract-added keratinocytes.
As shown in Table 1, it can be seen that the gene expression levels of both syntaxin3 and SNAP-23 were increased by the addition of enamel extract.

<Example 2> Confirmation of pigmentation improvement effect by extract that promotes mRNA expression level of exocytosis-related factor Isolated melanosomes were obtained in the same manner as in Reference Example 1.
Neonatal human normal keratinocytes were seeded on a ^4- well chamber slide at 7.0 × 10 ⁴ cells / well, respectively. 24 hours after seeding, the medium was replaced with KG2 medium (1000 μL / well) supplemented with isolated melanosomes to a final concentration of 0.01% (v / v). Twenty-four hours later, the melanosomes attached to the cell membrane were removed by pipetting well with PBS. As a positive control, PMA was added to a final concentration of 100 nM and exposed for 15 minutes to activate exocytosis, and further cultured for 24 hours. On the other hand, as a test substance, a sample in which the mRNA expression levels of both syntaxin3 and SNAP-23 were increased in Example 1 (Emisodia extract) was added to a final concentration of 1% (10 μL / well) for another 24 hours. Cultured. After the culture, the medium was removed, the cells were washed with PBS, fixed and encapsulated with 4% paraformaldehyde. A melanin distribution image was acquired with a confocal microscope (FIG. 6). The image was binarized using image analysis software ImageJ, and the total area of the black region in the same visual field area was measured.
As shown in FIG. 7, it can be seen that the keratinocytes to which the enamel extract was added had a small amount of melanin distribution and improved pigmentation.

  Since the screening method of the present invention can search for an effective component as a whitening agent based on a new mechanism of action, it is very useful industrially.

Claims (7)

A method of screening for a pigmentation-improving agent using the activity of an exocytosis-related factor in keratinocytes as an index ,
The method wherein the exocytosis-related factor is syntaxin-3 or SNAP-23 . The activity of the exocytosis-related factor is the expression level of a gene encoding a protein constituting the exocytosis-related factor;
Determining the expression level of the gene in keratinocytes adding the test substance, as compared to the expression level of the gene in keratinocytes was not added to the test substance when large heard, the test substance to have a pigmentation improving effect The method of claim 1. When the expression level of the gene encoding the protein constituting the exocytosis factor in the keratinocyte added with the test substance is 10% or more larger than the expression level of the gene in the keratinocyte not added with the test substance, The method according to claim 1 or 2 , wherein the test substance is determined to have a pigmentation improving action. A step of performing the method according to any one of claims 1 to 3 , and a step of adding a substance determined to have a pigmentation-improving action in the step to the composition. Design method. The design method according to claim 4 , wherein the composition is an external preparation for skin. The design method according to claim 4 or 5 , wherein the composition is a cosmetic (including a quasi-drug). The design method according to any one of claims 4 to 6 , wherein the composition is for whitening.