JP2019004748A - Screening method for pigmentation inhibitor - Google Patents

Abstract

To provide screening methods for pigmentation inhibitors.SOLUTION: The present invention provides a screening method for a pigmentation inhibitor using the activity of a regulator of cell migration of melanocytes as an index. Preferably, the activity of the regulator is a gene encoding a protein constituting the regulator or an expression amount of the protein, and the screening method comprises adding a test substance to cells, and selecting a test substance in which the expression amount in cells added with the test substance varies compared with the expression amount in cells to which the test substance is not added.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for screening for a pigmentation inhibitor.

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, 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.
Aiming at the development of whitening cosmetics that can still achieve higher effects, the search for new ingredients that are effective in improving pigmentation and the study of new mechanisms of action that can be targets for pigmentation inhibitors are being made. .

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

Control of melanin pigment and development of whitening agent (Fragrance Journal, extra edition), No. 14, P.I. 118-126 (1995) T.A. Yamada et al. , Journal of Dermatological Science, Volume 73, Issue 3, March 2014, Pages 251-257.

  By the way, it has been reported that melanocytes are densely present in the epidermis at the site of skin spots (Non-patent Document 2). Therefore, the skin tone can be reduced overall by suppressing melanin production for each melanocyte, but the contrast of the spot portion is maintained. In addition, nearby melanocytes stimulate each other, the production of melanin is promoted, and the problem is that the spots become darker and larger.

It is an object of the present invention to search for an effective component as a pigmentation inhibitor that approaches a stain by a new mechanism, and an object thereof is to establish a new screening method therefor.

As a result of earnest research, the present inventors have found a gene group highly correlated with pigmentation such as spots in yellow races, and also found that there are regulators of cell migration of melanocytes among them.
And where melanocytes are densely present in the spots of spots, moving the melanocytes to disperse them eliminates the appearance of relatively dark skin spots and spots, or suppresses excessive production of melanin Assuming that the activity of the regulator of cell migration of melanocytes is used as an index, the inventors came up with the idea that a material capable of suppressing pigmentation such as spots could be screened.

That is, the present invention is as follows.
[1] A screening method for a pigmentation inhibitor using as an index the activity of a regulator of cell migration of melanocytes.
[2] The activity of the regulatory factor is the gene encoding the protein constituting the regulatory factor or the expression level of the protein, the step of adding a test substance to the cell, and the expression level in the cell to which the test substance is added The method for screening according to [1], comprising a step of selecting a test substance that changes in comparison with the expression level in cells to which the test substance has not been added.
[3] The control factor is a factor that suppresses cell migration of melanocytes, the change is a decrease in expression level, and the expression level in a cell to which a test substance is added is expressed in a cell to which no test substance is added. The screening method according to [2], wherein a test substance that is 90% or less of the amount is selected.
[4] The screening method according to [3], wherein the control factor is selected from the group consisting of an NCAM2 gene, an ABR gene, an SMYD3 gene, and a KIAA0825 gene.
[5] The control factor is a factor that promotes cell migration of melanocytes, the change is an increase in expression level, and the expression level in a cell to which a test substance is added is expressed in a cell to which no test substance is added. The screening method according to [2], wherein a test substance that is 110% or more of the amount is selected.
[6] The screening method according to any one of [2] to [5], wherein the cell is a melanocyte.
[7] A method for designing a composition, comprising a step of performing the screening method according to any one of [1] to [6], and a step of containing a substance selected by the step.
[8] The design method according to [7], wherein the composition is a whitening cosmetic.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided a screening method for a pigmentation inhibitor that uses a novel mechanism to eliminate stains using the activity of a regulator of cell migration of melanocytes as an index.

The microscope picture ((A) control, (B) ABR gene expression suppression) showing the 24 hour positional change of the melanocyte which suppressed the expression of ABR gene. In both cases, the magnification is 40, the circle indicates the position of the cell nucleus at the start of tracking, and the arrow indicates the movement path at 24 hours. The graph showing the movement distance for 24 hours of the melanocyte which suppressed expression of each gene. (A) NCAM2 gene, (B) ABR gene, (C) SMYD3 gene, (D) KIAA0825 gene. The graph showing the movement distance for 24 hours of the melanocyte which added the test substance which suppresses expression of an ABR gene.

The screening method of the present invention uses the activity of a regulator of cell migration of melanocytes as an index.
As described above, melanocytes are densely present at the spot. Therefore, by moving and dispersing the melanocytes, it is possible to eliminate the appearance of relatively dark skin color and stains, or to suppress the excessive production of melanin. It can be a pigmentation inhibitor.
The factor that controls cell movement of melanocytes in the present invention may be either a factor that controls the cell movement of melanocytes or a factor that controls the cell movement of melanocytes. For example, as a factor controlling in a direction to suppress cell migration of melanocytes, NCAM2 gene, ABR gene, SMYD3 gene, and KIAA0825 gene, which are genes that the present inventors have newly found an association with stains, are preferably mentioned. It is done. These genes are known to be expressed in human skin or other organs (eg Nucleic Acids Research, 1989 Nov 11; 17 (21): 8821-31), but can also be expressed in melanocytes. It was also not known to be associated with skin pigmentation. In addition, human base sequences of these genes and amino acid sequences of proteins are known and can be obtained from GenBank and the like. However, the present invention is not limited to these, and the gene of a factor that regulates cell migration of melanocytes can be used as an index in the screening method of the present invention.

In the present invention, the activity of the regulator preferably refers to the gene encoding the protein constituting the regulator or the expression level of the protein.
The screening method of the present invention usually comprises a step of adding the test substance of the present invention to the cell, and the expression level in the cell to which the test substance is added is compared with the expression level in the cell to which the test substance is not added. Selecting a test substance that changes.

When the control factor is a factor that suppresses cell migration of melanocytes, the change is a decrease in the expression level in the screening method of the present invention. Here, the degree of decrease in the expression level may be small as long as the expression level in the cell to which the test substance is added is smaller than the expression level in the cell to which the test substance is not added, preferably 12 or 24 after the addition of the test substance. It can be said that it has changed when it has decreased to 90% or less after the lapse of time, more preferably 80% or less, and even more preferably 70% or less.
When the regulatory factor is a factor that suppresses cell migration of melanocytes, for example, when the regulatory factor is selected from the NCAM2 gene, ABR gene, SMYD3 gene, and KIAA0825 gene, suppression of expression of these genes, Increased cell migration correlates and consequently correlates with pigmentation inhibition. Therefore, by adding a test substance to a cell, a test substance in which expression suppression of these genes has occurred can be selected as a component having a pigmentation inhibitory action.

When the regulatory factor is a factor that enhances cell migration of melanocytes, the change is an increase in the expression level in the screening method of the present invention. Here, the degree of increase in the expression level is sufficient if the expression level in the cells to which the test substance is added is larger than the expression level in the cells to which the test substance is not added, and preferably 12 or 24 after the test substance is added. It can be said that it has changed when it has increased to 110% or more after a lapse of time, more preferably 120% or more, and even more preferably 130% or more.
When the regulatory factor is a factor that enhances cell migration of melanocytes, increased expression of these genes correlates with increased cell migration of melanocytes, and consequently correlates with suppression of pigmentation. Therefore, by adding a test substance to a cell, the test substance in which the expression of these genes is enhanced can be selected as a component having a pigmentation-inhibiting action.

In the present specification, the “pigmentation suppression” action usually means an action that eliminates the fact that melanocytes are densely packed and the color of that part of the skin looks relatively dark, and / or by suppressing melanin production. It refers to the action of preventing and / or improving the occurrence of pigmentation such as spots on the skin, resulting in a whitening effect on the skin.

The expression level of the melanocyte cell migration regulator can be measured by 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. In addition, when the regulator of cell migration of melanocytes is a known gene, for example, the base sequences of the NCAM2 gene, ABR gene, SMYD3 gene, KIAA0825 gene, etc. described above are disclosed, respectively, and those skilled in the art Primers can be appropriately designed and used for PCR.
In addition, for example, the expression level of the gene may be determined by quantitatively measuring the intracellular abundance of the protein encoded by the gene by a conventional method.

  In the screening method of the present invention, the cell to which the test substance is added is not particularly limited as long as it is a cell in which a gene for a cell migration regulator of melanocytes is present, but is usually a melanocyte.

The test substance targeted by the screening method of the present invention may be any of a pure substance, an extract derived from animals and 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.

Since the pigmentation inhibitor selected by the screening method of the present invention has an action of dispersing melanocytes by enhancing cell migration of melanocytes, it has an action of eliminating a portion where the skin color looks relatively dark due to the denseness of melanocytes. Have. Moreover, since activation by melanocytes adjoining and stimulating each other is suppressed, it also has an action of suppressing melanin production. Therefore, as a result of these, it has an action of suppressing pigmentation.
Therefore, the pigmentation inhibitor selected by the screening method of the present invention can be suitably blended as an active ingredient in a whitening composition such as a cosmetic.

That is, the present specification also provides a method for designing a composition, which includes the step of carrying out the screening method of the present invention and containing a substance (pigmentation inhibitor) selected thereby. Such compositions include compositions that are administered transdermally (for example, skin preparations such as cosmetics, quasi-drugs, and pharmaceuticals) and compositions that are orally administered / ingested (for example, foods and beverages, pharmacy). Oral compositions such as foreign products and pharmaceuticals).
Moreover, the dosage form of a composition is not specifically limited. For example, the dosage form in the case of being designed as a cosmetic includes a commonly known lotion preparation, emulsion preparation, essence preparation, cream preparation, powder-containing preparation, and the like.

When designing a composition containing a pigmentation inhibitor selected by the screening method of the present invention, the content (blending amount) of the pigmentation inhibitor is usually 0.00001% by mass or more, preferably 0.0001. It is at least mass%, more preferably at least 0.001 mass%, usually at most 80 mass%, preferably at most 30 mass%, more preferably at most 10 mass%. By setting it as the said range, it can be set as the composition which show | plays the pigmentation inhibitory effect suitably.
Moreover, the kind of pigmentation inhibitor contained in the composition may be not only one but also two or more kinds.

When designing a composition containing a pigmentation inhibitor selected by the screening method of the present invention, it may be designed so that necessary components are appropriately blended according to the above-described embodiment and application.
For example, when designing as a cosmetic composition, it is possible to broadly mix components that are usually used in cosmetics, and the dosage form and application are not limited at all. Hereinafter, components that can be contained in the cosmetic when mainly applied to the cosmetic will be described.

Examples of the active ingredient include other whitening ingredients, wrinkle improving ingredients, anti-inflammatory ingredients, extracts derived from animals and plants, and the like. In addition, you may mix | blend overlapping with the pigmentation inhibitor selected by the screening method of this invention.
Other whitening components are not particularly limited as long as they are generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, ellagic acid, kojic acid, linoleic acid, nicotinamide, 5,5′-dipropylbiphenyl-2, 2'-diol, 5'-adenylic acid disodium, cetyl tranexamic acid, 4-methoxysalicylic acid potassium salt, hydroquinone, pantothenic acid and the like.
Content of the whitening component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 0.3-5 mass% is more preferable.

The wrinkle improving component is not particularly limited as long as it is generally used in cosmetics. For example, sodium isopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate, nicotinamide, vitamin A or a derivative thereof (retinol, retinal, retinoic acid, tretinoin, isotretinoin, retinoic acid tocopherol, retinol palmitate And retinol acetate), ursolic acid benzyl ester, ursolic acid phosphoric acid ester, betulinic acid benzyl ester, and benzyl acid phosphoric acid ester.
Content of the wrinkle improvement component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.

The extracts derived from animals and plants are not particularly limited as long as they are generally used for pharmaceuticals, cosmetics, foods and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, achacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo biloba extract, Indian mushroom extract, fennel extract, udo extract, ages extract, sorghum extract , Enmezo extract, Ogon extract, Oat extract, Auren extract, Panax ginseng extract, Hypericum extract, Adrianthus extract, Orange extract, Oyster extract, Cuckoo extract, Chamomile extract, Carrot extract, Kawara mugwort extract, Licorice extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, Gardenia extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Black rice , Chlorella extract, mulberry extract, caquette extract, gentian extract, gentian extract, Gennosho extract, tea extract, burdock extract, rice extract, fermented rice extract, rice fermented extract, rice germ oil, bilberry extract, salvia extract, bonito extract , Sasa extract, Hawthorn extract, Sansha extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, Perilla extract, Linden extract, Citrus extract, Peonies extract, Pepper extract, Ginger root extract, Birch extract, Japanese cedar extract, Stevia extract, Stevia fermentation , Kizuta extract, Hawthorn extract, Elderberry extract, Yarrow extract, Pepper extract, Sage extract, Mallow Kiss, Senkyu Extract, Sembura Extract, Sakuhakuhi Extract, Daiou Extract, Soybean Extract, Taiso Extract, Thyme Extract, Dandelion Extract, Tea Extract, Clove Extract, Chimpi Extract, Green Tea Extract, Capsicum Extract, Toki Extract, Tokinsenka Extract, Tonin Extract, Spruce Extract , Dokudami extract, tomato extract, natto extract, carrot extract, garlic extract, wild rose extract, hibiscus extract, bacmond extract, lotus extract, parsley extract, birch extract, hammamellis extract, cypress extract, hinoki extract, loquat extract, dandelion extract, fukinotou extract , Bukuryo extract, Butcher bloom extract, Grape extract, Grape seed extract, Loofah extract, Safflower extract, Peppermint extract, Bo Daiju extract, button extract, hop extract, pine extract, mayonnaise extract, maronier extract, mizuba sho extract, mukuroji extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, yukinoshita extract, yuzu extract, lily extract, yakuinin extract, mugwort extract, lavender Extracts such as extract, green tea extract, apple extract, rooibos tea extract, litchi extract, lettuce extract, lemon extract, forsythia extract, forsythia extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract, and bitumen extract are preferred. Can be mentioned.
Content of the plant and animal origin extract in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.
Content of the plant and animal origin extract in foodstuffs is 0.01-80 mass% normally, 0.1-50 mass% is preferable and 1-30 mass% is more preferable.

Examples of the anti-inflammatory component include clarinone, glabrizine, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol, and the like, and preferably glycyrrhizic acid and its salt, glycyrrhetinic acid alkyl and its salt, and glycyrrhetic acid and its salt.
Content of the anti-inflammatory component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.

Examples of the oil component include polar oil and volatile hydrocarbon oil.
Polar oils include synthetic ester oils such as isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, lactic acid Cetyl, myristyl lactate, lanolin acetate, 2-ethylhexyl isononanoate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, monoisostearic acid N- Alkyl glycol, neopentyl glycol dicaprate, diisostearyl malate, glyceryl di-2-heptylundecanoate, tri-2-ethyl It may be mentioned hexanoic acid trimethylolpropane, trimethylolpropane triisostearate, tetra-2-ethylhexanoate pentane erythritol, tri-2-ethylhexanoate glyceryl, a trimethylolpropane triisostearate.

Furthermore, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptyl undecanoic acid glyceride, castor oil fatty acid methyl ester, oleic acid oil, cetostearyl alcohol, acetoglyceride, palmitic acid 2 -Heptylundecyl, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyl myristate Decyl, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, octylme Kishishin'nameto, etc. may also be mentioned.

  Natural oils such as avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, southern oil, castor oil, flaxseed oil, Safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, cinnagari oil, Japanese kiri oil, jojoba oil, germ oil, triglycerin, glyceryl trioctanoate, glyceryl triisopalmitate Etc.

  Examples of the volatile hydrocarbon oil include isododecane and isohexadecane.

As surfactants, fatty acid soap (sodium laurate, sodium palmitate, etc.), anionic surfactants such as potassium lauryl sulfate, alkylsulfuric triethanolamine ether, stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide Cationic surfactants such as, betaine surfactants (alkyl betaines, amide betaines, sulfobetaines, etc.), imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy 2) Sodium salts, etc.), amphoteric surfactants such as acylmethyltaurine,
Sorbitan fatty acid esters (such as sorbitan monostearate, sorbitan sesquioleate), glycerin fatty acid esters (such as glyceryl monostearate), propylene glycol fatty acid esters (such as propylene glycol monostearate), hydrogenated castor oil derivatives, glycerin alkyl Ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polysoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso) Stearates, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-O) Cutyl decyl ether, etc.), POE alkyl phenyl ethers (POE nonyl phenyl ether, etc.), Pluronic types, POE / POP alkyl ethers (POE / POP 2-decyl tetradecyl ether, etc.), Tetronics, POE castor oil / cured Castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid ester, alkyl glucoside, and the like.

  Polyhydric alcohols include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4 -Hexylene glycol, 1,2-hexanediol, 1,2-octanediol and the like.

Thickeners include guar gum, quince seed, carrageenan, galactan, gum arabic, pectin, mannan, starch, xanthan gum, curdlan, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, methylhydroxypropylcellulose, chondroitin sulfate, dermatan sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl Chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, Kill modified carboxyvinyl polymers, sodium polyacrylate, polyethylene glycol, and bentonite.

  As powders, the surface may be treated, mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc. May be treated, bengara, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine, bitumen, titanium oxide, zinc oxide inorganic pigments, surface may be treated, mica titanium, fish phosphorus foil, Pearl agents such as bismuth oxychloride, which may be raked Red 202, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223 No., Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, organic dyes, polyethylene powder, polymeta Methyl acrylic acid, nylon powder, organic powders such as organopolysiloxane elastomers, and the like.

  Examples of the UV absorber include paraaminobenzoic acid UV absorbers, anthranilic acid UV absorbers, salicylic acid UV absorbers, cinnamic acid UV absorbers, benzophenone UV absorbers, sugar UV absorbers, 2- (2 UV absorbers such as' -hydroxy-5'-t-octylphenyl) benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane, and the like.

  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> Effect of each regulatory factor on cell migration of melanocytes by inhibiting gene expression The following procedure examined the effect of melanocytes on cell migration by inhibiting the gene expression of each regulatory factor in melanocytes with siRNA. .
The siRNA described in Table 1 was introduced into normal human melanocytes (newborn, Dark / African American, Lot. 1223150, manufactured by Thermo Fisher Scientific) using NEPA21 manufactured by Nepagene. Non-coding siRNA was used as a control. The melanocytes were seeded in a 24-well plate (4.0 × 10 ⁴ cells / well), and O / N cultured at 37 ° C. for 24 hours in Medium 254 + HMGS medium (manufactured by Gibco).
The medium was replaced with Medium 254 + HMGS medium containing a cell nucleus staining reagent (0.1% Cellstein Hoechst 33342 solution, H342, manufactured by Dojindo) and incubated for 15 minutes. The cell nucleus staining reagent-containing medium was removed, and Medium 254 + HMGS medium was added (500 μL / well).

A microscope (an inverted research microscope IX83, manufactured by Olympus Corporation) and a moving image shooting software (Multi Dimensional Acquisition, manufactured by Molecular Devices Corporation) were activated, and a 24-well plate was placed on the stage. With the following settings, a time-lapse movie of melanocytes was taken, and the movie taken 24 to 48 hours after the start of shooting was saved as Multi-plane TIFF data.
Shooting conditions: DAPI, shooting frequency: every 4 minutes, shooting time: 48 hours Using the video analysis software (manufactured by Multi Dimensional Motion Analysis Device), the detection sensitivity of cell nucleus is maximized for the video data. The fluorescence threshold was adjusted so as to minimize the above, and various settings were made as shown below in Search Options, the cell nucleus was tracked, and the numerical data was stored. From the numerical data, arbitrary 21 cells were selected from the cells detected in all the captured images, the moving distance of each cell was measured, and the average value of each group was calculated.
Algorithm: Auto-find Objects
Segmentation method: Adaptive threshold
XY diameter: min 5, max 30
Local intensity above bkgd: 100
Split touching objects: Existence Object may skip frames along path: Existence

  In addition, for each melanocyte into which the above-described siRNA was introduced, the cells were collected using the QIAshredder (manufactured by QIAGEN) and RNeasy Mini Kit (manufactured by QIAGEN) after the O / N culture, and QIAcube (manufactured by QIAGEN) RNA was extracted with. CDNA was synthesized using SuperScript VILO cDNA Synthesis Kit (manufactured by Thermo Fisher SCIENTIFIC), RT-PCR was performed using the reagents listed in Table 2, and the mRNA amount of each gene was measured.

FIG. 1 shows a photomicrograph showing a 24-hour positional change of melanocytes in which expression of the ABR gene is suppressed. Moreover, in FIG. 2, the movement distance for 24 hours of the melanocyte which suppressed the expression of each gene is shown as a relative value when making a control group 100%. Enhanced cell migration was observed in melanocytes that suppressed the expression of the NCAM2 gene, ABR gene, SMYD3 gene, and KIAA0825 gene.
Separately, it was confirmed that melanocytes also move similarly in melanocytes in an environment surrounded by keratinocytes like biological skin.
Moreover, it was confirmed from the results of RT-PCR that the expression level of each gene was significantly suppressed in melanocytes into which siRNA was introduced (vs. control: 30 to 50%).

<Example 1> Screening using mRNA expression level of ABR gene using cultured human normal melanocytes as an index Screening of pigmentation production inhibitors was performed using the mRNA expression level of ABR gene in melanocytes as an index.
Normal human melanocytes (newborn, Dark / African American, Lot. 1223150, manufactured by Thermo Fisher Scientific Co., Ltd.) were seeded in a 24-well plate at 8.0 × 10 ⁴ cells / well, and medium 254 + HMGS medium medium at 37 ° C. for 24 hours at 37 ° C. Cultured. After culturing, a test substance having a final concentration of 1.0% by mass was added to form a test group, and a control group was prepared by adding the same amount of the solvent in place of each test substance, followed by further O / N culture at 37 ° C. for 24 hours. did. After culturing, mRNA was extracted from the collected melanocytes in the same manner as in Reference Example, and RT-PCR was performed using the synthesized cDNA to measure the amount of mRNA of each gene.

In Table 3, the mRNA expression level of the ABR gene in melanocytes is shown as a relative value when the same expression level in the test substance-free (control) melanocytes is set to “1”.
In the rosemary extract, it was confirmed that the expression of the ABR gene was suppressed in 40% of the control, and it was suggested that this extract has an effect of promoting cell migration of melanocytes, and as a result, has an effect of suppressing pigmentation.

<Example 2> Evaluation of cell migration promoting effect The following procedure evaluated the cell migration promoting effect of melanocytes of a substance that suppresses the expression of the ABR gene.
Normal human melanocytes (newborn, Dark / African American, Lot. 1223150, manufactured by Thermo Fisher Scientific Co., Ltd.) were seeded in a 24-well plate at 4.0 × 10 ⁴ cells / well, and cultured in Medium 254 + HMGS medium at 37 ° C. for 24 hours at 37 ° C. did. After culturing, the same test substance as in Example 1 having a final concentration of 1.0% by mass was added to form a test group, and a control group was prepared by adding the same amount of the solvent in place of each test substance. The medium was replaced with Medium 254 + HMGS medium containing a cell nucleus staining reagent (0.1% Cellstein Hoechst 33342 solution, H342, manufactured by Dojindo) and incubated for 15 minutes. The cell nucleus staining reagent-containing medium was removed, and the test substance-containing Medium 254 + HMGS medium was added (500 μL / well). Similar to the reference example, a time-lapse movie of the cells was taken and the cell migration was analyzed.

  In FIG. 3, the movement distance of melanocytes for 24 hours is shown as a relative value when the control group is 100%. In rosemary extract, significant enhancement of cell migration was observed.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided a new method for screening for a pigmentation inhibitor using the activity of a regulator of cell migration of melanocytes as an index. This makes it possible to search for materials that can be useful in the design and manufacture of whitening cosmetics and the like, which is industrially useful.

Claims (8)

  A screening method for a pigmentation inhibitor using the activity of a regulator of cell migration of melanocytes as an index. The activity of the regulator is the gene encoding the protein constituting the regulator or the expression level of the protein,
A step of adding a test substance to the cell, and a step of selecting a test substance in which the expression level in the cell to which the test substance is added is changed compared to the expression level in the cell to which the test substance is not added,
The screening method according to claim 1, comprising: The control factor is a factor that suppresses cell migration of melanocytes,
The change is a decrease in the expression level;
The screening method according to claim 2, wherein the test substance is selected such that the expression level in the cells to which the test substance is added is 90% or less of the expression level in the cells to which the test substance is not added.   The screening method according to claim 3, wherein the control factor is selected from the group consisting of an NCAM2 gene, an ABR gene, an SMYD3 gene, and a KIAA0825 gene. The regulator is a factor that enhances cell migration of melanocytes,
The change is an increase in the expression level,
The screening method according to claim 2, wherein the test substance is selected such that the expression level in the cells to which the test substance is added is 110% or more of the expression level in the cells to which the test substance is not added.   The screening method according to any one of claims 2 to 5, wherein the cell is a melanocyte. A step of performing the screening method according to any one of claims 1 to 6, and a step of containing a substance selected by the step;
A method for designing a composition, comprising:   The design method according to claim 7, wherein the composition is a whitening cosmetic.