JP2019004746A - Screening method for pigmentation inhibitor - Google Patents

Abstract

To provide screening methods for pigmentation inhibitors.SOLUTION: A pigmentation inhibitor is screened using the activity of a regulator of plural melanocyte activators as an index. Preferably, the plural melanocyte activators are selected from the group consisting of endothelin 1, IL-1α, GRO, NRG1, SCF, TNF, ADM, IL-6, and GM-CSF, and the regulator is preferably EHF gene.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.

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)

Aiming at the development of whitening cosmetics that can still achieve higher effects, the search for new ingredients effective in suppressing pigmentation and the study of new mechanisms of action that can be targets for pigmentation inhibitors are being made. .
The object of the present invention is to search for an effective component as a pigmentation inhibitor and to establish a new screening method therefor.

As a result of diligent research, the present inventors have found a group of genes highly correlated with pigmentation such as spots in yellow races, and among them, there are also factors that control multiple melanocyte activators. I found it.
Conventionally, when trying to search for a pigmentation inhibitor based on the mechanism of melanocyte activation, an experiment was conducted by setting up a test system for each individual melanocyte activator. By using as an index the activity of regulators of a plurality of melanocyte activators found by the present inventors, a material capable of suppressing melanocyte activation that can have a higher action can be screened more efficiently. I came up with it and came to complete it.

That is, the present invention is as follows.
[1] A screening method for a pigmentation inhibitor using as an index the activity of regulators of a plurality of melanocyte activators.
[2] The plurality of melanocyte activators are selected from the group consisting of endothelin 1, IL-1α, GRO, NRG1, SCF, TNF, ADM, IL-6, and GM-CSF. Screening method.
[3] 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] or [2], comprising a step of selecting a test substance that changes in comparison with the expression level in a cell to which the test substance is not added.
[4] The control factor is a factor that suppresses a melanocyte activator, 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 [3], wherein a test substance that is 110% or more of the amount is selected.
[5] The screening method according to [4], wherein the regulatory factor is an EHF gene.
[6] The control factor is a factor that enhances a melanocyte activator, 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 [3], wherein a test substance that is 90% or less of the amount is selected.
[7] The screening method according to any one of [3] to [6], wherein the cell is a keratinocyte.
[8] A method for designing a composition, comprising a step of performing the screening method according to any one of [1] to [7], and a step of containing a substance selected by the step.
[9] The design method according to [8], wherein the composition is a whitening cosmetic.

  According to the present invention, there is provided a new method for screening for a pigmentation inhibitor using the activity of a regulator of a plurality of melanocyte activators as an index.

The graph showing the amount of melanin production in the melanocyte which added the culture supernatant of the keratinocyte which suppressed the expression of the EHF gene. The graph showing the amount of melanin production in the melanocyte which added the culture supernatant of the keratinocyte which added the test substance which promotes the expression of an EHF gene.

The screening method of the present invention uses the activity of regulators of a plurality of melanocyte activators as an index.
In general, when melanocytes are activated, it is known that melanin production is enhanced by tyrosinase. As factors that activate melanocytes, endothelin 1, IL-1α, GRO, NRG1, SCF, TNF, ADM, IL -6, GM-CSF, and the like are known, and these are known to activate melanocytes by promoting their expression and promote melanin production. The factor that controls a plurality of melanocyte activators in the present invention may be either a factor that controls two or more of the melanocyte activators, or a factor that controls it. For example, as a factor controlling in a direction to suppress two or more melanocyte activators, the EHF gene, which is a gene that the present inventors have newly found an association with a stain, is preferable. However, the present invention is not limited thereto, and a gene that is a known gene and controls a plurality of melanocyte activators can be used as an index in the screening method of the present invention.
The EHF gene is known to be expressed in human keratinocytes, and the DNA base sequence of the gene and the amino acid sequence of the protein are known (Gene ID: 2).
6298), GenBank et al.

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.
For example, when the regulator of a plurality of melanocyte activators used as the index of the screening method of the present invention is the EHF gene, the enhanced expression of the EHF gene correlates with the suppression of pigmentation. Therefore, by adding a test substance to a cell, the test substance in which expression enhancement of the EHF gene occurs can be selected as a component having a pigmentation inhibitory action.

When the regulatory factor is a factor that suppresses the melanocyte activator, 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 the elapse of time, more preferably 115% or more, and still more preferably 120% or more.
When the regulatory factor is a factor that suppresses melanocyte activator, for example, when the regulatory factor is an EHF gene, the melanocyte activator is suppressed by the increased expression of the regulatory factor gene, and melanin production is suppressed. Therefore, the enhanced expression correlates with suppression of pigmentation as a result. Therefore, by adding a test substance to a cell, the test substance in which the expression of the regulatory factor gene is increased can be selected as a component having a pigmentation-inhibiting action.

When the regulatory factor is a factor that enhances the melanocyte activator, 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. It can be said that it has changed when it has decreased to 90% or less after the passage of time, more preferably it has decreased to 85% or less, and even more preferably to 80% or less.
When the regulatory factor is a factor that enhances the melanocyte activator, suppression of gene expression of the regulatory factor suppresses the melanocyte activator and suppresses melanin production. Correlates with suppression. Therefore, by adding a test substance to a cell, the test substance in which the expression of the regulatory factor gene is suppressed can be selected as a component having a pigmentation-inhibiting action.

  In the present specification, the “pigmentation suppression” action refers to the action of preventing or improving pigmentation such as spots on the skin, usually by inhibiting melanin production, resulting in a whitening action on the skin. Is.

The expression level of the regulators of a plurality of melanocyte activators 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. In addition, the base sequence of the gene of the regulator of the several known melanocyte activator is each published, and those skilled in the art can design a primer suitably and can use 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 plurality of regulatory factor genes for melanocyte activators are present, but is usually a keratinocyte.

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.

The pigmentation inhibitor selected by the screening method of the present invention has a pigmentation inhibitory action through a melanin production inhibitory action.
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, red 202, red 228, red 226, yellow 4, blue 404, yellow 5, red 505, red 230, red 223 which may be raked 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 1> Effect of melanocyte activator on gene expression by suppressing expression of EHF gene In the following procedure, the expression of various melanocyte activators is inhibited by inhibiting the expression of EHF gene in keratinocytes with siRNA. The influence of the EHF gene was examined.
Normal human keratinocytes (NHEK, Kurabo Industries, Lot. 00702) were seeded in a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2 (
And 24 hours at 37 ° C. and 5% CO ₂ .
After incubation, change to HuMedia KG2 medium without antibiotics (450 μL / well)
, SiRNA mix was added at 50 μL / well, and the mixture was added under conditions of 37 ° C. and 5% CO _{2 for} 24 hours.
Incubate for hours. In addition, siRNA mix mixed the required amount so that it may become Lipofectamine 3000 1 microliter with Opti-mem 25 microliter, and the necessary amount so that siRNA (20 micromol) might become 1.5 microliter with Opti-mem 25 microliter. It was prepared by mixing equal amounts with each other and allowing to stand at room temperature for 5 minutes. The siRNA reagent is Hs_EHF_5_ Flexi Tube siRNA (QIAGEN
Cat No. SI03171077) or Allstars Negative Control siRNA (NC siRNA) (QIAGEN, Cat No.
. SI0365031) was used.
48 hours after introduction of siRNA, QIAshredder (QIAGEN) and RN
Cells were collected using easy Mini Kit (QIAGEN) and QIAcub
e RNA was extracted with (QIAGEN).
The obtained RNA and the resulting RNA were subjected to microarray analysis by a one-color method using an Agilent microarray (8 × 60K). C. Changes in expression of various genes in keratinocytes into which EHF siRNA was introduced when keratinocytes into which siRNA was introduced were used as controls were evaluated.

Table 1 shows N.I. C. The gene expression level in keratinocytes introduced with siRNA is 10
The expression levels of various genes in keratinocytes into which EHF siRNA was introduced when 0% is shown.
In keratinocytes that suppressed gene expression of the EHF gene, gene expression of melanocyte activator was promoted.

<Reference example 2> Influence on melanin production amount by suppression of expression of EHF gene In the following procedure, the influence on melanin production amount in melanocytes was examined by inhibiting the expression of EHF gene in keratinocytes with siRNA.
(Keratinocyte culture)
Normal human keratinocytes (NHEK, Kurabo Industries, Lot. 00702) were seeded in a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2
(Kurabo Co., Ltd.) for 24 hours at 37 ° C. and 5% CO ₂ .
After culture, the medium was replaced with an evaluation medium (HuMediaKG2 minus HC, hEGF, BPE, antibiotics, phenol red) (900 μL / well), and siRNA mix
Was added (100 μL / well). In addition, siRNA mix is a mixture of the necessary amount so as to be 2 μL of Lipofectamine 3000 with 50 μL of Opti-mem, and a mixture of siRNA (20 μM) with a required amount so as to be 3 μL of Opti-mem 50 μL. Were mixed in equal amounts and further allowed to stand at room temperature for 5 minutes. siRNA reagent is Hs_EHF_5_ Flexi Tube siRN
A (QIAGEN, Cat No. SI03171077) or Allstars Negative Control siRNA (NC siRNA) (QIAGEN
Cat No. SI0365031) was used.
After culturing for 48 hours, the collected culture supernatant was centrifuged (1,000 rpm, 3 mi
n, 25 ° C.) and the supernatant was recovered.

(Culture of melanocytes)
Normal human melanocyte cells (NHEM, Life Technologies, Lot. 122
3150) was seeded in a 24-well plate (8.0 × 10 ⁴ cells / well) and cultured under conditions of 37 ° C. and 5% CO _{2 for} 24 hours. The medium is Medium 254 (Thermo Fisher SCIENTIFIC) and HMGS (Thermo Fish).
er SCIENTIFIC) was used.
After the culture, the melanocyte cells were washed with PBS, the above-mentioned normal human keratinocyte culture supernatant was added at 800 μL / well, and 2- [2- ¹⁴ C] thiouracil was added at 1.0 μm.
Ci / well was added.
After culturing at 37 ° C. and 5% CO _{2 for} about 48 hours, 40 μL of WST-8 reagent was added, and after culturing for 2 hours, absorbance at 450 nm and 650 nm was measured with a plate reader,
% / Control was calculated using the difference in the measured values (Abs. 450-Abs. 650) as the number of cells.
After removing the medium and washing the melanocyte cells with PBS, 100% trichloroacetic acid was added to each well at 120 μL / well to lyse the cells. 500 μL of ice-cold water was added to each well and transferred to a 1.5 mL tube. Further, 500 μL of ice-cold water was added to each well, and after washing the well, this solution was also transferred to the 1.5 mL tube. The 1.5 mL tube was allowed to stand in the refrigerator for 30 minutes or more, and then centrifuged (15,000 rpm, 5 mi
n, 4 ° C.) and the supernatant was removed. After adding 1 mL of 10% trichloroacetic acid solution to each 1.5 mL tube, the mixture was centrifuged (15,000 rpm, 5 min, 4 ° C.), and the supernatant was removed. Add 1 mL of liquid scintillation cocktail to each 1.5 mL tube,
After stirring, the radioactivity was measured, and% / control was calculated as the amount of melanin produced. Furthermore, the amount of melanin per cell (= melanin production amount / number of cells) was calculated using the calculated cell value and melanin production amount value.

In FIG. C. Melanin per cell in melanocytes added with culture supernatant of keratinocytes introduced with EHF siRNA when melanin production per cell in melanocytes added with culture supernatant of keratinocytes introduced with siRNA was taken as control (100%) The production amount is shown.
In the keratinocytes that suppressed the expression of the EHF gene, the expression of the melanocyte activator was promoted, and thus the melanin production in the melanocytes was promoted.

Example 1 Screening Using EHF Gene mRNA Expression Level as an Index Using Cultured Normal Human Keratinocytes In the following procedure, screening for pigmentation inhibitors was performed using the EHF gene mRNA expression level in keratinocytes as an index.
Normal human keratinocytes (NHEK, Kurabo Industries, Lot. 00702) were seeded in a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2
(Kurabo Co., Ltd.) for 24 hours at 37 ° C. and 5% CO ₂ .
After the cultivation, the medium was replaced with an evaluation medium (HuMediaKG2 excluding HC, hEGF, BPE, antibiotics, and phenol red) (900 μL / well).
Furthermore, the test substance was added to each well so as to have a final concentration of 1.0 or 0% by mass, and cultured for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, the cells were collected using QIAshredder (QIAGEN) and RNeasy Mini Kit (QIAGEN), and RNA was extracted using QIAcube (QIAGEN).
SuperScript VILO cDNA Synthesis Kit (Ther
The expression level of the EHF gene was analyzed by real-time PCR using cDNA synthesized using mo Fisher SCIENTIFIC.

In Table 2, the mRNA expression level of the EHF gene in keratinocytes is shown as a relative value when the mRNA expression level of the EHF gene in the keratinocytes with no test substance added (control) is 100%.
In yeast extract and chimpi extract, expression of EHF gene of 110% or more of the control was observed, suggesting that these extracts have a melanin production inhibitory action.

<Example 2> Evaluation of melanin production inhibitory effect The following procedure evaluated the melanin production inhibitory effect of a substance that enhances the expression of the EHF gene.
Normal human keratinocytes (2.0 × 10 ⁵ cells / well) and normal human melanocytes (1.0 × 10 ⁵ cells / well) were converted into 24 well plates for RI evaluation (Vi
siPlate-24TC, manufactured by WallAC). The medium is a mixture of HuMediaKG2: Medium254 + HMGS = 2: 1 and 1.5 m.
L / well was used. After culturing at 37 ° C. and 5% CO _{2 for} 24 hours, the medium was replaced with the evaluation medium (900 μL / well).
Furthermore, the same test substance as in Example 1 was added to each well so as to have a final concentration of 1.0 or 0% by mass, and cultured for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After the culture, the medium was replaced with an evaluation medium (1.0 mL / well), and 2- [2- ¹⁴ C] thiouracil was added at 1.0 μCi / well.
After culturing at 37 ° C. for about 48 hours, 50 μL of WST-8 reagent was added, and after culturing for 2 hours, absorbance at 450 nm and 650 nm was measured with a plate reader, and the difference between the measured values (Abs. 450-Abs. %) / Control was calculated using 650) as the number of cells.
After removing the medium and washing the cells with PBS, 1 mL of a liquid scintillation cocktail was added to each well, and after stirring for 15 minutes, radioactivity was measured and% / control was calculated as the amount of melanin produced. Furthermore, the amount of melanin per cell (= melanin production amount / number of cells) was calculated using the calculated cell value and melanin production amount value.

FIG. 2 shows the amount of melanin production per cell when the test substance non-addition (control) is 100%.
A significant melanin production inhibitory effect was observed in the yeast extract and the chimney extract.

  According to the present invention, there is provided a new method for screening for a pigmentation inhibitor using the activity of a regulator of a plurality of melanocyte activators 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 (9)

  A screening method for a pigmentation inhibitor using as an index the activity of regulators of a plurality of melanocyte activators.   The screening method according to claim 1, wherein the plurality of melanocyte activators are selected from the group consisting of endothelin 1, IL-1α, GRO, NRG1, SCF, TNF, ADM, IL-6, and GM-CSF. . 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 of Claim 1 or 2 containing these. The regulator is a factor that suppresses the melanocyte activator,
The change is an increase in the expression level,
The screening method according to claim 3, 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 claim 4, wherein the control factor is an EHF gene. The regulator is a factor that enhances the melanocyte activator;
The change is a decrease in the expression level;
The screening method according to claim 3, wherein the test substance is selected such that the expression level in the cell to which the test substance is added is 90% or less of the expression level in the cell to which the test substance is not added.   The screening method according to any one of claims 3 to 6, wherein the cell is a keratinocyte. A step of performing the screening method according to any one of claims 1 to 7, and a step of containing a substance selected by the step;
A method for designing a composition, comprising:   The design method according to claim 8, wherein the composition is a whitening cosmetic.

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