JP6992220B2 - Screening method for pigmentation inhibitors - Google Patents

Description

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

Post-sunburn pigmentation, chloasma, chloasma, senile pigmented spots, etc. on the skin are states in which melanin production is significantly enhanced by activation of melanocytes (pigment cells) present in the skin. Since the occurrence or deterioration of these skin pigmentation-related troubles interferes with the beauty of the skin, research on ingredients having the effect of preventing or improving these has been actively conducted, and various mechanisms of action have been carried out. A whitening ingredient based on is created.

For example, ascorbic acids, colloidal sulfur, glutathione, hydroquinone, catechol and the like (Non-Patent Document 1) are well known as whitening ingredients. Furthermore, in recent years, various whitening ingredients based on a new mechanism of action have been developed. Targets for suppressing melanin production include tyrosinase-related protein inhibitors (Patent Document 1), endothelin action inhibitors (Patent Document 2), proton pump inhibitors (Patent Document 3), and stem cell factor binding inhibitors (Patent Document 1). 4) and so on. Further, as targets for suppressing the movement of the produced melanin to the epidermis, there are melanocyte dendrite elongation inhibitor (Patent Document 5), melanin transfer or release inhibitor (Patent Document 6) and the like.

Japanese Unexamined Patent Publication No. 2010-195732 Japanese Unexamined Patent Publication No. 2012-02950 Japanese Unexamined Patent Publication No. 2011-178760 Japanese Unexamined Patent Publication No. 2008-031094 Japanese Unexamined Patent Application Publication No. 2003-11302 Japanese Unexamined Patent Publication No. 2008-143796

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

With the aim of developing whitening cosmetics that are still more effective, the search for new ingredients that are effective in suppressing pigmentation and the new mechanism of action that can be the target of pigmentation inhibitors are being investigated. ..
An object of the present invention is to search for an effective component as a pigmentation inhibitor, and to establish a new screening method for that purpose.

As a result of diligent research, the present inventors have found a group of genes highly correlated with pigmentation such as age spots in the yellow race, and it is also possible that there are factors that control multiple melanocyte activators among them. I found it.
Conventionally, when searching for a pigmentation inhibitor based on the mechanism of melanocyte activation, a test system was set up for each individual melanocyte activating factor and an experiment was conducted. By using the activity of the regulators of a plurality of melanocyte activators found by the present inventors as an index, it is possible to more efficiently screen a material capable of suppressing melanocyte activation which may have a higher action. I came up with the idea and came to complete it.

That is, the present invention is as follows.
[1] A method for screening a pigmentation inhibitor using the activity of a regulator of a plurality of melanocyte activators as an index.
[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, according to [1]. Screening method.
[3] The activity of the regulator is the expression level of the gene encoding the protein constituting the regulator or the protein, and the step of adding the test substance to the cells and the expression level in the cells to which the test substance is added. [1] or [2].
[4] The regulator is a factor that suppresses the melanocyte activator, the change is an increase in the expression level, and the expression level in the cells to which the test substance is added is the expression in the cells to which the test substance is not added. The screening method according to [3], wherein a test substance having an amount of 110% or more is selected.
[5] The screening method according to [4], wherein the regulator is an EHF gene.
[6] The regulator is a factor that enhances the melanocyte activator, the change is a decrease in the expression level, and the expression level in the cells to which the test substance is added is the expression in the cells to which the test substance is not added. The screening method according to [3], wherein a test substance having an amount of 90% or less is selected.
[7] The screening method according to any one of [3] to [6], wherein the cells are keratinocytes.
[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 incorporating a substance selected by the step.
[9] The design method according to [8], wherein the composition is a whitening cosmetic.

INDUSTRIAL APPLICABILITY The present invention provides a new screening method for a pigmentation inhibitor using the activity of a regulator of a plurality of melanocyte activators as an index.

The graph which shows the melanin production amount in the melanocyte to which the culture supernatant of the keratinocyte which suppressed the expression of the EHF gene was added. The graph which shows the melanin production amount in the melanocyte which added the culture supernatant of the keratinocyte which added the test substance which enhances the expression of an EHF gene.

The screening method of the present invention uses the activity of a regulator of a plurality of melanocyte activators as an index.
Generally, it is known that melanin production is enhanced by tyrosinase when melanocytes are activated, and the factors that activate melanocytes include endothelin 1, IL-1α, GRO, NRG1, SCF, TNF, ADM, and IL. -6, GM-CSF and the like are known, and it is known that these activate melanocytes by enhancing their expression and promote melanin production. The factor that controls a plurality of melanocyte activating factors in the present invention may be either a factor that controls two or more of the melanocyte activating factors in a direction of suppressing them or a factor that controls them in a direction of increasing them. For example, as a factor that controls two or more of melanocyte activating factors in a direction of suppressing them, the EHF gene, which is a gene that the present inventors have newly found to be associated with age spots, is preferable. However, the activity is not limited to this, and a known gene that 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 and the like.

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.
In the screening method of the present invention, usually, the step of adding the test substance of the present invention to cells and the expression level in the cells to which the test substance is added are compared with the expression level in the cells to which the test substance is not added. Includes the step of selecting a subject to change.
For example, when the regulator of a plurality of melanocyte activators used as an index of the screening method of the present invention is an EHF gene, the upregulation of EHF gene expression and the suppression of pigmentation correlate with each other. Therefore, the test substance in which the expression of the EHF gene is enhanced by adding the test substance to the cells can be selected as a component having an action of suppressing pigmentation.

When the regulator is a factor that suppresses the melanocyte activator, the change in the screening method of the present invention is an increase in the expression level. Here, the degree of increase in the expression level may be as long as 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, preferably 12 or 24 after the addition of the test substance. It can be said that it changed when it increased to 110% or more after the lapse of time, more preferably to 115% or more, and further preferably to 120% or more.
When the regulator is a factor that suppresses the melanocyte activator, for example, when the regulator is the EHF gene, the melanocyte activator is suppressed by the enhanced expression of the gene of the regulator, and the melanin production is suppressed. As a result, the upregulation correlates with the suppression of pigmentation. Therefore, the test substance in which the expression of the gene of the regulator is enhanced by adding the test substance to the cells can be selected as a component having an action of suppressing pigmentation.

When the regulator is a factor that enhances the melanocyte activator, the change in the screening method of the present invention is a decrease in the expression level. Here, the degree of decrease in the expression level may be as long as the expression level in the cells to which the test substance is added is smaller than the expression level in the cells 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 changed when it decreased to 90% or less after the lapse of time, more preferably to 85% or less, and further preferably to 80% or less.
When the regulator is a factor that enhances the melanocyte activator, the suppression of the expression of the gene of the regulator suppresses the melanocyte activator and suppresses the production of melanin, so that the suppression of the expression results in pigmentation. Correlates with suppression. Therefore, the test substance in which the expression of the gene of the regulator is suppressed by adding the test substance to the cells can be selected as a component having a pigmentation-suppressing action.

As used herein, the "pigmentation-suppressing" action usually means an action of preventing or ameliorating pigmentation such as age spots on the skin by suppressing melanin production, resulting in a whitening action on the skin. It is a thing.

The expression level of the regulator of a plurality of melanocyte activators 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. The base sequences of the genes of the regulators of a plurality of known melanocyte activators have been published, and those skilled in the art can appropriately design primers and use them for PCR.
Further, for example, the intracellular abundance of the protein encoded by the gene may be quantitatively measured by a conventional method and used as the expression level of the gene.

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 genes of a plurality of regulators of melanocyte activators are present, but is usually keratinocyte.

The test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals and plants, a mixture thereof, or the like.
The animal or plant-derived extract shall mean not only the animal or plant-derived extract itself, but also the fraction of the extract, the purified fraction, the extract or the fraction, and the solvent-removed extract of the purified product. Examples of the plant-derived extract include native or grown plants, extracts using those sold as raw materials for Chinese herbal medicine, and commercially available extracts.
In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the above-ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the spikes, and the flower buds can be used, but these are crushed or finely divided in advance. It is preferable to cut it to improve the extraction efficiency. 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. One or more selected from polar solvents such as, etc. 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 a part used for extraction of a plant or the like or 1 mass of a dried product thereof, and at room temperature for several days at a temperature near the boiling point. If there is, a method of immersing for several hours, cooling to room temperature, removing insoluble matter and / or solvent if desired, and fractionally purifying by column chromatography or the like can be mentioned.

The pigmentation inhibitor selected by the screening method of the present invention has a pigmentation inhibitory action via 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 whitening compositions such as cosmetics.

That is, the present specification also provides a method for designing a composition, which comprises a step of performing the screening method of the present invention and incorporating a substance (pigmentation inhibitor) selected by the screening method. Such compositions include compositions that are transdermally administered (eg, cosmetics, quasi-drugs, external skin preparations such as pharmaceuticals), and compositions that are orally administered / ingested (eg, foods and drinks, quasi-drugs, etc.). Oral compositions such as quasi-drugs and pharmaceuticals) can be used.
The dosage form of the composition is not particularly limited. For example, the dosage form when designed as a cosmetic includes a commonly known lotion formulation, emulsion formulation, essence formulation, cream formulation, powder-containing formulation and the like.

When designing a composition containing the 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 0% by mass or more, more preferably 0.001% by mass or more, usually 80% by mass or less, preferably 30% by mass or less, and more preferably 10% by mass or less. Within the above range, a composition that preferably exerts an effect of suppressing pigmentation can be obtained.
Further, the type of the pigmentation inhibitor contained in the composition may be not only one type but also two or more types.

When designing a composition containing a pigmentation inhibitor selected by the screening method of the present invention, it may be designed to appropriately contain necessary components according to the above-mentioned aspects and uses.
For example, when designing as a cosmetic composition, it is possible to widely blend ingredients normally used in cosmetics, and the dosage form and use thereof are not limited at all. Hereinafter, the ingredients that can be contained in the cosmetics when mainly applied to the cosmetics will be described.

Examples of the active ingredient include other whitening ingredients, wrinkle improving ingredients, anti-inflammatory ingredients, animal and plant-derived extracts and the like. In addition, it may be blended in duplicate with the pigmentation inhibitor selected by the screening method of the present invention.
The other whitening ingredients are not particularly limited as long as they are generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-О-ethylascorbic acid, tranexamic acid, arbutin, ellagic acid, kodi acid, linoleic acid, nicotinic acid amide, 5,5'-dipropylbiphenyl-2, Examples thereof include 2'-diol, 5'-disodium adenylate, cetyl tranexamic acid, potassium 4-methoxysalicylic acid salt, hydroquinone, pantothenic acid and the like.
The content of the whitening component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 0.3 to 5% by mass.

The wrinkle improving ingredient is not particularly limited as long as it is generally used in cosmetics. For example, isopropyl trifluoride oxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate sodium, nicotinic acid amide, vitamin A or a derivative thereof (retinol, retinal, retinoic acid, trethinoin, isotretinoin, tocopherol retinoate, retinol palmitate). , Retinyl acetate, etc.), ursoleic acid benzyl ester, ursoleic acid phosphate ester, bethuric acid benzyl ester, benzylic acid phosphate ester.
The content of the wrinkle improving component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass.

The animal and plant-derived extracts are not particularly limited as long as they are generally used in pharmaceuticals, cosmetics, foods and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, amacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo extract, indokino extract, uikyo extract, udo extract, agetsu extract, elephant kogi extract. , Enmeisou extract, Ogon extract, Oubaku extract, Ouren extract, Otaneninjin extract, Otogirisou extract, Odorikosou extract, Orange extract, Kakyoku extract, Kakkon extract, Chamomile extract, Carrot extract, Kawarayomogi extract, Kanzo extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, Kuchinashi extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Black rice extract, Chlorella extract, Kwa extract, Keiketto extract, Gettoyo extract, Gentiana extract, Gennoshoco extract, Tea extract, Gobo extract, Rice extract, Fermented rice extract, fermented rice bran extract, rice germ oil, kokemomo extract, salvia extract, sabonsou extract, sasa extract, sanzashi extract, sansha extract, sansho extract, shiitake extract, dioscorea extract, shikon extract, shiso extract, shinanoki extract, shimotsukesou extract, shakuyaku Extract, ginger extract, ginger root extract, white birch extract, sugina extract, stevia extract, stevia fermented product, ginger extract, ginger extract, ginger extract, ginger extract, ginger extract, sage extract, zenia oyster extract, senkyu Extract, Senburi extract, Souhakuhi extract, Daiou extract, Soybean extract, Taiso extract, Thyme extract, Dandelion extract, Tea extract, Chouji extract, Chinpi extract, Jincha extract, Togarashi extract, Touki extract, Tokinsenka extract, Tounin extract, Tohi extract, Dokudami extract , Tomato extract, Natto extract, Carrot extract, Garlic extract, Novara extract, Hibiscus extract, Bakumondou extract, Hass extract, Parsley extract, Birch extract, Hamamelis extract, Hikiokoshi extract, Hinoki extract, Biwa extract, Fukitanpopo extract, Fukinotou extract, Bukuryo extract , Butcher Bloom Extract, Grape Extract, Grape Seed Extract, Hechima Extract, Benibana Extract, Peppermint Extract, Bo Daiju extract, button extract, hop extract, pine extract, mayonara extract, marronnier extract, mizubasho extract, mukuroji extract, melissa extract, mozuku extract, peach extract, yagurumagiku extract, eucalyptus extract, yukinoshita extract, yuzu extract, lily extract, yokunin extract, yomogi extract, lavender Extracts such as extract, green tea extract, apple extract, louisbos tea extract, reishi extract, lettuce extract, lemon extract, lotus extract, lotus extract, rose extract, rosemary extract, Roman chamomile extract, royal jelly extract, and crackle extract are preferable. Can be mentioned.
The content of the animal and plant-derived extract in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass.
The content of the animal and plant-derived extract in the food is usually 0.01 to 80% by mass, preferably 0.1 to 50% by mass, and more preferably 1 to 30% by mass.

Examples of the anti-inflammatory component include clarinone, glabridin, glycyrrhizinic acid, glycyrrhetinic acid, pantothenyl alcohol and the like, preferably glycyrrhizinic acid and its salt, alkyl glycyrrhetinate and its salt, and glycyrrhetinic acid and its salt.
The content of the anti-inflammatory component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass.

Examples of the oily component include polar oils and volatile hydrocarbon oils.
As polar oils, synthetic ester oils include isopropyl myristate, cetyl octanate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyl dimethyl octanoate, and 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, N-monoisostearate Alkyl glycol, neopentyl glycol dicaprate, diisostearyl malate, glyceryl di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane tetra-2-ethylhexanoate Examples thereof include erythritol, glyceryl tri-2-ethylhexanoate, and trimethylolpropane triisostearate.

In addition, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptyl undecanoate 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 adipylate, ethyllaurate, di-2-ethylhexyl sebatate, 2-hexyl myristate Examples thereof include decyl, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebatate, 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, and octylmethoxycinnamate.

Also, as natural oils, 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 ka oil, castor oil, flaxseed oil, Saflower oil, cotton seed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, cinnamon oil, Japanese millet oil, jojoba oil, germ oil, triglycerin, glyceryl trioctanoate, glyceryl triisopalmitate And so on.

Examples of the volatile hydrocarbon oil include isododecane and isohexadecane.

Examples of the surfactant include anionic surfactants such as fatty acid sekken (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, triethanolamine alkyl sulfate ether, stearyltrimethylammonium chloride, benzalconium chloride, laurylamine oxide. Cationic surfactants such as, betaine-based surfactants (alkylbetaine, amide betaine, sulfobetaine, etc.), imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxyside-1-carboxyethyroxy 2) (Sodium salt, etc.), amphoteric surfactants such as acylmethyltaurine,
Solbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acid esters (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hardened castor oil derivative, glycerin alkyl Ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, monostearate polyoxyethylene sorbitan, etc.), POE sorbit fatty acid esters (POE-Sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso) Stearetes, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), pluronic types, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ethers, etc.), Tetronics, POE castor oil / hardened castor oil derivatives (POE castor oil, POE cured castor oil, etc.), sucrose fatty acid esters, alkyl glucosides, etc. Non-ionic surfactants, etc. may be mentioned.

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

As thickeners, guar gum, quince seed, carrageenan, galactan, arabic gum, pectin, mannan, starch, xanthan gum, curdran, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, methyl hydroxypropyl cellulose, chondroitin sulfate, dermatane sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, tragant gum, keratane sulfate, chondroitin, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, keratosulfate, locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl Examples thereof include chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, sodium polyacrylate, polyethylene glycol, bentonite and the like.

As the powders, the surface may be treated, such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, barium sulfate and the like, and the surface. May be treated, red iron oxide, black iron oxide, cobalt oxide, ultramarine, dark blue, 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. Organic pigments such as No. 201, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, Polyethylene powder, Polymethyl methacrylate, Nylon powder, Examples thereof include organic powders such as organopolysiloxane elastomers.

Examples of UV absorbers include paraaminobenzoic acid-based UV absorbers, anthranylic acid-based UV absorbers, salicylic acid-based UV absorbers, cinnamic acid-based UV absorbers, benzophenone-based UV absorbers, sugar-based UV absorbers, 2- (2). Examples thereof include ultraviolet absorbers such as'-hydroxy-5'-t-octylphenyl) benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples as long as the gist thereof is not exceeded.

<Reference Example 1> Effect of suppression of EHF gene expression on gene expression of melanosite activating factors By inhibiting the expression of EHF gene in keratinocytes with siRNA by the following procedure, the expression of various melanosite activating factors can be achieved. The effect of the EHF gene was investigated.
Normal human keratinocytes (NHEK, Kurabo Industries, Ltd., Lot. 00702) were sown on a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2 (HuMediaKG2) (
It was cultured in Kurabo Industries) for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, replace with antibiotic-free HuMedia KG2 medium (450 μL / well).
, SiRNA mix 50 μL / well and 24 at 37 ° C. under 5% CO ₂ conditions.
Incubated for hours. The siRNA mix was obtained by mixing a required amount of LiPofectamine3000 with 1 μL of Opti-mem and 1.5 μL of siRNA (20 μM) with 25 μL of Opti-mem. It was prepared by mixing the same amount with each other in equal amounts and allowing it to stand at room temperature for 5 minutes. The siRNA reagent is Hs_EHF_5_ Flexi Tube siRNA (QIAGEN).
Made by Cat No. SI03171077) or Allstars Negative Control siRNA (NC siRNA) (manufactured by QIAGEN, Cat No.)
.. SI0365031) was used.
48 hours after siRNA introduction, QIAshredder (manufactured by QIAGEN) and RN
Cells were harvested using an easy Mini Kit (manufactured by QIAGEN) and QIAcub.
RNA was extracted with e (manufactured by QIAGEN).
The obtained RNA and the microarray were analyzed by a monochromatic method using an Agilent microarray (8 × 60K), and N.I. C. The expression fluctuations of various genes in the EHF siRNA-introduced keratinocytes were evaluated when the siRNA-introduced keratinocytes were used as controls.

In Table 1, N. C. The gene expression level in keratinocytes into which siRNA was introduced was 10.
The expression levels of various genes in keratinocytes into which EHF siRNA was introduced are shown at 0%.
In keratinocytes that suppressed the gene expression of the EHF gene, promotion of gene expression of the melanocyte activator was observed.

<Reference Example 2> Effect of suppression of EHF gene expression on melanin production The effect on melanin production in melanocytes was investigated by inhibiting the expression of the EHF gene in keratinocytes with siRNA by the following procedure.
(Culturing keratinocytes)
Normal human keratinocytes (NHEK, manufactured by Kurabo Industries, Ltd., Lot. 00702) were sown on a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2.
The cells were cultured in (Kurabo Industries, Ltd.) for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, the medium was replaced with an evaluation medium (HuMediaKG2 excluding HC, hEGF, BPE, antibiotics, and phenol red) (900 μL / well), and siRNA mix.
Was added (100 μL / well). The siRNA mix was prepared by mixing 50 μL of Opti-mem with a required amount so as to be Lipofectamine 3000 2 μL and 50 μL of Opti-mem mixed with a required amount of 3 μL of siRNA (20 μM). Was mixed in equal amounts and allowed to stand at room temperature for 5 minutes to prepare the mixture. The siRNA reagent is Hs_EHF_5_ Flexi Tube siRN
A (Cat No. SI03171077, manufactured by QIAGEN) or Allstars Negative Control siRNA (NC siRNA) (QIAGEN)
Made by Cat No. SI0365031) was used.
After culturing for 48 hours, centrifuge the collected culture supernatant (1,000 rpm, 3 mi).
n, 25 ° C.) was performed, and the supernatant was collected.

(Culturing of melanocytes)
Human normal melanocyte cells (NHEM, Life Technologies, Ltd., Lot. 122)
3150) was seeded on a 24-well plate (8.0 × 10 ⁴ cells / well) and cultured for 24 hours at 37 ° C. under 5% CO ₂ conditions. The medium was Medium 254 (manufactured by Thermo Fisher SCIENTIFIC) and HMGS (Thermo Fisher).
The one to which er SCIENTIFIC (manufactured by SCIENTIFIC) was added was used.
After culturing, the melanocyte cells were washed with PBS, 800 μL / well of the culture supernatant of the above-mentioned normal human keratinocyte was added, and 1.0 μm of 2- [ ^2-14 C] thiouracil was added.
Ci / well was added at a time.
After culturing under 37 ° C. and 5% CO ₂ conditions for about 48 hours, 40 μL of WST-8 reagent was added, and after culturing for 2 hours, the absorbances at 450 nm and 650 nm were measured with a plate reader.
The% / control was calculated using the difference between the measured values (Abs.450-Abs.650) as the number of cells.
After removing the medium and washing the melanocyte cells with PBS, 120 μL / well of 100% trichloroacetic acid was added to each 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 wells, this solution was also transferred to the 1.5 mL tube. After allowing the 1.5 mL tube to stand in the refrigerator for 30 minutes or more, centrifuge (15,000 rpm, 5 mi).
n, 4 ° C.) was performed, and the supernatant was removed. After adding 1 mL of 10% trichloroacetic acid solution to each 1.5 mL tube, centrifugation (15,000 rpm, 5 min, 4 ° C) was performed, 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. 1, N. C. When the amount of melanin produced per cell in melanocytes supplemented with siRNA-introduced keratinocyte culture supernatant was controlled (100%), melanin per cell in melanocytes supplemented with EHF siRNA-introduced keratinocyte culture supernatant. Shows the amount of production.
In keratinocytes in which the expression of the EHF gene was suppressed, the expression of the melanocyte activator was promoted, so that the promotion of melanin production in the melanocytes was observed.

<Example 1> Screening using the mRNA expression level of the EHF gene using cultured normal human keratinocytes as an index A pigmentation inhibitor was screened using the mRNA expression level of the EHF gene in keratinocytes as an index according to the following procedure.
Normal human keratinocytes (NHEK, manufactured by Kurabo Industries, Ltd., Lot. 00702) were sown on a 24-well plate (4.0 × 10 ⁴ cells / well), and HuMediaKG2.
The cells were cultured in (Kurabo Industries, Ltd.) for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, the medium was replaced with an evaluation medium (HuMediaKG2 excluding HC, hEGF, BPE, antibiotics, and phenol red) (900 μL / well).
Further, the test substance was added to each well to a final concentration of 1.0 or 0% by mass, and the cells were cultured for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, cells were collected using QIAshredder (manufactured by QIAGEN) and RNeasy Mini Kit (manufactured by QIAGEN), and RNA was extracted using QIAcube (manufactured by 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).

Table 2 shows the mRNA expression level of the EHF gene in keratinocytes as a relative value when the mRNA expression level of the EHF gene in the keratinocyte to which the test substance was not added (control) was 100%.
Expression of 110% or more of the EHF gene was observed in yeast extract and chimpi extract, suggesting that these extracts have an inhibitory effect on melanin production.

<Example 2> Evaluation of melanin production inhibitory effect The melanin production inhibitory effect of a substance that enhances the expression of the EHF gene was evaluated by the following procedure.
Normal human keratinocytes (2.0 × 10 ⁵ cells / well) and normal human melanocytes (1.0 × 10 ⁵ cells / well) are placed on a 24-well plate (Vi) for RI evaluation.
The seeds were sown in the same well of siPlate-24TC (manufactured by WallAC). The medium was a mixture of HuMediaKG2: Medium254 + HMGS = 2: 1 and 1.5 m.
L / well was used. After culturing under the conditions of 37 ° C. and 5% CO ₂ for 24 hours, the cells were replaced with an evaluation medium (900 μL / well).
Further, the same test substance as in Example 1 was added to each well to a final concentration of 1.0 or 0% by mass, and the cells were cultured for 24 hours at 37 ° C. under 5% CO ₂ conditions.
After culturing, the medium was replaced with an evaluation medium (1.0 mL / well), and 2- [ ^2-14 C] thiouracil was added at a rate of 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, the absorbances at 450 nm and 650 nm were measured with a plate reader, and the difference between the measured values (Abs. 450-Abs. % / Control was calculated with 650) as the number of cells.
After removing the medium and washing the cells with PBS, 1 mL of liquid scintillation cocktail was added to each well, and after stirring for 15 minutes, 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.

FIG. 2 shows the amount of melanin produced per cell when 100% is the case where the test substance is not added (control).
A significant melanin production inhibitory effect was observed in yeast extract and chimpi extract.

INDUSTRIAL APPLICABILITY The present invention provides a new screening method 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 may be useful for the design and manufacture of whitening cosmetics and the like, which is industrially useful.

Claims (6)

A screening method for a pigmentation inhibitor using the activity of the EHF gene as an index. The activity of the EHF gene is the expression level of the EHF gene or the protein encoded by the EHF gene .
The step of adding the test substance to the cells, and the step of selecting the test substance whose expression level in the cells to which the test substance is added increases as compared with the expression level in the cells to which the test substance was not added.
The screening method according to claim 1 . Claimed to select a test substance whose expression level in cells to which the test substance is added is 110% or more of the expression level in cells to which the test substance is not added.2The screening method described in. The screening method according to claim 2 or 3 , wherein the cells are keratinocytes. A step of performing the screening method according to any one of claims 1 to 4 , and a step of containing a substance selected by the step.
A method of designing a composition, including. The design method according to claim 5 , wherein the composition is a whitening cosmetic.

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