JP6880434B2 - Screening method for pigmentation inhibitors - Google Patents

Description

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

Hyperpigmentation, spots, chloasma, senile pigmented spots, etc. after sunburn 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 and deterioration of these skin pigmentation-related troubles interfere 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 a target for suppressing the movement of the produced melanin to the epidermis, there are a dendrite elongation inhibitor (Patent Document 5) of melanocytes, a melanin transfer or release inhibitor (Patent Document 6), and the like.

JP-A-2010-195732 Japanese Unexamined Patent Publication No. 2012-02920 Japanese Unexamined Patent Publication No. 2011-178760 Japanese Unexamined Patent Publication No. 2008-031094 Japanese Unexamined Patent 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) T. Yamada et al. , Journal of Dermatologic Science, Volume 73, Issue 3, March 2014, Pages 251-257

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. ..
By the way, it has been reported that melanocytes are densely present in the epidermis at the spots on the skin (Non-Patent Document 2). Therefore, by suppressing the production of melanin for each melanocyte, the skin color tone can be lightened as a whole, but the contrast of the spot portion is maintained. In addition, nearby melanocytes stimulate each other to promote the production of melanin, which causes a problem that the stain becomes darker or larger.
An object of the present invention is to search for an effective ingredient as a pigmentation inhibitor, which approaches the elimination of stains by a new mechanism, and an object of the present invention is to establish a new screening method for that purpose.

As a result of diligent research, the present inventors have found a new phenomenon in which melanocytes gather to form agglomerates. In addition, the present inventors have found a group of genes highly correlated with pigmentation such as stains in the yellow race, and found that among them, a regulator of cell aggregation of melanocytes is present.
Then, when there is an agglomerate of melanocytes, the stain becomes large or dark, and it is considered that the stain can be prevented or ameliorated by suppressing such aggregation, and if the activity of the regulator of cell aggregation of melanocytes is used as an index, We came up with the idea of being able to screen for materials that can prevent or improve pigmentation such as stains, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for screening a pigmentation inhibitor using the activity of a regulator of cell aggregation of melanocytes as an index.
[2] 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 cell and the expression level in the cell to which the test substance is added. The screening method according to [1], wherein the method comprises 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 regulator is a factor that suppresses cell aggregation of melanocytes, 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 [2], wherein a test substance having an amount of 110% or more is selected.
[4] The screening method according to [3], wherein the regulator is the DENND1A gene. [5] The regulatory factor is a factor that promotes cell aggregation of melanocytes, 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 [2], wherein a test substance having an amount of 90% or less is selected.
[6] The screening method according to any one of [2] to [5], wherein the cells are melanocytes.
[7] A method for designing a composition, which comprises a step of performing the screening method according to any one of [1] to [5], and a step of incorporating a substance selected by the step.
[8] The design method according to [7], wherein the composition is a whitening cosmetic.

INDUSTRIAL APPLICABILITY The present invention provides a screening method for a pigmentation inhibitor that approaches the elimination of stains by a new mechanism using the activity of a regulator of cell aggregation of melanocytes as an index.

The figure which shows the imaging part in the reference example. A photomicrograph showing cell aggregation of melanocytes in which the expression of the DENND1A gene is suppressed. The graph which shows the number of cell aggregates of the melanocyte which suppressed the expression of the DENND1A gene. The graph which shows the total area of the cell aggregate of the melanocyte which suppressed the expression of the DENND1A gene. The graph which shows the number of cell aggregates of the melanocyte to which the test substance which enhances the expression of the DENND1A gene was added.

The screening method of the present invention uses the activity of a regulator of cell aggregation of melanocytes as an index.
The phenomenon that melanocytes aggregate to form agglomerates, which was newly discovered by the present inventors, is considered to be one of the causes for making pigmented sites such as stains larger or darker. Therefore, by suppressing the aggregation of melanocytes, pigmentation such as stains can be prevented or ameliorated, and a substance having such an action can be a pigmentation inhibitor.

The factor that controls the cell aggregation of melanocytes in the present invention may be either a factor that controls the cell aggregation of melanocytes in a direction of suppressing the cell aggregation or a factor that controls the cell aggregation of melanocytes in a direction of increasing the cell aggregation. For example, as a factor that controls the cell aggregation of melanocytes in a direction of suppressing cell aggregation, the DENND1A gene, which is a gene newly found to be associated with stains by the present inventors, is preferably mentioned. This gene is known to be an activator of Rab35, a factor involved in intracellular vesicle transport in humans, and has also been reported to be involved in polycystic ovary syndrome (PCOS). So far, nothing has been known about the function in stains and skin.
The DNA base sequence of the DENND1A gene and the amino acid sequence of the protein are known (Gene ID 57706, Mol Cell. 2010 Feb 12; 37 (3): 370-82), and are disclosed in GenBank and the like. However, the activity of a known gene that controls cell aggregation of melanocytes can be used as an index in the screening method of the present invention.

In the present invention, the activity of the regulatory factor preferably refers to a gene encoding a protein constituting the regulatory factor or an 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. The step of selecting a test substance to be changed is included.

When the regulator is a factor that suppresses cell aggregation of melanocytes, the change is an increase in expression level in the screening method of the present invention. Here, the degree of increase in the expression level may be such that 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 120% or more, and further preferably 130% or more.
When the regulator is a factor that suppresses melanocyte cell aggregation, for example, when the regulator is the DENND1A gene, the upregulation of this gene correlates with the suppression of melanocyte cell aggregation, resulting in pigmentation. Correlates with suppression. Therefore, the test substance in which the expression of these genes is enhanced by adding the test substance to the cells can be selected as a component having a pigmentation inhibitory effect.

When the regulator is a factor that promotes cell aggregation of melanocytes, 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 such that 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 the change occurs when the amount decreases to 90% or less after the lapse of time, more preferably 80% or less, and further preferably 70% or less.
When the regulator is a factor that promotes cell aggregation of melanocytes, the suppression of expression of these genes correlates with the suppression of cell aggregation of melanocytes, and as a result, it correlates with the suppression of pigmentation. Therefore, the test substance in which the expression of these genes 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 "inhibition of cell aggregation" action usually means that the number of aggregates (aggregates) formed by aggregation of melanocytes does not decrease or increase, and / or the total area of aggregates decreases or It means that it does not increase.
As used herein, the "pigmentation-suppressing" action usually refers to an action of preventing or ameliorating the increase or darkening of pigmentation such as stains by suppressing cell aggregation of melanocytes. As a result, it has a whitening effect on the skin.

The expression level of the regulator of cell aggregation of melanocytes 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. When the regulator of cell aggregation of melanocytes is a known gene, for example, the nucleotide sequences of the above-mentioned DENND1A gene and the like are publicly available, and those skilled in the art should appropriately design primers and use them for PCR. Can be done.
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 a gene for a regulator of cell aggregation of melanocytes is present, but is usually melanocytes.

The test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals and plants, or a mixture thereof.
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 fraction, and the solvent-removed product of the purified product. Examples of plant-derived extracts 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 1 mass of a part used for extraction of a plant or the like or a dried product thereof, and the solvent is added 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 an effect of preventing or ameliorating the formation of melanocyte aggregates by suppressing cell aggregation of melanocytes, so that pigmentation such as stains increases or becomes darker. It has the effect of preventing or ameliorating swelling.
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 (for example, external preparations for skin such as cosmetics, quasi-drugs, and pharmaceuticals) and compositions that are orally administered / ingested (for example, food and drink, quasi-drugs). It can be an external product, an oral composition of a pharmaceutical product, etc.).
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, and 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 in any way. 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, extracts derived from animals and plants, 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, arbutine, ellagic acid, succinic acid, linoleic acid, nicotinamide, 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, isopropyloxopropylaminocarbonylpyrrolidin carbonylmethylpropylaminocarbonylbenzoylaminoacetate sodium, nicotinic acid amide, vitamin A or derivatives thereof (retinol, retinal, retinoic acid, tretinoin, 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, Gennoshouko 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, Jio 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, ginger extract, sage extract, zegna oyster extract, senkyu Extract, Senburi extract, Souhakuhi extract, Daiou extract, Soybean extract, Taisou 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, louis boss tea extract, leishi 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 extracts 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.
Polar oils include isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, and lactic acid as synthetic ester oils. Cetyl, myristyl lactate, lanolin acetate, 2-ethylhexyl isononanoate, isosetyl stearate, isosetyl 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-heptyl undecanoate, trimethyl propane tri-2-ethylhexanoate, trimethyl propane triisostearate, pentan tetra-2-ethylhexanoate Examples thereof include erythritol, glyceryl tri-2-ethylhexanoate, and trimethylolpropane triisostearate.

In addition, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, glyceride tri-2-heptylundecanoate, castor oil fatty acid methyl ester, oleic acid oil, cetostearyl alcohol, acetoglyceride,
2-Heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundesyl adipate, ethyllaurate, di-2-ethylhexyl sebatate, myristic acid Examples thereof include 2-hexyldecyl, 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.

Surfactants include anionic surfactants such as fatty acid surfactants (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 hydroxide-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, polyoxyethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso) Steerates, etc.), POE fatty acid esters (polyethylene glycol monoolate, 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 ether, etc.), tetronics, POE castor oil / hardened castor oil derivative (POE castor oil, POE cured castor oil, etc.), sucrose fatty acid ester, alkyl glucoside, etc. Nonionic surfactants, etc.

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, dermatan sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, traganth gum, keratan 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, silicic acid anhydride (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 201, Red 213, Yellow 204, Yellow 203, Blue 1, Green 201, Purple 201, Red 204, Polyethylene powder, Polymethyl methacrylate, Nylon powder, Examples include organic powders such as organopolysiloxane elastomers.

Examples of UV absorbers include para-aminobenzoic acid-based UV absorbers, anthranilic 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> Effect of suppression of DENND1A gene expression on melanocyte cell aggregation The effect of melanocyte on cell aggregation was examined by inhibiting the expression of DENND1A gene in melanocytes with siRNA by the following procedure.
SiRNA was introduced into normal human melanocytes (newborn, Dark / African American, Male, Lot. 1223150, manufactured by Life Technologies) using NEPA 21 (manufactured by Neppagene) by electroporation. As the siRNA reagent, Hs_DENND1A_2_ Flexi Tube siRNA (QIAGEN, Cat No. SI04134879) was used, and as a control, Allstars Negative Control siRNA (QIAGEN, Cat No. 31) was used. The melanocytes were seeded on a 24-well plate (8.0 × 10 ⁴ cells / well) and cultured in Medium 254 + HMGS medium (manufactured by Gibco) at 37 ° C. for 24 hours at O / N. After culturing, the medium was replaced with a preparation medium (Medium 254 + HMGS medium from which BPE, PMA, and heparin were removed), and O / N culture was further carried out at 37 ° C. for 48 hours. After culturing, bright-field images were taken at three locations (three squares in FIG. 1) per well at a magnification of 40. Using the image analysis software "ImageJ", the captured image data was analyzed, and the number and total area of melanocyte cell aggregates in each image were calculated and used as the measurement result per well.

Further, for the melanosite into which the above-mentioned siRNA was introduced, cells were collected using QIAshredder (manufactured by QIAGEN Co., Ltd.) and RNeasy Mini Kit (manufactured by QIAGEN Co., Ltd.) after the O / N culture, and the cells were collected into QIAcube (manufactured by QIAGEN Co., Ltd.). RNA was extracted. CDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (manufactured by Thermo Fisher SCIENTIFIC), and Hs_DENND1A_1_SG QuantiTect Primer.
RT-PCR was performed by a conventional method using Assay (QT00059311, manufactured by QIAGEN Co., Ltd.), and the amount of mRNA of the DENND1A gene was measured.

FIG. 2 shows a micrograph showing cell aggregation of melanocytes in which the expression of the DENND1A gene was suppressed. In addition, FIGS. 3 and 4 show the number and total area of cell aggregates of melanocytes that suppressed the expression of the DENND1A gene, respectively. The total area of the agglomerates is shown as a relative value when the control group is 100%. In melanocytes in which the expression of the DENND1A gene was suppressed, a significant increase was observed in both the number of cell aggregates and the total area.
In addition, it was confirmed from the results of RT-PCR that the expression level of the DENND1A gene was significantly suppressed in the melanocytes into which siRNA was introduced (against control: 10%).

<Example 1> Screening using cultured human normal melanocytes using the mRNA expression level of the DENND1A gene as an index A pigmentation inhibitor was screened using the mRNA expression level of the DENND1A gene in melanocytes as an index according to the following procedure.
Normal human melanocytes (newborn, Dark / African American, Male, Lot. 1223150, manufactured by Life Technologies) were ^{seeded on a 24-well plate at 8.0 × 10 4} cells / well, and cultivated in Medium254 + HMGS medium (Gibco) for 24 hours 37. O / N culture was performed at ° C. After culturing, a test substance having a final concentration of 1.0% by mass was added to form a test group, and the same amount of the solvent was added in place of each test substance to form a control group, which was further subjected to O / N culture at 37 ° C. for 24 hours. did. After culturing, mRNA was extracted from the collected melanocytes and RT-PCR was performed using the synthesized cDNA to measure the amount of mRNA of each gene, as in the reference example.

Table 1 shows the mRNA expression level of the DENND1A gene in melanocytes as a relative value when the same expression level in melanocytes without the addition of the test substance (control) was set to “1”.
In comfrey extract, the expression of the DENND1A gene was found to be enhanced in 160% or more of the control, suggesting that this extract has an inhibitory effect on cell aggregation of melanocytes, and as a result, an inhibitory effect on pigmentation.

<Example 2> Evaluation of cell aggregation inhibitory effect The cell aggregation inhibitory effect of melanocytes of a substance that enhances the expression of the DENND1A gene was evaluated by the following procedure.
Similar to the reference example, normal human melanocytes (newborn, Dark / African American, Male, Lot. 1223150, manufactured by Life Technologies) into which siRNA was introduced were ^{seeded on a 24-well plate at 8.0 × 10 4} cells / well, and Medium 254 + HMGS medium (Medium 254 + HMGS medium). O / N culture was carried out at 37 ° C. for 24 hours in a medium (manufactured by Gibco). 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 the same amount of the solvent was added in place of each test substance to form a control group, and the temperature was 37 ° C. for 24 hours. O / N culture was performed in. After culturing, the number and total area of cell aggregates were measured in the same manner as in the reference example.

Tables 2 and 5 show the number and total area of melanocyte cell aggregates as relative values when the control group is 100%. Significant suppression of cell aggregation was observed in comfrey extract.

INDUSTRIAL APPLICABILITY The present invention provides a new screening method for a pigmentation inhibitor using the activity of a regulator of cell aggregation of melanocytes 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 method for screening a pigmentation inhibitor using the activity of the DENND1A gene as an index. Activity of the gene, an expression amount of the protein encoded by said gene or said 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. The screening method according to claim 2, wherein the test substance whose 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 is selected. The screening method according to claim 2 or 3 , wherein the cells are melanocytes. 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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