JP2017176086A - Method of screening pge2 production inhibitor using clock gene as index - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods of screening PGEproduction inhibitor.SOLUTION: A method of screening PGEproduction inhibitor comprises adding test substances to cells, and selecting a test substance in which the expression level of the clock gene in the cells which the test substances are added has changed as compared with the expression level of the clock gene in the cells which the test substances are not added. The clock gene is preferably Bmal1 or Dec1.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for screening for a PGE ₂ production inhibitor.

Skin is affected in various ways by exposure to ultraviolet radiation. At that time, various chemical mediators and the like produced in the skin cause inflammation and cause great damage to the skin tissue. Examples of such chemical mediators include prostaglandins and leukotrienes which are arachidonic acid metabolites, interleukins and tumor necrosis factor-α which are a kind of cytokine, among which prostaglandins E ₂ (PGE ₂ ) are It is widely known as a representative inflammatory mediator.

By the way, it is known that a “clock gene” present in cells in a living body has a function of maintaining a periodicity of about 24 hours called the daily rhythm in the living body. A plurality of specific genes are known as clock genes. For example, Period and Cry whose expression is activated at daytime, and Bmal1 and Clock whose expression is activated at night are known.
Although the regulation of clock gene expression is still under study, it has been reported so far that a specific plant extract or the like has a function of regulating the expression of a clock gene such as Bmal1 (Patent Document 1, etc.). In addition, a method for screening whitening materials using changes in the expression level of clock genes in melanocytes as an index has been proposed (Patent Document 2).

International publication 2011/122040 pamphlet Japanese Patent Laying-Open No. 2015-208317

While PGE ₂ plays an important role in maintaining the homeostasis of the living body, when PGE ₂ production is increased by exposure to ultraviolet rays, inflammation is caused, and skin tissue is greatly damaged.
Since skin aging such as wrinkles and spots may progress, it is desirable to suppress PGE ₂ production in the skin.
In view of this situation, an object of the present invention is to provide a new method for screening for a PGE ₂ production inhibitor.

The present inventors proceeded with research on clock genes, and found a relationship between clock genes and PGE ₂ production, which was not conventionally known. That is, it was found that fluctuations in the expression level of clock genes in keratinocytes (keratinocytes) in the skin affect the production of PGE ₂ levels. Based on this finding, the inventors have conceived that a PGE ₂ production inhibitor can be screened by using a clock gene as an index, and have been completed.

That is, the present invention is as follows.
[1] A step of adding a test substance to a cell, and a test in which the expression level of a clock gene in a cell to which the test substance is added is changed compared to the expression level of the clock gene in a cell to which the test substance is not added Selecting a substance,
A method for screening a PGE ₂ production inhibitor.
[2] The screening method according to [1], wherein the cell is a keratinocyte.
[3] The screening method according to [1] or [2], wherein the clock gene is Dec1, and the change is suppression of Dec1 expression.
[4] The clock gene is Bmal1, and the change is increased expression of Bmal1. [
The screening method according to [1] or [2].
[5] The screening method according to any one of [1] to [4], wherein the PGE ₂ production inhibitor has an action of inhibiting PGE ₂ production during ultraviolet irradiation.
[6] A step of performing the screening method according to any one of [1] to [5], and a step of containing a substance selected by the step,
A method for designing a composition, comprising:
[7] The design method according to [6], wherein the composition is for whitening, anti-inflammatory, or anti-wrinkle.

The present invention provides a novel screening method for suppressing PGE ₂ production using a clock gene as an index.

Graph showing the PGE ₂ production amount of time the ultraviolet irradiation in keratinocytes that inhibited expression of clock genes.

<Clock gene>
The screening method of the present invention uses a clock gene as an index. The clock gene is known to have a function of maintaining a periodicity of about 24 hours called “circadian rhythm” in the living body, and a plurality of specific genes are known. The clock gene used as an index in the screening method of the present invention is a clock gene that has some relationship with the PGE ₂ production function in skin, particularly keratinocytes. Specifically, a clock gene selected from the group consisting of Bmal1 and Dec1 is preferable. However, the present invention is not limited to these, and is a known clock gene that is expressed in the cell when ultraviolet rays are irradiated to the cell or when a component or compound having a known PGE ₂ production activity is added to the cell. The changing clock gene can be used as a clock gene serving as an index in the screening method of the present invention. Examples of components and compounds having a PGE ₂ production action that can be used when selecting a clock gene as an index include non-steroidal anti-inflammatory agents (NSAIDs).

That is, the screening method of the present invention comprises the steps of adding a compound having a PGE ₂ production inhibitory action to a cell and selecting a clock gene whose expression level has been changed by the addition as an indicator of a screening method for a PGE ₂ production inhibitor. But you can.

The screening method of the present invention comprises a step of selecting a test substance in which the expression level of the clock gene in the cell to which the test substance is added is changed compared to the expression level of the clock gene in the cell to which the test substance is not added. Have.
The relationship between the clock gene and PGE ₂ production varies depending on the type of clock gene. That is, in the screening method of the present invention, when the expression level of the clock gene in the cell to which the test substance is added changes through the addition step of adding the test substance to the cell, the expression level of the clock gene changes. Selected test substances as PGE ₂ production inhibitors,
The present inventors have confirmed that the change in gene expression depends on the type of clock gene.

For example, when the clock gene used as an index of the screening method of the present invention is Bmal1, the enhancement of Bmal1 expression correlates with the suppression of PGE ₂ production. Therefore, by adding a test substance to a cell, a test substance in which increased expression of Bmal1 occurs can be selected as a component having a PGE ₂ production inhibitory action.

Moreover, when the clock gene used as the index of the screening method of the present invention is Dec1, suppression of expression of Dec1 and suppression of PGE ₂ production correlate. Therefore, by adding the test substance to the cells, the test substance in which the Dec1 expression is suppressed can be selected as a component having a PGE ₂ production inhibitory action.

In the present specification, the “suppression of PGE ₂ production” refers to an action of reducing the amount of PGE ₂ in cells, particularly keratinocytes, or an action of suppressing an increase in the amount of PGE ₂ in cells, particularly keratinocytes. More preferably, it refers to the action of reducing the amount of PGE ₂ in cells, particularly keratinocytes, or the action of suppressing the increase in the amount of PGE ₂ in cells, particularly keratinocytes, when irradiated with ultraviolet rays.

  In the screening method of the present invention, the expression level of the clock gene in the cell to which the test substance is added may be changed as compared with the expression level of the clock gene in the cell to which the test substance is not added. It can be said that it has changed when there is a change of 3% or more after 12 or 24 hours from the addition of the test substance, preferably a change of 5% or more, more preferably a change of 10% or more, The change is preferably 15% or more, particularly preferably 20% or more, and most preferably 25% or more.

  More specifically, when the clock gene is Bmal1, enhanced expression of Bmal1 means that the expression level is usually 10% or more, preferably 15% or more, more preferably 20% or more. When the clock gene is Dec1, the suppression of Dec1 expression means that the expression level is usually 90% or less, preferably 85% or less, more preferably 80% or less.

The expression level of the clock gene can be measured using any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer, and quantitative detection is performed. The base sequences of known clock genes are publicly disclosed, and those skilled in the art can appropriately design primers and use them for PCR.
In addition, for example, the expression level of the clock gene may be determined by quantitatively measuring the intracellular abundance of the protein encoded by the clock 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 contains a clock gene, but is preferably 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, 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, the solvent is used for 1 to 3 parts of a dry matter or a part used for extraction of a plant or the like.
Add 0 parts by mass, immerse for several days at room temperature, immerse for several hours at temperatures near the boiling point, cool to room temperature, remove insoluble matter and / or solvent as desired, and fractionate and purify by column chromatography etc. A method is mentioned.

The PGE ₂ production inhibitor selected by the screening method of the present invention has a PGE ₂ production inhibitory action.
It is known that when PGE ₂ production is increased, inflammation occurs in the skin, which may cause wrinkles and sagging. Therefore, PGE ₂ selected by the screening method of the present invention is used.
The production inhibitor can be suitably blended as an active ingredient of a composition for anti-inflammatory, anti-wrinkle, anti-aging and the like.
In addition, it is known that when PGE ₂ production is enhanced, melanocytes are activated, and tyrosinase enhances melanin production, which may cause spots, buckwheat, and dullness. Therefore, it was selected by the screening method of the present invention. The PGE ₂ production inhibitor can be suitably blended as an active ingredient in the whitening composition.

That is, this specification also provides a method for designing a composition, which includes a step of carrying out the screening method of the present invention and containing a substance (PGE ₂ production 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 PGE ₂ production inhibitor selected by the screening method of the present invention, the content (blending amount) of the PGE ₂ production inhibitor is usually 0.00001% by mass or more, preferably 0. 0.0001% 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, more preferably 10% by mass or less. Within the above range, suitably it may be of a composition that exhibits the PGE ₂ production inhibitory effect.
The type of PGE ₂ production inhibitors to be contained in the composition may be two or more, not only one type.

When designing a composition containing a PGE ₂ production 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 active ingredients include whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, animal and plant extracts. It is also possible to blend a duplicate of PGE ₂ production inhibitor selected by the screening method of the present invention.
The whitening component is not particularly limited as long as it is generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N- (p-toluyl) cysteic acid, N- (p -Methoxybenzoyl)
Examples include cysteic acid, panthenol, pantothenic acid and the like. Furthermore, as other whitening components, N-benzoyl-serine, N- (p-methylbenzoyl) serine, N- (p-ethylbenzoyl) serine, N- (p-methoxybenzoyl) serine, N- (p-fluorobenzoyl) ) Serine, N- (p-trifluoromethylbenzoyl) serine, N- (2-naphthoyl) serine, N- (4-phenylbenzoyl) serine, N- (p-methylbenzoyl) serine methyl ester, N- (p -Methylbenzoyl) serine ethyl ester, N- (2-naphthoyl) serine methyl ester, N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O -Acetylserine, N- (2-naphthoyl) -O-methylserine 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, vitamin A or a derivative thereof is retinol, retinal, retinoic acid, tretinoin, isotretinoin, retinoic acid tocopherol, retinol palmitate, retinol acetate, ursolic acid benzyl ester, ursolic acid phosphate ester, betulinic acid benzyl ester, benzylic acid A phosphate ester is mentioned.
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 animal and plant 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-hydroxystearylate, ethylene glycol di-2-ethylhexylate, dipentaerythritol fatty acid ester, N-alkyl monoisostearate Glycol, neopentyl glycol dicaprate, diisostearyl malate, glycerin di-2-heptylundecanoate, tri-2-ethyl Hexyl trimethylolpropane, may be mentioned trimethylolpropane triisostearate, tetra-2-ethylhexyl acid pentane erythritol, tri-2-ethylhexyl acid glycerin, trimethylolpropane triisostearate.

  Furthermore, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, glyceride tri-2-heptylundecanoate, 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 include 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, sasanca oil, castor oil, flaxseed oil, Safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, cinnagar oil, Japanese kiri oil, jojoba oil, germ oil, triglycerin, trioctanoic acid glycerin, triisopalmitic acid glycerin 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 and sorbitan sesquioleate), glycerin fatty acids (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, polyoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisosteare) Rate), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-octylide) Decyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), pluronic types, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ether, etc.), tetronics, POE castor oil / cured castor Examples include oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid esters, alkyl glucosides, 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.

In <Reference Example> effects following procedure clock genes into PGE ₂ production amount of UV stimulation, by inhibiting the expression of various clock genes in keratinocytes in siRNA, the clock genes to PGE ₂ production amount by UV stimulated The impact was examined.
Using a keratinocyte basal medium (Humedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human keratinocyte cultured cells are seeded at a density of 6.0 × 10 ⁴ cells / well in a 24-well plate, and the temperature is 37 ° C. and 5% carbon dioxide. Incubated in. On the next day, the medium was changed for all wells under the following conditions. That is, 500 μL of fresh keratinocyte culture medium (control) was added to 3 wells, and 3 μl each of 500 μL of keratinocyte culture complete medium containing siRNA of the clock gene prepared to a final concentration of 4 to 6 nM was added to the remaining wells. Added. Bmal1, Dec1, Per2, and Cry1 were targeted as clock genes that inhibit expression. After culturing for 24 hours, all wells were replaced with 500 μL of keratinocyte basic medium (Humedia-KG2, manufactured by Kurabo Industries Co., Ltd.) supplemented with dexamethasone (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 100 nM. After culturing for 2 hours, the cells were washed with PBS (phosphate buffered saline) and irradiated with ultraviolet rays so as to be 50 mJ / cm ² using an ultraviolet lamp. The irradiated cells were replaced with a new complete medium for keratinocyte culture and cultured for another 24 hours. The culture supernatant was collected and used as a sample, and the amount of PGE ₂ was measured using a human PGE ₂ Biotrak Easy ELISA kit (manufactured by GE Healthcare). Calculated as a percentage of the amount of PGE ₂ clock gene silencing group for PGE ₂ quantity produced in the control group, it was PGE ₂ production amount (%).

The results are shown in FIG.
It was confirmed that the amount of PGE ₂ produced at the time of UV irradiation decreased due to the inhibition of Dec1 expression compared to the control. Moreover, it was confirmed that the inhibition of Bmal1 expression increases the amount of PGE ₂ produced during UV irradiation compared to the control.
On the other hand, inhibition of Per2 and Cry1 expression was found not to affect the amount of PGE ₂ produced during UV irradiation.

<Example 1: Screening using Dec1 mRNA expression level as an index using cultured human normal keratinocytes>
In cultured cells, the biological rhythm is reset by culturing in a dexamethasone-containing medium for 2 hours. Using this reaction, the action of the plant extract on biological rhythm in keratinocytes was evaluated by the following procedure, and a PGE ₂ production inhibitor was screened using the expression level of the clock gene Dec1 as an index.
Using a keratinocyte basal medium (Humedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human keratinocytes (manufactured by Kurabo Industries Co., Ltd.) were seeded at a density of 6.0 × 10 ⁴ cells / well in a 24-well plate. Culturing was performed at 37 ° C. for 24 hours under 5% carbon dioxide. Thereafter, the medium was changed under the following conditions for all wells. That is, 500 μL of fresh keratinocyte culture medium is added to 3 wells (control), and the remaining wells are 500 μL of keratinocyte culture complete medium containing various plant extracts prepared as test substances to a final concentration of 1% by mass. Was added in 3 wells. After culturing for 24 hours, all wells were replaced with 500 μL of keratinocyte complete culture medium (Kurabo) supplemented with dexamethasone (Wako Pure Chemical Industries) to a final concentration of 100 nM. After culturing for 2 hours, the plate was washed with PBS (phosphate buffered saline), and RLT buffer of RNeasyMiniKit (manufactured by QIAGEN) was added to the keratinocytes collected in 3 wells for each test substance to extract mRNA.
RT-PC using QuantiTect Primer Assay (QIAGEN) using the extracted mRNA
R was performed and the amount of Dec1 mRNA was measured. Dec1 mRNA expression level was calculated by the comparative CT method using β-actin as an endogenous control, and expressed as a relative value when the Dec1 mRNA expression level in the control keratinocytes was set to “1”. When the expression of Dec1 was lower than that of the control, it was judged that there was an action of suppressing the expression of Dec1.
The results are shown in Table 1. Decl expression suppression action was recognized in the amacha extract and gardenia extract.

<Test Example 1: Evaluation of PGE ₂ production inhibitory action upon UV irradiation>
The PGE ₂ production inhibitory effect of the plant extract that suppresses the expression of the clock gene Dec1 was evaluated by the following procedure.
Using a keratinocyte basal medium (Humedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human keratinocyte cultured cells are seeded at a density of 6.0 × 10 ⁴ cells / well in a 24-well plate, and the temperature is 37 ° C. and 5% carbon dioxide. Incubated in. On the next day, the medium was changed for all wells under the following conditions. That is, 500 μL of fresh keratinocyte culture medium (control) was added to 3 wells, and 3 μl of keratinocyte culture complete medium containing plant extract prepared to a final concentration of 1% by mass was added to the remaining wells. Wells were added one by one. After culturing for 24 hours, all wells were replaced with 500 μL of keratinocyte basic medium (Humedia-KG2, manufactured by Kurabo Industries Co., Ltd.) supplemented with dexamethasone (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 100 nM. After culturing for 2 hours, the cells were washed with PBS (phosphate buffered saline) and irradiated with ultraviolet rays so as to be 50 mJ / cm ² using an ultraviolet lamp.
The cells after irradiation are further treated with a new complete keratinocyte culture medium (control) or a basic keratinocyte medium containing a plant extract (Humedia-KG2, Kurabo Industries).
Using the culture supernatant cultured for 4 hours as a sample, the amount of PGE ₂ was measured using a human PGE ₂ Biotrak Easy ELISA kit (manufactured by GE Healthcare). It calculates the amount of PGE ₂ plant extracts added group with respect to PGE ₂ quantity produced by the control group in percentage, was PGE ₂ production amount (%).
Table 2 shows the results. It was found that the amacha extract and gardenia extract have an inhibitory effect on PGE ₂ production by ultraviolet rays.

<Example 2: Screening using Bmal1 mRNA expression level as an index using cultured human normal keratinocytes>
A PGE ₂ production inhibitor was screened by the same procedure as in Example 1 except that the clock gene for which the amount of mRNA was measured was Bmal1. When the expression of Bmal1 in cultured human normal keratinocytes was higher than that of the control, it was judged that there was an action of enhancing the expression of Bmal1.
The results are shown in Table 3. The Bmal1 expression enhancing action was observed in the murasakikibu fruit extract and the red ginseng extract.

<Test Example 2: Evaluation of PGE ₂ production inhibitory action upon UV irradiation>
In the same procedure as in Test Example 1, the PGE ₂ production inhibitory effect of the plant extract in which the Bmal1 expression enhancing action was observed in Example 2 was evaluated.
Table 4 shows the results. It has been found that the murasakixibu fruit extract and the red ginseng extract have a PGE ₂ production inhibitory effect by ultraviolet rays.

The present invention provides a novel screening method for suppressing PGE ₂ production using a clock gene as an index. This makes it possible to search for materials that can be useful for designing and manufacturing anti-inflammatory compositions and the like, which is industrially useful.

Claims (7)

A test substance is added to the cell, and a test substance in which the expression level of the clock gene in the cell to which the test substance is added is changed compared to the expression level of the clock gene in the cell to which the test substance is not added is selected. The process of
A method for screening a PGE ₂ production inhibitor.   The screening method according to claim 1, wherein the cell is a keratinocyte.   The screening method according to claim 1, wherein the clock gene is Dec1 and the change is Dec1 expression suppression.   The screening method according to claim 1 or 2, wherein the clock gene is Bmal1, and the change is enhanced expression of Bmal1. The PGE ₂ production inhibitor has an action of suppressing PGE ₂ production at the time of UV irradiation, the screening method according to any one of claims 1-4. Performing the screening method according to any one of claims 1 to 5, and a step of containing a substance selected by the step,
A method for designing a composition, comprising:   The design method according to claim 6, wherein the composition is for whitening, anti-inflammatory, or anti-wrinkle.

Images