JP6993076B2 - Screening method for components that improve subcutaneous tissue structure - Google Patents

Description

The present invention relates to a method for screening a component for improving subcutaneous tissue structure.

It is thought that one of the causes of skin tarmi and wrinkles, which become noticeable with aging, is a decrease in skin elasticity. Conventionally, as a method for improving the elasticity of the skin, for example, a technique for promoting the production or suppressing decomposition of extracellular matrix such as collagen and hyaluronic acid, which play an important role in maintaining the elasticity of the skin, and (Patent Document 1). , A technique for promoting the proliferation of dermal fibroblasts that produce extracellular matrix (Patent Document 2) is known. However, there has been no report on a method for improving tarmi and wrinkles by improving the structure of the subcutaneous tissue existing directly under the skin.

The present inventors focused on a reticulated fibrous structure called a skin zonule (Retinacula cutis) existing in the subcutaneous tissue, and examined the relationship with facial tarmi. As a result, the skin zonule became sparse. It has been found that the elasticity of the deep part of the skin is reduced and the tarmi may be caused (Non-Patent Document 1). In addition, it is known that the skin zonule density is reduced in the subcutaneous tissue immediately below the deep wrinkle site on the forehead and the outer corner of the eye (Non-Patent Document 2). From the description of Non-Patent Documents 1 and 2, it is presumed that if the density of the skin band can be increased, it will lead to improvement of tarmi and wrinkles. However, until now, the components of the skin zonule, the cells that produce it, and the mechanism of the change in the density of the skin zonule have not been known, and the components that enhance the cause of the change in the density of the skin zonule have also been searched for. I wasn't.

Japanese Unexamined Patent Publication No. 2006-257101 Japanese Unexamined Patent Publication No. 2008-105983

Sakata A., et al.: ”Breakthrough in improving the skin sagging with focusing on the subcutaneous tissue structure, retinacula cutis”, 23rd IFSCC, Zurich (2015) Tsukahara K. et al., Arch Dermatol. (2012), 148, 39-46

An object of the present invention is to provide a novel method for screening an improving component of subcutaneous tissue structure. An object of the present invention is to provide a method for screening an ingredient useful for anti-aging, particularly an ingredient effective for improving skin tarmi and wrinkles. Another object of the present invention is to provide an agent for promoting the expression of skin support components.

As a result of diligent research, the present inventors have identified the constituents of the skin band and the cell types that produce the constituents. Furthermore, they have found that certain components that make up the skin zonules decrease with age. Then, they have found that it is possible to screen for an improving component of the subcutaneous tissue structure by using the expression level of the skin brace component that decreases with aging as an index, and completed the present invention.

The present invention, which solves the above-mentioned problems, is a screening method characterized in that a component for improving the subcutaneous tissue structure is screened using the expression level of the skin brace component in cells as an index.
In particular, the present invention is useful for screening components having an anti-aging effect, particularly a tarmi-improving component and a wrinkle-improving component.

In a preferred embodiment of the invention, the cell is at least one selected from fibroblasts and tendon cells.

In a preferred embodiment of the invention, the cells are cells present in the skin zonule.

In a preferred embodiment of the invention, the skin tress components, which decrease with age, are Mimecan, Annexin A2, Biglycan, Vinculin and Microfibril-binding protein 4. At least one selected from assisted protein 4, MFAP4) is selected.
By using the expression level of any of these skin zonal components as an index, it is possible to efficiently screen the components for improving the subcutaneous tissue structure.

According to the present invention, it is possible to search for an effective component for improving the subcutaneous tissue structure based on a new mechanism of action, that is, to make the skin zonules dense.

<1> Screening Method The screening method of the present invention is characterized in that a component for improving the subcutaneous tissue structure is screened using the expression level of the skin brace component in cells as an index. Here, the screening includes searching for an improving component of the subcutaneous tissue structure or a candidate thereof.

Examples of the component for improving the subcutaneous tissue structure include a component having an anti-aging effect, particularly a component having an effect of improving wrinkles, improving tarmi, preventing a decrease in firmness, and preventing a decrease in skin elasticity. Here, Talmi refers to the deformation of the skin caused by the decrease in elasticity of the skin and the subcutaneous tissue.

As the cells used in the screening method of the present invention, at least one selected from fibroblasts and tendon cells is preferable. We have found that cells are present in the skin zonules and that these cells contain both fibroblasts and tendon cells. Therefore, it is efficient to use at least one of these cells for measuring the expression level of the skin brace component. It is also preferable to use both of them.

Examples of the skin brace component include at least one selected from the following.
Mimecan, Annexin A2, Biglycan, Vinculin, Microfibril-Associated Protein 4, MFAP4, Type 6 Collagen (Col), Type 6 Collagen Collagen type 1), Annexin A5, Decorin, Lumican, Prolagin.
Among them, Mimecan, Annexin A2, Biglycan, Vinculin, and Microfibril-binding protein 4 (microfibril-associated protein 4, MFAP4) are reduced with age. Therefore, it is preferable to use the expression level of at least one selected from these components as an index from the viewpoint of screening for components that improve the subcutaneous tissue structure.

The screening method of the present invention comprises adding a test substance to a cell culture system and measuring the expression level of the skin zonal component in the cell. Specifically, the expression level of the skin brace component in the cells cultured with the addition of the test substance is statistically significantly higher than the expression level of the skin brace component in the cells cultured without the addition of the test substance. When it is large, the test substance can be determined as a candidate for an improving component of the subcutaneous tissue structure. Specifically, when it is larger than 1 times the expression level of the skin brace component in the cells cultured without adding the test substance, it can be selected as a candidate for the component for improving the subcutaneous tissue structure.

The expression level of the skin brace component can be measured by a conventional method such as mRNA measurement and immunohistochemical analysis.
For example, the expression level of the gene encoding the skin zonule component is quantitatively detected by performing PCR using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer. Since the gene sequence encoding the skin brace component is open to the public, those skilled in the art can appropriately design primers.
In addition, since there are commercially available antibodies for each of the skin brace components, the expression level in cells can be measured by using them.

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.

<2> Promotion agent for expression of skin brace component The expression promoter for skin brace component of the present invention shall be used for quasi-drugs, cosmetics and foods among the components searched for by the screening method of the present invention. Consists of possible ingredients. As a specific example, the following plant extracts can be exemplified.
Carrot extract, perilla extract, Houttuynia cordata extract, Swertia japonica extract, Izayoi rose extract, Ouren extract, Kiwi extract, Aloe extract, Kuwa extract, Damask rose flower water A mode in which two or more kinds of components having an expression-promoting effect of the above are contained in combination is also preferable.

The expression-promoting agent for the skin brace component of the present invention is preferably in the form of an external preparation or an oral preparation in appropriate combination with any component used for formulation. Examples of the external preparation include cosmetics, quasi-drugs, skin external medicines and the like. Moreover, those dosage forms are not particularly limited. Among them, the form of a cosmetic that can be used continuously is preferable because of its use for promoting the expression of skin brace components, and above all, the form of a lotion, a beauty essence, a milky lotion, a cream, a gel, a sun care product, etc. Is preferable.
Further, as the oral preparation, the form of a supplement having a dosage form such as a tablet, a granule, or a drink is preferable.

The content of the skin support component expression-promoting agent in the external preparation (dry mass in the case of an extract) is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, and 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, wrinkle improvement, tarmi improvement, firmness reduction prevention, and skin elasticity reduction prevention effect can be achieved.
Further, the type of the expression-promoting agent for the skin brace component may be not only one type but also two or more types.

In the case of an oral preparation, the amount of the dry mass of the extract is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, and usually, depending on the dosage form. It is 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less.

The skin band plays an important role in maintaining the shape of the skin. We have also found that the sparseness of the skin zonules correlates with age and the degree of skin tarmi.
Therefore, the expression-promoting agent for the skin brace component of the present invention can be used as an agent for improving the subcutaneous tissue structure.
In particular, the expression-promoting agent for skin brace components of the present invention is useful as an anti-aging agent, and is particularly preferably used as an agent for improving wrinkles, improving tarmi, preventing elasticity, and preventing skin elasticity. It is preferably useful as a skin tarmi improving agent.

When an agent for promoting the expression of skin brace components is used in cosmetics, it is possible to widely blend the components normally used in cosmetics, and the dosage form and use thereof are not limited at all. Hereinafter, other ingredients that can be contained in cosmetics when applied to cosmetics will be described.

In cosmetics, in addition to the expression-promoting agent for skin brace components searched for as an active ingredient by the screening method described above, a whitening component, other wrinkle-improving components, anti-inflammatory components and the like can be blended.
The whitening ingredient is not particularly limited as long as it is generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-О-ethylascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidin, 2- (triphenylmethyloxy) ethanol, 2- (Triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N- (o-toluoil) cysteine acid, N- (m) -Toll oil) cysteine acid, N- (p-toluyl) cysteine acid, N- (p-methoxybenzoyl) cysteine acid and the like can be mentioned. Still other whitening ingredients include 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 can be mentioned.
Some of these whitening ingredients are already on the market, and some can be obtained synthetically. For example, 3-О-ethylascorbic acid can be synthesized by a known method described in JP-A-8-134005. There are also commercially available products (“VC Ethyl” manufactured by Nippon Fine Chemical Co., Ltd.), which can be obtained and used. 1-Triphenylmethylpiperidin, 1-triphenylmethylpyrrolidin, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, tri For phenylmethanol, triphenylmethane, and aminodiphenylmethane, see Patent Document WO2010-074052 in the pamphlet of N- (o-toluoil) cysteine acid, N- (m-toluoil) cysteine acid, N- (p-toluyl) cysteine acid, N. -(P-methoxybenzoyl) cysteine acid, N- (4-phenylbenzoyl) cysteine acid, N- (p-toluoil) homocysteine acid, are described in WO2011-058730 pamphlet, 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 Esters, N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O-acetylserine, N- (2-naphthoyl) -O-methylserine, etc. Since the synthesis method thereof is disclosed in the WO2011 / 074643 pamphlet, the synthesis can be performed according to the disclosure.
The content of the whitening component in cosmetics is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, more preferably 0.01 to 5% by mass (dry mass in the case of an extract). ..

The wrinkle improving ingredient is not particularly limited as long as it is generally used in cosmetics. However, it is preferable to use a component having a wrinkle improving effect by a mechanism different from that for promoting the expression of the skin zonule, from the viewpoint of fully utilizing the action and effect of each component. For example, vitamin A or a derivative thereof includes retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, and retinol acetate. In addition, ursoleic acid benzyl ester, ursoleic acid phosphate ester, bethuric acid benzyl ester, benzylic acid phosphate ester can be mentioned. The content of other wrinkle-improving components of the skin band development promoter in cosmetics is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, preferably 0.01 to 5% by mass. More preferred (dry mass for extracts).

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. Will be.
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, more preferably 1 to 5% by mass (dry mass in the case of an extract). ..

In addition, it is preferable to use extracts derived from animals and plants that 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, aronia extract, apricot extract, ginkgo extract, indokino extract, uikyo extract, udo extract, elephant kogi extract, emmeiso extract, sardine extract. Extract, Otaneninjin extract, Odorikosou extract, Orange extract, Kakyoku extract, Kakkon extract, Camomila extract, Carrot extract, Kawarayomogi extract, Kanzo extract, Kiwi extract, Cucab bar extract, Guava extract, Kuchinashi extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, black rice extract, chlorella extract, mulberry extract, keketo extract, gettoyo extract, genchiana extract, tea extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, moss peach extract, salvia extract, sabonsou extract, sasa extract , Sansha extract, Sansho extract, Shiitake extract, Jio extract, Shikon extract, Shiso extract, Shinanoki extract, Shimotsukesou extract, Shokyo extract, Shobu root extract, Sugina extract, Stevia extract, Stevia fermented product, Seiyousanzashi extract, Seiyouniwatoko extract, Seiyo Nokogirisou extract, Seiyouhakka extract, Sage extract, Zeniaoi extract, Senkyu extract, Senburi extract, Souhakuhi extract, Daiou extract, Soybean extract, Taiso extract, Thyme extract, Dandelion extract, Tea extract, Chouji extract, Chimpi extract, Jincha extract, Togarashi extract, Touki extract, Tokinsenka extract, Tounin 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, Grape extract, Grape seed extract, Hechima extract, Peppermint extract, Bodaiju extract, Pine extract, Mizubasho extract, Melissa extract, Mozuku extract, Peach extract, Yagurumagiku extract, Eucalyptus extract, Yuzu extract, Yuri extract, Yokuinin extract , Yomogi extract, lavender extract, green tea extract, apple extract, Louis Boss tea extract, Reishi extract, lettuce extract, Remo Extracts such as forsythia extract, forsythia extract, forsythia extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract, and crackle extract are preferable.
The content (dry mass) of the arbitrary animal and plant-derived extract in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 0.3 to 3% by mass. preferable.
The content (dry mass) of the arbitrary animal and plant 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.

In addition to the above-mentioned active ingredients, optional ingredients usually used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, martitol, propylene glycol, and di. Polyols such as propylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol, fatty acid sequen (sodium laurate, palmitic acid) (Sodium, etc.), anionic surfactants such as potassium lauryl sulfate, triethanolamine ether of alkyl sulfate, cationic surfactants such as stearyltrimethylammonium chloride, benzalconium chloride, and laurylamine oxide, and imidazoline-based amphoteric surfactants (imidazoline-based amphoteric surfactants) 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy 2-sodium salt, etc.), betaine-based surfactants (alkylbetaine, amidebetaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyltaurine, etc. , Solbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin 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, polyochiethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso) Stearate, 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 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, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, sodium lactate, surface treatment Powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic acid anhydride (silica), aluminum oxide, barium sulfate, etc., may be surface-treated, cobalt oxide, Ultramarine, dark blue, zinc oxide inorganic pigments, surface-treated, composite pigments such as iron oxide titanium dioxide sintered body, surface-treated, mica titanium, fish phosphorus foil, bismuth oxychloride, etc. Pearl agents, red 202, red 228, red 226, yellow 4, blue 404, yellow 5, red 505, red 230, red 223, orange 201, which may be raked. Organic pigments such as No., Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, Polyethylene powder, Polymethylmethacrylate, Nylon powder, Organopolysiloxane elastomer Organic powders such as, ethanol, lower alcohols such as isopropanol, vitamin A or its derivatives, vitamin B ₆ hydrochloride, vitamin B ₆ tripalmitate, vitamin B ₆ dioctanoate, vitamin B ₂ or its derivatives, vitamin B ₁₂ , Vitamin Bs such as Vitamin B ₁₅ or its derivatives, Vitamin Es such as α-tocopherol, β-tocopherol, γ-tocopherol, Vitamin E acetate, Vitamin Ds, Vitamin H, Pantothenic acid, Pantetin, Pyrroloquinolinquinone, etc. Vitamins include.

<Test Example 1> Screening using dermal-derived fibroblasts Human normal dermal fibroblasts were seeded in a 24-well plate with 10% FBS / DEME medium so as to be 2.0 × 10 ⁴ cells / well, and 37 The cells were cultured at 5% and CO ₂ for 24 hours.
Twenty-four hours after sowing, various plant extracts were added as test substances (final concentration 0.5% by mass).
After 24 hours, the medium was removed, the cells were washed with PBS, mRNA was collected using FastLane Cell RT-PCR Kit (manufactured by QIAGEN), and cDNA was synthesized with QuantiTect Reverse Traction Kit (manufactured by QIAGEN).
Quantitative RT-PCR was performed using the QuantiTect Primer Assay (manufactured by QIAGEN), and Mimecan, Annexin A2, Biglycan, and Vinculin, which are skin zonal constituent proteins that decrease with age, were performed. Vinculin), the amount of mRNA of the gene encoding the microfibril binding protein 4 (Microfibril-associated protein 4, MFAP4) was measured.
The above-mentioned mRNA expression level was calculated by a comparative CT method using β-actin as an endogenous control, and as a relative value when the target mRNA expression level in human normal dermal fibroblasts to which no test substance was added was set to "1". The results are shown in Table 1.

From Table 1, it was clarified that the expression of the Biglycan gene was promoted by adding the kiwi extract. In addition, it was clarified that the expression of the Microfibrillar-associated protein 4 (MFAP4) gene was promoted by adding the aloe extract and the quail extract, respectively. In addition, it was clarified that the expression of the Vinculin gene was promoted by adding Damask rose flower water. The above extract can be determined to be a candidate substance for an improving component of the subcutaneous tissue structure.

<Test Example 2> Screening using tendon cells Human tendon-derived tendon cells were seeded in a collagen I-coated 24-well plate in a special medium so as to have 2.0 × 10 ⁴ cells / well, and the temperature was 37 ° C., 5%. The cells were cultured for 24 hours in a CO ₂ environment.
Twenty-four hours after sowing, various plant extracts were added as test substances (final concentration 0.5% by mass).
After 24 hours, the medium was removed, the cells were washed with PBS, mRNA was collected using FastLane Cell RT-PCR Kit (manufactured by QIAGEN), and cDNA was synthesized with QuantiTect Reverse Traction Kit (manufactured by QIAGEN).
Quantitative RT-PCR was performed using the QuantiTect Primer Assay (manufactured by QIAGEN), and Mimecan, Annexin A2, Biglycan, and Vinculin, which are skin zonal constituent proteins that decrease with age, were performed. Vinculin), the amount of mRNA of the gene encoding the microfibril binding protein 4 (Microfibril-associated protein 4, MFAP4) was measured.
The above-mentioned mRNA expression level was calculated by a comparative CT method using β-actin as an endogenous control, and the result was as a relative value when the target mRNA expression level in human tendon-derived cells to which no test substance was added was set to "1". Is shown in Table 2.

From Table 2, it was clarified that the expression of the Biglycan gene was promoted by adding the carrot extract and the perilla extract, respectively. It was also clarified that the addition of Houttuynia cordata extract promotes the expression of the Microfibrillar-associated protein 4 (MFAP4) gene. In addition, it was clarified that the expression of the Vinculin gene was promoted by adding the Swertia japonica extract and the Izayoi rose extract, respectively. In addition, it was clarified that the expression of the Mimecan gene was promoted by adding Coptis chinensis extract. The above extract can be determined to be a candidate substance for an improving component of the subcutaneous tissue structure.

The present invention can be applied to prescription design of cosmetics and supplements.

Claims (4)

A screening method comprising screening a candidate substance for a component for improving the subcutaneous tissue structure using the expression level of the skin brace component in tendon cells as an index. The skin brace component is selected from Mimecan, Annexin A2, Biglycan, Vinculin and Microfibril-binding protein 4 (at least one selected from Microfibrillar-associated protein 4), MFAP4. The screening method according to claim 1, wherein there is. The screening method according to claim 1 or 2 , wherein the component for improving the subcutaneous tissue structure is a component having an anti-aging effect. The third aspect of the present invention, wherein the anti-aging effect is one or more effects selected from the group consisting of wrinkle improvement, tarmi improvement, firmness reduction prevention, and skin elasticity reduction prevention. Screening method.