JP2017218429A - Skin ligament-improving agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel skin ligament-improving agent having an action of increasing the expression level of protein that is a component of the skin ligament.SOLUTION: According to the present invention, there is provided a skin ligament-improving agent in which a plant extract having an action of increasing the expression level of protein is added.SELECTED DRAWING: None

Description

  The present invention relates to an agent for improving skin stroma that increases the expression level of a skin stroma component.

  It is thought that skin elasticity and wrinkles, which become prominent with aging, are caused by a decrease in skin elasticity. Conventionally, as a method for improving the elasticity of the skin, for example, a technique for promoting the production of an extracellular matrix such as collagen or hyaluronic acid that plays an important role in maintaining the elasticity of the skin or suppressing the degradation (Patent Document 1) A technique for promoting the growth of dermal fibroblasts that produce an extracellular matrix is known (Patent Document 2). 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 inventors of the present invention have focused on the mesh-like fiber structure called Retinacula cutis existing in the subcutaneous tissue, and as a result of studying the relationship with the facial talmi, It has been found that the elasticity of the deep part of the skin is lowered and that there is a possibility of causing talmi (Non-patent Document 1). Moreover, it is known that the skin brachial density is reduced in the subcutaneous tissue directly under the deep wrinkle site in the forehead and the corner of the eye (Non-Patent Document 2). From the descriptions in Non-Patent Documents 1 and 2, it can be estimated that if the density of the skin striation can be increased, it will lead to an improvement in tarmi and wrinkles. However, until now, there has been no knowledge about the components of the skin stroma, its producing cells, and the mechanism of the change in the density of the skin stroma, and no component has been known to increase the cause of the change in the density of the skin stroma. In addition, there has been no report so far on a skin stroma improving agent that increases the expression level of a constituent component of the skin stroma.

JP 2006-257101 A JP 2008-105983 A

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 skin stroma ameliorating agent having an action of increasing the expression level of a protein that is a skin stroma component in cells.

As a result of intensive studies, the present inventors have identified a component of the skin stroma and a cell tumor that produces the component. In addition, mimecan, biglycan, vinculin, and microfibril-associated protein 4 (MFAP4), annexin A2 (Annex A2), which are proteins constituting the skin stroma. We found that it decreases with time. Furthermore, skin stroma can be improved by a plant extract having an action of increasing the expression level of mimecan, biglycan, vinculin, and microfibril-associated protein 4, MFAP4. I found it.
Based on the above findings, the present inventors have completed the present invention.

The present invention for solving the above-mentioned problems is a cell comprising at least one protein selected from mimecan, biglycan, vinculin, and microfibrillar-associated protein 4, MFAP4. It is a skin stroma improving agent comprising a plant extract having an action of increasing the expression level.
According to the present invention, the skin stroma can be improved by increasing the expression level of the protein.

In the preferable form of this invention, it consists of two or more plant extracts chosen from the plant extract which has the effect | action which increases the expression level of a different protein among the said protein.
By making it the form which increases the expression level of a different protein, the improvement effect of a skin stroma can be improved.

The present inventors have found that the protein constituting the skin stroma is produced mainly in tendon cells and fibroblasts.
Therefore, the present invention is preferably used for increasing the expression level of the protein in tendon cells and / or fibroblasts.

  Moreover, it is particularly preferable that the present invention is in the form of a plant extract having an action of increasing the expression level of the total protein in tendon cells.

The present invention is preferably in the form of a plant extract selected from plant extracts belonging to the following.
Geraniaceae Geranoum, Rosaceae Roses (Rosa), Camellia, Camellia, Asperaceus, Aspalaeus, Aspalaeus The genus Uncaria.

Furthermore, the present invention is preferably in the form of an extract of a plant selected from the following plants.
Geraniaceae Geranium Genberium (Rosaceae) Rosea (Rosa multisera), Camellia family (Theaceae) Fabaceae Aspalathus linearias, Rubiaceae Uncaria gambarir.
By using extracts of these plants as plant extracts, the expression level of the total protein can be increased, and a higher skin branching amelioration effect can be obtained.

  The present invention is preferably in the form of an external preparation or an oral preparation.

  The skin stroma improving agent of this invention is excellent in the effect | action which increases the expression level of the protein which is a skin stroma component in a cell.

<1> Skin branching band component The subcutaneous tissue directly under the skin has a network-like fiber structure called skin branching band (Retinacula cutis), and its constituents include mimecan and Annexin A2 (Annexin A2). , Biglycan, Vinculin, Microfibrillar-associated protein 4, MFAP4, Collagen type 5 (Collagen type 5), Collagen type 5 (Collagen type 5) , Decorin, Lumican, Prolargin and the like are known.

<2> Plant extract (active ingredient)
The skin stroma improving agent of the present invention is at least one of these proteins selected from mimecan, biglycan, vinculin, microfibril-associated protein 4, and MFAP4. It consists of a plant extract that increases the expression level of protein.
The plant extract in this invention will not be specifically limited if it has the effect | action which increases the expression level of the said protein. Examples of the genus to which the plant used in the present invention belongs include Geraniaceae, Geranium, Rosaceae, Rosea, Rosaceae, Crataegus, and Fabaceae. Genus (Sophora), Cannabeceae (Humulus), Azalea (Ericaceae), Vaccinium, Saxifragaacea (Saxifraceae), Saxifragae (Saxifragae) Oxaliidaceae, Averrhoa, Asteraceae, Arcti mu), Camellia, Theaceae, Phylacestachys, Gutiferae, Hypericum, Citaceae (Ruaceae) Fabaceae, Asparathus, Rubiaceae, Uncaria, Lamiaceae, Scutellaria, Roseacee, Rubiaceae, Rubaceae, Rubaceae, Rubaceae Cinchona). Preferably, Geraniaceae, Geranium, Rosaceae, Rosea, Theaceae, Camellia, Fabaceae, Asparasus, Aspalaeus (Rubiaceae) Uncaria, Ranunculaceae, Aurora (Coptis), Bean subfamily (Faboideae), Indocalin (Pterocarpus), Vitaceae (Vitaceae) Grapes (Vitacee)

  Specific plant species include Geraniaceae, Geranium thunbergii, Rosaceae, Rosea, Rosea, and Rosea e. Crataegus hawthorn (Craaegus cuneata), legume (Fabaceae) Clara (Sophora) Clara (Sophora flavesens), Asaaceae (Cannabaceae) genus (Humulus) Vaccinium myrtillu ), Saxifragaceae (Saxifragaceae) Saxifraga (Saxifraga) saxifrage (Saxifraga stolonifera), onagraceae (Onagraceae) evening primrose genus (Oenothera) evening primrose (Oenothera biennis), oxalidaceae (Oxalidaceae) carambola genus (Averrhoa) star fruit (Averrhoa carambola) , Asteraceae burdock (Arctiumu) burdock (Arcium lappa), camellia (Camellia) camellia (Camellia sinensis), genus (Poaceae chrysalis) P ostachys edulis, hypericumaceae (Guttiferae) hypericum (hypericum) hypericum (hypericum erectum), citrus (Rutaceae) genus (Citrus) mandarin orange (Citrus reticulaceae) Aspalathus linearias, Rubiaceae spp. (Uncaria) Gambir (Uncaria gambir), Lamiaceae spp. (Rutaceae) Rutaceae (Scutellaria) us) European strawberry (Rubus idaeus), Rubiaceae genus (Cinchona) red cypress (Cinchona pubescens), Ranunculaceae genus (Coptis) genus (Coptis) genus (Coptida) (Pterocarpus) Indian mushroom (Pterocarpus marsupium), Grapeaceae (Vitaceae) Grapes (Vitaceae) Grapes (Vitis spp), etc. can be illustrated.

  The above plant extracts have the effect of improving the expression of at least one of mimecan, biglycan, vinculin, microfibril-associated protein 4, MFAP4. Tables 1 and 2 show which protein expression of each plant extract improves in fibroblasts or tendon cells.

As shown in Tables 1 and 2, there are those that show an expression improving action only on a part of the four kinds of proteins. In this invention, it is preferable to set it as the form which combines two or more plant extracts which show an expression improvement effect only with respect to some of these four types of proteins. In other words, the present invention is preferably an embodiment comprising a combination of two or more plant extracts having an action of increasing the expression level of different proteins among the proteins.
More specifically, among the plant extracts shown in Tables 1 and 2, an embodiment comprising a combination of two or more plant extracts having an action of increasing the expression level of different proteins is preferable.
By setting it as such embodiment, the higher skin branching improvement effect is realizable.

In order to achieve a higher skin stroma improving action, it is preferable to use a form of plant extract having an action of increasing the expression level of the total protein.
As shown in Table 2, Geraniaceae, Geranium thunbergii, Rosaceae, Rosea multiflora, Camellia ai (Camellia sinensis), Fabaceae, Asparathus, Aspalathus linearias, Rubiaceae, Uncaria, Uncariaria, Uncaria To improve the expression of all fibril binding protein 4 There is action.
Therefore, it is particularly preferable to use an embodiment using at least one of these plant extracts.

The present inventors have found that fibroblasts or tendon cells are cells constituting the skin stroma, and that these cells produce the above-mentioned proteins that are constituents of the skin stroma.
Therefore, the present invention is preferably used in a form used for increasing the expression level of the protein in tendon cells and / or fibroblasts, particularly preferably in tendon cells.

  The extract of the plant in the present invention can be prepared using a plant grown or grown in Japan, a Japanese product sold as a herbal medicine raw material, etc., or a plant extract such as Maruzen Co., Ltd. It is also possible to purchase and use a commercially available extract sold by a company that handles products.

The plant extract means not only the plant extract itself but also the extract fraction, the purified fraction, the extract or the fraction, and the solvent-removed product of the purified product. Examples include natural or grown plants, extracts that are sold as raw materials for herbal medicines, and extracts that are commercially available.
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, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. If so, there may be mentioned a method of immersing for several hours, cooling to room temperature, removing insoluble matters and / or solvents if desired, and fractionating and purifying by column chromatography or the like.

<3> Skin stroma improving agent It is preferable that the skin stroma improving agent of the present invention is appropriately combined with an arbitrary component used for formulation to be in the form of an external preparation or an oral preparation.
As an external preparation, forms, such as cosmetics, a quasi-drug, and a skin external medicine, are mentioned, for example. Moreover, those dosage forms are not particularly limited. Among them, the form of cosmetics that can be used continuously is preferable from the relationship with the use of promoting the expression of skin stroma components, and among them, the forms of lotions, cosmetic liquids, emulsions, creams, gels, suncare products, etc. Is preferred.

  Moreover, when setting it as an oral preparation, it is preferable to set it as the form of the composition for foods which contains the skin branching remedy of this invention as an active ingredient. More specifically, it is preferable to use a supplement form having dosage forms such as general foods, tablets, granules, and drinks.

The content of the plant extract in the external preparation (dry mass in the case of the extract) is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, Usually, it is 80 mass% or less, Preferably it is 30 mass% or less, More preferably, it is 10 mass% or less. By setting it as the said range, there exists a wrinkle improvement, a sagging improvement, prevention of a fall of elasticity, and the fall prevention effect of skin elasticity suitably.
Moreover, the kind of expression promoter of a skin branching component may be not only one type but two or more types.

  In the case of oral preparations, the amount of intake per dose is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, depending on the dosage form. It is 2000 mg or less, preferably 1000 mg or less, more preferably 500 mg or less.

  Skin struts play an important role in maintaining the shape of the skin. Therefore, the skin stroma improving agent of the present invention can be used as an agent for improving subcutaneous tissue structure.

In addition, the density of the skin stroma has a correlation with age and the degree of skin talmi, and among the proteins constituting the skin stroma, mimecan, biglycan, vinculin, and microfibril binding The present inventors have found that protein 4 (Microfibrillar-associated protein 4, MFAP4) is a component that decreases with aging, and that the decrease in these proteins causes wrinkles and sagging.
Therefore, the skin stroma improving agent of the present invention is useful as an anti-aging agent, and is particularly preferably used as a wrinkle improving agent, a sagging agent improving agent, a reduction preventing elasticity reduction, and a skin elasticity reducing agent.

  In the case where the skin stroma improving agent is in the form of a cosmetic, 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, when it applies to cosmetics, the other component which can be contained in cosmetics is demonstrated.

In cosmetics, in addition to the plant extract described above, a whitening component, other wrinkle improving components, anti-inflammatory components and the like can be blended.
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- (o-toluoyl) cysteic acid, N- (m -Toluoyl) cysteic acid, N- (p-toluyl) cysteic acid, N- (p-methoxybenzoyl) cysteic 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.

Some of these whitening components are already on the market, or they can be obtained by synthesis. For example, 3-O-ethylascorbic acid can be synthesized by a known method described in JP-A-8-134055. There are also commercially available products (“VC ethyl” manufactured by Nippon Seika Co., Ltd.), and these can be obtained and used. 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenyl Phenylmethanol, triphenylmethane and aminodiphenylmethane are disclosed in pamphlet of WO2010-074052 as N- (o-toluoyl) cysteic acid, N- (m-toluoyl) cysteic acid, N- (p-toluyl) cysteic acid, N -(P-methoxybenzoyl) cysteic acid, N- (4-phenylbenzoyl) cysteic acid and N- (p-toluoyl) homocysteic acid are disclosed in WO 2011-058730, N-benzoyl-serine, N- (p -Mechi Benzoyl) 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 are disclosed in WO2011 / No. 074643 pamphlet, the synthesis method of which is open to the public, It is possible to have synthetic.
Content of the whitening component in cosmetics is 0.0001-30 mass% normally, 0.001-10 mass% is preferable, and 0.01-5 mass% is more preferable (in the case of an extract, dry mass). .

  The wrinkle improving component 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 the promotion of the expression of the skin stroma from the viewpoint of making full use of the effects of each component. For example, vitamin A or a derivative thereof includes retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoic acid, retinol palmitate, and retinol acetate. Further, ursolic acid benzyl ester, ursolic acid phosphoric acid ester, betulinic acid benzyl ester, and benzyl acid phosphoric acid ester can be mentioned. The content of other wrinkle-improving components of the skin stroma expression promoter in cosmetics is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and 0.01 to 5% by mass. More preferred (dry mass in the case of an extract).

Examples of the anti-inflammatory component include clarinone, glabrizine, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol, preferably glycyrrhizic acid and its salt, glycyrrhetinic acid alkyl and its salt, and glycyrrhetinic acid and its salt It is done.
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 (in the case of an extract, dry mass). .

  Moreover, it is preferable to use the extract derived from animals and plants 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, aronia extract, apricot extract, ginkgo biloba extract, fennel extract, udo extract, sorghum extract, enmio extract, peony extract, ginseng extract , Nettle extract, oyster extract, cuckoo extract, chamomile extract, carrot extract, kawara mugwort extract, licorice extract, kiwi extract, cucumber extract, guava extract, gardenia extract, kumazasa extract, walnut extract, black rice extract, chlorella extract, mulberry extract, caquette Extract, gentian extract, gentian extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, salvia extract, Bonso extract, Sasa extract, Sansha extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, Perilla extract, Linden extract, Citrus extract, Ginger extract, Ginger root extract, Horsetail extract, Stevia extract, Stevia fermented product, Elderberry extract, Achillea millefolium Mint extract Tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, bacmond extract, lotus root , Parsley extract, Birch extract, Clam extract, Cypress extract, Cypress extract, Biwa extract, Japanese dandelion extract, Japanese cypress extract, Bukuryo extract, Loofah extract, Peppermint extract, Bodaiju extract, Pine extract, Citrus extract, Melissa extract, Mozuku extract, Peach extract Extracts, Eucalyptus extract, Lily extract, Yakuinin extract, Artemisia extract, Lavender extract, Apple extract, Rooibos tea extract, Ganoderma extract, Lettuce extract, Forsythia extract, Forsythia extract, Rosemary extract, Roman chamomile extract, Royal jelly extract, Waremoko extract, etc. An extract is mentioned as a preferable thing.

Content (dry mass) of the said arbitrary animal and plant-derived extract in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable, and 0.3-3 mass% is more. preferable.
Content (dry mass) of the said arbitrary 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.

  For cosmetics, in addition to the above-mentioned active ingredients, optional ingredients commonly used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, diethylene glycol, Polyols such as propylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexylene glycol, 1,2-hexanediol, 1,2-octanediol, fatty acid soap (sodium laurate, palmitic acid) Sodium), anionic surfactants such as potassium lauryl sulfate and alkylsulfuric triethanolamine ether, and cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride and laurylamine oxide. Imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.), acylmethyl Amphoteric surfactants such as taurine, 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 derivative, glycerin alkyl ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (PO -Sorbit monolaurate, etc.), POE glycerol fatty acid esters (POE-glycerol monoisostearate, 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 derivatives ( POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid ester, alkyl glucoside, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, sodium lactate, surface treatment Mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc., surface treated, cobalt oxide, Ultraviolet pigments, bitumen, 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, which may be raked Red 202, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201 No., Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, etc., organic powder, polyethylene powder, polymethacrylic acid Organic powders such as chill, nylon powder, organopolysiloxane elastomer, lower alcohols such as ethanol and isopropanol, vitamin A or derivatives thereof, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or the like Vitamin B such as derivatives, vitamin B12, vitamin B15 or derivatives thereof, vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate, vitamin D, vitamin H, pantothenic acid, panthetin, pyrrolo Vitamins such as quinoline quinone can be mentioned.

<Test Example 1> Effect on gene expression level in dermis-derived fibroblasts by plant extract Human normal dermal fibroblasts were placed at 2.0 × 10 ⁴ cells / well in a 24-well plate with 10% FBS / DEME medium. The seeds were sowed and cultured at 37 ° C. in a 5% CO ₂ environment for 24 hours.
24 hours after sowing, plant extracts prepared using the extraction solvents shown in Tables 3 to 5 were added (final concentration 0.5% by mass).

After 24 hours, the medium was removed, the plate was washed with PBS, mRNA was recovered using FastLane Cell RT-PCR Kit (manufactured by QIAGEN), and cDNA was synthesized using Quantect Reverse Transfer Kit (manufactured by QIAGEN).
Quantitative RT-PCR is performed using QuantiTect Primer Assay (manufactured by QIAGEN). Biggercan (Viglycan), vinculin, and microfibrillar binding protein 4 (Microfibrillar-associate) are skin striatal constituent proteins that decrease with age. The amount of mRNA of the gene encoding protein 4, MFAP4) was measured.
The above-mentioned mRNA expression level is calculated by a comparative CT method using β-actin as an endogenous control, and is expressed as a relative value when the target mRNA expression level in human normal dermal fibroblasts to which no test substance is added is “1”. The results are shown in Tables 3-5.

As shown in Tables 3 to 5, the addition of Genokosho extract increased the Biglycan gene expression level ratio to 2.34 and the MFAP4 gene expression level ratio to 2.20. Moreover, the expression level ratio of the Biglycan gene increased to 1.28 by adding the aurene extract. Further, by adding the Asparagus linenaria extract, the expression level ratio of MFAP4 gene is 1.41, and by adding Gambiroki extract, the expression level ratio of Vinculin gene is 1.93, and the expression level ratio of MFAP4 gene By adding pterostilbene, the expression level ratio of Biglycan gene increased to 2.25 and the expression level ratio of MFAP4 gene increased to 2.28.
From these results, it was found that the above plant extract can improve the expression level of at least one of Biglycan, Vinculin, and MFAP4 in dermis-derived fibroblasts.

<Test Example 2> Effect of plant extract on gene expression level in tendon cells Human tendon-derived tendon cells were seeded in a collagen I-coated 24-well plate at a density of 2.0 × 10 ⁴ cells / well in a special medium. The cells were cultured at 37 ° C., 5% in a CO ₂ environment for 24 hours.
24 hours after sowing, various plant extracts extracted using the extraction solvents shown in Tables 6 to 9 were added (final concentration 0.5% by mass).
After 24 hours, the medium was removed, the plate was washed with PBS, mRNA was recovered using FastLane Cell RT-PCR Kit (manufactured by QIAGEN), and cDNA was synthesized using Quantect Reverse Transfer Kit (manufactured by QIAGEN).
Quantitative RT-PCR is performed using QuantiTect Primer Assay (manufactured by QIAGEN), and mimecan, biglycan, vinculin, and microfibril binding protein are skin striatal constituent proteins that decrease with age. The amount of mRNA of the gene encoding 4 (Microfibrillar-associated protein 4, MFAP4) was measured.
The above-mentioned mRNA expression level was calculated by the comparative CT method using β-actin as an endogenous control, and the result was expressed as a relative value when the target mRNA expression level in human tendon-derived cells to which no test substance was added was “1”. Are shown in Tables 6-9.

According to Tables 6-9, when the Genokosho extract, Agetsu extract, tea leaf extract, Asparagus linenalis extract, Gambir tree extract is added, the expression ratio of all of the Mimecan, Biglycan, Vinculin, and MFAP4 genes is increased. I found out that I could do it.
Even when other plant extracts were added, it was found that the expression level ratio of at least one of Mimecan, Biglycan, Vinculin and MFAP4 can be increased.
The above extract has been found to increase the expression ratio of at least one of Mimecan, Biglycan, Vinculin, and MFAP4 in tendon cells, and is suitable as an active ingredient of a skin stroma ameliorating agent. I understood.

The present invention can be applied to external preparations and oral preparations.

Claims (7)

  A plant having an effect of increasing the expression level of at least one protein selected from mimecan, biglycan, vinculin, and microfibril-associated protein 4, MFAP4 in cells. An agent for improving skin support, consisting of an extract.   The skin branching remedy according to claim 1, comprising two or more selected from plant extracts having an action of increasing the expression level of different proteins among the proteins.   The skin stroma improving agent according to claim 1 or 2, wherein the cells are tendon cells and / or fibroblasts.   The skin stroma improving agent according to claim 1, wherein the cell is a tendon cell, and the plant extract has an action of increasing the expression level of the total protein. The skin branching remedy according to claim 4, wherein the plant extract is selected from plant extracts belonging to the following:
Geraniaceae Geranoum, Rosaceae Roses (Rosa), Camellia, Camellia, Asperaceus, Aspalaeus, Aspalaeus The genus Uncaria. The skin branching remedy according to claim 5, wherein the plant is the following plant:
Geraniaceae Geranium Genberium (Rosaceae) Rosea (Rosa multisera), Camellia family (Theaceae) Fabaceae Aspalathus linearias, Rubiaceae Uncaria gambarir. The skin stroma improving agent according to any one of claims 1 to 6, which is an external preparation or an oral preparation.