JP7706499B2 - Protein Expression Enhancers - Google Patents

Description

The present invention relates to a protein expression enhancer that increases the expression level of skin retinaculum components.

One of the causes of sagging skin and wrinkles that become prominent with age is thought to be a decrease in skin elasticity. Conventional methods for improving skin elasticity include, for example, a technique for promoting the production or inhibiting the decomposition of extracellular matrices such as collagen and hyaluronic acid, which play an important role in maintaining skin elasticity (Patent Document 1), and a technique for promoting the proliferation of dermal fibroblasts that produce extracellular matrices (Patent Document 2). However, there have been no reports of a method for improving sagging skin and wrinkles by improving the structure of the subcutaneous tissue located directly under the skin.

The present inventors have focused on a mesh-like fibrous structure called the retinaculum cutis present in subcutaneous tissue, and have investigated its relationship with facial sagging. As a result, they have found that sagging may be caused by a decrease in the elasticity of the deeper skin due to the sparseness of the retinaculum (Non-Patent Document 1). It is also known that the density of the retinaculum is reduced in the subcutaneous tissue directly below deep wrinkles on the forehead and corners of the eyes (Non-Patent Document 2). From the descriptions in Non-Patent Documents 1 and 2, it is estimated that if the density of the retinaculum can be increased, it will lead to the improvement of sagging and wrinkles. However, there has been no knowledge of the components of the retinaculum, the cells that produce it, or the mechanism of change in the density of the retinaculum, and the components that increase the cause of change in the density of the retinaculum have not been known. In addition, there have been no reports of a retinaculum improvement agent that increases the expression amount of the components of the retinaculum.

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

The objective of the present invention is to provide a novel skin retinaculum improving agent that has the effect of increasing the expression level of a protein that is a component of the skin retinaculum in cells.

As a result of intensive research, the present inventors have identified the components of the cutaneous retinaculum and the cell tumors that produce the components. Furthermore, they have found that the proteins that constitute the cutaneous retinaculum, mimecan, biglycan, vinculin, microfibrillar-associated protein 4 (MFAP4), and annexin A2, decrease with age. Furthermore, they have found that the cutaneous retinaculum can be improved by a plant extract that has the effect of increasing the expression levels of mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4).
Based on the above findings, the present inventors have completed the present invention.

The present invention, which solves the above problems, provides a skin retinaculum improving agent comprising a plant extract having an effect of increasing the expression amount of at least one protein selected from mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4) in cells.
According to the present invention, the expression level of the protein can be increased to improve the cutaneous retinaculum.

In a preferred embodiment of the present invention, the composition comprises two or more plant extracts selected from plant extracts having the effect of increasing the expression levels of different proteins among the above proteins.
By making the formulation into one that increases the expression levels of different proteins, the effect of improving the cutaneous retinaculum can be improved.

The present inventors have discovered that the proteins constituting the cutaneous retinaculum are produced mainly in tendon cells and fibroblasts.
Therefore, the present invention is preferably used to increase the expression level of the protein in tendon cells and/or fibroblasts.

Furthermore, it is particularly preferable that the present invention be in the form of a plant extract that has the effect 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 the extracts of plants belonging to the following categories:
Geraniaceae (Geraniaceae) genus, Rosaceae (Rosa) family, Theaceae (Theaceae) genus, Camellia (Camellia) family, Fabaceae (Aspalathus) family, Rubiaceae (Rubiaceae) genus, Uncaria (Uncaria).

Furthermore, the present invention is preferably in the form of an extract of a plant selected from the following plants:
Geraniaceae family (Geraniaceae) Geranium genus (Geraniumthunbergii), Rosaceae family (Rosa) Rosa genus (Rosa multiflora), Theaceae family (Theaceae) Camellia genus (Camellia sinensis), Fabaceae family (Aspalathus linearis), Rubiaceae family (Rubiaceae) Uncaria genus (Uncaria gambir).
By using extracts from these plants as the plant extract, the expression amount of the total protein can be increased, and a higher effect of improving the skin retinaculum can be obtained.

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

The skin retinaculum improving agent of the present invention has an excellent effect of increasing the expression level of proteins that are components of the skin retinaculum in cells.

<1> Retinacula cutis components The subcutaneous tissue located just below the skin contains a mesh-like fibrous structure called the retinacula cutis, and its components include mimecan, annexin A2, biglycan, vinculin, microfibrillar-associated protein 4 (MFAP4), collagen type 6, collagen type 1, annexin A5, decorin, lumican, and prolargin.

<2> Plant extract (active ingredient)
The skin retinaculum improvement agent of the present invention comprises a plant extract that increases the expression level of at least one protein selected from among these proteins, mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4).
The plant extract in the present invention is not particularly limited as long as it has the effect of increasing the expression level of the protein. Examples of the genera to which the plant used in the present invention belongs include the genus Geraniaceae (Geraniaceae), the genus Rosa (Rosaceae), the genus Crataegus (Rosaceae), the genus Sophora (Fabaceae), the genus Cannabaceae (Cannabaceae), and the genus Calamus (Cannabaceae). Humulus, Ericaceae Vaccinium, Saxifragaceae Saxifraga, Onagraceae Oenothera, Oxalidaceae Averrhoa, Asteraceae steraceae Arctium, Theaceae Camellia, Poaceae Phyllostachys, Guttiferae Hypericum, Rutaceae Citrus, Fabaceae Fructus Examples of the genus Aspalathus of the family Rubiaceae, the genus Uncaria of the family Rubiaceae, the genus Scutellaria of the family Lamiaceae, the genus Rubus of the family Rosaceae, and the genus Cinchona of the family Rubiaceae are preferred.
Examples include the genus Rosa of the family Rosaceae, the genus Camellia of the family Theaceae, the genus Aspalathus of the family Fabaceae, the genus Uncaria of the family Rubiaceae, the genus Coptis of the family Ranunculaceae, the genus Pterocarpus of the subfamily Faboideae, and the genus Vitaceae of the family Vitaceae.

Specific plant species include Geraniaceae (Geraniaceae) Geranium thunbergii, Rosa multiflora (Rosa) ... lupulus), Ericaceae Vaccinium Vaccinium myrtillus, Saxifragaceae Saxifraga Saxifraga stolonifera, Onagraceae Oenothera Oenothera biennis, Oxalidaceae Averrhoa Starfruit carambola), Asteraceae Arctium Arctium lappa, Theaceae Camellia Camellia sinensis, Poaceae Phyllostachys edulis, Guttiferae Hypericum Hypericum erectum, Rutaceae Citrus Citrus mandarin orange Citrus reticulata), Fabaceae Aspalathus linearis, Rubiaceae Uncaria gambir, Lamiaceae Scutellaria baicalensis, Rosaceae Rubus Rubus idaeus, Rubiaceae Cinchona Cinchona pubescens), Ranunculaceae, Coptis japonica, Faboideae, Pterocarpus marsupium, Vitaceae, Vitis spp., etc. are examples.

The above plant extracts have the effect of increasing the expression of at least one of mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4). Tables 1 and 2 show which proteins each plant extract increases in fibroblasts or tendon cells.

As shown in Tables 1 and 2, there are plant extracts that exhibit an expression-enhancing effect only on some of the four proteins. In the present invention, it is preferable to combine two or more plant extracts that exhibit an expression-enhancing effect only on some of the four proteins. That is, it is preferable to implement the present invention in an embodiment that comprises a combination of two or more plant extracts that have the effect of increasing the expression levels of different proteins among the proteins.
More specifically, it is preferable that the embodiment be a combination of two or more plant extracts, among those shown in Tables 1 and 2, that have the effect of increasing the expression levels of different proteins.
By adopting such an embodiment, a higher effect of improving the skin retinaculum can be achieved.

In order to achieve a higher effect of improving the skin retinaculum, it is preferable to use a plant extract that has the effect of increasing the expression level of the total protein.
As shown in Table 2, Geraniaceae, Geranium thunbergii, Rosaceae, Rosa, Camellia, Theaceae, Camellia
Plant extracts from Aspalathus linearis of the Fabaceae family, and Uncaria gambir of the Rubiaceae family have the effect of increasing the expression of all of mimecan, biglycan, vinculin, and microfibril-associated protein 4.
Therefore, embodiments using at least one of these plant extracts are particularly preferred.

The present inventors have found that fibroblasts or tendon cells are cells that constitute the cutaneous retinaculum, and that these cells produce the above-mentioned proteins that are components of the cutaneous retinaculum.
Therefore, the present invention is preferably in a form used to increase the expression level of the protein in tenocytes and/or fibroblasts, particularly preferably in tenocytes.

The plant extracts of the present invention can be prepared from plants that grow wild or are grown in Japan, or from plants that are sold in Japan as raw materials for herbal medicines, or commercially available extracts sold by companies that deal in plant extracts, such as Maruzen Co., Ltd., can be purchased and used.

The plant extract is a general term not only for the plant extract itself, but also for fractions of the extract, purified fractions, and products from which the solvent has been removed from the extract, fraction, or purified product. Examples of plant-derived extracts include extracts made from wild or cultivated plants, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts.
In the extraction operation, the whole plant can be used, and also the plant body, above-ground parts, roots and stems, trunks, leaves, stems, inflorescences, flower buds, etc. can be used, but it is preferable to crush or chop them in advance to improve the extraction efficiency. Suitable examples of the extraction solvent include one or more polar solvents selected from 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. Specific extraction methods include, for example, adding 1 to 30 parts by mass of a solvent to 1 mass of the part or dried product of the plant body to be extracted, immersing the part for several days at room temperature or several hours at a temperature near the boiling point, cooling to room temperature, removing insoluble matter and/or solvent as desired, and fractionating and purifying the part by column chromatography or the like.

<3> Skin retinaculum improving agent The skin retinaculum improving agent of the present invention is preferably in the form of an external preparation or an oral preparation in appropriate combination with any component used in formulation.
Examples of external preparations include cosmetics, quasi-drugs, and skin external medicines. There are no particular limitations on the dosage form. Among them, in terms of the purpose of promoting the expression of skin retinaculum components, the form of cosmetics that can be used continuously is preferred, and among them, the form of lotion, beauty essence, milky lotion, cream, gel, sun care product, etc. is preferred.

When the agent is to be administered orally, it is preferably in the form of a food composition containing the skin retinaculum improving agent of the present invention as an active ingredient. More specifically, it is preferably in the form of a supplement having a dosage form such as a general food, tablet, granule, or drink.

The content of the plant extract in the topical 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, and usually 80% by mass or less, preferably 30% by mass or less, more preferably 10% by mass or less. By keeping it within the above range, it is possible to preferably achieve the effects of improving wrinkles, improving sagging, preventing loss of firmness, and preventing loss of skin elasticity.
Furthermore, the type of the expression promoter for the skin retinaculum component may be not only one type but two or more types.

In the case of oral preparations, the amount to be taken per dose is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, and usually 2000 mg or less, preferably 1000 mg or less, more preferably 500 mg or less, in terms of the dry mass of the extract, depending on the dosage form.

The retinaculum plays an important role in maintaining the shape of the skin. Therefore, the retinaculum improvement agent of the present invention can be used as an agent for improving the subcutaneous tissue structure.

The inventors have also discovered that the density of the cutaneous retinaculum correlates with age and the degree of skin sagging, and further that, among the proteins that make up the cutaneous retinaculum, mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4) are components that decrease with age, and that the decrease in these proteins is the cause of wrinkles and sagging.
Therefore, the skin retinaculum improving agent of the present invention is useful as an anti-aging agent, and is particularly preferably used as an agent for improving wrinkles, improving sagging, preventing loss of firmness, and preventing loss of skin elasticity.

When the skin retinaculum improvement agent is in the form of a cosmetic, it can contain a wide range of ingredients that are typically used in cosmetics, and there are no limitations on its formulation or use. Below, we will explain other ingredients that can be contained in the cosmetic when it is applied to cosmetics.

In addition to the above-mentioned plant extracts, cosmetics may contain whitening ingredients, other wrinkle-improving ingredients, anti-inflammatory ingredients, and the like.
The whitening ingredient is not particularly limited as long as it is one that is generally used in cosmetics, and examples thereof include 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethyl ascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2-(triphenylmethyloxy)ethanol, 2-(triphenylmethylamino)ethanol, 2-(triphenylmethyloxy)ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane, aminodiphenylmethane, N-(o-toluoyl)cysteic acid, N-(m-toluoyl)cysteic acid, N-(p-toluoyl)cysteic acid, N-(p-methoxybenzoyl)cysteic acid, and the like. Further examples of other whitening ingredients include N-benzoylserine, 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 ingredients are already commercially available, while others can be obtained by synthesis. For example, 3-O-ethyl ascorbic acid can be synthesized by the known method described in JP-A-8-134055. Some are commercially available ("VC Ethyl" manufactured by Nippon Fine Chemicals), so these can be obtained and used. 1-Triphenylmethylpiperidine, 1
In Patent Document WO2010-074052, N-(o-toluoyl)cysteic acid, N-(m-toluoyl)cysteic acid, N-(p-toluoyl)cysteic acid, N-(p-methoxybenzo ... In WO2011-058730, 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, N-(p-toluoyl)homocysteic acid, N-benzoyl-serine, N-(p-methylbenzoyl)serine, N-(p-toluo ... Methods for synthesizing N-(2-naphthoyl)-O-methylserine, N-(p-methylbenzoyl)-O-acetylserine, N-(2-naphthoyl)-O-methylserine and the like are disclosed in WO2011/074643 and can be synthesized according to the disclosure therein.
The content of the whitening ingredient in the cosmetic is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (dry mass in the case of an extract).

There are no particular limitations on the wrinkle-improving ingredient, so long as it is one that is generally used in cosmetics. However, it is preferable to use an ingredient that has a wrinkle-improving effect through a mechanism different from that of promoting the expression of the retinaculum, from the viewpoint of making full use of the action and effect of each ingredient. For example, vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, and retinol acetate. Other examples include ursolic acid benzyl ester, ursolic acid phosphate ester, benzylic acid phosphate ester. The content of the wrinkle-improving ingredient other than the expression promoter of the retinaculum in the cosmetic is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass, and more preferably 0.01 to 5% by mass (in the case of an extract, the dry mass).

Anti-inflammatory components include clarinone, glabridin, glycyrrhizinic acid, glycyrrhetinic acid, pantothenyl alcohol, and the like, and preferred examples include glycyrrhizinic acid and its salts, alkyl glycyrrhetinates and their salts, and glycyrrhetinic acid and its salts.
The content of the anti-inflammatory component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry mass in the case of an extract).

It is also preferable to use extracts derived from animals and plants that are generally used in medicines, cosmetics, foods, etc. For example, Akebia extract, Asunaro extract, Asparagus extract, Avocado extract, Amacha extract, Almond extract, Arnica extract, Aronia extract, Apricot extract, Ginkgo extract, Fennel extract, Aralia extract, Eleuthero extract, Enmeiso extract, Phellodendron bark extract, Panax ginseng extract, Lamium extract, Kakyoku extract, Pueraria lobata extract, Chamomilla extract, Carrot extract, Artemisia capillaris extract, Licorice extract, Kiwi extract, Cucumber extract, Guava extract, Gardenia extract, Sasa veitchii extract, Kudzu extract, Lumi extract, black rice extract, chlorella extract, mulberry extract, kaempfera extract, alpinia speciosa extract, gentiana extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, salvia extract, soapwort extract, bamboo extract, sanshou extract, zanthoxylum extract, shiitake mushroom extract, rehmannia extract, lithospermum extract, perilla extract, linden extract, meadowsweet extract, ginger extract, calamus root extract, horsetail extract, stevia extract, stevia fermentation product, elderberry extract, yarrow extract, sei Mentha extract, sage extract, mallow extract, cnidium extract, swertia bristlecone extract, sophora japonica extract, rhubarb extract, soybean extract, tsinga extract, thyme extract, dandelion extract, clove extract, chili pepper extract, angelica extract, calendula extract, peach kernel extract, tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, burdock extract, lotus extract, parsley extract, birch extract, witch hazel extract, hyacinth extract, cypress extract, loquat extract, coltsfoot extract Preferred examples include butterbur extract, poria extract, loofah extract, peppermint extract, linden extract, pine extract, skunk cabbage extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, lily extract, coix seed extract, mugwort extract, lavender extract, apple extract, rooibos tea extract, lychee extract, lettuce extract, forsythia extract, astragalus extract, rosemary extract, Roman chamomile extract, royal jelly extract, and sanguisorba extract.

The content (dry mass) of the optional animal or plant-derived extract in the cosmetic is usually 0.01 to 30 mass%, preferably 0.1 to 10 mass%, and more preferably 0.3 to 3 mass%.
The content (dry mass) of the optional animal or plant extract in the food is usually 0.01 to 80 mass%, preferably 0.1 to 50 mass%, more preferably 1 to 30 mass%.

In addition to the above-mentioned active ingredients, optional ingredients that are usually used in cosmetics include polyols such as 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, and 1,2-octanediol, fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and anionic surfactants such as alkyl sulfate triethanolamine ether, cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, and laurylamine oxide, imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), and beta surfactants (alkyl betaine, amido betaine, sulfobetaine, etc.), amphoteric surfactants such as acyl methyl taurine, sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonyl phenyl ether, etc.), Pluronic (registered trademark) types, POE/POP alkyl ethers (POE/POP 2-decyl tetradecyl ether, etc.), Tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, and sodium lactate, powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate, which may be surface-treated, inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, and zinc oxide, which may be surface-treated, composite pigments such as iron oxide titanium dioxide sintered bodies, which may be surface-treated, pearling agents such as titanium mica, fish phosphorus foil, and bismuth oxychloride, which may be surface-treated, laked pigments Organic dyes such as Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, and Red No. 204 that may be present; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomers; lower alcohols such as ethanol and isopropanol. vitamins such as vitamin A or its derivatives, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or its derivatives, vitamin B12, vitamin B15 or its derivatives, vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate, vitamin D, vitamin H, pantothenic acid, pantethine, pyrroloquinoline quinone, etc.

<Test Example 1> Effect of plant extract on gene expression level in dermis-derived fibroblasts Normal human dermis fibroblasts were seeded in a 24-well plate in 10% FBS/DEME medium at 2.0 x ¹⁰⁴ cells/well and cultured at 37°C in a 5% _CO2 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 and the cells were washed with PBS. mRNA was recovered using a FastLane Cell RT-PCR Kit (QIAGEN), and cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN).
Quantitative RT-PCR was performed using QuantiTect Primer Assay (QIAGEN) to measure the amount of mRNA of genes encoding biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4), which are proteins that constitute the dermal retinaculum and decrease with age.
The above-mentioned mRNA expression levels were calculated by the comparative CT method using β-actin as an endogenous control, and the results are shown in Tables 3 to 5 as relative values when the expression level of the target mRNA in normal human dermal fibroblasts to which the test substance was not added was set to "1."

As shown in Tables 3 to 5, the addition of Geranium Herb extract increased the expression ratio of the Biglycan gene to 2.34 and the expression ratio of the MFAP4 gene to 2.20. The addition of Coptis japonica extract increased the expression ratio of the Biglycan gene to 1.28. The addition of Aspalathus linearis extract increased the expression ratio of the MFAP4 gene to 1.41, the addition of Gambir extract increased the expression ratio of the Vinculin gene to 1.93 and the expression ratio of the MFAP4 gene to 2.75, and the addition of Pterostilbene increased the expression ratio of the Biglycan gene to 2.25 and the expression ratio of the MFAP4 gene to 2.28.
These results demonstrate that the above plant extracts are capable of increasing the expression level of at least one of the genes biglycan, vinculin, and MFAP4 in dermis-derived fibroblasts.

Test Example 2: Effect of plant extract on gene expression level in tendon cells Tendon cells derived from human tendon were seeded in a collagen I-coated 24-well plate in a dedicated medium at 2.0 x ¹⁰⁴ cells/well, and cultured at 37°C in a 5% _CO2 environment for 24 hours.
24 hours after sowing, various plant extracts extracted with the extraction solvents shown in Tables 6 to 9 were added (final concentration: 0.5% by mass).
After 24 hours, the medium was removed and the cells were washed with PBS. mRNA was recovered using a FastLane Cell RT-PCR Kit (QIAGEN), and cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN).
Quantitative RT-PCR was performed using QuantiTect Primer Assay (QIAGEN) to measure the amount of mRNA of genes encoding the cutaneous retinaculum constituent proteins that decrease with age, namely mimecan, biglycan, vinculin, and microfibrillar-associated protein 4 (MFAP4).
The above-mentioned mRNA expression levels were calculated by the comparative CT method using β-actin as an endogenous control, and the results are shown in Tables 6 to 9 as relative values when the expression level of the target mRNA in human tendon-derived cells without the addition of the test substance was set to "1."

As seen from Tables 6 to 9, when Geranium Herb extract, Star Anemone extract, Tea leaf extract, Aspalathus linearis extract, or Gambir extract was added, the expression ratios of all of the Mimecan, Biglycan, Vinculin, and MFAP4 genes could be increased.
It was found that the addition of other plant extracts also enabled an increase in the expression ratio of at least one of the genes Mimecan, Biglycan, Vinculin, and MFAP4.
It was revealed that the above extracts increase the expression ratio of at least one of the genes mimecan, biglycan, vinculin, and MFAP4 in tendon cells, and were found to be suitable as active ingredients for skin retinaculum improvement agents.

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

Claims (3)

It is used to increase the total protein expression of Mimecan, Biglycan, Vinculin and Microfibrillar-associated protein 4 (MFAP4) in tendon cells.
A protein expression enhancer comprising all of (A)' to (D)' as active ingredients:
(A)' A protein expression enhancer used to increase the expression level of mimecan in tendon cells, comprising an extract obtained from one or more plants selected from the following group using a polar solvent as an extraction solvent , wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
Fabaceae, Sophora genus, Sophora flavesens,
Cannabaceae, Humulus genus, hops (Humulus lupulus),
Ericaceae family, Vaccinium genus, Vaccinium myrtillus,
Asteraceae (Asteraceae) Arctium genus (Arctium) Arctium lappa,
Hypericum erectum,
Family Rutaceae, Genus Citrus, Mandarin orange (Citrus reticulata),
Family Rosaceae, Genus Rubus, Rubus idaeus

(B)' A protein expression enhancer used to increase the expression level of biglycan in tendon cells, comprising an extract obtained from one or more plants selected from the following group using a polar solvent as an extraction solvent , wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
Family Rosaceae, Genus Crataegus, Crataegus cuneata,
Saxifragaceae family (Saxifragaceae) Saxifraga genus (Saxifraga) Saxifraga stolonifera,
Family Rutaceae: Citrus genus (Citrus) Mandarin orange (Citrus reticulata), and Family Lamiaceae: Scutellaria genus (Scutellaria) Scutellaria baicalensis

(C)' A protein expression enhancer used to increase the expression level of vinculin in tendon cells, comprising an extract obtained from one or more plants selected from the following group using a polar solvent as an extraction solvent , wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
Family Rosaceae, Genus Crataegus, Crataegus cuneata,
Cannabaceae, Humulus genus, hops (Humulus lupulus),
Saxifragaceae family (Saxifragaceae) Saxifraga genus (Saxifraga) Saxifraga stolonifera,
Onagraceae family (Oenothera genus) Oenothera biennis,
Oxalidaceae (Averrhoa) Starfruit (Averrhoa carambola),
Arctium lappa (Arctium lappa) from the Asteraceae family, and Rubus idaeus (Rubus idaeus) from the Rosaceae family

(D)' A protein expression enhancer used for increasing the expression level of MFAP4 in tendon cells, comprising an extract obtained from one or more plants selected from the following group using a polar solvent as an extraction solvent , wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
Family Rosaceae, Genus Crataegus, Crataegus cuneata,
Cannabaceae, Humulus genus, hops (Humulus lupulus),
Saxifragaceae family (Saxifragaceae) Saxifraga genus (Saxifraga) Saxifraga stolonifera,
Onagraceae family (Oenothera genus) Oenothera biennis,
Oxalidaceae (Averrhoa) Starfruit (Averrhoa carambola),
Asteraceae (Asteraceae) Arctium genus (Arctium) Arctium lappa,
Poaceae, Phyllostachys, Phyllostachys edulis,
Hypericum erectum,
Family Rutaceae, Genus Citrus, Mandarin orange (Citrus reticulata),
Rosaceae family, Rubus genus, Rubus idaeus, and Rubiaceae family, Cinchona genus, Cinchona pubescens The protein expression enhancer according to claim 1, which satisfies any one of the following conditions (1) to (14):
(1) An extract of Sophora flavesens of the genus Sophora in the family Fabaceae obtained by using a polar solvent as an extraction solvent , and the above-mentioned (B)' to (D)' are contained as active ingredients, and the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(2) The composition contains, as active ingredients , an extract of hops (Humulus lupulus) of the genus Humulus of the family Cannabaceae (Cannabaceae) obtained using a polar solvent as an extraction solvent, and the above (B)', wherein the polar solvent is one or more selected from the group consisting of water, ethanol, and 1,3-butanediol .
(3) An extract of Vaccinium myrtillus of the genus Vaccinium of the family Ericaceae obtained using a polar solvent as an extraction solvent , and the above-mentioned (B)' to (D)' are contained as active ingredients, and the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(4) The extract of Arctium lappa of the Asteraceae family (Asteraceae) obtained using a polar solvent as an extraction solvent , and the (B)' as an active ingredient , wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(5) An extract of Hypericum erectum of the family Hypericaceae (Guttiferae) obtained using a polar solvent as an extraction solvent, and the above (B)' and (C)' are contained as active ingredients, wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(6) An extract of mandarin orange (Citrus reticulata) of the genus Citrus of the family Rutaceae (Rutaceae) obtained using a polar solvent as an extraction solvent , and the above (C)' are contained as active ingredients, and the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(7) An extract of Rubus idaeus of the genus Rubus of the family Rosaceae obtained by using a polar solvent as an extraction solvent , and the above (B)' are contained as active ingredients, and the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(8) An extract of Crataegus cuneata of the genus Crataegus of the family Rosaceae obtained by using a polar solvent as an extraction solvent , and the above (A)' are contained as active ingredients, and the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(9) The composition contains, as active ingredients , an extract of Saxifraga stolonifera of the Saxifragaceae family obtained using a polar solvent as an extraction solvent, and the above-mentioned (A)', wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(10) An extract of Scutellaria baicalensis of the genus Scutellaria of the family Lamiaceae obtained using a polar solvent as an extraction solvent , and the above-mentioned (A)', (C)' and (D)' are contained as active ingredients, wherein the polar solvent is one or more selected from water, ethanol and 1,3-butanediol .
(11) An extract of Oenothera biennis of the genus Oenothera of the family Onagraceae obtained by using a polar solvent as an extraction solvent, and the above (A)' and (B)' are contained as active ingredients, wherein the polar solvent is one or more selected from the group consisting of water, ethanol, and 1,3-butanediol .
(12) An extract of Averrhoa carambola, Averrhoa genus, Oxalidaceae family , obtained using a polar solvent as an extraction solvent, and (A)' and (B)' as active ingredients, wherein the polar solvent is one or more selected from water, ethanol, and 1,3-butanediol .
(13) An extract of Phyllostachys edulis of the genus Phyllostachys in the family Poaceae obtained using a polar solvent as an extraction solvent , and the above-mentioned (A)' to (C)' are contained as active ingredients, wherein the polar solvent is one or more selected from the group consisting of water, ethanol, and 1,3-butanediol .
(14) An extract of Cinchona pubescens of the Cinchona genus of the Rubiaceae family obtained by using a polar solvent as an extraction solvent , and the above (A)' to (C)' are contained as active ingredients, wherein the polar solvent is one or more selected from the group consisting of water, ethanol, and 1,3-butanediol . the extract of Sophora flavesens, the extract of hops (Humulus lupulus), the extract of St. John's wort (Hypericum erectum), the extract of mandarin orange (Citrus reticulata), the extract of hawthorn (Crataegus cuneata), and the extract of Scutellaria baicalensis are extracts obtained by using one or more kinds of extractants selected from water and ethanol as an extraction solvent;
The extract of the Vaccinium myrtillus, the extract of the burdock, the extract of the European raspberry, the extract of the Saxifraga stolonifera, the extract of the evening primrose, the extract of the star fruit, the extract of the Phyllostachys edulis, and the extract of the red cinchona pubescens are obtained using one or more selected from water and 1,3-butanediol as an extraction solvent, the protein expression enhancer according to claim 1 or 2.