A screening method for agents that increase the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, and for skin tone improving components.

Abstract

To provide a novel agent for increasing the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, and to provide a screening method of a component for increasing the expression of the adhesion-related factors.SOLUTION: Provided are an agent for increasing the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, containing the extract of S.officinalis as an active ingredient; and a screening method comprising selecting a skin color-improving component using the adhesion state between vascular pericytes and vascular endothelial cells as an index.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to an agent for increasing the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, and to a method for screening skin tone improving components. [Background technology]

[0002] Pericytes are cells that surround the capillary walls and regulate the diameter and permeability of capillaries. Specifically, platelet-derived growth factor (PDGF) secreted by vascular endothelial cells is received by vascular pericytes via receptors (PDGFRβ), causing it to accumulate around endothelial cells. Additionally, angiopoietin 1 (ANGPT1) secreted by vascular pericytes is received by vascular endothelial cells via receptors, strengthening the adhesion between vascular pericytes and vascular endothelial cells and controlling vessel diameter and permeability (see Non-Patent Literature 1).

[0003] Here, a known prior art focusing on vascular pericytes is a vascular pericyte protective agent containing a prostanic acid derivative as an active ingredient (Patent Document 1). Furthermore, a known prior art focusing on vascular endothelial cells is a method for screening vascular endothelial cell activators based on the expression level of LINC00942 in fibroblasts (Patent Document 2). Furthermore, as a conventional technology focusing on the adhesion of vascular cells, an agent for increasing the expression of intercellular adhesion factors (tight junction-related factors) in blood vessels and / or lymphatic vessels is known (Patent Document 3). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2000-119184 [Patent Document 2] Japanese Patent Publication No. 2021-185818 [Patent Document 3] Japanese Patent Publication No. 2022-077456 [Non-patent literature]

[0005] [Non-Patent Document 1] Molecular mechanisms of vascular maturation, Nobuyuki Takakura, Journal of Thrombosis and Hemostasis 22(6):343~347, 2011. [Overview of the project] [Problems that the invention aims to solve]

[0006] The causes of decreased adhesion between vascular pericytes and vascular endothelial cells have not been fully elucidated. Furthermore, focusing on vascular pericytes, the components that increase factors related to this adhesion state have also been unknown. In light of the above situation, the present invention aims to provide an agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells. Furthermore, the present invention aims to provide a screening method for skin tone improving components. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have revealed that low-molecular-weight hyaluronic acid, which is produced by UV light and oxidation reactions, weakens the adhesion between vascular pericytes and vascular endothelial cells. Furthermore, they have revealed that Sanguisorba officinalis extract, or a complex extract of Sanguisorba officinalis extract and Aspathus linearis extract, increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells.

[0008] In other words, the present invention, which solves the above problems, is an agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, with Sanguisorba officinalis extract as the active ingredient. According to the present invention, the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells can be increased.

[0009] In a preferred embodiment of the present invention, the adhesion-related factor expression enhancer further comprises rooibos (Aspalathus linearis) extract. According to the present invention, by combining the extract of *Paeonia lactiflora* Pall. and the extract of *Aspalathus linearis*, the expression of adhesion-related factors can be further improved as compared with the addition of the extract of *Paeonia lactiflora* Pall. alone.

[0010] In a preferred embodiment of the present invention, the adhesion-related factor is platelet-derived growth factor receptor (PDGFRβ) and / or angiopoietin 1 (ANGPT1) of vascular pericytes.

[0011] In a preferred embodiment of the present invention, the expression enhancer of the adhesion-related factor is for suppressing the weakening of the adhesion between vascular pericytes and vascular endothelial cells caused by low molecular weight hyaluronic acid. According to the present invention, it is possible to suppress the weakening of the adhesion of the cells caused by low molecular weight hyaluronic acid.

[0012] In a preferred embodiment of the present invention, the expression enhancer of the adhesion-related factor is for suppressing vascular permeability. According to the present invention, it is possible to suppress vascular permeability.

[0013] Furthermore, the present invention for solving the above problems is a screening method including selecting a skin color improving component using the adhesion state between vascular pericytes and vascular endothelial cells as an index. Conventionally, it is known that vascular permeability is related to the leakage of inflammatory substances and this can affect skin color. According to the present invention, it is possible to screen a skin color improving component using the adhesion state between vascular pericytes and vascular endothelial cells as an index.

[0014] In a preferred embodiment of the present invention, the screening method includes selecting a component that increases the adhesion state as a skin color improving component.

[0015] In a preferred embodiment of the present invention, the adhesion state is the expression level of adhesion-related factors between vascular pericytes and vascular endothelial cells.

[0016] In a preferred embodiment of the present invention, the adhesion state is the number of pericytes adhered to vascular endothelial cells.

[0017] In a preferred embodiment of the present invention, the adhesion-related factor is platelet-derived growth factor receptor (PDGFRβ) and / or angiopoietin 1 (ANGPT1) of pericytes.

[0018] In a preferred embodiment of the present invention, the skin color improving component is a component for improving at least any one of redness, unevenness, and pigmentation.

[0019] In a preferred embodiment of the present invention, the skin color improving component is a component for suppressing vascular permeability. According to the present invention, as a component for suppressing vascular permeability, a component for improving skin color can be screened.

[0020] In a preferred embodiment of the present invention, the screening method uses the adhesion state in the presence of low molecular weight hyaluronic acid as an index. As described above, the present inventors have clarified that the adhesion state between pericytes and vascular endothelial cells is reduced by low molecular weight hyaluronic acid. That is, according to the present invention, a component effective for the reduction of the adhesion state caused by low molecular weight hyaluronic acid can be screened.

Effects of the Invention

[0021] According to the present invention, an expression enhancer of an adhesion-related factor between pericytes and vascular endothelial cells containing waremokou extract or a composite extract of waremokou extract and rooibos extract as an active ingredient can be provided. Furthermore, a screening method for selecting a skin color improving component can be provided using the adhesion state between pericytes and vascular endothelial cells as an index.

Brief Description of the Drawings

[0022] [Figure 1]This graph shows the results of the gene expression levels of ANGPT1 and PDGFRβ in vascular pericytes cultured in culture media containing each plant extract in Test Example 1. [Figure 2] a) is a graph showing the results of the gene expression levels of ANGPT1 and PDGFRβ in vascular pericytes cultured in a medium containing Sanguisorba officinalis extract and each plant extract in Test Example 2. b) is a graph showing the results of the gene expression levels of ANGPT1 and PDGFRβ in vascular pericytes cultured in a medium containing Sanguisorba officinalis extract and a medium containing a complex extract of Sanguisorba officinalis extract and Rooibos extract (hereinafter simply referred to as "complex extract") in Test Example 2. [Figure 3] This graph shows the results of the gene expression levels of ANGPT1 and PDGFRβ in pericytes cultured in a medium containing low molecular weight hyaluronic acid or a medium containing low molecular weight hyaluronic acid and a complex extract in Test Example 3. [Figure 4] In Test Example 4, the number of vascular pericytes adhering to vascular endothelial cells in a low molecular weight hyaluronic acid-containing medium and the number of the aforementioned adhering vascular pericytes in a medium containing low molecular weight hyaluronic acid and a complex extract are shown. [Figure 5] This graph shows the inhibitory effect of the complex extract on vascular permeability in Test Example 5. [Modes for carrying out the invention]

[0023] The present invention is described below. <1> An agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells, and <2> The screening method for skin tone improving ingredients is divided into sections and explained in detail.

[0024] <1> An agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells. The adhesion-related factor expression enhancer according to the present invention contains Sanguisorba officinalis extract. In a preferred form, it further contains Rooibos extract. As will be described later in the examples, Sanguisorba officinalis extract alone has the effect of increasing the expression of the above-mentioned adhesion-related factors, but a combined extract of Sanguisorba officinalis extract and Rooibos extract can improve the expression of the above-mentioned adhesion-related factors compared to Sanguisorba officinalis extract alone.

[0025] Here, "Sanguisorba officinalis extract" and "Rooibos extract" refer not only to the extracts derived from Sanguisorba officinalis and Rooibos, but also to fractions of these extracts, purified fractions, and solvent-removed extracts or fractions or purified products. Furthermore, extracts derived from Sanguisorba officinalis and Rooibos include extracts from plants that grow wild or cultivated, extracts from plants sold as raw materials for herbal medicines, and commercially available extracts. When extracting the above extract, the extraction process can use the entire plant, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flower spikes, and flower buds. However, it is preferable to crush or finely chop these parts beforehand to improve the extraction efficiency. Furthermore, when extracting Sanguisorba officinalis extract, it is preferable to use the roots and rhizomes, and when extracting Rooibos extract, it is preferable to use the whole plant. Suitable extraction solvents include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol; polyhydric alcohols such as 1,3-butylene glycol, 1,3-butanediol, and polypropylene glycol; ketones such as acetone and methyl ethyl ketone; and ethers such as diethyl ether and tetrahydrofuran. In particular, 50% ethanol is preferred as the extraction solvent for Sanguisorba officinalis extract, and 50% 1,3-butylene glycol is preferred as the extraction solvent for Rooibos extract. Specific methods for extracting the above extract include, for example, adding 1 to 30 parts by mass of solvent to 1 mass of the plant body or its dried material to be used for extraction, immersing it for several days at room temperature or for several hours at a temperature near the boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography, but the extraction method is not limited to this.

[0026] The adhesion-related factor expression enhancer of the present invention is used to increase the expression of factors involved in the adhesion between vascular pericytes and vascular endothelial cells. In a preferred embodiment, the adhesion-related factor is a factor expressed in vascular pericytes, more preferably PDGFRβ and / or ANGPT1, and particularly preferably both PDGFRβ and ANGPT1.

[0027] The present invention's adhesion-related factor expression enhancer is preferably used to suppress the weakening of adhesion between vascular pericytes and vascular endothelial cells caused by low molecular weight hyaluronic acid. Low molecular weight hyaluronic acid is generated when hyaluronic acid in the body is broken down by oxidative stress caused by UV rays and daily stress. In other words, it is also preferable to use it to suppress the weakening of the adhesive state caused by UV rays and oxidative stress.

[0028] The adhesion-related factor expression enhancer of the present invention can be appropriately combined with any component used in formulation to take the form of an oral preparation or a topical skin preparation. In oral preparations, the amount of Sanguisorba officinalis extract per dose, depending on the dosage form, is usually 0.1 mg or more, preferably 1 mg or more, and more preferably 10 mg or more, as dry mass of the extract. Furthermore, it is usually 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less. In the case of oral preparations containing a complex extract, the amount of Sanguisorba officinalis extract is preferably as described above, and the amount of Rooibos extract per dose, depending on the dosage form, is usually 0.1 mg or more, preferably 1 mg or more, and more preferably 10 mg or more, as dry mass of the extract. Furthermore, it is usually 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less.

[0029] When the adhesion-related factor expression enhancer of the present invention is used as a topical skin preparation, examples include cosmetics, quasi-drugs, and topical skin medicines. Furthermore, the dosage form is not particularly limited. Specifically, forms such as facial cleansers, makeup removers, lotions, serums, emulsions, creams, gels, and sun care products are preferred. Forms such as face masks are also preferred.

[0030] In topical skin preparations, the content (dry weight) of Sanguisorba officinalis 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. Also, it is usually 1% by mass or less, preferably 0.1% by mass or less, more preferably 0.01% by mass or less, even more preferably 0.001% by mass or less, even more preferably 0.0001% by mass or less, and particularly preferably 0.00005% by mass or less. In the case of oral preparations containing a complex extract, the content of Sanguisorba officinalis extract is preferably as described above, and the content of Rooibos extract, depending on the dosage form, is usually 0.0000001% by mass or more, preferably 0.0000005% by mass or more, more preferably 0.000001% by mass or more, and even more preferably 0.00001% by mass or more, based on the dry weight of the extract per dose. Furthermore, the amount is usually 1% by mass or less, preferably 0.1% by mass or less, more preferably 0.01% by mass or less, even more preferably 0.001% by mass or less, even more preferably 0.0001% by mass or less, even more preferably 0.00001% by mass or less, and particularly preferably 0.000005% by mass or less.

[0031] In a preferred embodiment of the present invention, the preferred ratio (dry mass) of Sanguisorba officinalis extract and Rooibos extract contained in the adhesion-related factor expression enhancer is 1:0.1 to 1:1. More preferably, it is 1:0.2 to 1:1.

[0032] When incorporating Sanguisorba officinalis extract, or a complex extract of Sanguisorba officinalis extract and Rooibos extract, into a cosmetic product, other ingredients commonly used in cosmetics, in addition to whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, plant and animal extracts, and active ingredients, may be included as optional ingredients, provided that they do not impair the effect of these extracts on increasing the expression of adhesion-related factors. These optional ingredients may be obtained from the market or synthesized using known methods. Furthermore, each optional ingredient may have two or more effects (for example, a whitening effect and a wrinkle-improving effect).

[0033] As for whitening ingredients, ingredients commonly used in cosmetics can be used without particular restrictions. For example, 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 and aminodiphenylmethane, N-(o-toluyl)cysteic acid, N-(m-toluyl)cysteic acid, N-(p-toluyl)cysteic acid, N-(p-methoxybenzoyl)cysteic acid, N-benzoyl-serine, N-(p-methylben Examples include zoyl)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, dexpanthenol W, niacinamide, etc.

[0034] The amount of whitening ingredients in cosmetics 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 weight in the case of extracts). As wrinkle-improving ingredients, ingredients commonly used in cosmetics can be used without particular restrictions. For example, vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoic acid, retinyl palmitate, and retinyl acetate. Other examples include benzyl ursolate, ursolic acid phosphate, benzyl betulinate, benzyl acid phosphate, sodium trifluoride isopropyl oxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate, and niacinamide.

[0035] The amount of wrinkle-improving ingredients in cosmetics 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 weight in the case of extracts).

[0036] Examples of anti-inflammatory ingredients include clarinon, glabridin, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol, niacinamide, and tranexamic acid, with glycyrrhizic acid and its salts, alkyl glycyrrhetinate and its salts, and glycyrrhetinic acid and its salts being particularly preferred. The content of anti-inflammatory ingredients in cosmetics is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 1 to 5% by mass (dry weight in the case of extracts).

[0037] Examples of plant and animal-derived extracts include akebia extract, thunbergii extract, asparagus extract, avocado extract, hydrangea extract, almond extract, arnica extract, aronia extract, apricot extract, ginkgo extract, fennel extract, Japanese angelica extract, Eleutherococcus senticosus extract, Enmeisou extract, Phellodendron amurense extract, Panax ginseng extract, Lamium album extract, Arctium moniliforme extract, Pueraria lobata extract, chamomile extract, carrot extract, Artemisia capillaris extract, licorice extract, kiwi extract, cucumber extract, and gua gravy. Bamboo extract, gardenia extract, bamboo grass extract, walnut extract, black rice extract, chlorella extract, mulberry extract, keiketto extract, ginger extract, gentian extract, rice extract, rice fermentation extract, rice bran fermentation extract, rice germ oil, salvia extract, soapwort extract, bamboo extract, sunflower extract, sansho extract, shiitake mushroom extract, rehmannia extract, lithospermum extract, perilla extract, linden extract, meadowsweet extract, ginger extract, calamus root extract, horsetail extract, stevia extract, stevia extract Fermented product, yarrow extract, peppermint extract, sage extract, mallow extract, Cnidium officinale extract, Swertia japonica extract, mulberry bark extract, rhubarb extract, soybean extract, jujube extract, dandelion extract, clove extract, chili pepper extract, angelica extract, calendula extract, peach kernel extract, Houttuynia cordata extract, tomato extract, natto extract, carrot extract, garlic extract, hibiscus extract, Ophiopogon japonicus extract, lotus extract, parsley extract, birch extract, witch hazel extract, toad lily Preferred extracts include those of Isodon japonicus, Hinoki cypress, Loquat, Coltsfoot, Butterbur, Poria cocos, Luffa gourd, Peppermint, Linden, Pine, Skunk cabbage, Melissa, Mozuku seaweed, Peach, Cornflower, Lily, Coix seed extract, Artemisia princeps, Lavender, Apple, Reishi mushroom, Lettuce, Forsythia extract, Astragalus membranaceus extract, Rosemary extract, and Roman chamomile extract.

[0038] The content (dry mass) of the aforementioned arbitrary animal or plant-derived extracts in the cosmetic composition is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, and more preferably 0.3 to 3% by mass.

[0039] In addition to the active ingredient, ingredients commonly used in cosmetics include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, polyols such as 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 alkyl sulfate triethanolamine A. Anionic surfactants such as tel, cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, and laurylamine oxide, amphoteric surfactants such as imidazoline (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine surfactants (alkyl betaine, amide betaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyltaurine, sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (monostearate) Glycerin phosphate, 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 sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (e.g., POE2-octyldodecyl ether), POE alkylphenyl ethers (e.g., POE nonylphenyl ether), Pluronic® type, POE·POP alkyl ethers (e.g., POE·POP2-decyltetradecyl ether), Tetronic type, POE castor oil / hydrogenated castor oil derivatives (e.g., POE castor oil, POE hydrogenated castor oil), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid, and sodium lactate, which may be surface-treated.Powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, barium sulfate, etc., which may be surface-treated; inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, zinc oxide, which may be surface-treated; composite pigments such as iron oxide titanium dioxide sintered bodies, which may be surface-treated; pearlescent agents such as titanium mica, fish scale foil, bismuth oxychloride, which may be lake-formed; Red 202, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue 1, Green 201, Violet 20 Examples include organic dyes such as No. 1 and Red No. 204, organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomers, lower alcohols such as ethanol and isopropanol, vitamin A or its derivatives, vitamin B derivatives such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or its derivatives, vitamin B12, vitamin B15 or its derivatives, vitamin E derivatives such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate, vitamin D derivatives, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0040] <2> Screening methods The screening method according to the present invention will be described in detail below. The screening method according to the present invention is a screening method that includes selecting skin tone improving components using the adhesion state between vascular pericytes and vascular endothelial cells as an indicator. The skin tone improving components include components that improve at least one of redness, unevenness, and pigmentation, and preferably include components that improve at least one of unevenness and pigmentation. Furthermore, it is preferable that the components to be selected are those that suppress vascular permeability. That is, it is preferable to select skin tone improving components that improve skin tone by suppressing vascular permeability. Here, the terms "selection" or "screening" refer to a concept that includes searching for skin tone improving ingredients or their candidates.

[0041] The substance to be tested in the screening method of the present invention may be a pure substance, an extract of biological origin, or a mixture thereof. Biological extracts refer to not only the extracts themselves derived from animals or plants, but also to fractions of extracts, purified fractions, and solvent-removed products of extracts, fractions, or purified products. Plant-derived extracts include extracts made from plants that grow naturally or are cultivated, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts. The extraction process can use the whole plant, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flower spikes, and flower buds. However, it is preferable to crush or finely chop these parts beforehand to improve extraction efficiency. Suitable extraction solvents include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol; polyhydric alcohols such as 1,3-butylene glycol, 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 solvent to 1 mass of the plant tissue or its dried material to be extracted, immersing it for several days at room temperature or for several hours at a temperature near its boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography or the like.

[0042] In the screening method of the present invention, the cell type used is preferably normal human vascular endothelial cells and / or pericytes. Preferably, the cells are derived from normal humans. Alternatively, commercially available cells may be used. A culture medium commonly used by those skilled in the art can be used for culturing the aforementioned cells. Furthermore, the culture conditions for culturing the aforementioned cells can be appropriately set depending on the cells used.

[0043] In the present invention, preferably, the expression levels of adhesion-related factors between vascular pericytes and vascular endothelial cells can be used as the adhesion state indicator. The test substance is added to the cell culture system, and the expression levels of the adhesion-related factors are measured and used as the indicator. Preferably, the cells are vascular pericytes, more preferably the adhesion-related factors are PDGFRβ and / or ANGPT1, and particularly preferably both PDGFRβ and ANGPT1.

[0044] Specifically, if the expression levels of PDGFRβ and / or ANGPT1 in cells cultured with the test substance added are higher than those in cells cultured without the test substance, the test substance can be selected as a skin tone improving ingredient. More specifically, if the expression levels of PDGFRβ and / or ANGPT1 in cells cultured with the test substance added are 1.2 times greater than the expression levels in cells cultured without the test substance, and more preferably 1.5 times greater, then it is preferable to select them as candidates for skin tone improving ingredients.

[0045] The expression levels of the adhesion-related factors mentioned above can be measured by conventional methods such as mRNA measurement and immunohistochemical analysis. For example, the expression levels of PDGFRβ and / or ANGPT1 can be quantitatively detected by PCR using a DNA fragment containing a sequence that specifically binds to the sequence of the gene in question as a primer. Furthermore, since the gene sequences encoding PDGFRβ and / or ANGPT1 in vascular pericytes are publicly available, those skilled in the art can design appropriate primers. Alternatively, commercially available primers may be used.

[0046] A preferred embodiment of the present invention includes the step of adding low molecular weight hyaluronic acid and the test substance to a cell culture system. Then, pericytes are cultured together with the test substance in the presence of low molecular weight hyaluronic acid, and the expression level of the factor is evaluated. By including the above steps, it is possible to select a skin tone improving ingredient that prevents the deterioration of skin tone caused by low molecular weight hyaluronic acid, or improves the aforementioned skin tone.

[0047] Furthermore, in the present invention, the number of perivascular cells adhering to vascular endothelial cells can be used as the adhesion state, which is the aforementioned indicator. That is, in a preferred embodiment of the present invention, the test substance is added to the cell culture system and the number of perivascular cells adhering to vascular endothelial cells is measured. Specifically, the process includes culturing stained pericytes in a culture medium containing the test substance, seeding the cultured pericytes onto vascular endothelial cells, washing them multiple times with phosphate buffer or the like, and then counting the number of adhering pericytes using a fluorescence microscope.

[0048] Furthermore, if the number of perivascular cells adhering to vascular endothelial cells in cells cultured with the test substance added is greater than the number of adhering perivascular cells in cells cultured without the test substance added, the test substance can be selected as a candidate for a skin tone improving ingredient. [Examples]

[0049] The present invention will be described in detail below with reference to the following examples, but the technical scope of the present invention is not limited to the following examples.

[0050] <Test Example 1> We measured the effects of various extracts on the gene expression levels of ANGPT1 and PDGFRβ in vascular pericytes. The extraction solvents used for each active ingredient extract in this example are as follows:

[0051] [Table 1] Furthermore, the same extraction solvent was used for the extracts in the subsequent test examples.

[0052] (method) 1) Place human placental-derived vascular pericytes (Promo Cell) in a 24-well plate in 3.0 × 10⁶ units. 4 Seeds were seeded in cells / well and cultured for 24 hours at 37°C in a 5% CO2 environment. The following reagents and culture media were used for cell culture. • Pericyte Growth Medium 2 (Promo Cell, C-28041) • Dulbecco's PBS (-) for biochemistry (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795) 0.05% Trypsin-EDTA (GIBCO) TM (Manufactured by 25300-062) 0.4% trypan blue aqueous solution (GIBCO TM (Manufactured by 15250-061) 2) Remove the culture medium and replace it in each well with the medium containing each extract (final concentration 0.1%). 3) After 24 hours, the gene expression levels of ANGPT1 and PDGFRβ in pericytes were measured by qRT-PCR and compared with those of the control group. The following methods were used for mRNA expression analysis. ANGPT1 primer (Hs_ANGPT1_1_SG QuantiTect Primer Assay 1, Qiagen Co., Ltd., QT00046865), PDGFR primer (Hs_PDGFR_1_SG QuantiTect Primer Assay, Qiagen Co., Ltd., QT00082327), ACTB primer (Hs_ACTB_2_SG QuantiTect Primer Assay, Qiagen Co., Ltd., QT01680476), Superscript VILO cDNA Synthesis kit 250T (Invitrogen K.K., 11754-250), RNeasy Mini Kit-8 (250) (Qiagen Co., Ltd., 74106), Fast SYBR® Green Master Mix (Applied Biosystems, Inc., 4385617)

[0053] (result) Figure 1 shows the results of the gene expression levels of ANGPT1 and PDGFRβ in Test Example 1. As shown in Figure 1, the culture medium containing Sanguisorba officinalis extract showed an effect of improving the expression of the aforementioned gene compared to culture media containing other extracts.

[0054] <Test Example 2> Using the method of Test Example 1, Sanguisorba officinalis extract was combined with other plant extracts, and the gene expression levels of ANGPT1 and PDGFRβ in vascular pericytes were measured and compared. The extraction solvents used for each plant extract in this example are as follows:

[0055] [Table 2] Furthermore, the same extraction solvent was used for the extracts in the subsequent test examples.

[0056] (result) Figure 2a) shows the results of gene expression levels for complex extracts of Sanguisorba officinalis extract and various plant extracts. Among the combinations of plant extracts, the complex extract of Sanguisorba officinalis extract and rooibos extract showed increased gene expression levels compared to the control group. Furthermore, to confirm the results obtained above, the expression levels of the aforementioned gene were measured under the conditions described above, both for Sanguisorba officinalis alone and for a combined extract of Sanguisorba officinalis extract and Rooibos extract. The results are shown in Figure 2b). As shown in Figure 2b), it was confirmed that the expression level of the aforementioned gene increased in the case of a combined extract of Sanguisorba officinalis extract and Rooibos extract compared to the case of Sanguisorba officinalis extract alone. Based on these results, it was confirmed that combining Sanguisorba officinalis extract and Rooibos extract has the effect of improving the expression of adhesion-related factors compared to adding them individually.

[0057] <Test Example 3> Next, in order to clarify the effect of low molecular weight hyaluronic acid on the adhesion of pericytes and endothelial cells, and to clarify the effect of the complex extract (hereinafter simply referred to as "complex extract") whose effect was confirmed in the above-mentioned test example on the effect of low molecular weight hyaluronic acid, the following tests were conducted.

[0058] (method) 1) Human placental-derived vascular pericytes were cultured using the reagents and culture medium described in Test Example 1. 2) The culture medium was replaced with one containing low molecular weight hyaluronic acid and a complex extract, and incubated for 24 hours. The final concentrations were 0.2 mg / mL for low molecular weight hyaluronic acid and 0.1% for each extract. Hyaluronan Ultra-Low Molecular Weight (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 513-69521) was used as the low molecular weight hyaluronic acid reagent. 3) Similar to Test Example 1, the gene expression levels of ANGPT1 and PDGFRβ in pericytes were measured. The same reagents as in Test Example 1 were used for the mRNA expression analysis.

[0059] (result) The results of Test Example 3 are shown in Figure 3. The results showed that, in the presence of low molecular weight hyaluronic acid, a significant decrease in the expression of ANGPT1 and PDGFRβ genes, which are adhesion factors between vascular pericytes and vascular endothelial cells, was observed compared to the control group. Furthermore, the addition of a complex extract suppressed the decrease in the expression of both factors.

[0060] <Test Example 4> Next, the following tests were conducted to clarify the effect of low molecular weight hyaluronic acid on the adhesion between vascular pericytes and vascular endothelial cells, and the effect of the complex extract on this effect.

[0061] (method) In the presence of low molecular weight hyaluronic acid, we conducted a study to measure and compare the number of perivascular cells adhering to vascular endothelial cells after washing with PBS(-), depending on whether or not a complex extract was present. 1) After nuclear staining of normal human placental pericytes (Promo Cell), 2.5 × 10¹⁶ cells were placed in a 75 mL flask. 5 Cells were seeded and cultured for 48 hours at 37°C under a 5% CO2 environment in a medium supplemented with a complex extract. The following reagents and culture media were used for cell culture. • Pericyte Growth Medium 2 (Promo Cell, C-28041) • Dulbecco's PBS (-) for biochemistry (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795) 0.05% Trypsin-EDTA (GIBCO) TM (Manufactured by 25300-062) 0.4% trypan blue aqueous solution (GIBCO TM (Manufactured by 15250-061) For nuclear staining, Hoechest 33342 (Thermofisher Scientific) was used. 2) Normal human-derived vascular endothelial cells (manufactured by KURABO Corporation) are placed on a 4-well chamber slide in a 1.0 × 10⁶ size. 5 Cells were seeded and cultured for 24 hours. The following culture media were used for cell culture. • Humedia EG2 (manufactured by KURABO Corporation, KE-2150S) 3)1) Place the pericyte cells on top of the endothelial cells at a rate of 1.0 × 10 4 Cells were seeded and cultured for 24 hours. 4) The culture medium was replaced with one containing low molecular weight hyaluronic acid and a complex extract (final concentration: low molecular weight hyaluronic acid 0.2 mg / mL, each extract 0.1%) and incubated for 72 hours. 5) After removing the culture medium and washing with 1 mL of PBS(-), the number of perivascular cells was observed using a fluorescence microscope.

[0062] (result) The results of Test Example 4 are shown in Figure 4. The white dots in the figure represent stained pericytes that are in adhesion to vascular endothelial cells. When compared with the Control group, the addition of low molecular weight hyaluronic acid was confirmed to reduce the number of pericytes adhering to vascular endothelial cells (Figure 4, central figure, low molecular weight hyaluronic acid). In addition, when low molecular weight hyaluronic acid and the complex extract were added together, it was confirmed that the decrease in the number of pericytes adhering to vascular endothelial cells was suppressed (Figure 4, right figure, low molecular weight hyaluronic acid + extract).

[0063] <Test Example 5> It is known that when endothelial cells receive ANGPT1, the expression of genes related to tight junction between endothelial cells is induced and vascular permeability is suppressed. In order to confirm whether the complex extract is effective in suppressing vascular permeability, the following test was conducted.

[0064] (Method) The cell lines used in Test Example 5 are shown below. · Normal human placenta-derived pericytes (hPC-PL) (manufactured by Promo cell) · Normal human-derived vascular endothelial cells (manufactured by KURABO Industries Ltd.) In addition, the reagents and media for cell culture used in this test example are as follows. · Pericyte Growth Medium 2 (manufactured by Promo cell, C-28041) · Dulbecco's PBS(-) for biochemistry (manufactured by FUJIFILM Wako Pure Chemical Corporation, 045-29795) · 0.05% Trypsin-EDTA (manufactured by GIBCO TM , 25300-062) · 0.4% Trypan blue aqueous solution (manufactured by GIBCO TM , 15250-061) · HumediaEG2 (manufactured by KURABO Industries Ltd., KE-2150S) · Collagen acidic solution (Atelocell (registered trademark), IPC-30) · Hydrochloric acid (manufactured by FUJIFILM Corporation, :083-01095)

[0065] [Collagen coating process] 1) A collagen solution diluted 10-fold with hydrochloric acid was added 200 μL to the outside of each Transwell insert, and the excess solution was removed with a pipette. 2) The inserts were placed in a 12-well plate, 300 μL of collagen solution was added to the inside of each insert, and excess collagen solution was removed with a pipette. 3) The samples were left standing for at least 30 minutes at 37°C in a 5% CO2 environment. 4) 1 mL of PBS was added to the outside of the insert, and 300 μL to the inside. 5) Remove PBS using an aspirator. 6) Repeat steps 4) and 5) above, and leave the mixture standing after removing the PBS.

[0066] [Pericyte cell seeding - complex extract addition process] 1) 6.7 × 10 4 A cell suspension was prepared to achieve a concentration of cells / mL. 2) Place the insert upside down on the top lid of a 12-well plate, remove any excess PBS, and add 150 μL of cell suspension to each insert. 3) Place the lid of a new 12-well plate on top and incubate at 37°C and 5% CO2 for 4 hours. 4) 1 mL of pericyte medium was added to each of the 12 wells. 5) After removing the culture medium from the insert with a pipette, it was placed in a 12-well container. 6) 300 μL of culture medium was added to the insert, and overnight incubation was performed at 37°C and 5% CO2. 7) Remove the culture medium, add 300 μL of the medium containing the complex extract to the inside of the insert and 1 mL to the plate, and incubate at 37°C and 5% CO2 for 24 hours.

[0067] [Endothelial cell seeding process] 1) Remove the culture medium and wash the insert and plate with 300 μL and 1 mL of PBS(-), respectively. 2) Prepare a culture medium (hereinafter referred to as co-culture medium) by mixing pericyte medium and endothelial cell medium in a 1:1 ratio, and add 1 mL to the outside of the insert. 3) 2 × 10 5After preparing a cell suspension in co-culture medium to a concentration of cells / mL, 500 μL of the endothelial cell suspension was added to the insert, and the cells were cultured for 96 hours at 37°C under 5% CO2 conditions.

[0068] [Permeability test] 1) Solution preparation step: Reagents were prepared according to the procedure described below. (i) Create Basolateral Buffer Basolateral buffer was prepared by mixing 1.45 mM CaCl2 (40.2 mg) and 10 mM glucose (450 mg) in PBS(-) (250 mL). The following reagents were used. • Calcium chloride (CaCl2) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 038-24985) • D(+)-glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 049-31165) (ii) Creation of an Apical Buffer 1 mg / mL of FITC-Dextran (additional amount 10 mg) was mixed with Basolateral Buffer (-) (10 mL) and stored at 4°C in a light-shielded container. The following reagents were used. • FITC-Dextran (manufactured by Sigma Aldrich, FD40-100MG) 2) Remove the culture medium and wash the insert and plate with 500 μL of PBS and 1 mL of PBS in the lower layer, respectively. 3) 1.5 mL of Basolateral Buffer was added to the 12-well plate prepared for measurement, and 500 μL of Apical Buffer was added to the inserted insert. 4) Incubated for 3 hours at 37°C in a 5% CO2 environment. 5) Gently remove the insert, shake the basolateral buffer in the plate, and then pipette. 6) A standard series of standard curves was fabricated using Apical Buffer on a 96-well plate. Solutions with FITC-Dextran concentrations ranging from 250 μg / ml to 0 μg / ml were prepared with a common ratio of 2 and 8. Dilution was performed using basolateral buffer. 7) 200 ml of the recovered Basolateral Buffer was added to each of the 96 wells. 8) Measurements were performed using a spectrophotometer with n=3 (standards were n=2). (Excitation was at 485 / 535 nm.)

[0069] (result) The results of Test Example 5 are shown in Figure 5. As shown in Figure 5, when comparing the amount of FITC-Dextran that permeated from the insert to the plate without the complex extract (left in the graph, no extract), a significant decrease in the amount of FITC-Dextran that permeated was observed when the complex extract was added (right in the graph, with extract). Therefore, it was confirmed that the extract, which is the active ingredient of the present invention, suppresses vascular permeability. [Industrial applicability]

[0070] According to the present invention, it is possible to provide an agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells. Furthermore, it is possible to provide a method for screening skin tone improving components based on the suppression of vascular permeability.

Claims

1. This product contains Sanguisorba officinalis extract as its active ingredient and is an agent that increases the expression of adhesion-related factors between vascular pericytes and vascular endothelial cells.

2. Furthermore, the adhesion-related factor expression enhancer according to claim 1 further comprises an extract of rooibos (Aspalathus linearis).

3. The adhesion-related factor expression enhancer according to claim 1 or 2, wherein the adhesion-related factor is platelet-derived growth factor receptor (PDGFRβ) and / or angiopoietin 1 (ANGPT1) of vascular pericytes.

4. An expression enhancer for adhesion-related factors according to claim 1 or 2, for suppressing the weakening of adhesion between vascular pericytes and vascular endothelial cells caused by low molecular weight hyaluronic acid.

5. An agent for increasing the expression of adhesion-related factors according to claim 1 or 2, for suppressing vascular permeability.

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