Cell death inhibitor and GATA6-AS1 expression promoter, their use and screening method

Abstract

To provide agents for suppressing cell death, preferably cell death induced by AGEs, to provide methods for suppressing cell death, and to provide methods for screening components that suppress cell death.SOLUTION: Provided is a cell death inhibitor containing one or more extracts selected from Paeonia suffruticosa extract, Eucalyptus globulus extract, and Hypericum erectum extract. Provided is a GATA6-AS1 expression promoter containing one or more extracts selected from Paeonia suffruticosa extract, Eucalyptus globulus extract, and Hypericum erectum extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for suppressing cell death and / or promoting the expression of GATA6-AS1, a method for using the agent, and a screening method for the agent. [Background technology]

[0002] Advanced glycation endproducts (AGEs) accumulate in the body through the Maillard reaction or by consuming cooked foods. AGEs are known to have various effects in the body, including the induction of cell death. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways (Zoubin Alikhani, Mani Alikhani, Coy M. Boyd, Kiyoko Nagao, Philip C. Trackman,and Dana T. Graves, J Biol Chem. 2005 1;280(13)) Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the above circumstances, an object of the present invention is to provide an agent for suppressing cell death, preferably cell death induced by AGEs. Further objects of the present invention are to provide a method for suppressing cell death and a method for screening for components that suppress cell death.

[0005] As a result of extensive research, the inventors of the present invention have revealed that a decrease in the expression level of GATA6-AS1, a type of lncRNA, promotes the expression of AGE receptors. Therefore, a further object of the present invention is to provide an agent for promoting the expression of GATA6-AS1, a method for promoting the expression of GATA6-AS1, and a method for screening for components that promote the expression of GATA6-AS1 and / or components that suppress the expression of AGEs receptors. [Means for solving the problem]

[0006] The present invention, which solves the above-mentioned problems, is a cell death inhibitor containing one or more extracts selected from peony extract, eucalyptus extract, and Hypericum extract. In a preferred embodiment of the present invention, at least Paeonia suffruticosa extract is contained. According to the present invention, cell death can be suppressed.

[0007] In a preferred embodiment of the present invention, the cell death inhibitor is an agent for inhibiting cell death in skin tissue. In a preferred embodiment of the present invention, the skin tissue is a vascular endothelial cell. According to the present invention, cell death of skin tissue, particularly vascular endothelial cells, can be suppressed.

[0008] In a preferred embodiment of the present invention, the cell death inhibitor is an agent for inhibiting cell death caused by advanced glycation endoproducts (AGEs). Cell death is induced by various factors such as viral infection of cells, DNA damage, AGEs, etc. According to the present invention, cell death induced particularly by AGEs can be suppressed.

[0009] In a preferred embodiment of the present invention, the cell death inhibitor for inhibiting cell death caused by advanced glycation endoproducts further comprises an AGE production inhibitor. In a preferred embodiment of the present invention, the AGE production inhibitor contains mugwort extract and / or Houttuynia cordata extract. According to the present invention, the inhibitory effect on cell death caused by AGEs can be enhanced by inhibiting cell death induced by AGEs and also inhibiting the production of AGEs themselves. In particular, when the AGE production inhibitor contains mugwort extract and Houttuynia cordata extract, the inhibitory effect on cell death caused by AGEs can be enhanced.

[0010] The present invention, which solves the above-mentioned problems, provides a non-therapeutic method for inhibiting cell death, which comprises applying the above-mentioned cell death inhibitor to skin tissue. According to the present invention, cell death in skin tissue can be suppressed.

[0011] A preferred embodiment of the present invention is a non-therapeutic method for inhibiting cell death, which comprises applying the above-mentioned cell death inhibitor to skin tissue and then massaging the skin tissue. According to the present invention, the cell death inhibitor applied to skin tissue can be efficiently penetrated into the skin tissue, and cell death in the skin tissue can be inhibited.

[0012] The present invention, which solves the above-mentioned problems, provides a GATA6-AS1 expression promoter comprising one or more extracts selected from peony extract, eucalyptus extract, and Hypericum extract. In a preferred embodiment of the present invention, at least Paeonia suffruticosa extract is contained. According to the present invention, the expression of GATA6-AS1 can be promoted.

[0013] In a preferred embodiment of the present invention, the GATA6-AS1 expression promoter is an agent for promoting the expression of GATA6-AS1 in skin tissue. In a preferred embodiment of the present invention, the skin tissue is a vascular endothelial cell. According to the present invention, it is possible to promote the expression of GATA6-AS1 in skin tissues, particularly in vascular endothelial cells.

[0014] The present invention, which solves the above-mentioned problems, provides a non-therapeutic method for promoting GATA6-AS1 expression, which involves applying the above-mentioned GATA6-AS1 expression promoter to skin tissue. According to the present invention, the expression of GATA6-AS1 in skin tissue can be promoted.

[0015] A preferred embodiment of the present invention is a non-therapeutic method for promoting GATA6-AS1 expression, which comprises applying the above-mentioned GATA6-AS1 expression promoter to skin tissue, followed by massaging the skin tissue. According to the present invention, the GATA6-AS1 expression promoter applied to skin tissue can be efficiently penetrated into the skin tissue, thereby promoting the expression of GATA6-AS1 in the skin tissue.

[0016] The present invention, which solves the above-mentioned problems, provides a screening method for screening cell death-inhibiting components using the expression level of GATA6-AS1 and / or the expression level of an AGEs receptor in skin tissue as an index. The present inventors have found that there is a correlation between the expression level of GATA6-AS1 and the expression level of the AGEs receptor. According to the present invention, cell death-suppressing components can be screened using the expression level of GATA6-AS1 and / or the expression level of an AGEs receptor as an index.

[0017] In a preferred embodiment of the present invention, a component that increases the expression level of GATA6-AS1 is selected as the cell death-suppressing component. In a preferred embodiment of the present invention, a component that reduces the expression level of an AGE receptor is selected as the cell death-inhibiting component. The present inventors found that a decrease in the expression level of GATA6-AS1 increases the expression level of AGE receptors, thereby promoting cell death caused by AGEs. According to the present invention, a component that increases the expression level of GATA6-AS1 and / or a component that decreases the expression level of an AGE receptor can be screened as a cell death-suppressing component.

[0018] In a preferred embodiment of the present invention, the skin tissue is a vascular endothelial cell. According to the present invention, it is possible to screen for components that have an effect of inhibiting cell death, particularly in vascular endothelial cells.

[0019] In a preferred embodiment of the present invention, the cell death-inhibiting component is a component that inhibits cell death induced by advanced glycation endoproducts in the skin tissue. As described above, cell death is induced by various factors such as viral infection of cells, DNA damage, AGEs, etc. According to the present invention, a component that inhibits cell death induced by AGEs in particular can be selected as a cell death-inhibiting component. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide an agent for inhibiting cell death, preferably cell death induced by AGEs, a method for inhibiting cell death, and a method for screening for a component that inhibits cell death.

[0021] Furthermore, the present invention can provide an agent for promoting GATA6-AS1 expression and a method for promoting GATA6-AS1 expression. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a graph showing the expression level of GATA6-AS1 in cells transfected with GATA6-AS1 siRNA in Test Example 1. [Figure 2] 1 is a graph showing the expression level of AGE receptor mRNA in cells transfected with GATA6-AS1 siRNA in Test Example 1. [Figure 3]Figure 3(A) shows the experimental results of Test Example 1, in which cells transfected with control siRNA or GATA6-AS1 siRNA were cultured in the presence of collagen (control) or collagen-AGEs. Figure 3(B) shows the results of cell staining. In Figure 3(B), the open arrow indicates dead cells. [Figure 4] 1 is a graph showing the expression level of GATA6-AS1 in vascular endothelial cells cultured in the presence of AGEs in Test Example 2. [Figure 5] 1 is a graph showing the experimental results of the extract in Test Example 3, which was found to promote the expression of GATA6-AS1 in vascular endothelial cells. [Figure 6] FIG. 1 is a graph showing the AGE production inhibitory effects of mugwort extract and Houttuynia cordata extract in Test Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0023] <1> cell death inhibitors The cell death inhibitor of the present invention contains one or more extracts selected from the group consisting of peony extract, eucalyptus extract, and St. John's wort extract. In a preferred embodiment, the cell death inhibitor of the present invention contains at least peony extract. The present inventors have found that the above-mentioned peony extract, eucalyptus extract and St. John's wort extract have the effect of suppressing cell death. That is, according to the present invention, cell death can be suppressed.

[0024] In a preferred embodiment of the present invention, the cell death inhibitor is used to inhibit cell death in skin tissue, and in a more preferred embodiment, to inhibit cell death in vascular endothelial cells. In the present invention, "skin tissue" refers to tissue that constitutes the skin, and includes, for example, epidermal cells, dermal cells, vascular endothelial cells, and the like.

[0025] In a preferred embodiment of the present invention, the cell death inhibitor is an agent for inhibiting cell death caused by advanced glycation endoproducts (AGEs). Cell death is known to occur due to various factors, such as DNA damage caused by trauma or ultraviolet radiation, viral infection of cells, oxidative stress in vivo (increase in reactive oxygen species, etc.), and AGEs. According to the present invention, it is possible to inhibit cell death, particularly cell death induced by AGEs.

[0026] Here, peony extract, eucalyptus extract, and St. John's wort extract refer collectively to not only the extracts derived from peony, eucalyptus, and St. John's wort themselves, but also fractions of these extracts, purified fractions, and products from which the solvent has been removed from the extracts, fractions, and purified products. Extracts derived from peony, eucalyptus, and St. John's wort include extracts from wild-growing or cultivated plants, extracts sold as raw materials for herbal medicines, and commercially available extracts. When extracting the above-mentioned extract, the extraction procedure can use the whole plant or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, inflorescences, and flower buds, but it is preferable to crush or shred these beforehand to improve extraction efficiency. Suitable examples of extraction solvents include one or more selected from polar solvents such as 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 for the above-mentioned extract include, for example, a method in which 1 to 30 parts by mass of solvent is added to 1 mass of the part of the plant body or its dried material used for extraction, followed by immersion for several days at room temperature or for 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 by column chromatography or the like, but the extraction method is not limited to this.

[0027] The cell death inhibitor of the present invention can be appropriately combined with any ingredients used in formulation and can be in the form of an oral agent or an external agent for skin application. From the viewpoint of exhibiting an inhibitory effect on cell death of skin tissues, particularly vascular endothelial cells, the cell death inhibitor of the present invention is preferably in the form of an external preparation for skin.

[0028] When the cell death inhibitor of the present invention is formulated as an oral preparation, it is preferably in the form of a food composition containing one or more extracts selected from the group consisting of Paeonia lactiflora extract, Eucalyptus extract, and Hypericum perforatum extract as an active ingredient. Specifically, it is preferably in the form of a supplement in the form of a general food, tablet, granule, drink, or the like.

[0029] The content of one or more extracts selected from peony extract, eucalyptus extract, and St. John's wort extract in the oral preparation is usually 0.1 mg or more, preferably 1 mg or more, and more preferably 10 mg or more, in terms of dry mass of the extract per dose, depending on the dosage form. Also, it is usually 2000 mg or less, preferably 1000 mg or less, more preferably 500 mg or less. In the case of an oral preparation containing two or more extracts selected from peony extract, eucalyptus extract, and St. John's wort extract, the total content may be within the above numerical range.

[0030] When used as an external preparation for skin, examples of the form include cosmetics, quasi-drugs, external medicines for skin, etc. Furthermore, there are no particular limitations on the dosage form. In view of its intended purpose of inhibiting cell death of skin tissues, particularly vascular endothelial cells, the cosmetic product is preferably in the form of a cosmetic that can be used continuously. Specifically, the cosmetic product is preferably in the form of a facial cleanser, cleanser, lotion, serum, emulsion, cream, gel, sunscreen, or the like. Furthermore, the cosmetic product is preferably in the form of a face pack. In particular, in relation to the cell death inhibition method described below, it is more preferable that the topical skin preparation be a topical skin preparation that can be used in combination with massage, such as a facial cleanser, cleanser, lotion, emulsion, cream, or serum.

[0031] The content (dry weight) of paeonia extract, eucalyptus extract and hypericum extract in the external skin preparation is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more. Also, it is usually 80% by mass or less, preferably 30% by mass or less, and more preferably 10% by mass or less. By setting the concentration within the above range, cell death can be suppressed. In addition, when the topical skin preparation contains two or more extracts selected from peony extract, eucalyptus extract, and St. John's wort extract, the total content may be within the above numerical range.

[0032] When the peony extract, eucalyptus extract, and St. John's wort extract are blended into cosmetics, whitening ingredients, wrinkle-reducing ingredients, anti-inflammatory ingredients, extracts derived from plants and animals, and ingredients normally used in cosmetics other than active ingredients may be blended as optional ingredients within a range that does not impair the cell death inhibitory effect. These optional ingredients may be commercially available and used, or may be synthesized by known methods and used. Each optional ingredient may have two or more effects (for example, a whitening effect and an anti-wrinkle effect).

[0033] As for whitening ingredients, ingredients commonly used in cosmetics can be used without any 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-toluoyl)cis Theic acid, N-(m-toluoyl)cysteic acid, N-(p-toluoyl)cysteic acid, N-(p-methoxybenzoyl)cysteic acid, 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 Examples of suitable benzoylserine include 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, and niacinamide.

[0034] 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).

[0035] As the wrinkle-reducing component, any component commonly used in cosmetics can be used without any particular limitation. Examples of vitamin A or its derivatives include retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate, and retinol acetate.Further examples include ursolic acid benzyl ester, ursolic acid phosphate, betulinic acid benzyl ester, benzilic acid phosphate, trifluoroisopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetic acid sodium, and niacinamide.

[0036] The content of the wrinkle-reducing 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).

[0037] Examples of anti-inflammatory ingredients include kurarinone, glabridin, glycyrrhizinic acid, glycyrrhetinic acid, pantothenyl alcohol, niacinamide, and tranexamic acid, 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).

[0038] Examples of extracts derived from plants and animals include akebia extract, asparagus extract, avocado extract, hydrangea extract, almond extract, arnica extract, aronia extract, apricot extract, ginkgo extract, fennel extract, udo extract, Siberian ginseng extract, emmeiso extract, phellodendron bark extract, Panax ginseng extract, white nettle extract, kakyō extract, pueraria root extract, chamomile extract, carrot extract, artemisia capillaris extract, licorice extract, kiwi extract, cucumber extract, and guava extract. , gardenia extract, kumazasa extract, walnut extract, black rice extract, chlorella extract, mulberry extract, kaempfer extract, alpinia zerumbet extract, gentian extract, rice extract, fermented rice extract, fermented rice bran extract, rice germ oil, salvia extract, soapwort extract, bamboo extract, zanthoxylum extract, Japanese pepper extract, shiitake mushroom extract, rehmannia root extract, lithospermum root extract, perilla extract, linden extract, meadowsweet extract, ginger extract, calamus root extract, horsetail extract, stevia extract, fermented stevia, yarrow Watermelon extract, peppermint extract, sage extract, mallow extract, cnidium extract, Swertia bristle extract, Morus alba extract, rhubarb extract, soybean extract, Chinese laurel 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, burdock extract, lotus extract, parsley extract, birch extract, witch hazel extract, holly extract, cypress extract Preferred examples of extracts include loquat extract, coltsfoot extract, butterbur extract, poria extract, loofah extract, peppermint extract, linden extract, pine extract, skunk cabbage extract, melissa extract, mozuku extract, peach extract, cornflower 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, and burnet extract. Among these, it is preferable to contain an extract derived from an animal or plant that has the effect of suppressing the production of AGEs, and it is preferable to contain, for example, Artemisia capillaris extract, Artemisia princeps extract, or Houttuynia cordata extract.

[0039] The content (dry mass) of the optional animal and plant-derived extract in the cosmetic is usually 0.01 to 30 mass %, preferably 0.1 to 10 mass %, more preferably 0.3 to 3 mass %.

[0040] In addition to active ingredients, 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 triethanolamine alkyl sulfate. anionic surfactants such as esters, cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide, imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based 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 (monostearate, Glycerin phosphate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitan fatty acid esters (POE-sorbitan 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 nonylphenyl ether, etc.), Pluronic (registered trademark) types, POE·POP alkyl ethers (POE·POP 2-decyltetradecyl 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, sodium lactate, which may be surface-treated,Powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, and barium sulfate; inorganic pigments such as cobalt oxide, ultramarine, Prussian blue, and zinc oxide, which may be surface-treated; composite pigments such as sintered iron oxide and titanium dioxide, which may be surface-treated; pearling agents such as titanium dioxide, fish phosphate foil, and bismuth oxychloride, which may be surface-treated; and laked pigments such as 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, and Purple 20. Examples of suitable organic pigments include organic dyes such as Red No. 1 and Red No. 204; organic powders such as polyethylene powder, polymethyl methacrylate, nylon powder, and organopolysiloxane elastomer; lower alcohols such as ethanol and isopropanol; vitamin A or a derivative thereof; vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or a derivative thereof, vitamin B12, and vitamin B15 or a derivative thereof; vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate; vitamin D, vitamin H, pantothenic acid, pantethine, and pyrroloquinoline quinone.

[0041] In a preferred embodiment of the present invention, the cell death inhibitor for inhibiting cell death caused by AGEs further comprises an AGE production inhibitor. A commonly available composition having an AGE production inhibitory effect can be used as the AGE production inhibitor. For example, it may be a commercially available composition known to have an AGE production inhibitory effect, or a plant extract. Examples of plant extracts that can be used include peony extract, chamomile extract, witch hazel leaf extract, Bergenia ligulata root extract, and silk tree bark extract. Commercially available extracts sold by Ichimaru Pharcos Co., Ltd., for example, can be used. In a preferred embodiment, the AGE production inhibitor includes mugwort extract and / or Houttuynia cordata extract, and in a more preferred embodiment, the AGE production inhibitor includes mugwort extract and Houttuynia cordata extract. According to the present invention, the cell death-inhibiting effect can be enhanced by suppressing cell death induction by AGEs and by suppressing the production of AGEs.

[0042] The above-mentioned methods can be applied to the extraction of mugwort extract and Houttuynia cordata extract. Furthermore, when the cell death inhibitor of the present invention contains an AGE production inhibitor, the content thereof is generally 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). Furthermore, the ratio of the content of mugwort extract to the content of Houttuynia cordata extract in the AGE production inhibitor can be 2:3 to 3:2, and is preferably 1:1.

[0043] <2> Cell death suppression method The method for inhibiting cell death of the present invention comprises the steps of: <1> This is a method of applying the cell death inhibitor described in the above to skin tissue. According to the present invention, cell death in particular of skin tissue, particularly of vascular endothelial cells, can be suppressed. The method for inhibiting cell death of the present invention is a non-therapeutic method, preferably a cosmetic method.

[0044] In the present invention, "applying a cell death inhibitor to skin tissue" means applying a cell death inhibitor to skin tissue at any part of the body, such as the face or hands, and the above-mentioned <1> This refers to contacting the cell death inhibitor described in the above with the subject, and can be in the form of applying it to the skin, for example. Furthermore, when the above-mentioned cell death inhibitor is applied to skin tissue, the cell death inhibitor applied to the skin tissue may not be removed, or the above-mentioned cell death inhibitor may be applied to the skin and then, after a certain period of time (e.g., 1 to 10 minutes), removed by wiping or rinsing with water.

[0045] In a preferred embodiment of the present invention, the cell death inhibitor is applied to skin tissue, and then the skin tissue is massaged. Here, in the present invention, "massaging skin tissue" refers to applying pressure to the skin tissue to which the cell death inhibitor has been applied using hands or a tool, regardless of the strength of the pressure. For example, a cream may be used as the dosage form of the cell death inhibitor, and after applying it to the face, the person who applied the cell death inhibitor or a third party may massage the face with the hands or a tool. Also, since it is sufficient to apply pressure to the skin tissue, for example, a facial cleanser may be used as the dosage form of the cell death inhibitor, allowing for face washing and massage at the same time. According to the present invention, the stimulation provided by massage allows the cell death inhibitor to penetrate further into skin tissue, thereby enhancing the cell death inhibitory effect.

[0046] <3> GATA6-AS1 expression promoter The GATA6-AS1 expression promoter according to the present invention contains one or more extracts selected from the group consisting of peony extract, eucalyptus extract, and Hypericum extract. In a preferred embodiment, it contains at least peony extract. The present inventors have found that the above-mentioned peony extract, eucalyptus extract, and St. John's wort extract have the effect of promoting the expression of GATA6-AS1. That is, according to the present invention, the expression of GATA6-AS1 can be promoted.

[0047] In a preferred embodiment of the present invention, the GATA6-AS1 expression promoter is used to promote GATA6-AS1 expression in skin tissues, and more preferably, in vascular endothelial cells.

[0048] The same points as those stated above regarding the cell death inhibitor can be applied to the extraction method of the peony extract, eucalyptus extract, and St. John's wort extract used in the GATA6-AS1 expression promoter, the dosage form of the GATA6-AS1 expression promoter, the amount of peony extract, eucalyptus extract, and St. John's wort extract in the GATA6-AS1 expression promoter, and the optional ingredients that may be contained in the GATA6-AS1 expression promoter and their amounts.

[0049] <4> Method for promoting GATA6-AS1 expression The method for promoting GATA6-AS1 expression of the present invention comprises the steps of: <3> This is a method of applying the cell death inhibitor described in the above to skin tissue. According to the present invention, the expression of GATA6-AS1 can be promoted particularly in skin tissue, and in particular in vascular endothelial cells. The method for promoting GATA6-AS1 expression of the present invention is a non-therapeutic method, preferably a cosmetic method.

[0050] For the method of promoting GATA6-AS1 expression, see above. <2> The matters mentioned in the previous paragraph can be applied. In a preferred embodiment of the present invention, the GATA6-AS1 expression promoter is applied to skin tissue, and then the skin tissue is massaged. According to the present invention, the stimulation provided by massage allows the GATA6-AS1 expression promoter to penetrate more deeply into skin tissue, thereby enhancing the GATA6-AS1 expression promoting effect.

[0051] <5> Screening Methods The screening method according to the present invention is a screening method for screening cell death-suppressing components using the expression level of GATA6-AS1 and / or the expression level of an AGEs receptor as an index. Here, screening includes searching for cell death-suppressing components or candidates thereof. The present inventors discovered a correlation between the expression levels of GATA6-AS1 and AGE receptors. As mentioned above, AGEs are known to induce cell death. According to the present invention, cell death-suppressing components can be screened using the expression level of GATA6-AS1 and / or the expression level of an AGEs receptor as an index.

[0052] In a preferred embodiment of the present invention, a component that increases the expression level of GATA6-AS1 is selected as the cell death-suppressing component. In another preferred embodiment of the present invention, a component that reduces the expression level of an AGE receptor is selected as the cell death-inhibiting component. The present inventors discovered that when the expression level of GATA6-AS1 decreases, the expression level of the AGEs receptor increases. Therefore, a component that increases the expression level of GATA6-AS1 and / or a component that decreases the expression level of an AGE receptor can be selected as a cell death-suppressing component.

[0053] In a preferred embodiment of the present invention, the skin tissue is a vascular endothelial cell. According to the present invention, it is possible to select a component capable of suppressing cell death in skin tissue.

[0054] In a preferred embodiment of the present invention, the cell death-inhibiting component is a component that inhibits cell death induced by advanced glycation endoproducts in skin tissue. According to the present invention, a component that inhibits cell death caused by AGEs can be selected as the cell death-inhibiting component.

[0055] The substance to be tested in the screening method of the present invention may be any of a pure substance, an extract derived from a living organism, or a mixture thereof. The term "biological extract" refers to not only the extract itself derived from animals or plants, but also a collective term for fractions of extracts, purified fractions, and solvent-removed extracts, fractions, and purified products. Examples of plant extracts include extracts made from wild or cultivated plants, extracts sold as herbal medicine ingredients, and commercially available extracts. For the extraction procedure, the whole plant may be used, or parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, inflorescences, and flower buds may be used. However, it is preferable to crush or shred these in advance to improve the extraction efficiency. Suitable examples of extraction solvents include one or more selected from polar solvents such as 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 solvent to 1 mass of the part of the plant body or its dried material used for extraction, immersing for several days at room temperature or for 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 by column chromatography or the like.

[0056] The screening method of the present invention comprises adding a test substance to a cell culture system and measuring the expression levels of GATA6-AS1 and / or AGEs receptor in the cells. The cells that can be used here include normal human keratinocytes, normal human fibroblasts, normal human vascular endothelial cells, etc.

[0057] Specifically, if the expression level of GATA6-AS1 in cells cultured with the addition of a test substance is statistically significantly higher than the expression level of GATA6-AS1 in cells cultured without the addition of the test substance, the test substance can be determined to be a candidate for a cell death-inhibiting component. More specifically, if the expression level of GATA6-AS1 in cells cultured with the addition of a test substance is more than one-fold higher than the expression level of GATA6-AS1 in cells cultured without the addition of the test substance, the substance can be selected as a candidate anti-aging ingredient.

[0058] Furthermore, if the expression level of AGE receptors in cells cultured with the addition of a test substance is statistically significantly lower than the expression level of AGE receptors in cells cultured without the addition of the test substance, the test substance can be determined to be a candidate for a cell death-inhibiting component. More specifically, when the expression level of the AGE receptor in cells cultured with the addition of the test substance is less than 1-fold the expression level of the AGE receptor in cells cultured without the addition of the test substance, the substance can be selected as a candidate for cell death-inhibiting components.

[0059] The expression levels of GATA6-AS1 and AGEs receptor can be measured by standard methods such as mRNA measurement and immunohistochemical analysis. For example, the expression levels of GATA6-AS1 and the gene encoding an AGEs receptor are quantitatively detected by PCR using, as primers, DNA fragments having sequences that specifically bind to the sequences of the genes. The GATA6-AS1 sequence and the gene sequence encoding the AGEs receptor have been made public, so those skilled in the art can design appropriate primers. Furthermore, since antibodies are also commercially available, they can be used to measure the expression level in cells.

[0060] <6> AGE production inhibitor The present invention also relates to an AGE production inhibitor. The AGE production inhibitor of the present invention contains mugwort extract and Houttuynia cordata extract. According to the present invention, the production of AGEs in the body is inhibited, thereby making it possible to suppress the effects of AGEs on the body.

[0061] In a preferred embodiment of the present invention, the AGE production inhibitor is combined with the above-mentioned cell death inhibitor and / or GATA6-AS1 expression promoter. The cell death-inhibiting effect and / or GATA6-AS1 expression-promoting effect can be enhanced by the synergistic effect with the AGE production-inhibiting effect.

[0062] The extraction methods for mugwort extract and Houttuynia cordata extract, the dosage form of the AGE production inhibitor, the content of mugwort extract and Houttuynia cordata extract, the extraction methods for each extract, the dosage form of the AGE production inhibitor, and optional ingredients that may be contained in the AGE production inhibitor and their content are described above. <1> The points mentioned above regarding AGE production inhibitors can be applied. [Example]

[0063] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0064] <Test Example 1> Relationship between the amount of GATA6-AS1 expression in cells and cell death induction by AGEs In this test example, the relationship between the expression level of GATA6-AS1 in cells and the induction of cell death by AGEs was examined.

[0065] [1] Preparation of collagen AGEs (1) A collagen solution and a D-(-)-ribose solution were mixed according to the composition shown in Table 1 below and incubated at 37°C for one week. (2) The mixture was filtered to remove gel-like lumps. (3) OxiSelect TM The AGE concentration was measured using an AGE Competitive ELISA Kit (Cell Bio Labs) according to the product protocol, and it was confirmed that AGEs were not produced in the collagen solution, but were produced in the collagen-AGE solution.

[0066] [Table 1]

[0067] [2] Effect of GATA6-AS1 siRNA transfection on AGE-induced cell death (1) Normal human umbilical vein endothelial cells (HUVEC) were cultured in a 24-well plate at a density of 2.5 × 10 4 The cells were seeded at 100 cells / well. A low-serum liquid medium for growing normal human vascular endothelial cells (hereinafter referred to as HuMedia-EG2, manufactured by Kurabo Industries, Ltd.) was used as the medium. (2) After overnight culture at 37°C in a 5% CO2 environment, siRNA transfection was carried out to a final concentration of 10 nM. (3) After further culturing for 24 hours in a 37°C, 5% CO2 environment, the collagen solution or collagen AGEs solution prepared in [1] above was added to the cells to a concentration of 10%. (4) After further culturing for 24 hours at 37°C in a 5% CO2 environment, WST-8 assay was performed using Cell Counting Kit-8 (Dojindo Laboratories, Inc.) to confirm cytotoxicity. Live and dead cells were stained using Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque, Inc.), and the number of each was counted. Furthermore, mRNA was extracted using standard methods, and the mRNA expression of GATA6-AS1 and AGE receptor (AGER) was analyzed by qRT-PCR.

[0068] The siRNA introduced into the cells was All star negative control siRNA (Qiagen) as the control siRNA and FlexiTube GeneSolution GS100128893 for GATA6-AS1 (Qiagen) as the GATA6-AS1 siRNA. For analysis of mRNA expression, we used the GATA6-AS1 primer (Hs_GATA6-AS1_1_SG QuantiTect Primer, Qiagen, QT01864940), AGER primer (Hs_AGER_1_SG QuantiTect Primer, Qiagen, QT02448138), and ACTB primer (Hs_ACTB_2_SG QuantiTect Primer, Qiagen, QT01680476).

[0069] (siRNA transfection method) (1) Lipofectamine 3000 (Thermo Fisher Scientific) was diluted with Opti-MEM (Thermo Fisher Scientific), vortexed for 2 to 3 seconds, and then gently centrifuged. (2) The siRNA was diluted with Opti-MEM and then mixed well by pipetting. (3) Equal amounts of each solution were mixed and incubated at room temperature for 5 minutes. (4) 50 μL of the solution prepared in (3) was added dropwise to 450 μL of HuMedia-EG2 medium. Although the data is not shown, there is no cytotoxicity due to siRNA introduction using this method.

[0070] (Method for observing live and dead cells) (1) Floating cells in the culture medium were collected. (2) The remaining adherent cells were recovered by trypsinization and mixed with the floating cells recovered in (1). (3) The collected cells were washed twice with PBS and then diluted to 1 × 10 6 The cells were suspended in Annexin V binding solution (Nacalai Tesque) at a concentration of 100 cells / mL. (4) To 100 μL of the solution prepared in (3), 5 μL of propidium iodide solution (hereinafter referred to as PI solution) and 0.5 μL of Hoechst 33342 solution (manufactured by Dojindo Laboratories, Inc.) were added, and the mixture was incubated for 15 minutes at room temperature in the dark. (5) The solution prepared in (4) was dropped onto a glass slide and observed under a fluorescence microscope as soon as possible.

[0071] The expression level of GATA6-AS1 in the GATA6-AS1 siRNA-introduced cells is shown in FIG. 1, with the expression level of GATA6-AS1 in the control siRNA-introduced cells set at 1. The expression level of AGE receptor (AGER) mRNA in GATA6-AS1 siRNA-introduced cells is shown in FIG. 2, with the expression level of AGE receptor (AGER) mRNA in control siRNA-introduced cells set at 1. Figure 3(A) shows the cell viability of control sRNA-transfected cells cultured in the presence of collagen-AGEs and the cell viability of GATA6-AS1 siRNA-transfected cells cultured in the presence of collagen-AGEs, with the cell viability of control siRNA-transfected cells cultured in the presence of collagen-AGEs set to 1. The staining results for control siRNA-transfected cells cultured in the presence of collagen, control siRNA-transfected cells cultured in the presence of collagen-AGEs, and GATA6-AS1 siRNA-transfected cells cultured in the presence of collagen-AGEs are shown in Figure 3(B).

[0072] As shown in Figure 1, the expression level of GATA6-AS1 was reduced in the GATA6-AS1 siRNA-transfected cells prepared by the above method, compared to the control siRNA-transfected cells. As shown in Figure 2, the expression level of AGE receptor (AGER) mRNA in GATA6-AS1 siRNA-introduced cells was reduced compared to the expression level of AGE receptor (AGER) mRNA in control siRNA-introduced cells. These results demonstrate that there is a correlation between the expression levels of GATA6-AS1 and AGE receptors (AGER) in cells. Specifically, it was found that a decrease in GATA6-AS1 expression in cells leads to an increase in the expression level of AGE receptors (AGER).

[0073] As shown in Figure 3(A), when the cell viability of control siRNA-introduced cells cultured in the presence of collagen (left center of Figure 3(A)) was set to 1, the cell viability of control siRNA-introduced cells cultured in the presence of collagen AGEs (center center of Figure 3(A)) was reduced. Furthermore, as shown in Figure 3(A), when control siRNA-introduced cells cultured in the presence of collagen and control siRNA-introduced cells cultured in the presence of collagen AGEs were stained with Hoechst 33342 for live cells and with PI solution for dead cells, the control siRNA-introduced cells cultured in the presence of collagen AGEs (center of Figure 3(B)) showed a decreased number of live cells and an increased number of dead cells (white arrows in Figure 3(B)) compared to the control siRNA-introduced cells cultured in the presence of collagen (left of Figure 3(B)). From the above, it was confirmed that AGEs induce cell death, as described in Non-Patent Document 1 above.

[0074] As shown in Figure 3(A), when the cell viability of control siRNA-introduced cells cultured in the presence of collagen (left center of Figure 3(A)) was set to 1, the cell viability of GATA6-AS1 siRNA-introduced cells cultured in the presence of collagen AGEs (right center of Figure 3(A)) was even lower than the cell viability of control siRNA-introduced cells cultured in the presence of collagen AGEs (center center of Figure 3(A)). Furthermore, as shown in Figure 3(B), the number of live cells further decreased and the number of dead cells further increased in GATA6-AS1 siRNA-transfected cells cultured in the presence of collagen AGEs (Figure 3(B) center right, open arrow). These findings suggest that a decrease in the expression level of GATA6-AS1 promotes cell death induced by AGEs. Furthermore, Figures 1 to 3 suggest that when the expression level of GATA6-AS1 decreases, the expression level of AGE receptors increases, promoting the induction of cell death by AGEs.

[0075] <Test Example 2> Factors that decrease the expression level of GATA6-AS1 In this study, we examined the effect of the presence of AGEs on GATA6-AS1 expression in cells.

[0076] Test Example 2 was carried out as follows. (1) Place 2.5 × 10 HUVECs in a 24-well plate. 4 Cells were seeded at 1000 cells / well. HuMedia-EG2 medium was used. (2) After culturing for 24 hours in a 37°C, 5% CO2 environment, a collagen solution or a collagen-AGEs solution prepared in the same manner as in Test Example 1 was added at a concentration of 10%. (3) The cells were further cultured at 37°C in a 5% CO2 environment for 48 hours. (4) mRNA was extracted by a conventional method, and GATA6-AS1 expression was analyzed by qRT-PCR in the same manner as in Test Example 1. The same primers as in Test Example 1 were used for qRT-PCR. The results are shown in Figure 4.

[0077] As shown in Figure 4, when the expression level of GATA6-AS1 in cells cultured in the presence of collagen ("Collagen" in Figure 4) was set to 1, the expression level of GATA6-AS1 in cells cultured in the presence of collagen AGEs ("Collagen AGEs" in Figure 4) was significantly lower. These findings suggest that the presence of AGEs reduces the expression level of GATA6-AS1 in cells.

[0078] <Test Example 3> Selection of an extract that promotes GATA6-AS1 expression in cells In this test example, an extract that promotes the expression of GATA6-AS1 in cells was selected.

[0079] Test Example 3 was carried out as follows. (1) Place 2.5 × 10 HUVECs in a 24-well plate. 4 Cells were seeded at 1000 cells / well. HuMedia-EG2 medium was used. (2) After culturing for 24 hours in a 37°C, 5% CO2 environment, the extracts listed in Table 2 below were added to a final concentration of 0.1% or 0.2%. (3) The cells were further cultured at 37°C in a 5% CO2 environment for 48 hours. (4) Cytotoxicity was confirmed by WST-8 measurement in the same manner as in Test Example 1, and then mRNA was extracted by a standard method, and GATA6-AS1 expression was analyzed by qRT-PCR in the same manner as in Test Example 1. The same primers as in Test Example 1 were used in qRT-PCR. As a result of the analysis, the extracts that showed increased expression levels of GATA6-AS1 are shown in Figure 5.

[0080] [Table 2]

[0081] As shown in Figure 5, the expression level of GATA6-AS1 in cells cultured in the presence of Hypericum perforatum extract, Paeonia suffruticosa extract, and Eucalyptus extract (final concentration 0.1% by mass) among the extracts listed in Table 2 above was increased compared to the expression level of GATA6-AS1 in cells cultured in the absence of the above extracts. From the above, it was revealed that St. John's wort extract, peony extract, and eucalyptus extract have the effect of promoting GATA6-AS1 expression.

[0082] Furthermore, the results of Test Example 1 above revealed that when the expression level of GATA6-AS1 in cells decreases, the expression level of AGE receptor (AGER) increases. In other words, increasing the expression level of GATA6-AS1 can decrease the expression level of AGE receptors (AGER). Furthermore, the results of Test Example 1 above revealed that a decrease in the expression level of GATA6-AS1 in cells promotes cell death induction by AGEs. In other words, it can be said that increasing the expression level of GATA6-AS1 can suppress cell death caused by AGEs. Combining the results of Test Example 1 and this Test Example, it was suggested that St. John's wort extract, peony extract, and eucalyptus extract have the effect of promoting GATA6-AS1 expression, and therefore also have the effect of increasing the expression level of AGE receptors (AGER) and suppressing cell death (especially cell death caused by AGEs).

[0083] Furthermore, combining the results of this test with those of Test Example 1, it is possible to screen for cell death-inhibiting components using the expression levels of GATA6-AS1 and AGE receptor as indicators. Specifically, components that increase the expression level of GATA6-AS1 and components that suppress cell death can be screened. Furthermore, because a decrease in the expression level of GATA6-AS1 increases the expression level of AGE receptors, it is predicted that increasing the expression level of GATA6-AS1 will decrease the expression level of AGE receptors. Therefore, components that decrease the expression level of AGE receptors can be selected as cell death-suppressing components.

[0084] <Test Example 4> Verification of the AGE production inhibitory effect of mugwort extract and Houttuynia cordata extract In this test example, the AGE production inhibitory effects of mugwort extract and Houttuynia cordata extract were tested.

[0085] This test example was carried out as follows. (1) A collagen solution and a collagen-AGEs solution were prepared with the compositions shown in Table 1 of Test Example 1. (2) Four types of test systems were prepared: (a) 100 μL of milliQ water was added to 500 μL of the collagen AGEs solution prepared in (1) above; (b) 50 μL of mugwort extract (hereinafter also referred to as YAC extract) and 50 μL of milliQ water were added; (c) 50 μL of Houttuynia cordata extract and 50 μL of milliQ water were added; and (d) 50 μL of YAC extract and 50 μL of Houttuynia cordata extract were added. (3) The collagen solution prepared in (1) above and each collagen AGE solution prepared in (2) above were incubated at 37°C for one week. (4) OxiSelect TM The AGE concentrations in (a) to (d) were compared using an AGE Competitive ELISA Kit (manufactured by Cell Biolabs) according to the product protocol. The results are shown in Figure 6.

[0086] As shown in Figure 6, when the concentration of AGEs produced in the collagen solution was set to 1, the AGE concentration was high even when 100 μL of milliQ water was added (A). In other words, a large amount of AGEs was produced in (A). In contrast, the AGE concentrations in (B) the mixture containing 50 μL of YAC extract and 50 μL of milliQ water, and (C) the mixture containing 50 μL of Houttuynia cordata extract and 50 μL of milliQ water, were significantly lower than those in (A) above. This indicates that YAC extract and Houttuynia cordata extract each have the effect of inhibiting AGE production on their own. Furthermore, the AGE concentration in (d) the mixture containing 50 μL of YAC extract and 50 μL of Houttuynia cordata extract was even lower than in (b) and (c) above. In other words, it was revealed that the combination of YAC extract and Houttuynia cordata extract has an even greater effect in inhibiting the production of AGEs.

[0087] Furthermore, as mentioned above, AGEs promote cell death. It was suggested that cell death caused by AGEs could be suppressed by combining a cell death inhibitor containing the cell death-inhibiting components screened using the above method with an AGE production inhibitor containing YAC extract and / or Houttuynia cordata extract.

[0088] Examples of formulations for the cell death inhibitor and / or GATA6-AS1 expression promoter according to the present invention are shown below.

[0089] [Table 3] [Industrial Applicability]

[0090] According to the present invention, it is possible to provide a cell death inhibitor, a cell death inhibition method, and a method for screening a cell death inhibition component. Furthermore, the present invention can provide a GATA6-AS1 expression promoter, a method for promoting GATA6-AS1 expression, and a method for screening a component that promotes GATA6-AS1 expression. Furthermore, the present invention can provide an AGE production inhibitor.

Claims

1. A cell death inhibitor containing one or more extracts selected from the group consisting of peony extract, eucalyptus extract, and St. John's wort extract, and intended to inhibit cell death of vascular endothelial cells.

2. A cell death inhibitor containing one or more extracts selected from peony extract, eucalyptus extract, and St. John's wort extract, for inhibiting cell death caused by AGEs resulting from a decrease in the expression level of GATA6-AS1.

3. The cell death inhibitor according to claim 1 or 2, comprising at least a Paeonia suffruticosa extract.

4. The cell death inhibitor according to any one of claims 1 to 3, for inhibiting cell death caused by advanced glycation end-products.

5. The cell death inhibitor according to any one of claims 1 to 4, further comprising an AGEs production inhibitor.

6. The cell death inhibitor according to claim 5, wherein the AGEs production inhibitor comprises mugwort extract and / or Houttuynia cordata extract.

7. A GATA6-AS1 expression promoter comprising one or more extracts selected from the group consisting of peony extract, eucalyptus extract and St. John's wort extract.

8. The GATA6-AS1 expression promoter according to claim 7, comprising at least a peony extract.

9. The GATA6-AS1 expression promoter according to claim 7 or 8, for promoting the expression of GATA6-AS1 in skin tissue.

10. The GATA6-AS1 expression promoter according to claim 9, wherein the skin tissue is a vascular endothelial cell.

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