Sirtuin-3 gene expression activator

Abstract

This provides a novel material for activating the Sirtuin-3 gene. [Solution] A Sirtuin-3 gene expression activating material according to one aspect of the present disclosure contains peony root or an ethanol-containing solvent extract thereof.

Description

Technical Field

[0001] The present invention relates to a material for activating Sirtuin-3 gene expression.

Background Art

[0002] The Sirtuin gene, known as a longevity gene, encodes a Sirtuin protein that is a NAD + -dependent deacetylase, and the Sirtuin protein is known to be a protein having an anti-aging effect. It is known that when the Sirtuin gene is activated, various anti-aging effects occur, including activation of cells and enhancement of metabolic functions.

[0003] So far, as a method for activating the Sirtuin gene in mammals, a method of making the body in a starvation state by calorie restriction through diet therapy has been proposed (Non-Patent Document 1).

[0004] However, in recent years, physiologically active components capable of activating the Sirtuin gene by being formulated in food and drink products or cosmetics have been reported (Patent Documents 1 to 5).

[0005] In mammals, there are a total of seven types of Sirtuin proteins from Sirtuin-1 to Sirtuin-7, which are classified according to their localization in cells and their functionality. Among these, the Sirtuin-3 protein is localized in the mitochondria in cells and is involved in the synthesis and metabolic control of reactive oxygen species (ROS). Gene deletion of Sirtuin-3 has been reported to cause an increase in ROS in mitochondria (Non-Patent Document 2).

[0006] So far, as a Sirtuin gene activation material, materials containing polyphenol compounds such as resveratrol contained in grapes or fisetin contained in strawberries have been known (Patent Documents 1, 4).

[0007] Furthermore, Patent Document 1 discloses a Sirtuin-1 activator characterized by comprising the plant body or solvent extract of Peucedanum japonicum Thunb.

[0008] Patent Document 2 discloses a topical skin preparation composition for preventing skin aging, which contains an ethanol extract of Japanese red pine root as an active ingredient. It also discloses that this composition is for inducing the expression of the SIRT1 gene, which repairs damaged DNA, and the LMNA1 gene, which maintains the structure of the nuclear membrane.

[0009] Patent Document 3 discloses a Sirt1 activator containing a specific linoleic acid derivative as an active ingredient.

[0010] Patent Document 4 discloses a Sirtuin-1 activator characterized by comprising the plant body of Isodon japonicus Hara or a solvent extract thereof.

[0011] Patent Document 5 discloses a composition for promoting SIRT production containing theaflavin and / or theaflavin gallate. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] Patent No. 5666053 [Patent Document 2] Special Publication No. 2012-519164 [Patent Document 3] Japanese Patent Publication No. 2015-189693 [Patent Document 4] International Publication No. 2018 / 003035 [Patent Document 5] Japanese Patent Publication No. 2024-3280 [Non-patent literature]

[0013]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0014] One aspect of the present invention aims to provide a novel Sirtuin-3 gene activation material.

Means for Solving the Problems

[0015] The inventors of the present invention have conducted intensive research to achieve the above object. As a result, it has been found that an ethanol-containing solvent extract of Paeonia lactiflora root can activate Sirtuin-3 gene expression, leading to the completion of the present invention.

[0016] That is, the Sirtuin-3 gene expression activation material according to one aspect of the present invention is characterized by containing Paeonia lactiflora root or its ethanol-containing solvent extract.

Effects of the Invention

[0017] According to one aspect of the present invention, a novel Sirtuin-3 gene activation material can be provided.

Modes for Carrying Out the Invention

[0018] The present invention will be described in detail below. Unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more and B or less".

[0019] [1. Sirtuin-3 Gene Expression Activation Material] A Sirtuin-3 gene expression activating material according to one aspect of the present invention is characterized by containing peony root or an ethanol-containing solvent extract thereof. In this disclosure, the "Sirtuin-3 gene expression activating material" may be referred to as the "Sirtuin-3 gene activating material" for convenience.

[0020] Here, "Sirtuin-3 gene expression activating material" in this disclosure means a substance containing a component capable of activating Sirtuin-3 gene expression (hereinafter referred to as "Sirtuin-3 gene activating component"). In this disclosure, "Sirtuin-3 gene expression activating material" may itself possess a Sirtuin-3 gene expression activating function (hereinafter referred to as "Sirtuin-3 gene activation function") as long as it contains a Sirtuin-3 gene activating component, or it may express the Sirtuin-3 gene activation function after a treatment that expresses the Sirtuin-3 gene activation function. In other words, a substance before undergoing a treatment that expresses the Sirtuin-3 gene activation function is also included in the category of "Sirtuin-3 gene expression activating material" in this disclosure. Examples of treatments that express such a Sirtuin-3 gene activation function include solvent extraction using an ethanol-containing solvent and concentration.

[0021] A Sirtuin-3 gene activating material according to one aspect of the present invention may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). "Mixture" is also referred to as "composition". If the Sirtuin-3 gene activating material according to one aspect of the present invention is a mixture, each component constituting the mixture may individually have a Sirtuin-3 gene activating function, or the mixture may contain components that do not have such a function.

[0022] The expression of the Sirtuin-3 gene can be activated by applying a Sirtuin-3 gene activating material according to one aspect of the present invention to cells. In this disclosure, Sirtuin-3 gene activation means that when the Sirtuin-3 gene activating material according to one aspect of the present invention is applied to cells, the expression of the Sirtuin-3 gene is promoted, and the amount of Sirtuin-3 gene expression is increased compared to a state in which the Sirtuin-3 gene activating material according to one aspect of the present invention is not applied (steady state). In this case, the degree of increase in the amount of Sirtuin-3 gene expression is not particularly limited.

[0023] The method for evaluating Sirtuin-3 gene activation is not particularly limited. For example, in this specification, Sirtuin-3 gene activation can be evaluated by statistically analyzing the expression level of the Sirtuin-3 gene measured by real-time PCR using Dunnett's test.

[0024] Activating Sirtuin-3 gene expression is thought to be useful in suppressing aging phenomena caused by increased reactive oxygen species (ROS) in mitochondria. Skin aging phenomena caused by oxidative stress due to ROS include wrinkle formation due to accelerated collagen breakdown and decreased skin moisture content, and the formation of age spots due to abnormal melanin synthesis. Abnormal melanin synthesis and decreased collagen production are triggered by internal stimuli such as aging and external stimuli such as ultraviolet rays. As we age, intracellular metabolic function and energy production decrease, and the skin's natural metabolic mechanisms do not function properly, thereby accelerating these skin aging processes. Therefore, in order to maintain healthy skin that is less prone to skin problems, it is important to improve mitochondrial function decline due to aging and enhance intracellular metabolic function. By activating Sirtuin-3 gene expression, it is expected that the increase in ROS in the body will be suppressed, and skin aging due to oxidative stress will be suppressed.

[0025] Based on the effects described above, Sirtuin-3 gene activating materials are useful as active ingredients in topical skin preparations. For example, the skincare effects of cosmetics containing Sirtuin-3 gene activating materials are expected to include improving aging signs such as wrinkle formation due to collagen breakdown caused by oxidative stress on cells, and the formation of age spots due to abnormal melanin synthesis.

[0026] (Peony root and ethanol-containing solvent extract of peony root) The following describes peony root and ethanol-containing solvent extracts of peony root.

[0027] The peony (Paeonia lactiflora) used in this invention is a plant belonging to the genus Paeonia, family Paeoniaceae, order Saxifragales.

[0028] The peony root contained in the Sirtuin-3 gene activation material according to one aspect of the present invention may be a fresh root containing moisture, or a dried root. Dried peony root is preferably used from the viewpoint of quality stability, supply, and distribution. The peony root may be in the form of lumps, granules, or powder. Peony root powder can be produced by grinding methods commonly used in the art. For example, methods include drying the peony root and then shredding or grinding it, or shredding the peony root, drying it, and then grinding it. Commercially available peony root powder may also be used.

[0029] An ethanol-containing solvent extract of peony root according to one aspect of the present invention can be prepared using the procedures of extraction methods commonly used in the art. For example, it can be prepared by immersing peony root in an ethanol-containing solvent and allowing it to stand at a predetermined temperature for a predetermined time.

[0030] There are no particular restrictions on the form of the peony root used in the preparation of the extract; it may be a fresh root containing moisture, or a dried root. It is preferable to use dried peony root so that the solvent can easily penetrate it. The peony root may also be used in the form of lumps, granules, or powder, but it is preferable to use it in powder form from the viewpoint of increasing the specific surface area and thereby improving the extraction efficiency.

[0031] As shown in the examples described later, the components of peony root extracted in ethanol include a Sirtuin-3 gene activating component. Therefore, any ethanol-containing solvent can be used to extract the Sirtuin-3 gene activating component from peony root, and the concentration of ethanol can be appropriately selected by those skilled in the art. For example, the ethanol concentration in the ethanol-containing solvent should be 1% by volume or more per 100% by volume of the ethanol-containing solvent, and can be, for example, 5% by volume or more, 10% by volume or more, 20% by volume or more, 30% by volume or more, 40% by volume or more, 50% by volume or more, 60% by volume or more, 70% by volume or more, 80% by volume or more, or 90% by volume or more. The ethanol-containing solvent may also be anhydrous ethanol (99.5% by volume or more of ethanol).

[0032] Theoretically, the higher the ethanol concentration in the ethanol-containing solvent, the greater the total amount of Sirtuin-3 gene-activating components that can be extracted into the ethanol-containing solvent extract. However, from the standpoint of extraction efficiency and cost, the ethanol concentration in the ethanol-containing solvent is preferably 50% by volume or less, and more preferably 10-30% by volume or less.

[0033] Furthermore, the ethanol-containing solvent may also contain at least one of an organic solvent other than ethanol and an aqueous solvent. Examples of such organic solvents include methanol, lower alcohols such as 1,3-butylene glycol, and ethers. Examples of aqueous solvents include water.

[0034] Furthermore, during extraction, a sufficient amount (volume) of ethanol-containing solvent should be added to immerse the extractant (peony root). For example, extraction may be carried out by mixing peony root and ethanol-containing solvent in a mass ratio of 1 to 10:20.

[0035] The extraction temperature can be selected to allow extraction of the Sirtuin-3 gene activating component without damaging it, and the ethanol-containing solvent may be cooled or heated as needed. The lower limit of such an extraction temperature is, for example, 0°C or higher, 10°C or higher, 20°C or higher, or 30°C or higher, and the upper limit of the extraction temperature is preferably 40°C or lower. The extraction time should be selected appropriately, taking into account factors that affect the extraction rate, such as the morphology of the peony root and the temperature of the ethanol-containing solvent.

[0036] The extract may be in the form of the extract itself, or it may be diluted or concentrated as appropriate by commonly used methods. Furthermore, the extract may be in the form of a powder or a solid mass obtained by drying the extract by commonly used methods.

[0037] (Content of peony root or its ethanol-containing solvent extract) A Sirtuin-3 gene activating material according to one aspect of the present invention may contain peony root or an ethanol-containing solvent extract thereof, but the content is not particularly limited. A higher content of peony root or an ethanol-containing solvent extract in a Sirtuin-3 gene activating material according to one aspect of the present invention is preferable because it increases the content of Sirtuin-3 gene activating components contained in the Sirtuin-3 gene activating material. The content of peony root or its ethanol-containing solvent extract in the Sirtuin-3 gene activating material according to one aspect of the present invention can be, for example, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 5.0% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, 99% by mass or more, or 100% by mass, based on 100% by mass of the Sirtuin-3 gene activating material.

[0038] Furthermore, the peony root or its ethanol-containing solvent extract in the Sirtuin-3 gene activating material according to one aspect of the present invention can be confirmed by the confirmation test method specified in the standards for quasi-drug ingredients.

[0039] (Other substances) A Sirtuin-3 gene activating material according to one aspect of the present invention may contain substances other than peony root or its ethanol-containing solvent extract, to the extent that it does not impair the effects of the present invention. Examples of such other substances include various parts of the peony other than the root (e.g., leaves, stems, flowers, etc.) or their ethanol-containing solvent extracts.

[0040] (Form of Sirtuin-3 gene activating material) The form of the Sirtuin-3 gene activating material according to one aspect of the present invention is not particularly limited, and may be a liquid, or a powder or a solid mass.

[0041] [2. Topical skin preparations] As one aspect of the present invention, a topical skin preparation containing the Sirtuin-3 gene activating material according to the above-described aspect of the present invention is provided. The Sirtuin-3 gene activating material according to the above aspect of the present invention, which is included in the topical skin preparation according to the above aspect of the present invention, has already been described, so it will not be described again here.

[0042] A topical skin preparation according to one aspect of the present invention contains a Sirtuin-3 gene activating material according to one aspect of the present invention, and therefore has a Sirtuin-3 gene activating function.

[0043] (Form of topical skin preparation) A topical skin preparation according to one aspect of the present invention can be directly administered transdermally to the whole body or locally. Examples of forms of a topical skin preparation according to one aspect of the present invention include pharmaceuticals; quasi-drugs; topical or systemic topical skin preparations; medicinal and / or cosmetic preparations for application to the scalp and hair; bath additives for use in bathwater; antiperspirants, deodorants, antiperspirants, sanitary products, sanitary cotton, wet wipes, etc.

[0044] Specific examples of topical or systemic skin preparations include, for example, basic cosmetics such as lotions, emulsions, creams, ointments, oils, and packs; facial cleansers and skin cleansers such as bar soaps, liquid soaps, and hand washes; massage agents, cleansing agents, depilatory agents, hair removal agents, shaving agents, aftershave lotions, preshave lotions, shaving creams; makeup cosmetics such as foundations, lipsticks, blushes, eyeshadows, eyeliners, and mascaras; perfumes; nail care products, nail polish, nail polish removers, poultices, plasters, tapes, sheets, patches, and aerosols.

[0045] Specific examples of medicinal and / or cosmetic preparations applied to the scalp and hair include, for example, shampoos, conditioners, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and nourishing products, poultices, plasters, tapes, sheets, aerosols, and the like.

[0046] The form of the topical skin preparation according to one aspect of the present invention is not particularly limited, and suitable forms for use can be selected, such as ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mists, sprays, etc.

[0047] The area to which the topical skin preparation according to one aspect of the present invention is applied is not particularly limited, and may be, for example, the face, arms, elbows, etc.

[0048] (Concentration of Sirtuin-3 gene activating material) The concentration of the Sirtuin-3 gene activating material according to one aspect of the present invention in a topical skin preparation according to one aspect of the present invention can be appropriately adjusted by a person skilled in the art depending on the form of the topical skin preparation, the desired use, etc.

[0049] (Other ingredients) A topical skin preparation according to one aspect of the present invention may further contain, if necessary, the following components and / or additives, to the extent that they do not impair the effects of the present invention.

[0050] (1) Various oils and fats: For example, avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange rafur oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peach oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japanese wax, coconut oil, beef tallow, lard, squalene, squalane, pristane, or hydrogenated versions of these oils and fats (hydrogenated oils, etc.).

[0051] (2) Waxes: For example, beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0052] (3) Mineral oils: For example, liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystalline wax, etc.

[0053] (4) Fatty acids: For example, natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolinic acid, and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

[0054] (5) Alcohols: For example, natural alcohols such as isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, and phenoxyethanol, and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.

[0055] (6) Polyhydric alcohols: For example, ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, treitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[0056] (7) Esters: For example, isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

[0057] (8) Metal soaps: For example, aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0058] (9) Gums, sugars or water-soluble polymer compounds: for example, gum arabic, gum benzoin, gum dammar, guaiac fat, Irish moss, gum karaya, gum tragacanth, gum carob, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide have been added, low molecular weight chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, a Luginic acid or its salts, hyaluronic acid or its salts, chondroitin sulfate or its salts, heparin, ethylcellulose, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, sodium carboxyethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylates, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their crosslinked polymers, carboxyvinyl polymers, polyethyleneimines, etc.

[0059] (10) Surfactants: For example, anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkylamines, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium and silicon, fluorinated carbon surfactants), etc.

[0060] (11) Various vitamins: • Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), etc. • Vitamin B group: Thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin, choline, inositol; • Vitamin C group: Vitamin C acid or its derivatives; • Vitamin D group: Ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachisterol; • Vitamin E group: Vitamin E or its derivatives, ubiquinones; • Vitamin K group: Phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4); Other nutrients include essential fatty acids (vitamin F), carnitine, ferulic acid, gamma-oryzanol, orotic acid, vitamin P compounds (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0061] (12) Various amino acids: for example, valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., and their sulfates, phosphates, nitrates, or citrates, or amino acid derivatives such as pyrrolidone carboxylic acid.

[0062] (13) Additives: For example, UV protection agents, stabilizers, chelating agents, pH adjusters, compressed gases (nitrogen, carbon dioxide), and liquefied gases (LPG, DME), etc.

[0063] These components and / or additives can be conventionally known components and / or additives that have been approved for incorporation into topical skin preparations. These components and / or additives may be arbitrarily selected and added to a topical skin preparation according to one embodiment of the present invention. Furthermore, these components and / or additives may be used individually or in combination of two or more.

[0064] When additives derived from plant or animal raw materials are used in topical preparations or cosmetics for the whole body or topically according to one aspect of the present invention, in addition to cosmetic effects such as protection of the skin and hair, moisturizing, improvement of feel and texture, imparting flexibility, alleviation of irritation, stress relief through fragrance, cell activation (prevention of cell aging), suppression of inflammation, improvement of skin and hair quality, prevention and improvement of rough skin, hair growth, hair nourishment, prevention of hair loss, imparting of shine, cleansing effect, fatigue relief, promotion of blood flow, and warming effect, effects such as fragrance, deodorization, thickening, preservative, and buffering can also be expected.

[0065] Furthermore, by combining the various cosmetic and pharmaceutical effects of each known raw material with a topical skin preparation according to one aspect of the present invention, it is possible to enhance the effects targeted by the present invention and create a product with multifunctional effects.

[0066] For such purposes, for example, a topical skin preparation according to one aspect of the present invention may further contain niacinamide.

[0067] Besides niacinamide, other such raw materials are known, for example, heparinoids, tranexamic acid, and dipotassium glycyrrhizinate, which are active ingredients in quasi-drugs.

[0068] (Method of manufacturing a topical skin preparation) A topical skin preparation according to one aspect of the present invention can be formulated by known methods using a Sirtuin-3 gene activating material according to one aspect of the present invention, which is an active ingredient, and other components as raw materials.

[0069] A topical skin preparation according to one aspect of the present invention may be subjected to conventional processing (for example, any selection and combination of processes such as crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.) depending on the type or form of the product. Such processing may be aimed at expressing the Sirtuin-3 gene activation function of the Sirtuin-3 gene activation material according to one aspect of the present invention.

[0070] [3. Methods for activating Sirtuin-3 gene expression] One aspect of the present invention provides a method for activating Sirtuin-3 gene expression, comprising the following steps: A step of administering a topical skin preparation according to one aspect of the present invention transdermally to a subject.

[0071] As has already been described, a topical skin preparation according to one aspect of the present invention will not be repeated here. The recipient of this topical skin preparation is not particularly limited and may be a human or a non-human mammal (e.g., laboratory animals, pets, and livestock). Examples of non-human mammals include monkeys, chimpanzees, mice, rats, dogs, cats, rabbits, cattle, pigs, sheep, goats, and horses.

[0072] When administering a topical skin preparation according to one aspect of the present invention to a subject, there is no limit to the amount administered to the subject, as long as the desired effect is obtained. Furthermore, when administering a topical skin preparation according to one aspect of the present invention to a subject, there is no limit to the administration interval, as long as the desired effect is obtained.

[0073] Furthermore, in another aspect of the present invention, a method for activating Sirtuin-3 gene expression is provided, comprising the following steps: A step of applying a Sirtuin-3 gene activating material according to one aspect of the present invention to cells.

[0074] As has already been described, the Sirtuin-3 gene activating material according to one aspect of the present invention will not be repeated here. The cells to which the Sirtuin-3 gene activating material is applied are not particularly limited and may be human cells or non-human mammalian cells. Furthermore, the cells may be cells isolated from living organisms or cultured cells in established lines.

[0075] The concentration of the Sirtuin-3 gene activating material applied to the cells can be appropriately selected by those skilled in the art, depending on the type of cell, etc., within a range that yields the effects of the present invention.

[0076] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in each embodiment are also included within the technical scope of the present invention.

[0077] 〔summary〕 One aspect of the present invention is as follows: [1] A Sirtuin-3 gene expression activating material characterized by containing peony root or an ethanol-containing solvent extract thereof, and [2] A topical skin preparation containing the Sirtuin-3 gene expression activating material described in [1] above. [Examples]

[0078] The present invention will be specifically described by the following examples.

[0079] <Preparation of various extracts> (1) Preparation of ethanol-containing solvent extract of peony root Peony roots were immersed in anhydrous ethanol and allowed to stand at 40°C for 48 hours. After ultrafiltration, an ethanol-containing solvent extract of peony roots (hereinafter referred to as "peony root ethanol extract") was obtained. The peony root ethanol extract was designated as test sample 1. In subsequent experiments, the undiluted peony root ethanol extract was used after being appropriately diluted with water, water / ethanol, or water / butylene glycol (BG).

[0080] (2) Preparation of aqueous extract and butylene glycol (BG) extract of peony root Solvent extracts of peony root were obtained in the same manner as for ethanol-containing solvent extracts, except that water or BG was used instead of anhydrous ethanol as the extraction solvent. The aqueous extract of peony root was designated as test sample 2, and the BG extract of peony root was designated as test sample 3. In subsequent experiments, the stock solutions of each solvent extract were used after being appropriately diluted with water, water / ethanol, or water / BG.

[0081] (3) Preparation of peony flower butylene glycol (BG) extract A BG extract of peony flowers was obtained using the same method as described in (2) above, except that peony flowers were used instead of peony roots. The BG extract of peony flowers was designated as test sample 4. In subsequent experiments, the undiluted BG extract of peony flowers was used after being appropriately diluted with water, water / ethanol, or water / BG.

[0082] [Example 1] Experiment 1: Preparation of cell samples Normal human epidermal keratinocytes (Human Epidermal Keratinocytes, Adult) were seeded at a rate of 10,000 cells per well in serum-free liquid medium for epidermal keratinocyte proliferation (Humedia-KB2) on a 96-well microplate CELLSTAR® (Griner, TC, flat bottom, #655180), and cultured for 24 hours in a 5% CO2 incubator at 37°C. Subsequently, test sample 1 was added to the serum-free liquid medium to achieve a final concentration of 0.1% (w / v) of test sample 1, and further incubated for 48 hours in a 5% CO2 incubator at 37°C (number of test cases n=3).

[0083] Experiment 2: Ethanol extract of peony root activates Sirtuin-3 gene expression. To evaluate the Sirtuin-3 gene expression activating effect of ethanol extract from peony root, the Sirtuin-3 gene expression level in normal human epidermal keratinocytes treated with test sample 1 was measured using real-time PCR.

[0084] After 48 hours of incubation in Experiment 1, the serum-free liquid medium supernatant containing test sample 1 was discarded by aspiration using an aspirator, and 5 μL of DNase I-containing Lysis Beffer (TaqMan, Thermo Fisher Scientific) was added to each well. TM The Fast Advanced Cells-to-CT™ Kit accessory was added and incubated at room temperature for 5 minutes. Then, Stop Solution (TaqMan) was added. TM The Fast Advanced Cells-to-CT™ Kit (included) was added and incubated at room temperature for 2 minutes.

[0085] To the obtained Lysis Solution (10 μL), add 25 μL of 2×RT Buffer, 2.5 μL of 20×RT Enzyme Mix, and 12.5 μL of Nuclease-free Water (all from Thermo Fisher Scientific TaqMan). TMThe Fast Advanced Cells-to-CT™ Kit (accessories) were mixed to a volume of 50 μL. This mixture was reacted using a T100™ thermal cycler (BIO-RAD) at 37°C for 30 minutes and then at 95°C for 5 minutes to synthesize the reverse transcript product.

[0086] Real-time PCR requires TaqMan per reaction mixture. TM Gene Expressin Master Mix (2×) 5 μL, solution of the obtained reverse transcript 2 μL, TaqMan TM A 10 μL solution was prepared using 0.5 μL each of probes (SIRT3 and ACTB (β-Actin, internal standard)) and 2 μL of nucleotide-free water (all from Thermo Fisher Scientific).

[0087] The reaction was analyzed using the real-time PCR analysis system CFX Connect. TM Using a BIO-RAD device, measurements were taken by heating at 50°C for 2 minutes, then at 95°C for 10 minutes, followed by a reaction of heating at 95°C for 15 seconds and then at 60°C for 60 seconds, repeated 40 times.

[0088] [Examples 2-4, Comparative Examples 1-9] Experiments 1 and 2 described above were carried out in the same manner as in Example 1, except that test samples 1 to 4 shown in Table 1 were applied to the final concentrations shown in Table 1, and the Sirtuin-3 gene expression levels in normal human epidermal keratinocytes to which each test sample was applied were measured.

[0089] [Comparative Examples 10-12] Experiments 1 and 2 were conducted in the same manner as in Example 1, except that in Experiment 1 described above, only ethanol, water, or BG was applied as a control instead of the test sample to obtain the final concentrations shown in Table 1. The Sirtuin-3 gene expression levels in normal human epidermal keratinocytes treated with the control were measured.

[0090] <Analysis> The relative expression levels of the SIRT3 gene were calculated from the cycle threshold Ct values ​​obtained in each test in Examples 1-4 and Comparative Examples 1-12. Statistical analysis was performed using the Dunnett test with ΔCt (blank) as the control group.

[0091] <Result> Table 1 shows the results of Sirtuin-3 gene expression levels in Examples 1-4 and Comparative Examples 1-12. Each Sirtuin-3 gene expression level is expressed as a relative value with the expression level of the control group (Comparative Example 10) set to 100 ("STD" in Table 1).

[0092] [Table 1]

[0093] As shown in Table 1, normal human epidermal keratinocytes treated with ethanol extract of peony root (test sample 1) showed increased Sirtuin-3 gene expression compared to those treated with BG extract or water extract. This result indicates that ethanol extract of peony root contains a component that activates Sirtuin-3 gene expression in normal human epidermal keratinocytes.

[0094] [Example 5] The effects of topical skin preparations containing ethanol extract of peony root on the skin. To evaluate the effects of peony root ethanol extract on the skin when applied, an application test was conducted using a cream containing peony root ethanol extract.

[0095] Specifically, nine male and female subjects aged 20-50 applied a topical skin preparation containing peony root ethanol extract (Cream A) and a topical skin preparation without peony root extract (Cream B), as shown in Table 2, to the inner side of each upper arm. Application was performed once a day for four consecutive weeks. In Table 2, "-" indicates that the ingredient was not included. Test sample 1 was used for the peony root ethanol extract. The amounts for test sample 1 in Table 2 represent the amount of undiluted pure extract.

[0096] [Table 2]

[0097] For each subject, the stratum corneum moisture content (skin moisture content) was measured using a multi-skin analyzer (MPA6 Corneometer CM825) in the areas where topical skin preparation (cream A) was applied, the areas where topical skin preparation (cream B) was applied, and the areas where no preparation was applied.

[0098] <Result> Table 3 shows the measurement results for various skin moisture levels. The values ​​in Table 3 represent the dielectric constant measured by a multi-skin analyzer. The values ​​in Table 3 also represent the average values ​​for the subjects. A higher dielectric constant indicates a higher moisture content.

[0099] [Table 3]

[0100] As shown in Table 3, skin treated with cream A, which contains peony root ethanol extract, showed an increase in stratum corneum moisture content compared to skin treated with cream B, which does not contain peony root ethanol extract, or untreated skin. This result indicates that activation of the Sirtuin-3 gene in skin cells increases stratum corneum moisture content. [Industrial applicability]

[0101] This invention can be used as a Sirtuin-3 gene expression activator or as a topical skin preparation.

Claims

1. A Sirtuin-3 gene expression activating material characterized by containing peony root or an ethanol-containing solvent extract thereof.

2. A topical skin preparation containing the Sirtuin-3 gene expression activating material described in claim 1.

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