Antioxidant

Abstract

To provide an antioxidant for safely and efficiently reducing oxidative stress in vivo by promoting the expression of an antioxidative enzyme or a redox-related factor in vivo, thereby maintaining good health and preventing various diseases and symptoms caused by oxidative stress.SOLUTION: An antioxidant contains an extract of Albizia julibrissin and has, in particular, an effect of promoting the expression of an antioxidative enzyme gene and / or a redox-related factor gene.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antioxidant containing an extract of Zelkova serrata as an active ingredient. In particular, the present invention relates to an antioxidant useful for improving the antioxidant ability of cells by acting on the cells.

Background Art

[0002] In recent years, various reactive oxygen species have attracted attention as factors that oxidize biological components, and their adverse effects on living organisms have become a problem. Reactive oxygen species refer to oxygen molecules and their related molecular species with increased chemical reactivity compared to oxygen molecules in the steady state, and are generated in the energy metabolism process in vivo. Reactive oxygen species include radical species such as superoxide (·O 2- ₂), hydroxyl radical (·OH), hydroperoxyl radical (HHO·), nitric oxide (NO), and non-radical species such as hydrogen peroxide (H₂O₂), singlet oxygen ( 1 ¹O₂), peroxynitrite (ONOO - ⁻), lipid hydroperoxide (LOOH), and the like. These reactive oxygen species serve as second messengers in the signal transduction system in vivo and are essential molecular species for life activities such as the control of cell growth and differentiation and the bactericidal action in macrophages and the like. On the other hand, excessive reactive oxygen species are considered to be harmful as they damage cells and tissues.

[0003] In response to the oxidative stress caused by these excessive reactive oxygen species, the living body has a reactive oxygen species production inhibitory mechanism as an antioxidant mechanism for defending against oxidative stress. The reactive oxygen species production inhibitory mechanism mainly consists of antioxidant-related enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX), and suppresses reactive oxygen species through enzymatic reactions. Reactive oxygen species change from superoxide to hydrogen peroxide and hydroxyl radicals, and in this process, SOD eliminates superoxide and catalase eliminates hydrogen peroxide. In addition, GPX, glutathione reductase (GRD), thioredoxin reductase (TRD), and peroxiredoxin (PRX) eliminate hydrogen peroxide and hydroxyl radicals by controlling the oxidation-reduction state of glutathione (GSH) and thioredoxin (TXN). By working in concert with these antioxidant-related enzymes and redox-related factors, the amount of reactive oxygen species in cells is maintained at an appropriate level.

[0004] Thus, in order to suppress cell damage caused by reactive oxygen species in the living body, it is considered effective to increase the expression levels of the above-mentioned antioxidant-related enzymes and redox-related factors. These antioxidant-related enzymes and redox-related factors are known to be enhanced by ultraviolet irradiation stress, but components that act safely and effectively on the living body and promote the expression of antioxidant-related enzymes and redox-related factors are expected.

[0005] As such components, for example, quercetin and quercetin belonging to flavonols widely distributed in tea plants have been reported to enhance the expression and activity of the glutathione system and the thioredoxin system in the living body (Patent Document 1).

[0006] Searching for components that act safely and effectively on antioxidant-related enzymes and / or redox-related factors in vivo is important not only from the perspective of providing safer and more effective components to the market, but also from the perspectives of diversification of pharmaceutical preparations, expansion of options in formulation compounding, and synergistic effects achieved by using different components in combination. Needless to say, there is a demand for the development of novel components that act on antioxidant-related enzymes and / or redox-related factors.

[0007] On the other hand, Nemunoki is a deciduous tall tree of the legume family that grows naturally in sunny wetlands such as the forest edges and fields of mountains south of the Tohoku region in Japan. It is also distributed and cultivated in China, Southeast Asia, etc. overseas. The bark is collected during the flowering period, washed with water, dried in the sun, and cut into appropriate lengths, which is called Acacia bark. Folk people take it expecting arthritis, low back pain, diuresis, edema, and tonic effects. Externally, the affected area is cold-compressed with the decoction for tumors, bruises, and joint pain, or it has been used as a bath additive. It is known that the bark contains triterpenoid saponins (numerous julibrosides), flavonoids (geraldone, isoocanin, luteolin, etc.), tannins, etc. (Non-Patent Document 1).

[0008] Regarding the effects of Nemunoki on the living body, an active oxygen scavenging effect and an aldose reductase inhibitory effect using components derived from plants of the genus Nemunoki as active ingredients have been reported. However, this effect focuses on the active oxygen scavenging effect, that is, its action remains at the scavenger effect, so it is not the kind that stimulates antioxidant-related enzymes and redox-related factors in the living body to achieve active oxygen scavenging (Patent Document 2). In addition, an inhibitory effect on melanin production and a promoting effect on collagen production for the skin of the bark extract of Nemunoki have been reported, but there is no mention of antioxidant-related factors (Patent Document 3).

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

[0010] [Non-Patent Document 1] Kampo Medicine No. 759 (August 2016) [Summary of the Invention] [Problems to be Solved by the Invention]

[0011] In view of the above background, an object of the present invention is to safely and efficiently reduce oxidative stress in a living body, maintain health, and prevent various diseases and symptoms caused by oxidative stress by promoting the expression of antioxidant-related enzymes and / or redox-related factors in the living body. [Means for Solving the Problems]

[0012] In view of the above circumstances, the present inventors have intensively studied and as a result, found that the Nemunoki extract has an antioxidant effect, and thus completed the present invention. Regarding the Nemunoki extract, it has been found that among antioxidant-related enzymes and / or redox-related factors in the living body, the gene expression of SOD, TXN, PRX, TRD, GRD, and glutaredoxin (GXN) is enhanced. By using the antioxidant of the present invention as an active ingredient for promoting the expression of antioxidant-related enzymes and / or redox-related factors in the living body, it is possible to eliminate excessive reactive oxygen species in the living body without stressing the applied living body, and reduce various oxidative stresses in the living body.

[0013] That is, the summary of the present invention is as follows. Item 1. An antioxidant containing a Nemunoki extract. Item 2. The antioxidant according to Item 1, wherein the Nemunoki extract is an aqueous extract from the plant body of a plant belonging to the genus Nemunoki. Item 3. The antioxidant according to claim 1 or 2, wherein the Nemunoki extract has an action of enhancing the gene expression of antioxidant-related enzymes and / or redox-related factors. Item 4. The antioxidant according to any one of claims 1 to 3, wherein the antioxidant-related enzyme and / or redox-related factor is at least one selected from the group consisting of superoxide dismutase (SOD), peroxiredoxin (PRX), thioredoxin (TXN), thioredoxin reductase (TRD), glutathione reductase (GRD), and glutaredoxin (GXN). Item 5. The antioxidant according to any one of claims 1 to 4, wherein the Nemunoki extract has an action of reducing intracellular reactive oxygen species. Item 6. An external preparation for skin containing the antioxidant according to any one of claims 1 to 5. Item 7. The external preparation for skin according to claim 6, wherein the Nemunoki extract is contained at a concentration of 0.00001 to 5.0% by weight.

Effects of the Invention

[0014] By using the antioxidant of the present invention, it can be expected to improve the antioxidant ability in vivo, and it is also possible to provide an external preparation for skin aimed at the effect of improving the antioxidant ability.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] Specific examples of the extraction method of the Nemunoki extract used in the present invention are shown below. The part of the plant body in the extract mainly uses the bark, but the whole plant body can also be used without specifying the bark. Needless to say, it is not limited to the examples described below.

[0017] In the present invention, the Nemunoki extract refers to the plant body of the Nemunoki genus plant itself, as well as its cuttings, dried powders, and its extracts. And this "extract" includes both the state containing the extraction solvent and the state after removing the extraction solvent, and also includes those further subjected to purification treatment after removing the extraction solvent.

[0018] The Nemunoki extract is extracted from the plant body of the Nemunoki genus plant, typically from its branches and leaves, either raw or dried and pulverized, and then obtained by performing extraction using water, preferably hot water at 70 to 100°C, or an organic solvent as the extraction solvent. The extraction can be carried out by combining water extraction and organic solvent extraction as necessary, but considering the influence when applied to a living body, it is preferably carried out only by water extraction. For example, water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridines; fats, waxes, and other oils can be mentioned. Among these, water, alcohols, and water-alcohol mixtures are preferred, and it is particularly preferred to use hot water as the extraction solvent.

[0019] When performing hot water extraction, typically, the pulverized plant body of the Nemunoki genus plant is extracted under heating and reflux. The extraction conditions vary depending on the solvent used. For example, when extracting with water, it is preferable to use 1 to 100 parts by weight of water per 1 part by weight of Nemunoki and extract at a temperature of 4 to 100°C for 10 minutes to 7 days. It is more preferable to use 5 to 20 parts by weight of water per 1 part by weight of the bark of Nemunoki and extract at a temperature of 70 to 100°C for 30 minutes to 2 hours. Also, from the viewpoint of further improving the extraction efficiency, pressurization, stirring, ultrasonic treatment, etc. may be carried out together during the extraction process. Furthermore, after such extraction treatment, it may be separated from the residue by filtration or centrifugation.

[0020] When the Nemunoki extract is distilled under reduced pressure to remove the extraction solvent, it usually gives a tarry, dark brown extract, and excipients can be added if necessary. When the extraction solvent is non-toxic, such as water, it can be used in the preparation as it is, containing the extraction solvent, or the extract from which the extraction solvent has been separated can also be used in the preparation. The extract after separation of the extraction solvent can be redissolved in a suitable solvent, such as water, and used in the preparation. Further, the extract thus obtained can be purified or the titer can be improved by purification means, such as fractional extraction, partitioning between two solvents, fractional adsorption / elution with a suitable adsorbent or chromatography, etc.

[0021] The Nemunoki extract obtained as described above can be used as an antioxidant as it is, but it is preferably formulated in an external preparation. The amount to be formulated in the external preparation is determined by factors such as the degree of absorption, the degree of action, the product form, the frequency of use, etc., and is not particularly limited, but it is desirable to be in a concentration range of 0.00001 to 5.0% by weight on a dry weight basis, preferably in the range of 0.00005 to 3.0% by weight, and particularly preferably in the range of 0.0001 to 1.0% by weight. If the content of the Nemunoki extract is less than 0.00001% by weight, sufficient effects cannot be exerted, and even if more than 5.0% by weight is added, the effects are almost stationary.

[0022] The antioxidant of the present invention contains the Nemunoki extract as an active ingredient. The antioxidant action in the present invention refers to, when acting on cells and tissues in a living body, in addition to the scavenger action by the antioxidant substances contained in the Nemunoki extract, the action of enhancing the expression of antioxidant-related enzymes and / or redox-related factors in the living body, particularly the action of enhancing the gene expression of antioxidant-related enzymes and redox-related factors. Further, by these actions, it exerts the action of reducing the amount of active oxygen in cells.

[0023] In the present invention, the action of enhancing the gene expression of antioxidant-related enzymes and redox-related factors can be evaluated by adding the antioxidant of the present invention to cultured cells and quantifying the amount of gene expression in the cells using real-time PCR. Also, in the present invention, the amount of reactive oxygen species in cells can be evaluated by incorporating a fluorescent substance that reacts with reactive oxygen species into cultured cells, adding the antioxidant of the present invention, and quantifying the fluorescence intensity emitted from the cells.

[0024] Examples of antioxidant-related enzymes and / or redox-related factors include superoxide dismutase (SOD), peroxidase, catalase, peroxiredoxin (PRX) that scavenge reactive oxygen species; glutathione reductase (GRD), thioredoxin reductase (TRD), glutaredoxin (GXN) that control the oxidation-reduction state of glutathione (GSH) and thioredoxin (TXN); enzymes that reduce and scavenge radicals; glutathione peroxidase that reduces hydroperoxides generated by lipid oxidation, α,β-hydrogenase that reduces intermediates with a carbonyl group (such as 4-hydroxynonenal), etc.; proteins that store and transport transition metal ions so that the Fenton reaction does not occur (ferritin, transferrin, etc.); proteases, lipases, nucleases, chlorophyll-degrading enzymes, pheophorbide a oxygenase, etc. that decompose molecules damaged by oxidation; methionine sulfoxide reductase, DNA repair enzymes, etc. that repair molecules oxidized by reactive oxygen species.

[0025] In the present invention, it is presumed that the Nemunoki extract enhances the gene expression levels of SOD, PRX, TXN, TRD, GRD, and GXN in particular, thereby increasing the amount of antioxidant-related enzymes and / or redox-related factors in vivo, reducing the reactive oxygen species generated in vivo, and exerting antioxidant ability.

[0026] In addition to the Nemunoki extract, the antioxidant of the present invention can contain other raw materials as long as the effects of the present invention are not impaired. Examples of such raw materials include water, excipients, antioxidants, preservatives, wetting agents, thickening agents, buffering agents, adsorbents, solvents, emulsifiers, stabilizers, surfactants, lubricants, water-soluble polymers, sweeteners, flavoring agents, acidulants, alcohols, and the like. Further, the antioxidant of the present invention can contain other active ingredients as long as the effects of the present invention are not impaired. Specific examples of the active ingredients include, for example, antioxidant components, anti-aging components, anti-inflammatory components, whitening components, cell-activating components, vitamins, blood circulation-promoting components, moisturizing components, components having a preventive and / or reparative effect on DNA damage, anti-glycation components, peptides or their derivatives, amino acids or their derivatives, hydroquinone glycosides and their esters, and the like.

[0027] In addition to the essential components of the Nemunoki extract, the antioxidant of the present invention can be formulated in combination with components and additives used in cosmetics, quasi-drugs, foods and drinks, pharmaceuticals, etc., as long as the effects of the present invention are not impaired within the necessary range.

[0028] For example, as oils and fats, there are avocado oil, almond oil, perilla oil, sesame oil, olive oil, orange oil, orange roughy oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow, beef tallow fatty acid, kukui nut oil, safflower oil, soybean oil, camellia oil, corn oil, rapeseed oil, peach kernel oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, cocoa butter, palm oil, palm kernel oil, beeswax, coconut oil, beef tallow, lard, hydrogenated oil, hydrogenated castor oil, and the like.

[0029] As waxes, there are beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, and the like.

[0030] As mineral oils, there are liquid paraffin, petrolatum, paraffin, ozokerite, ceresin, microcrystalline wax, polyethylene powder, squalene, squalane, pristane, and the like.

[0031] Examples of fatty acids include natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid, and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, isopentanoic acid, etc.

[0032] Examples of alcohols include natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol, polyhydric alcohols such as 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, glycerin, batyl alcohol, pentaerythritol, sorbitol, mannitol, glucose, sucrose, etc.

[0033] Examples of esters include 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.

[0034] Examples of the metal soaps include aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, and the like.

[0035] Examples of the gum and water-soluble polymer compounds include gum arabic, benzoin gum, dammar gum, guaiac resin, Irish moss, karaya gum, tragacanth gum, carob gum, quince seed, agar, casein, dextrin, gelatin, pectin, starch, carrageenan, carboxyalkyl chitin, chitosan, hydroxyalkyl chitin, low molecular weight chitosan, chitosan salt, sulfated chitin, phosphorylated chitin, alginic acid and its salts, hyaluronic acid and its salts, chondroitin sulfate, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate salts, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their cross-linked polymers, carboxyvinyl polymer, polyethyleneimine, and the like.

[0036] Examples of the surfactants include anionic surfactants (carboxylate salts, sulfonate salts, sulfate ester salts, phosphate ester salts), cationic surfactants (amine salts, quaternary ammonium salts), amphoteric surfactants (carboxylic acid type amphoteric surfactants, sulfate ester type amphoteric surfactants, sulfonate type amphoteric surfactants, phosphate ester 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 type nonionic surfactants), and other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), and the like.

[0037] As vitamins, in vitamin A group, there are retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A); in vitamin B group, there are thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin, choline, inositols; in vitamin C group, there are ascorbic acid and its derivatives; in vitamin D group, there are ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol; in vitamin E group, there are tocopherol and its derivatives, ubiquinones; in vitamin K group, there are phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), etc.

[0038] As amino acids, there are 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, citrates, or amino acid derivatives such as pyrrolidone carboxylic acid, etc.

[0039] As whitening agents, there are ascorbic acid or its derivatives, sulfur, placenta hydrolysate, ellagic acid or its derivatives, kojic acid or its derivatives, glucosamine or its derivatives, arbutin or its derivatives, hydroxycinnamic acid or its derivatives, glutathione, arnica extract, saffron extract, sophora flavescens extract, psoralea corylifolia extract, peony extract, ganoderma lucidum mycelium culture or its extract, cinnamon extract, peach leaf extract, polygonatum odoratum extract, polygala tenuifolia extract, dioscorea extract, button mushroom extract, witch hazel extract, paulownia extract, bupleurum falcatum extract, water-soluble glycyrrhiza extract, etc.

[0040] Examples of moisturizing agents include hyaluronic acid, polyglutamic acid, serine, glycine, threonine, alanine, collagen, hydrolyzed collagen, hydronectin, fibronectin, keratin, elastin, royal jelly, chondroitin sulfate heparin, glycerophospholipids, glyceroglycolipids, sphingophospholipids, sphingoglycolipids, linoleic acid or its esters, eicosapentaenoic acid or its esters, pectin, bifidobacterium ferment, lactic acid ferment, yeast extract, reishi mycelium culture or its extract, wheat germ oil, avocado oil, rice germ oil, jojoba oil, soybean phospholipids, γ-oryzanol, viola extract, yokukinin extract, angelica extract, ginseng extract, seaweed extract, aloe vera extract, burdock extract, beeswax extract, arnica extract, wheat bran, etc.

[0041] Examples of hair growth agents include glyceryl pentadecanoate, coleus extract, gentiana extract, cinnamon extract, royal jelly extract, miscanthus extract, t-flavanone, 6-benzylaminopurine, centella extract, calcium propionate chloride, minoxidil, finasteride, adenosine, nicotinamide, mulberry root extract, angelica extract, 5-aminolevulinic acid, etc.

[0042] As extracts or essences of animals, plants, or crude drugs, there are Acacia catechu, Ashitaba, Acerola, Althaea, Arnica, Avocado, Ama-cha (sweet tea), Aloe, Aloe vera, Nettles, Ginkgo biloba, Fennel, Turmeric, Asarum, Japanese plum, White mustard, Japanese sumac, Potentilla indica, Sedum aizoon, Astragalus membranaceus, Calendula officinalis, Cherry bark, Phellodendron amurense, Coptis chinensis, Ginseng, Lychnis flos-cuculi, Silene fortunei, Capsicum annuum, Orange, Polygala tenuifolia, Prunella vulgaris, Fallopia multiflora, Sophora japonica, Mugwort, Curcuma zedoaria, Kudzu root, Valerian root, Chamomile, Trichosanthes kirilowii, Artemisia capillaris, Licorice, Coltsfoot, Strawberry, Kiwifruit, Platycodon grandiflorum, Chrysanthemum, Catalpa ovata, Immature fruit of Poncirus trifoliata, Tangerine peel, Cucumber, Angelica pubescens or Notopterygium incisum, Apricot kernel, Wolfberry root bark, Lycium barbarum, Lycium chinense, Sophora flavescens, Camphor tree, Kalmia latifolia, Grapefruit, Cinnamon, Cinnamomum cassia, Cassia obtusifolia, Pharbitis nil or Pharbitis purpurea, Safflower, Chinese gall, Comfrey, Copaiba, Gardenia jasminoides, Gentiana scabra, Magnolia officinalis, Achyranthes bidentata, Evodia rutaecarpa, Burdock, Schisandra chinensis, Rice, Rice bran, Wheat, Bupleurum falcatum, Saffron, Soapwort, Japanese medlar, Prickly ash, Salvia officinalis, Panax notoginseng, Shiitake mushroom, Rehmannia glutinosa, Quisqualis indica, Purple root, Perilla frutescens, Persimmon calyx, Paeonia lactiflora, Plantago asiatica or Plantago major, Ginger, Acorus calamus, Glossy privet fruit, Stellaria media, White birch, Lonicera japonica, Vinca minor, Digitalis purpurea, Galium aparine, Sambucus williamsii, Cowpea, Chuanxiong rhizome, Swertia japonica, Mulberry bark,Mulberry leaves, Chinese date, soybean, oak, Japanese ginseng, anemarrhena rhizome, Sanguisorba officinalis, Houttuynia cordata, Cordyceps sinensis, capsicum, Physalis alkekengi, Viola philippica, green tea, black tea, clove, tangerine peel, camellia, Oxalis corniculata, capsicum, Angelica sinensis, Tagetes erecta, orange peel, Sanguisorba officinalis, maize, Eucommia ulmoides (Eucommia ulmoides leaves), tomato, Nandina domestica, garlic, malt, Dictamnus dasycarpus, Ophiopogon japonicus, parsley, sweet potato, mint, Hamamelis mollis, rose, loquat leaf, Poria cocos, grape or its leaves, Luffa cylindrica, Ficus carica, Paeonia suffruticosa, hops, rose flower, pine leaves, plane tree, beeswax, Akebia quinata, Melissa officinalis, Melilotus officinalis, papaya, mung bean sprouts, peach (peach kernel, peach leaf), Belamcanda chinensis, Areca catechu, Leonurus japonicus, Tagetes patula, Saxifraga stolonifera, Myrica rubra, Centaurea cyanus, Coix lacryma-jobi, Artemisia montana, Artemisia selengensis, lavender, apple fruit, Ganoderma lucidum, lemon fruit, Forsythia suspensa, Galium aparine, Geranium wilfordii, burdock root, chicken comb, bovine and human placenta extract, porcine and bovine stomach, duodenum, or intestinal extract or its degradation product, water-soluble collagen, water-soluble collagen derivative, collagen hydrolyzate, elastin, elastin hydrolyzate, water-soluble elastin derivative, silk protein, silk protein hydrolyzate, bovine hemoglobin hydrolyzate, etc.

[0043] Examples of microbial culture metabolites include yeast extract, zinc-containing yeast extract, germanium-containing yeast extract, selenium-containing yeast extract, magnesium-containing yeast extract, rice fermentation extract, Euglena extract, lactic acid fermented product of skim milk powder, etc.

[0044] Examples of α-hydroxy acids include glycolic acid, citric acid, malic acid, tartaric acid, lactic acid, etc.

[0045] Examples of inorganic pigments include anhydrous silicic acid, magnesium silicate, talc, kaolin, bentonite, mica, mica titanium, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium carbonate, yellow iron oxide, red iron oxide, black iron oxide, zinc white, chromium oxide, chromium hydroxide, carbon black, and karamin.

[0046] Examples of ultraviolet absorbers include p-aminobenzoic acid derivatives, salicylic acid derivatives, anthranilic acid derivatives, coumarin derivatives, amino acid-based compounds, benzotriazole derivatives, tetrazole derivatives, imidazoline derivatives, pyrimidine derivatives, dioxane derivatives, camphor derivatives, furan derivatives, pyrone derivatives, nucleic acid derivatives, allantoin derivatives, nicotinic acid derivatives, vitamin B6 derivatives, oxybenzone, benzophenone, guaiazulene, sikonin, baicalin, baicalein, and berberine.

[0047] Examples of astringents include lactic acid, tartaric acid, succinic acid, citric acid, allantoin, zinc chloride, zinc sulfate, zinc oxide, karamin, zinc p-phenolsulfonate, potassium aluminum sulfate, resorcinol, ferric chloride, and tannic acid.

[0048] Examples of antioxidants include ascorbic acid and its salts, stearic acid esters, tocopherol and its ester derivatives, nordihydroguaiaretic acid, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), para-hydroxyanisole, propyl gallate, sesamol, sesamolin, and gossypol.

[0049] Examples of anti-inflammatory agents include ichthyol, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d- or dl-camphor, hydrocortisone, guaiazulene, kamazulene, chlorpheniramine maleate, glycyrrhizic acid and its salts, and glycyrrhetinic acid and its salts.

[0050] Examples of bactericidal and disinfectant agents include acrinol, sulfur, benzalkonium chloride, benzethonium chloride, methylrosanilinium chloride, cresol, calcium gluconate, chlorhexidine gluconate, sulfamine, mercurochrome, lactoferrin or its hydrolyzate, and the like.

[0051] Examples of hair agents include selenium disulfide, alkylisquinolinium bromide solution, zinc pyrithione, bifenamine, thianthol, caster tincture, ginger tincture, capsicum tincture, quinine hydrochloride, strong ammonia water, potassium bromate, sodium bromate, thioglycolic acid, and the like.

[0052] Examples of fragrances include natural animal-derived fragrances such as civet, castoreum, ambergris, etc., essential oils of anise, angelica, iran, iris, wikyo, orange, cananga, karaway, cardamom, guaiacwood, cumin, blackletter, cinnamon, geranium, copaiba balsam, coriander, perilla, cedarwood, citronella, jasmine, gingergrass, cedar, spearmint, peppermint, star anise, tuberose, clove, neroli, wintergreen, tolu balsam, patchouli, rose, palmarosa, hinoki, sandalwood, petitgrain, bay, vetiver, bergamot, peru balsam, bois de rose, linalool, mandarin, eucalyptus, lime, lavender, linaloe, lemongrass, lemon, rosemary, Japanese mint, and other plant-derived fragrances, as well as other synthetic fragrances, and the like.

[0053] Examples of pigments and colorants include red cabbage pigment, red rice pigment, madder pigment, annatto pigment, squid ink pigment, turmeric pigment, safflower pigment, shrimp pigment, persimmon pigment, caramel, gold, silver, gardenia pigment, corn pigment, onion pigment, tamarind pigment, spirulina pigment, buckwheat herb pigment, cherry pigment, nori pigment, hibiscus pigment, grape juice pigment, marigold pigment, purple sweet potato pigment, purple yam pigment, lac dye, rutin, and the like.

[0054] Examples of sweeteners include sugar, sweet tea, fructose, arabinose, galactose, xylose, mannose, maltose, honey, glucose, miraculin, monellin, and the like.

[0055] Examples of nutritional fortifiers include calcined oyster shell calcium, cyanocobalamin, yeast, wheat germ, soybean germ, egg yolk powder, hemicellulose, heme iron, and the like.

[0056] In addition, hormones, metal ion sequestering agents, pH adjusters, chelating agents, preservatives and antibacterial agents, cooling agents, stabilizers, emulsifiers, animal and plant proteins and their decomposition products, animal and plant polysaccharides and their decomposition products, animal and plant glycoproteins and their decomposition products, blood flow accelerators, anti-inflammatory and anti-allergy agents, cell activators, keratolytic agents, wound healing agents, foaming agents, thickening agents, oral agents, deodorants and odor removers, bittering agents, seasonings, enzymes, and the like can be mentioned.

[0057] The dosage form of the present invention is arbitrary, and it can be used by being formulated into quasi-drugs such as ampoule form, capsule form, powder form, granule form, pill form, tablet form, solid form, liquid form, gel form, bubble form, emulsion form, cream form, ointment form, sheet form, mousse form, etc., cosmetics for skin and hair, bath agents, foods and drinks, and pharmaceuticals.

[0058] Specifically, examples of cosmetics and quasi-drugs include internal and external pharmaceutical preparations, lotions, milky lotions, creams, ointments, lotions, oils, packs and other basic cosmetics, facial washes and body washes, shampoos, rinses, hair treatments, hair creams, pomades, hair sprays, hair styling products, perming agents, hair tonics, hair dyes, hair growth and nourishing agents and other hair cosmetics, foundations, face powders, white face powders, lipsticks, blushes, eye shadows, eyeliners, mascaras, eyebrow pencils, false eyelashes and other makeup cosmetics, nail cosmetics and other finishing cosmetics, perfumes, bath agents, and other toothpastes, oral cooling agents and gargles, body odor and deodorant agents, sanitary products, sanitary napkins, wet tissues, etc.

[0059] Examples of food and beverages include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, frozen desserts such as ice cream, ice sherbet, shaved ice, noodles such as buckwheat noodles, udon noodles, glass noodles, gyoza wrappers, shumai wrappers, Chinese noodles, instant noodles, candies, chocolates, tablets, snacks, biscuits, jellies, jams, creams, baked goods, bread, etc., seafood such as crab, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whale, oysters, saury, squid, ark shell, scallop, abalone, sea urchin, ikura, sea grapes, etc., processed seafood and livestock products such as kamaboko, ham, sausage, dairy products such as powdered milk, processed milk, fermented milk, fats and oils and processed fat and oil foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, seasonings such as sauces, condiments, retort pouch foods such as curry, stew, oyakodon, rice porridge, mixed rice, Chinese bowl, cutlet bowl, tempura bowl, unadon, hayashi rice, oden, mapo tofu, beef bowl, meat sauce, egg soup, omurice, dumplings, shumai, hamburg, meatballs, etc., various forms of health and nutritional supplements, functional foods, tablets, capsules, drinkable agents, troches, etc.

[0060] The antioxidant of the present invention is preferably applicable to humans, but can also be applied to animals other than humans as long as the respective effects can be expected.

[0061] Aspects of the antioxidant of the present invention are not particularly limited. For example, they include facial cleansers, detergents, lotions (e.g., whitening lotions), creams (e.g., vanishing creams, cold creams), milky lotions, gels, beauty serums, packs (e.g., jelly-like peel-off type, paste-like wipe-off type, powder-like rinse-off type), face masks, cleansing products, foundations, lipsticks, lip creams, lip glosses, lip liners, blushes, makeup bases, shaving lotions, sunscreens, after-sun lotions, deodorant lotions, body lotions (including hand care lotions and foot care lotions), body oils, and the like.

[0062] Hereinafter, the present invention will be described in more detail based on examples. Note that the present invention is not particularly limited to the examples.

Examples

[0063] The aqueous extract of the nemunoki bark used in the following examples was prepared by the method disclosed in JP-A-2018-58783.

[0064] Example 1: Evaluation of the effect of enhancing the expression of target genes in human normal neonatal epidermal keratinocytes Using the nemunoki extract as a test sample, the effect of enhancing the expression of target genes was evaluated by the following test method. The target genes in this example mean SOD, PRX, TXN, TRD, GRD, and GXN.

[0065] Human normal neonatal epidermal keratinocytes (NHEK) were seeded on a T75 flask collagen-coated with Collagen Coating Solution (manufactured by Toyobo Co., Ltd.), and a medium for normal human epidermal keratinocyte growth (HuMedia-KG2) was added, followed by culturing at 37°C and 10% CO2. The medium was appropriately changed, and the culturing was continued until it became 80% confluent or more. After removing the medium and rinsing with PBS, trypsin treatment was performed to collect the cells. 1×10 cells were placed in a 96-well plate. 4Cells were seeded at a density of cells / well and cultured at 37 °C and 10% CO2 for 1 day. The medium was removed and replaced with a medium containing the test sample at a predetermined concentration, and incubated at 37 °C and 10% CO2 for 72 hours. After culturing, the cells were collected, and cDNA was prepared from the cells using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.).

[0066] Based on the obtained cDNA, real-time PCR analysis was performed using THUNDERBIRD SYBR (registered trademark) qPCR Mix (manufactured by Toyobo Co., Ltd.). The reaction solution composition followed the attached document. However, the reaction solution volume was 20 μl / well and the cDNA amount was 3 μl. The reaction cycle conditions were carried out as 95 °C for 1 minute → (95 °C for 15 seconds → 60 °C for 45 seconds) × 40 → 95 °C for 15 seconds → 60 °C for 1 minute → 95 °C for 15 seconds. Primers with the nucleotide sequences shown in SEQ ID NOs: 1 to 6 were used. The instrument used was 7500 Fast Real-Time PCR System (Applied Biosystems). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize the expression level of the target gene (SEQ ID NOs: 13 to 14). The expression level of each normalized target gene was divided by the expression level of the gene in the negative control without the test sample to calculate the relative value.

[0067] The results obtained are shown in Figures 1 to 3. As can be seen from Figures 1 to 3, in NHEK, it was confirmed that the gene expression levels of antioxidant-related enzymes and / or redox-related factors were improved compared to the negative control without the nemunoki extract added. From these results, it became clear that the nemunoki extract has the effect of enhancing the gene expression of antioxidant-related enzymes and / or redox-related factors in epidermal cells and improving the antioxidant capacity in vivo.

[0068] Example 2: Evaluation of the effect of enhancing the expression of target genes in human normal neonatal fibroblasts Human normal neonatal fibroblasts (NHDF) were seeded in a T75 flask, D-MEM (+10% FBS) was added, and the cells were cultured at 37°C and 5% CO2. The medium was changed as appropriate, and the culture was continued until the confluence reached 80% or more. After removing the medium and rinsing with PBS, trypsin treatment was performed to recover the cells. 4 The cells were seeded in a 96-well plate at a density of 1×10 cells / well and cultured at 37°C and 5% CO2 for 1 day.

[0069] After removing the medium, the medium containing the test sample at a predetermined concentration was exchanged, and the cells were incubated at 37°C and 5% CO2 for 72 hours. After culturing, the cells were recovered, and cDNA was prepared from the cells using SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.).

[0070] Based on the obtained cDNA, real-time PCR analysis was performed using THUNDERBIRD SYBR (registered trademark) qPCR Mix (manufactured by Toyobo Co., Ltd.). The reaction solution composition followed the attached document. However, the reaction solution volume was 20 μl / well and the cDNA volume was 3 μl. The reaction cycle conditions were carried out as 95°C for 1 minute → (95°C for 15 seconds → 60°C for 45 seconds) × 40 → 95°C for 15 seconds → 60°C for 1 minute → 95°C for 15 seconds. The primers used had the nucleotide sequences shown in SEQ ID NOs: 1 to 12. The instrument used was a 7500 Fast Real-Time PCR System (Applied Biosystems). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize the expression level of the target gene (SEQ ID NOs: 13 to 14). The expression level of each normalized target gene was divided by the expression level of the gene in the negative control without the test sample to calculate the relative value.The results obtained are shown in Figs. 4 to 9. As can be seen from Figs. 4 to 9, in NHDF, it was confirmed that the gene expression levels of antioxidant-related enzymes and redox-related factors were improved compared to the negative control without the addition of Nemunoki extract. From these results, it became clear that the Nemunoki extract has the effect of enhancing the gene expression of antioxidant-related enzymes and / or redox-related factors in epidermal cells and improving the antioxidant capacity in vivo.

[0071] Example 3: Evaluation of the effect of reducing reactive oxygen species in human normal neonatal fibroblasts Human normal neonatal fibroblasts (NHDF) were seeded in a T75 flask, D-MEM (+10% FBS) was added, and the cells were cultured at 37 °C and 5% CO2. The medium was changed appropriately, and the culture was continued until the confluence reached 80% or more. After removing the medium and rinsing with PBS, trypsin treatment was performed to recover the cells. The cells were seeded in a 96-well plate at 3×10 4 cells / well and cultured at 37 °C and 5% CO2 for 1 day. After removing the culture and washing with HBSS, the cells were replaced with 20 μM H2DCFDA (2’,7’-dichlorodihydrofluorescein diacetate) and incubated at 37 °C and 5% CO2 (in the dark) for 30 minutes. After removing H2DCFDA and washing with HBSS, the cells were replaced with HBSS containing the test sample and hydrogen peroxide at a predetermined concentration, and further incubated at 37 °C and 5% CO2 (in the dark). After 5 hours, the fluorescence intensity was measured with a fluorescence spectrometer (ARVO MX) at Ex / Em = 485 nm / 535 nm.

[0072] The results obtained are shown in Fig. 10. As can be seen from Fig. 10, in NHDF, the effect of reducing intracellular reactive oxygen species induced by oxidative stress caused by hydrogen peroxide was confirmed. This is considered to be due to the improvement of the body's own reactive oxygen species scavenging ability, which led to the reduction of reactive oxygen species in vivo, in addition to the antioxidant action contained in the Nemunoki extract and the enhanced gene expression of antioxidant-related enzymes and / or redox-related factors.

[0073] Example 4: Evaluation of the effect of enhancing the expression of antioxidant-related genes in cells collected from a living body An aqueous solution containing 2.0% by weight of the Nemunoki extract was used as a test sample, and the effect of enhancing the expression of the target gene was evaluated by the following test method. The target gene in this example means PRX.

[0074] The test sample was applied to the face of the subject, and cells were obtained from the whiskers in the applied area for evaluation. Specifically, before and 3 hours after the application of the test sample, the whiskers in the applied area were removed from the subject, and hair follicle cells were collected. The cells were stored by immersing them in RNAlater (manufactured by Qiagen) and stored at 4°C until RNA extraction. After the collection of the hair follicle cells, total RNA was extracted from the hair follicle cells using the RNeasy plus micro kit (manufactured by Qiagen) according to the attached protocol. For the extracted total RNA, cDNA was prepared using ReverTra Ace (registered trademark) qPCR RT Master Mix (manufactured by Toyobo Co., Ltd.) according to the attached protocol. Based on the obtained cDNA, real-time PCR analysis was performed using THUNDERBIRD SYBR (registered trademark) qPCR Mix (manufactured by Toyobo Co., Ltd.). The reaction solution composition was in accordance with the attached document. However, the reaction solution volume was 20 μl / well and the cDNA amount was 2 μl. The reaction cycle conditions were carried out at 95°C for 1 minute → (95°C for 15 seconds → 60°C for 45 seconds) × 40 → 95°C for 15 seconds → 60°C for 1 minute → 95°C for 15 seconds. Primers with the nucleotide sequences shown in SEQ ID NOs: 7-8 were used. The instrument used was the 7500 Fast Real-Time PCR System (Applied Biosystems). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize the expression level of the target gene (SEQ ID NOs: 13-14). The expression level of each normalized target gene was divided by the expression level of the gene in the negative control before the application of the test sample to calculate the relative value.

[0075] The results obtained are shown in Fig. 11. As can be seen from Fig. 11, in the cDNA obtained from hair follicle cells, it was confirmed that the gene expression level of PRX was improved 3 hours after application, as compared with the negative control before applying the Nemunoki extract. From these results, it became clear that the Nemunoki extract has the effect of enhancing the gene expression of antioxidant-related enzymes and / or redox-related factors even in cells collected from a living body, and improving the antioxidant ability in vivo.

[0076] Example 5: Formulation of External Preparation for Skin Formulation examples of the external preparation for skin in the present invention are shown below. All of these preparations are expected to have the effect of enhancing the expression of antioxidant-related enzyme genes due to the Nemunoki extract and are effective as antioxidants.

[0077] Lotion A lotion was produced by a conventional method according to the following composition. ·Purified water ··· 89.80 g ·Glycerin ··· 3.00 g ·Phenoxyethanol ··· 0.20 g ·Butylene glycol ··· 5.00 g ·Pentylene glycol ··· 1.00 g ·Nemunoki extract ··· 1.00 g

[0078] Gel A gel was produced by a conventional method according to the following composition. ·Purified water ··· 88.50 g ·Carbomer ··· 0.30 g ·Xanthan gum ··· 0.10 g ·Arginine ··· 0.40 g ·Glycerin ··· 5.00 g ·Phenoxyethanol ··· 0.20 g ·Butylene glycol ··· 5.00 g ·Nemunoki extract ··· 0.50 g

[0079] Cream A cream was produced by a conventional method according to the following composition. ·Purified water···58.50 g ·Butylene glycol···10.00 g ·Glycerin···5.00 g ·Phenoxyethanol···0.20 g ·Ethylhexylglycerin···0.20 g ·Squalane···10.00 g ·Olive oil···10.00 g ·Behenyl alcohol···2.50 g ·Polyglyceryl-10 pentastearate···1.90 g ·Sodium stearoyl lactate···0.60 g ·Cetyl palmitate···1.00 g ·Nemunoki extract···0.10 g

Industrial applicability

[0080] According to the present invention, by significantly promoting the expression of antioxidant-related enzymes and / or redox-related factors in vivo, it is possible to safely and efficiently reduce the oxidative stress of the living body, maintain health, and prevent various diseases and symptoms caused by oxidative stress. In particular, it is useful in the application to external skin preparations as cosmetics and quasi-drugs.

Claims

1. A method for producing an extract of Nemunoki bark for use as an antioxidant (excluding therapeutic acts on humans), which comprises a step of extracting from Nemunoki bark with water, and enhancing the gene expression of at least one antioxidant-related enzyme and / or redox-related factor selected from the group consisting of superoxide dismutase (SOD), peroxiredoxin (PRX), thioredoxin (TXN), thioredoxin reductase (TRD), glutathione reductase (GRD), and glutaredoxin (GXN) in fibroblasts, keratinocytes, or hair follicle cells.

2. The method for producing an extract of Nemunoki bark according to Claim 1, wherein the antioxidant is used as a cosmetic.

3. The method for producing an extract of Nemunoki bark according to Claim 1, wherein the cosmetic is any one selected from the group consisting of lotion, gel, and cream.

4. A method of use as an external preparation for skin (excluding therapeutic acts on humans), which is formulated with the extract of Nemunoki bark according to any one of Claims 1 to 3.

5. A method of use as an external preparation for skin (excluding therapeutic acts on humans) according to Claim 4, which contains the extract of Nemunoki bark at a concentration of 0.00001 to 5.0% by weight.

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