JP2023171681A - antioxidant - Google Patents

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 The present invention relates to an antioxidative agent having an active ingredient of an extract of Album alba. In particular, the present invention relates to an antioxidant that is useful for improving the antioxidant capacity of cells by acting on cells.

In recent years, various active 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 related molecular species that have increased chemical reactivity compared to steady-state oxygen molecules, and are generated during energy metabolic processes in living organisms. Active oxygen species include superoxide (・O ²⁻ ), hydroxyl radical (・OH), hydroperoxy radical (HHO・), and nitric oxide (NO) as radical species, and hydrogen peroxide (H ₂ O) as non-radical species. ₂ ), singlet oxygen ( ¹ O ₂ ), peroxynitrite (ONOO ⁻ ), lipid hydroperoxide (LOOH), and the like. These reactive oxygen species serve as second messengers in the signal transduction system in living organisms, and are essential molecular species for life activities such as control of cell proliferation and differentiation and bactericidal action in macrophages and the like. On the other hand, excessive reactive oxygen species are considered to be harmful and damage cells and tissues.

In response to oxidative stress caused by these excessive reactive oxygen species, living organisms are equipped with a reactive oxygen species production suppression mechanism as an antioxidant mechanism to protect against oxidative stress. The reactive oxygen species production suppression mechanism is mainly composed 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 scavenges superoxide and catalase scavenges hydrogen peroxide. In addition, GPX, glutathione reductase (GRD), thioredoxin reductase (TRD), and peroxiredoxin (PRX) control the oxidation and reduction states of glutathione (GSH) and thioredoxin (TXN), thereby generating hydrogen peroxide and hydroxyl radicals. Erase. By working in concert with these antioxidant-related enzymes and redox-related factors, the amount of intracellular reactive oxygen species is maintained at an appropriate level.

In this way, it is considered effective to increase the expression levels of the above-mentioned antioxidant-related enzymes and redox-related factors in order to suppress cell damage caused by reactive oxygen species in vivo. These antioxidant-related enzymes and redox-related factors are known to be enhanced by ultraviolet irradiation stress, but there are ingredients that act safely and effectively on living organisms and promote the expression of antioxidant-related enzymes and redox-related factors. It is expected.

As such components, for example, kaempferol and quercetin, which belong to flavonols widely distributed in tea leaf plants, have been reported to enhance the expression and activity of the glutathione system and thioredoxin system in vivo (Patent Document 1) .

Searching for ingredients that act safely and effectively on antioxidant-related enzymes and/or redox-related factors in the body is not only important from the perspective of providing safer and more effective ingredients to the market. This is important from the viewpoint of diversification of formulations, expansion of formulation options, and synergistic effects by using different ingredients in combination.New ingredients that act on antioxidant-related enzymes and/or redox-related factors are important. It goes without saying that the development of

On the other hand, Nemunoki is a deciduous tree of the Fabaceae family that grows naturally in sunny wetlands such as mountainous forest edges and fields south of the Tohoku region in Japan, and is distributed overseas in China, Southeast Asia, etc. cultivated. The bark is collected during the flowering period, washed with water, dried in the sun, and chopped into appropriate lengths and is called Hehuanpi.It is used in folk medicine for arthritis, back pain, diuresis, edema, and tonicity. Externally, it has been used to treat tumors, bruises, and joint pain by applying a decoction to the affected area as a cold compress, or as a bath additive. The bark is known to contain triterpene saponins (many julibrosides), flavonoids (geraldone, isoochanin, luteolin, etc.), and tannins. (Non-patent document 1)

It has been reported that the active oxygen scavenging effect and aldose reductase inhibitory effect using ingredients derived from plants of the genus Album as active ingredients are reported as effects of Alminophylla on the living body, but this effect focuses on the active oxygen scavenging effect; Since it only has a scavenger effect, it is not of the kind that stimulates antioxidant-related enzymes and redox-related factors in living organisms to achieve active oxygen elimination (Patent Document 2). In addition, although it has been reported that the melanin production suppressing effect and the collagen production promoting effect for the skin of the Alpine tree bark extract, there is no mention of antioxidant-related factors (Patent Document 3).

Patent No. 5833438 Japanese Patent Application Publication No. 2000-128798 Patent No. 5253862

Japanese and Chinese medicine No. 759 (August 2016)

In view of the above background, the present invention aims to safely and efficiently reduce oxidative stress in living organisms, maintain health, and promote the expression of antioxidant-related enzymes and/or redox-related factors in living organisms. The goal is to prevent various diseases and symptoms caused by oxidative stress.

In view of the above-mentioned circumstances, the present inventors have conducted intensive research and have found that the extract of Album tree has an antioxidant effect, and have completed the present invention. It has been found that the Album tree extract enhances gene expression of SOD, TXN, PRX, TRD, GRD, and glutaredoxin (GXN) among antioxidant-related enzymes and/or redox-related factors in vivo. 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 excess in the living body without stressing the living body to which it is applied. This makes it possible to eliminate active oxygen species and reduce a wide variety of oxidative stresses in living organisms.

That is, the outline of the present invention is as follows.
Item 1.
Antioxidant containing Alpine extract.
Item 2.
Item 2. The antioxidant according to item 1, wherein the Albatross extract is an aqueous extract from a plant of the genus Albatross.
Item 3.
Item 3. The antioxidant according to item 1 or 2, wherein the Album tree extract has an effect of enhancing gene expression of antioxidant-related enzymes and/or redox-related factors.
Item 4.
A group of antioxidant-related enzymes and/or redox-related factors consisting of superoxide dismutase (SOD), peroxiredoxin (PRX), thioredoxin (TXN), thioredoxin reductase (TRD), glutathione reductase (GRD), and glutaredoxin (GXN) Item 4. The antioxidant according to any one of Items 1 to 3, which is at least one selected from the following.
Item 5.
Item 5. The antioxidant according to any one of Items 1 to 4, wherein the Alpine extract has an action of reducing intracellular active oxygen.
Item 6.
Item 5. An external skin preparation containing the antioxidant according to any one of Items 1 to 5.
Section 7.
Item 7. The external preparation for skin according to item 6, which contains the Album tree extract at a concentration of 0.00001 to 5.0% by weight.

By using the antioxidant of the present invention, it is expected that the antioxidant ability in the living body will be improved, and it is also possible to provide an external preparation for skin intended for the effect of improving the antioxidant ability.

FIG. 3 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (SOD) in human normal neonatal epidermal keratinocytes in Example 1. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (TXN) in human normal neonatal epidermal keratinocytes in Example 1. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (GXN) in human normal neonatal epidermal keratinocytes in Example 1. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (SOD) in human normal neonatal fibroblasts in Example 2. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on a target gene (TXN) in human normal neonatal fibroblasts in Example 2. FIG. 3 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (GXN) in human normal neonatal fibroblasts in Example 2. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on a target gene (PRX) in human normal neonatal fibroblasts in Example 2. FIG. 3 is a diagram showing the results of evaluating the expression enhancement effect on target gene (TRD) in human normal neonatal fibroblasts in Example 2. FIG. 2 is a diagram showing the results of evaluating the expression enhancement effect on the target gene (GRD) in human normal neonatal fibroblasts in Example 2. FIG. 3 is a diagram showing the results of evaluating the active oxygen reducing effect in human normal neonatal fibroblasts in Example 3. FIG. 4 is a diagram showing the results of evaluating the expression enhancement effect on antioxidant-related genes in cells collected from living bodies in Example 4.

A specific example of the method for extracting the Album tree extract used in the present invention is shown below. The bark is mainly used as the part of the plant in the extract, but the whole plant can also be used without specifying the bark. Moreover, it goes without saying that the present invention is not limited to the examples described below.

In the present invention, the term "Album extract" refers to the entire body of a plant of the genus Album, as well as its cut products, dried powder, and extracts thereof. This "extract" includes both a state containing an extraction solvent and a state from which the extraction solvent has been removed, and also includes an extract that has been further subjected to a purification treatment after the extraction solvent has been removed.

Album extract is extracted from the plant body of a plant of the genus Album, typically its branches and leaves, and is either fresh or dried and ground, and then water is used as an extraction solvent, preferably heat at 70 to 100°C. It can be obtained by extraction using water or an organic solvent. Extraction can be carried out by a combination of water extraction and organic solvent extraction if necessary, but in consideration of the effects when applied to living organisms, it is preferable to carry out only 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; tetrahydrofuran and diethyl ether Chain and cyclic ethers such as; polyethers such as polyethylene glycol; hydrocarbons such as hexane, cyclohexane, petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridine; oils, fats, waxes, and other oils. Of these, water, alcohols, and water-alcohol mixtures are preferred, and it is particularly preferred to use hot water as the extraction solvent.

In the case of hot water extraction, the extraction is typically carried out by heating and refluxing the crushed plant body of a plant belonging to the genus Album. Extraction conditions vary depending on the solvent used, but for example, when extracting with water, use 1 to 100 parts by weight of water per 1 part by weight of Album, at a temperature of 4 to 100°C, for 10 minutes to 7 days. It is preferable to perform the extraction by using water in an amount of 5 to 20 parts by weight per 1 part by weight of the bark of the tree bark, and it is more preferable to perform the extraction at a temperature of 70 to 100° C. for 30 minutes to 2 hours. Further, from the viewpoint of further increasing the extraction efficiency, pressurization, stirring, ultrasonic treatment, etc. may be performed together with the extraction process. Furthermore, after such extraction treatment, the residue may be separated by filtration or centrifugation.

When the extraction solvent is distilled off under reduced pressure, a black-brown tarry extract is usually obtained from the Album tree extract, and excipients may be added if necessary. When the extraction solvent is non-toxic, for example water, it can be used for preparations while containing the extraction solvent, but the extract from which the extraction solvent has been separated can also be used for preparations. The extract after separation of the extraction solvent can also be redissolved in a suitable solvent, such as water, and used for preparation. In addition, the extract thus obtained can be purified or its titer improved by means of purification, such as fractional extraction, partition between two solvents, fractional adsorption/elution with a suitable adsorbent, or chromatography. .

Although the extract obtained as described above can be used as an antioxidant as it is, it is preferable to use it by blending it into an external preparation. The amount to be added to the external preparation is determined by the degree of absorption, degree of action, product form, frequency of use, etc., and is not particularly limited, but the concentration should be in the range of 0.00001 to 5.0% by dry weight. It is desirable that the amount is in the range of 0.00005 to 3.0% by weight, particularly preferably in the range of 0.0001 to 1.0% by weight. If the content of the Album tree extract is less than 0.00001% by weight, sufficient effects will not be exhibited, and even if 5.0% by weight or more is added, the effects will remain almost constant.

The antioxidant of the present invention contains an extract of Album alba as an active ingredient. Antioxidant action in the present invention refers to the scavenging action of antioxidants contained in the Album tree extract, as well as the scavenging action of antioxidant-related enzymes and/or redox-related factors in the living body by acting on cells and tissues in the living body. It also includes expression enhancing effects, particularly gene expression enhancing effects of antioxidant-related enzymes and redox-related factors. Furthermore, these effects exert an effect of reducing the amount of active oxygen within cells.

In the present invention, the effect of enhancing gene expression of antioxidant-related enzymes and redox-related factors is evaluated by adding the antioxidant of the present invention to cultured cells and quantifying the intracellular gene expression level using real-time PCR. be able to. Furthermore, in the present invention, the amount of intracellular active oxygen is evaluated by incorporating a fluorescent substance that reacts with active oxygen into cultured cells, adding the antioxidant of the present invention, and quantifying the fluorescence intensity emitted from within the cells. can do.

Antioxidant-related enzymes and/or redox-related factors include, for example, superoxide dismutase (SOD), peroxidase, catalase, and peroxiredoxin (PRX) that scavenge reactive oxygen species; oxidation and Glutathione reductase (GRD), thioredoxin reductase (TRD), and glutaredoxin (GXN) that control the reduction state; enzymes that reduce and scavenge radicals; glutathione peroxidase that reduces hydroperoxides generated in lipid oxidation; intermediates with carbonyl groups (4 - α, β-hydrogenases, etc. that reduce hydroxynonenal, etc.; proteins that store and transport transition metal ions to prevent the Fenton reaction (ferritin, transferrin, etc.); proteases, lipases, etc. that degrade molecules that have been damaged by oxidation. , nuclease, chlorophyll degrading enzyme, pheophorbide a oxygenase, etc.; methionine sulfoxide reductase, DNA repair enzyme, etc. which repair molecules oxidized by reactive oxygen species.

In the present invention, the Album tree extract increases the amount of antioxidant-related enzymes and/or redox-related factors in the body, particularly by increasing the gene expression levels of SOD, PRX, TXN, TRD, GRD, and GXN, thereby increasing the amount of antioxidant-related enzymes and/or redox-related factors in the body. It is assumed that it reduces active oxygen generated in the body and exhibits antioxidant ability.

The antioxidant of the present invention can contain other raw materials in addition to the Almania extract to the extent that the effects of the present invention are not impaired. Examples of such ingredients include water, excipients, antioxidants, preservatives, wetting agents, thickening agents, buffers, adsorbents, solvents, emulsifiers, stabilizers, surfactants, lubricants, Examples include water-soluble polymers, sweeteners, flavoring agents, acidulants, alcohols, and the like. Furthermore, the antioxidant of the present invention can contain other active ingredients within a range that does not impair the effects of the present invention.
Specific examples of active ingredients include antioxidant ingredients, anti-aging ingredients, anti-inflammatory ingredients, whitening ingredients, cell activation ingredients, vitamins, blood circulation promoting ingredients, moisturizing ingredients, and DNA damage prevention and/or repair effects. anti-glycation components, peptides or derivatives thereof, amino acids or derivatives thereof, hydroquinone glycosides and esters thereof, and the like.

The antioxidant of the present invention can be used in cosmetics, quasi-drugs, food and drink products, pharmaceuticals, etc., in addition to the essential components of the Album tree extract, if necessary, within the range that does not impair the effects of the present invention. It can be blended with other ingredients and additives.

For example, the oils and fats include avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange laffa oil, sesame oil, cacao 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, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, cocoa butter, palm oil, palm kernel oil, Japanese oak, coconut oil , beef tallow, lard, hydrogenated oil, hydrogenated castor oil, etc.

Examples of waxes include beeswax, carnauba wax, spermaceti wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, and the like.

Examples of the mineral oil include liquid paraffin, vaseline, paraffin, ozokeride, ceresin, microcrystalline wax, polyethylene powder, squalene, squalane, pristane, and the like.

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, isononanoic acid, caproic acid, 2- Examples include synthetic fatty acids such as ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

Alcohols include natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, and phytosterols, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol, and oxidized alcohols. Ethylene, 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, Examples include polyhydric alcohols such as glycerin, batyl alcohol, pentaerythritol, sorbitol, mannitol, glucose, and sucrose.

Esters include isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctoate, cetyl lactate, myristyl lactate, Examples include diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, and the like.

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

Gummy and water-soluble polymer compounds include gum arabic, benzoin gum, dammar gum, guaiac butter, Irish moss, karaya gum, tragacanth gum, carob gum, quinseed, agar, casein, dextrin, gelatin, pectin, starch, carrageenan, carboxyalkyl Chitin, chitosan, hydroxyalkyl chitin, low molecular 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, carboxy Sodium ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylate, polyalkylene oxide such as polyethylene oxide and polypropylene oxide, or crosslinked polymers thereof , carboxyvinyl polymer, polyethyleneimine, and the like.

Examples of surfactants include anionic surfactants (carboxylate, sulfonate, sulfate ester salt, phosphate ester salt), cationic surfactant (amine salt, quaternary ammonium salt), amphoteric surfactant (carboxylic acid ester salt, phosphate ester salt), cationic surfactant (amine salt, quaternary ammonium salt), type amphoteric surfactants, sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants), nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants) ionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants (natural surfactants, protein hydrolyzate derivatives, Polymer surfactants, titanium/silicon-containing surfactants, fluorocarbon surfactants), etc.

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

Amino acids include valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, and arginine. , ornithine, histidine, etc., their sulfates, phosphates, nitrates, citrates, and amino acid derivatives such as pyrrolidone carboxylic acid.

Whitening agents include ascorbic acid or its derivatives, sulfur, placenta hydrolyzate, 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, Scutellariae extract, Scutellariae extract, Saiko extract, Bofuu extract, Mycelium mycelium culture or its extract, Linna extract, Peach leaf extract, Eizu extract, Kujin extract, Jiyu extract, Angelica extract, Yokuinin extract, Persimmon leaf extract, Rhubarb extract , botanical extract, Hamamelis extract, horse chestnut extract, Hypericum perforatum extract, oil-soluble licorice extract, and the like.

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, and sphingophospholipids. , glycosphingolipid, linoleic acid or its esters, eicosapentaenoic acid or its esters, pectin, bifidobacteria fermented product, lactic acid fermented product, yeast extract, Ganoderma mycelium culture or its extract, wheat germ oil, avocado Examples include oil, rice germ oil, jojoba oil, soybean phospholipid, γ-oryzanol, velvet hyacinth extract, yokuinin extract, rhododendron extract, turmeric extract, turmeric extract, aloe extract, burdock extract, stone wax extract, arnica extract, wheat bran, and the like.

Hair growth agents include pentadecanoic acid glyceride, Coleus extract, Gentiana extract, Pinus cucumber extract, royal jelly extract, Kumazasa extract, t-flavanone, 6-benzylaminopurine, Jasperia japonica extract, carpronium chloride, minoxidil, finasteride, adenosine, nicotinamide, Examples include mulberry root extract, rhubarb extract, 5-aminolevulinic acid, and the like.

Extracts and extracts from animals, plants, and herbal medicines include Asenyaku, Ashitaba, Acerola, Althea, Arnica, Avocado, Amacha (Sweet Tea), Aloe, Aloe Vera, Nettle, Ginkgo Biloba, and Fennel ( Ingredients), Turmeric, Usubasaishin, Plum, Ujilogashi, Uwaurushi, Noibara, Hikiokoshi, Astragalus, Scutellaria, Yamazakura (cherry bark) ), yellowfin tuna (yellow oak), oren (yellow lily), panax ginseng (carrot), hypericum (brother cut grass), dead manflower, orchid, orange, yellow tuna (toshi), nepenthes (summer dry grass), tsudokudami (seven-headed crow), ange ), mugwort, gajutsu, kudzu, valerian, chamomile, currant gourd, mugwort, licorice, coltsfoot , raspberry, kiwi fruit, bellflower (bellflower), chrysanthemum (chrysanthemum), catalpa (catalpa), citrus fruit (continent), tachibana (tachibana peel), cucumber, udon or shishuido (long life, solitary life) , apricot (almond kernel), wolfberry (bone bark, nutmeg, nutmeg leaf), clara (bitter ginseng), camphor tree, kumazasa, grapefruit fruit, Nikkei (cinnamon bark), keigai (keigai), Ebisugusa (keiko), morning glory or Morning glory, safflower, gobaishi, comfrey, copaiba, gardenia, gentian, great tree, ox-knee, goshuyu, burdock, butterfly Misi, rice, rice bran, wheat, saiko, saffron, soapwort, hawthorn, sansho, salvia, ginseng, shiitake, Rhizophrenia, Shikunshi, Murasaki, Shiso, Persimmon, Peony, Plantain, Ginger, Calamus (iris), Japanese white birch (Female Sadako), meadowsweet, white birch, honeysuckle (Kinginka, Ninfuyu), Ivy, Yarrow, Sambucus, Azuki (red adzuki bean), Elderberry (boned tree), mallow, nebula (river cucumber) ), Aspergillus (this drug), Mulberry (mulberry bark, mulberry leaf), Jujube (large jujube), Soybean, Arata tree, Chikusetsu ginseng (Bamboo knot ginseng), Hanasuge (Chimo), Waremokou (Earth elm), Dokudami ( (10 medicines), Cordyceps sinensis (Cordyceps sinensis), chili pepper, Physalis (Torone), Thachimusou, Ryokucha (green tea), Koucha (black tea), Chouji (clove), Unshu mandarin orange (Chenpi), Camellia, Centella asiatica, Capsicum (Chinese peel) Japanese eucommia), Eucommia (Japanese persimmon), Japanese calendula (orange-skinned), Japanese elm (Japanese elm), corn (Nanbanmo), Eucommia (duchu, Eucommia), tomato, nandina (nantenji), garlic (large) barley (malt), hakusen (white moss), janohige (barley), parsley, batata, peppermint (thin), hamamelis, rose, loquat leaf (loquat leaf), pine hodo (buruyo), grape or Leaves, loofah, bodaiju, peonies, hops, maikai, pine needles, horse chestnut, stonecrop, Japanese starch, melissa, melilot, boké, bean sprouts, peaches, peach leaves, areca nut), areca nut, amethyst grass, knapweed, saxifrage (tiger ear grass), bayberry (yangbai skin), cornflower (cornhorn), pigeon berry (yokuinin), mugwort, sagebrush, lavender, apple fruit, stone mushroom (reishi), lemon fruit, forsythia, astragalus, gennoshoko, hashiridokoro (funnel root), chicken crest, cow/human placenta extract, pig/cow stomach, duodenum, or intestine. Extracts or decomposition products thereof, water-soluble collagen, water-soluble collagen derivatives, collagen hydrolysates, elastin, elastin hydrolysates, water-soluble elastin derivatives, silk proteins, silk protein decomposition products, bovine blood cell protein decomposition products, etc. It will be done.

Examples of microbial culture metabolites include yeast extract, zinc-containing yeast extract, germanium-containing yeast extract, selenium-containing yeast extract, magnesium-containing yeast extract, fermented rice extract, Euglena extract, lactic acid fermented product of skim milk powder, and the like.

Examples of α-hydroxy acids include glycolic acid, citric acid, malic acid, tartaric acid, and lactic acid.

Inorganic pigments include anhydrous silicic acid, magnesium silicate, talc, kaolin, bentonite, mica, titanium mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium carbonate, yellow iron oxide, Examples include red iron oxide, black iron oxide, gunjo, chromium oxide, chromium hydroxide, carbon black, and calamine.

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

Astringents include lactic acid, tartaric acid, succinic acid, citric acid, allantoin, zinc chloride, zinc sulfate, zinc oxide, calamine, zinc p-phenolsulfonate, potassium aluminum sulfate, resorcin, ferric chloride, tannic acid, etc. Can be mentioned.

Antioxidants include ascorbic acid and its salts, stearate esters, tocopherol and its ester derivatives, nordihydroguacerethenic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), parahydroxyanisole, propyl gallate. , sesamol, sesamolin, gossypol and the like.

Anti-inflammatory agents include ictamol, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d- or dl-camphor, hydrocortisone, guaiazulene, chamazulene, chlorpheniramine maleate, glycyrrhizic acid and its salts. , glycyrrhetinic acid and its salts.

Examples of sterilizing/disinfecting agents include acrinol, sulfur, benzalkonium chloride, benzethonium chloride, methylrosaniline chloride, cresol, calcium gluconate, chlorhexidine gluconate, sulfamine, mercurochrome, lactoferrin or its hydrolyzate.

Hair preparations include selenium disulfide, alkylisoquinolinium bromide solution, zinc pyrithione, biphenamine, thianthol, castari tincture, ginger tincture, capsicum tincture, quinine hydrochloride, strong aqueous ammonia, potassium bromate, sodium bromate, thio Examples include glycolic acid.

Fragrances include natural animal fragrances such as musk, civet, castoreum, and ambergris, anise essential oil, angelica essential oil, ylang essential oil, iris essential oil, fennel essential oil, orange essential oil, cananga essential oil, caraway essential oil, cardamom essential oil, guaiac wood essential oil, and cumin. Essential oils, Kuromoji essential oil, cinnamon essential oil, cinnamon essential oil, geranium essential oil, copaiba balsam essential oil, coriander essential oil, perilla essential oil, cedarwood essential oil, citronella essential oil, jasmine essential oil, ginger grass essential oil, cedar essential oil, spearmint essential oil, peppermint essential oil, large Boar essential oil, tuberose essential oil, clove essential oil, orange blossom essential oil, wintergreen essential oil, true balsam essential oil, baturi essential oil, rose essential oil, palmarosa essential oil, cypress essential oil, cypress essential oil, sandalwood essential oil, petitgrain essential oil, bay essential oil, vetiva essential oil, bergamot essential oil , Peruvian balsam essential oil, boad rose essential oil, camphor essential oil, mandarin essential oil, eucalyptus essential oil, lime essential oil, lavender essential oil, linaloe essential oil, lemongrass essential oil, lemon essential oil, rosemary essential oil, Japanese peppermint essential oil, and other synthetic botanical fragrances. Examples include fragrances and the like.

Pigments and coloring agents include red cabbage pigment, red rice pigment, madder pigment, annatto pigment, squid ink pigment, turmeric pigment, ange pigment, krill pigment, persimmon pigment, caramel, gold, silver, gardenia pigment, corn pigment, and onion pigment. , tamarind pigment, spirulina pigment, buckwheat whole plant pigment, cherry pigment, seaweed pigment, hibiscus pigment, grape juice pigment, marigold pigment, purple sweet potato pigment, purple yam pigment, lac pigment, rutin, and the like.

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

Examples of nutritional enrichers include calcined shell calcium, cyanocolabamine, yeast, wheat germ, soybean germ, egg yolk powder, hemicellulose, heme iron, and the like.

In addition, hormones, sequestering agents, pH adjusters, chelating agents, preservatives and anti-bacterial agents, cooling agents, stabilizers, emulsifiers, animal and vegetable proteins and their decomposition products, animal and vegetable polysaccharides and their Decomposition products, animal and vegetable glycoproteins and their decomposition products, blood flow promoters, anti-inflammatory and anti-allergic agents, cell activators, keratolytic agents, wound treatment agents, foaming agents, thickeners, oral preparations, Examples include deodorants/deodorants, bittering agents, seasonings, enzymes, etc.

The dosage form of the present invention is arbitrary and may be ampoule, capsule, powder, granule, pill, tablet, solid, liquid, gel, foam, emulsion, cream, ointment, or sheet. , quasi-drugs such as mousse, cosmetics for skin and hair, bath preparations, foods and beverages, and pharmaceuticals.

Specifically, cosmetics and quasi-drugs include, for example, medicinal preparations for internal and external use, basic cosmetics such as lotions, emulsions, creams, ointments, lotions, oils, and packs, face washes and skin cleansers, Shampoos, conditioners, hair treatments, hair creams, pomades, hair sprays, hair styling products, perming agents, hair tonics, hair dyes, hair cosmetics such as hair growth and nourishing products, foundations, whitening powders, face powder, lipsticks, blushers, eye shadows, Make-up cosmetics such as eyeliner, mascara, eyebrow ink, and eyelashes, finishing cosmetics such as nail polish, perfumes, bath additives, toothpaste, mouth fresheners and gargling agents, liquid odor and deodorizing agents, Examples include sanitary products, sanitary cotton, wet tissues, etc.

Examples of food and beverages include soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, frozen desserts such as ice cream, ice sherbet, shaved ice, soba, udon, hazelnut noodles, gyoza wrappers, shumai wrappers, and Chinese food. Noodles, instant noodles and other noodles, candy, candy, gum, chocolate, tablets, snack foods, biscuits, jelly, jam, cream, baked goods, bread and other confectionery, crab, salmon, clams, tuna, sardines, shrimp, Marine products such as bonito, mackerel, whale, oyster, saury, squid, red clam, scallop, abalone, sea urchin, salmon roe, tokobushi, processed seafood/livestock foods such as kamaboko, ham, sausage, milk such as powdered milk, processed milk, fermented milk, etc. Products, oils and fats and processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings, seasonings such as sauces and sauces, curry, stew, oyakodon, rice porridge, rice porridge, Chinese rice bowl, and katsudon , Tendon, Unadon, Hayashi rice, Oden, Marbodorf, Gyudon, Meat sauce, Egg soup, Omelet rice, Gyoza, Shumai, Hamburger, Meatball, etc., retort pouch foods, various forms of health/nutritional supplements, health functional foods, tablets , capsules, drinks, troches, etc.

Although the antioxidant of the present invention is preferably applied to humans, it can also be applied to animals other than humans as long as the respective effects can be expected.

Embodiments of the antioxidant of the present invention are not particularly limited, but include, but are not limited to, facial cleansers, cleansers, lotions (e.g., whitening lotions), creams (e.g., vanishing creams, cold creams), milky lotions, gels, and serums. , packs (e.g. jelly peel-off type, paste wipe type, powder wash-off type), face masks, cleansers, foundations, lipsticks, lip balms, lip glosses, lip liners, blushers, makeup bases, shaving lotions, sunscreens, Examples include after-sun lotion, deodorant lotion, body lotion (including hand care lotion and foot care lotion), and body oil.

Hereinafter, the present invention will be explained in more detail based on Examples. Note that the present invention is not particularly limited to the examples.

The aqueous extract of Nemunoki bark used in the following examples was prepared by the method disclosed in JP-A No. 2018-58783.

Example 1: Evaluation of the expression-enhancing effect on target genes in human normal neonatal epidermal keratinocytes Using the above-mentioned Album tree extract as a test sample, the expression-enhancing effect on target genes was evaluated by the following test method. Target genes in this example mean SOD, PRX, TXN, TRD, GRD, and GXN.

Human normal neonatal epidermal keratinocytes (NHEK) were seeded in a T75 flask coated with collagen using Collagen Coating Solution (manufactured by Toyobo Co., Ltd.), and normal human epidermal keratinocyte growth medium (HuMedia-KG2) was added. The cells were cultured at 37°C and 10% _CO2 . The culture medium was replaced as appropriate, and the culture was continued until it became 80% or more confluent. After removing the medium and rinsing with PBS, cells were collected by trypsin treatment. Cells were seeded in a 96-well plate at 1×10 ⁴ cells/well and cultured for 1 day at 37° C. and 10% CO ₂ .
The medium was removed, replaced with a medium containing the test sample at a predetermined concentration, and incubated at 37° C. and 10% CO ₂ 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.).

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 according to the attached document. However, the reaction solution volume was 20 μl/well, and the cDNA amount was 3 μl. The reaction cycle conditions were 95°C, 1 minute → (95°C, 15 seconds → 60°C, 45 seconds)×40 → 95°C, 15 seconds → 60°C, 1 minute → 95°C, 15 seconds. The base sequences shown in SEQ ID NOs: 1 to 6 were used as primers. The equipment 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 levels of the target genes (SEQ ID NOS: 13-14). The standardized expression level of each target gene was divided by the gene expression level of a negative control that did not contain the test sample, and a relative value was calculated.

The results obtained are shown in Figures 1 to 3. As can be seen from Figures 1 to 3, it was confirmed that the gene expression levels of antioxidant-related enzymes and/or redox-related factors were improved in NHEK compared to the negative control to which no Album tree extract was added. Ta. From the present results, it was revealed that the Album tree 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.

Example 2: Evaluation of expression enhancement effect on target genes in human normal neonatal fibroblasts Human normal neonatal fibroblasts (NHDF) were seeded in a T75 flask, and D-MEM (+10% FBS) was added. The cells were cultured at 37° C. and 5% CO ₂ . The culture medium was replaced as appropriate, and the culture was continued until it became 80% or more confluent. After removing the medium and rinsing with PBS, cells were collected by trypsin treatment. Cells were seeded in a 96-well plate at 1×10 ⁴ cells/well and cultured for 1 day at 37° C. and 5% CO ₂ .
The medium was removed, replaced with a medium containing the test sample at a predetermined concentration, and incubated at 37° C. and 5% CO ₂ 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.).

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 according to the attached document. However, the reaction solution volume was 20 μl/well, and the cDNA amount was 3 μl. The reaction cycle conditions were 95°C, 1 minute → (95°C, 15 seconds → 60°C, 45 seconds)×40 → 95°C, 15 seconds → 60°C, 1 minute → 95°C, 15 seconds. The base sequences shown in SEQ ID NOs: 1 to 12 were used as primers. The equipment 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 levels of the target genes (SEQ ID NOS: 13-14). The standardized expression level of each target gene was divided by the gene expression level of a negative control that did not contain the test sample, and a relative value was calculated.

The results obtained are shown in Figures 4 to 9. As can be seen from FIGS. 4 to 9, it was confirmed that the gene expression levels of antioxidant-related enzymes and redox-related factors were improved in NHDF compared to the negative control to which no Album tree extract was added. From the present results, it was revealed that the Album tree 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.

Example 3: Evaluation of active oxygen reducing effect in human normal neonatal fibroblasts Human normal neonatal fibroblasts (NHDF) were seeded in a T75 flask, D-MEM (+10% FBS) was added, and 37 The cells were cultured at 5% _CO2 . The culture medium was replaced as appropriate, and the culture was continued until it became 80% or more confluent. After removing the medium and rinsing with PBS, cells were collected by trypsin treatment. Cells were seeded in a 96-well plate at 3×10 ⁴ cells/well and cultured for 1 day at 37° C. and 5% CO ₂ .
After the culture was removed and washed with HBSS, it was replaced with 20 μM H ₂ DCFDA (2',7'-dichlorodihydrofluorescein diacetate) and incubated at 37° C. and 5% CO ₂ (in the dark) for 30 minutes. H ₂ DCFDA was removed and washed with HBSS, the test sample and hydrogen peroxide were replaced with HBSS containing a specified concentration, and further incubated at 37° C. and 5% CO ₂ (in the dark). After 5 hours, the fluorometer was used. (ARVO MX) and measured the fluorescence intensity at Ex/Em=485 nm/535 nm.

The obtained results are shown in FIG. As can be seen from FIG. 10, in NHDF, the effect of reducing intracellular active oxygen induced by oxidative stress due to hydrogen peroxide was confirmed. This is due to the fact that in addition to the antioxidant effect contained in the Album tree extract, gene expression of antioxidant-related enzymes and/or redox-related factors is enhanced, which improves the body's own active oxygen scavenging ability and increases the in-vivo activity. This is thought to have led to a reduction in oxygen levels.

Example 4: Evaluation of the effect of enhancing expression on antioxidant-related genes in cells collected from a living body Using an aqueous solution containing 2.0% by weight of the above-mentioned Nemunoki extract as a test sample, the effect of enhancing expression on target genes was evaluated by the following test method. did. The target gene in this example means PRX.

The test sample was applied to the subject's face, and cells were obtained from the beard in the applied area for evaluation. Specifically, before and 3 hours after application of the test sample, the beard in the application area was removed from the subject and hair follicle cells were collected. To preserve the cells, they were immersed in RNAlater (manufactured by Qiagen) and stored at 4°C until RNA extraction. After collecting the hair follicle cells, total RNA was extracted from the hair follicle cells using an RNeasy plus micro kit (manufactured by Qiagen) according to the attached protocol. cDNA was prepared from the extracted total RNA 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 according to the attached document. However, the reaction solution volume was 20 μl/well, and the cDNA amount was 2 μl. The reaction cycle conditions were 95°C, 1 minute → (95°C, 15 seconds → 60°C, 45 seconds)×40 → 95°C, 15 seconds → 60°C, 1 minute → 95°C, 15 seconds. The base sequences shown in SEQ ID NOs: 7 and 8 were used as primers. The equipment 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 levels of the target genes (SEQ ID NOS: 13-14). The standardized expression level of each target gene was divided by the gene expression level in the negative control before application of the test sample, and a relative value was calculated.

The results obtained are shown in FIG. As can be seen from Figure 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 compared to the negative control before application of Album tree extract. Ta. From these results, it is clear that the Album tree extract has the effect of enhancing the gene expression of antioxidant-related enzymes and/or redox-related factors even in cells collected from living organisms, and improving the antioxidant capacity in vivo. became.

Example 5: Prescription of external skin preparation The following is a formulation example of the external skin preparation of the present invention. All of these preparations are expected to have the effect of enhancing the expression of antioxidant-related enzyme genes due to the Album extract, and are effective as antioxidants.

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

Gel A gel was manufactured by a conventional method according to the following composition.
・Purified water...88.50g
・Carbomer...0.30g
・Xanthan gum...0.10g
・Arginine...0.40g
・Glycerin...5.00g
・Phenoxyethanol...0.20g
・Butylene glycol...5.00g
・Nemnoki extract...0.50g

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

According to the present invention, by significantly promoting the expression of antioxidant-related enzymes and/or redox-related factors in the living body, oxidative stress in the living body can be safely and efficiently reduced, health can be maintained, and oxidative stress It is possible to prevent various diseases and symptoms caused by , and it is particularly useful when applied to skin external preparations as cosmetics and quasi-drugs.

Claims (6)

1. A method for producing an extract of Almana tree bark for use as an antioxidant (excluding therapeutic actions on humans), comprising the step of extracting it from Almighty tree bark with water. 2. The method for producing the Album tree bark extract according to claim 1, wherein the extract is used as an antioxidant (excluding treatment for humans) by enhancing gene expression of antioxidant-related enzymes and/or redox-related factors. A group of antioxidant-related enzymes and/or redox-related factors consisting of superoxide dismutase (SOD), peroxiredoxin (PRX), thioredoxin (TXN), thioredoxin reductase (TRD), glutathione reductase (GRD), and glutaredoxin (GXN) 3. The method for producing a barley bark extract according to claim 2, wherein the extract is at least one selected from the following. 4. The method for producing an Album tree bark extract according to any one of claims 1 to 3, wherein the Album tree bark extract has an effect of reducing intracellular active oxygen. 5. A method of use as an external skin preparation (excluding therapeutic treatment for humans), which is a skin preparation containing the Album tree bark extract according to any one of claims 1 to 4. 6. The method of use as an external preparation for skin (excluding therapeutic treatment for humans) according to claim 5, which contains the Album tree bark extract at a concentration of 0.00001 to 5.0% by weight.