WO2020054204A1

WO2020054204A1 - Antioxidant

Info

Publication number: WO2020054204A1
Application number: PCT/JP2019/027489
Authority: WO
Inventors: 知宏菅原
Original assignee: 東洋紡株式会社
Priority date: 2018-09-14
Filing date: 2019-07-11
Publication date: 2020-03-19
Also published as: CN112367969A; JP2023171681A; JPWO2020054204A1

Abstract

Provided is 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 to thereby maintain good health and prevent various diseases and symptoms caused by oxidative stress. The 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.

Description

Antioxidant

(4) The present invention relates to an antioxidant containing a Neem tree extract as an active ingredient. In particular, the present invention relates to an antioxidant which is useful for improving the antioxidant ability of a cell by acting on the cell.

In recent years, various active oxygen species have attracted attention as a factor for oxidizing 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 that have increased chemical reactivity compared to steady-state oxygen molecules, and are generated during energy metabolism in vivo. The reactive oxygen species include superoxide (.O ^2- ), hydroxy radical (.OH), hydroperoxy radical (HHO.), 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 are second messengers of the signal transduction system in the living body, and are molecular species indispensable for vital activities such as control of cell growth / differentiation and bactericidal action in macrophages and the like. On the other hand, excessive reactive oxygen species damage cells and tissues and are considered harmful.

生体 The living body has a reactive oxygen species production suppression mechanism as an antioxidant mechanism that protects against oxidative stress caused by these excessive reactive oxygen species. The mechanism for suppressing the production of reactive oxygen species is mainly composed of antioxidant-related enzymes such as catalase and superoxide dismutase (SOD) glutathione peroxidase (GPX), and suppresses reactive oxygen species by an enzymatic reaction. Active oxygen species are transferred from superoxide to hydrogen peroxide and hydroxyl radical. In the process, SOD erases superoxide and catalase erases hydrogen peroxide. In addition, GPX, glutathione reductase (GRD), thioredoxin reductase (TRD), and peroxiredoxin (PRX) control hydrogen peroxide and hydroxyl radical by controlling the oxidation / reduction state of glutathione (GSH) and thioredoxin (TXN). To delete. By working in concert with these antioxidant-related enzymes and redox-related factors, the amount of reactive oxygen species in cells is kept at an appropriate level.

Thus, 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 the living body. It is known that these antioxidant-related enzymes and redox-related factors are enhanced by ultraviolet irradiation stress.However, components that act on living organisms safely and effectively and promote the expression of antioxidant-related enzymes and redox-related factors are included. Expected.

As such components, for example, it has been reported that kaempferol and quercetin belonging to flavonols widely distributed in tea leaf plants enhance the expression and activity of glutathione and thioredoxin systems in vivo (Patent Document 1). .

Searching for components that act safely and effectively on antioxidant-related enzymes and / or redox-related factors in the living body is not limited to providing safer and more effective components to the market. New ingredients that act on antioxidant-related enzymes and / or redox-related factors are important from the viewpoints of formulation diversification, expansion of formulation options, and synergistic effects of using different ingredients in combination. Needless to say, there is a need for development.

On the other hand, Japanese Alderwood is a legume deciduous tree that grows naturally in sunny wetlands such as forest margins and moorlands in the mountains south of the Tohoku region in Japan, and is also distributed overseas in China, Southeast Asia, etc. Cultivated. The bark collected during the flowering season, washed with water, dried in the sun and cut to an appropriate length is called happi skin, and is taken in the private sector in anticipation of arthritis, back pain, diuresis, edema, tonic, For external use, it has been used as a cold compress for affected areas with decoction for tumors, bruises and arthralgias, or as bath water. It is known that bark contains triterpene-based saponins (many julibrosides), flavonoids (geraldone, isookanin, luteolin, etc.), tannins and the like. (Non-Patent Document 1)

As the effects on the living body by the Alnus serrata, active oxygen scavenging activity and aldose reductase inhibitory activity have been reported that use components derived from plants of the family Sorghum spp. As active ingredients.However, this effect focuses on the active oxygen scavenging activity. Is not a kind of stimulating antioxidant-related enzymes and redox-related factors in living organisms to achieve scavenging of active oxygen (Patent Document 2). In addition, although the melanin production inhibitory effect and the collagen production promotion effect of skin of the Alnus bark extract have been reported, there is no mention of antioxidant-related factors (Patent Document 3).

Japanese Patent No. 5833438 JP-A-2000-128798 Japanese Patent No. 5,253,862

In view of the above background, the present invention safely and efficiently reduces oxidative stress in a living body by promoting the expression of antioxidant-related enzymes and / or redox-related factors in a living body, maintains health, and An object is to prevent various diseases and symptoms caused by oxidative stress.

In view of the above circumstances, the inventors of the present invention have conducted intensive studies and, as a result, have found that the extract of Alnus has an antioxidant effect, and have completed the present invention. It has been found that the extract of Alnus albicans 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 an antioxidant-related enzyme and / or a redox-related factor in a living body, excessive stress in the living body can be obtained without giving stress to the applied living body. It is possible to eliminate various reactive oxygen species and reduce various oxidative stresses in a living body.

That is, the outline of the present invention is as follows.
Item 1.
An antioxidant containing a Neem tree extract.
Item 2.
Item 2. The antioxidant according to Item 1, wherein the extract of Albinia is an aqueous extract from a plant of the Alnus plant.
Item 3.
Item 3. The antioxidant according to item 1 or 2, wherein the extract of Alnus serrata has an effect of enhancing gene expression of antioxidant-related enzymes and / or redox-related factors.
Item 4.
The antioxidant-related enzyme and / or redox-related factor is a group 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 group consisting of:
Item 5.
Item 5. The antioxidant according to any one of Items 1 to 4, wherein the extract of Alnus has an effect of reducing intracellular active oxygen.
Item 6.
Item 7. An external preparation for skin comprising the antioxidant according to any one of Items 1 to 5.
Item 7.
Item 7. An external preparation for skin according to Item 6, wherein the extract of Alnus serrata is contained at a concentration of 0.00001 to 5.0% by weight.

使用 By using the antioxidant of the present invention, the antioxidant ability in a living body can be expected to be improved, and a skin external preparation for the purpose of improving the antioxidant ability can also be provided.

FIG. 4 is a diagram showing the results of an evaluation of the effect of enhancing expression of a target gene (SOD) in human normal neonatal epidermal keratinocytes in Example 1. FIG. 4 is a diagram showing the results of an evaluation of the effect of enhancing expression of a target gene (TXN) in human normal neonatal epidermal keratinocytes in Example 1. FIG. 3 is a diagram showing the results of an evaluation of the effect of enhancing expression of a target gene (GXN) in human normal neonatal epidermal keratinocytes in Example 1. It is a figure which shows the result of having evaluated the expression enhancement effect with respect to a target gene (SOD) in a normal human neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the expression promotion effect with respect to a target gene (TXN) in a human normal neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the expression promotion effect with respect to a target gene (GXN) in a human normal neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the expression enhancement effect with respect to a target gene (PRX) in a human normal neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the expression promotion effect with respect to a target gene (TRD) in a human normal neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the expression enhancement effect with respect to a target gene (GRD) in a human normal neonatal fibroblast in Example 2. It is a figure which shows the result of having evaluated the active oxygen reduction effect in a normal human neonatal fibroblast in Example 3. It is a figure which shows the result of having evaluated the expression enhancement effect with respect to the antioxidant related gene in the cell collect | recovered from the living body in Example 4.

具体 Specific examples of the extraction method of the Alnus extract used in the present invention are shown below. Although the bark is mainly used as the site of the plant in the extract, the whole plant can be used without specifying the bark. It goes without saying that the present invention is not limited to the examples described below.

ネ In the present invention, the Alnus extract means not only a plant of the Alnus genus plant, but also a cut product, a dry powder, and an extract thereof. The “extract” includes both the state containing the extraction solvent and the state where the extraction solvent has been removed, and also includes the one that has been subjected to a purification treatment after the extraction solvent has been removed.

Neem tree extract is extracted from a plant of the genus Neem tree, typically the branches and leaves thereof, raw or dried and pulverized, and then used as an extraction solvent with water, preferably at a temperature of 70 to 100 ° C. It is obtained by performing extraction using water or an organic solvent. Extraction can be performed by combining water extraction and organic solvent extraction, if necessary. However, in consideration of the effect when applied to a living body, it is preferable to perform 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 polyethylene glycol; polyethers such as polyethylene glycol; hydrocarbons such as hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridines; fats and oils, waxes and other oils. Of these, water, alcohols, and a water-alcohol mixture are preferable, and it is particularly preferable to use hot water as the extraction solvent.

(4) In the case of hot water extraction, typically, a ground plant of the Alnus genus plant is extracted under heating and reflux. Although the extraction conditions vary depending on the solvent used, for example, in the case of extraction with water, 1 to 100 parts by weight of water is used for 1 part by weight of Almond at a temperature of 4 to 100 ° C. for 10 minutes to 7 days. Extraction is preferably performed, and more preferably, using 5 to 20 parts by weight of water with respect to 1 part by weight of bark of Alnus oleracea at a temperature of 70 to 100 ° C. for 30 minutes to 2 hours. Further, from the viewpoint of further improving the extraction efficiency, pressurization, stirring, ultrasonic treatment and the like may be performed together with the extraction processing. Furthermore, after the extraction treatment, the residue may be separated from the residue by filtration or centrifugation.

When the extraction solvent is distilled off under reduced pressure, it is usual to give a black-brown extract in the form of tar, and an excipient may be added as required. When the extraction solvent is non-toxic, for example, water, it can be used in the preparation while containing the extraction solvent, but the extract obtained by separating the extraction solvent can also be used in the preparation. The extract after separation of the extraction solvent can be used again for preparation by redissolving it in a suitable solvent, for example, water. The extract thus obtained can be purified or improved in titer by a purification means, for example, fractional extraction, partitioning between two solvents, fractional adsorption / elution with an appropriate adsorbent or chromatography. .

ネ The extract of Astragalus oleracea obtained as described above can be used as an antioxidant as it is, but it is preferable to use it by blending it with an external preparation. The amount added to the external preparation is determined by the degree of absorption, the degree of action, the form of the product, the frequency of use, etc., and is not particularly limited, but the concentration is in the range of 0.00001 to 5.0% by weight on a dry weight basis. It is 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 extract is less than 0.00001% by weight, a sufficient effect is not exhibited, and the effect is almost constant even if added at 5.0% by weight or more.

(4) The antioxidant of the present invention contains a Neem tree extract as an active ingredient. The antioxidant effect in the present invention means that, when acted on cells and tissues in a living body, in addition to the scavenger effect of the antioxidant contained in the Alnus serrata extract, the antioxidant-related enzyme and / or redox-related factor in the living body is reduced. It also includes the effect of enhancing expression, particularly the effect of enhancing gene expression of antioxidant-related enzymes and redox-related factors. In addition, these actions exert an action of reducing the amount of active oxygen in cells.

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

Examples of antioxidant-related enzymes and / or redox-related factors include superoxide dismutase (SOD), which eliminates reactive oxygen species, peroxidase, catalase, peroxiredoxin (PRX); and the oxidation and oxidation of glutathione (GSH) and thioredoxin (TXN). Glutathione reductase (GRD), thioredoxin reductase (TRD), glutaredoxin (GXN) that controls the reduction state; an enzyme that reduces and eliminates radicals; glutathione peroxidase that reduces hydroperoxide generated by lipid oxidation, an intermediate having a carbonyl group (4) Α, β-hydrogenase, etc. that reduces -hydroxynonenal; proteins that store and transport transition metal ions so that the Fenton reaction does not occur (ferritin, transferrin, etc.); oxidation Degrading proteases molecule that has undergone a wound, lipase, nuclease, chlorophyll degradation enzyme, pheophorbide a oxygenase and the like; methionine sulfoxide reductase that repair molecules oxidized by the active oxygen species include DNA repair enzymes and the like.

According to the present invention, the extract of Neem tree can enhance the amount of antioxidation-related enzymes and / or redox-related factors in a living body, particularly by enhancing the gene expression levels of SOD, PRX, TXN, TRD, GRD and GXN. It is presumed that it reduces active oxygen generated in the body and exerts antioxidant ability.

抗 The antioxidant of the present invention can contain other raw materials other than the extract of T. alder, within a range not to impair the effects of the present invention. Examples of such raw materials include water, excipients, antioxidants, preservatives, wetting agents, thickeners, buffers, adsorbents, solvents, emulsifiers, stabilizers, surfactants, lubricants, Water-soluble polymers, sweeteners, flavoring agents, acidulants, alcohols and the like can be mentioned. 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 ingredient include, for example, an antioxidant ingredient, an anti-aging ingredient, an anti-inflammatory ingredient, a whitening ingredient, a cell activating ingredient, a vitamin, a blood circulation promoting ingredient, a moisturizing ingredient, and a preventive and / or repair action of DNA damage. Components, 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 is used in cosmetics, quasi-drugs, foods and beverages, pharmaceuticals, and the like, as long as the effects of the present invention are not impaired, as necessary, in addition to the essential components of the Alnus extract. Components and additives in combination.

For example, as fats and oils, avocado oil, almond oil, fennel oil, sesame oil, olive oil, orange oil, orange raffer oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, tallow, tallow fatty acid, kukuinut 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, mocro, coconut oil , Beef tallow, lard, hardened oil, hardened castor oil and the like.

(5) Examples of waxes include beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax and the like.

Mineral oils include liquid paraffin, vaseline, paraffin, ozokeride, ceresin, microcrystalline wax, polyethylene powder, squalene, squalane, pristane and the like.

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.

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, and 2-octyldodecanol; 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, Glycerin, bacilia Call, pentaerythritol, sorbitol, mannitol, glucose, polyhydric alcohols such as sucrose, and the like.

Esters include isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyl dodecyl myristate, hexyl decyl dimethyloctanoate, cetyl lactate, myristyl lactate, Examples include diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, and propylene glycol dioleate.

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

As gums and water-soluble polymer compounds, gum arabic, benzoin gum, dammar gum, guaiac oil, Irish moss, karaya gum, tragacanth gum, carob gum, quinseed, 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, carboxy Ethyl cellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl chloride Alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylic acid salts, polyalkylene oxide or crosslinked polymer thereof such as polyethylene oxide or polypropylene oxide, carboxyvinyl polymer, polyethyleneimine and the like.

Examples of the surfactant include anionic surfactants (carboxylates, sulfonates, sulfates, phosphates), cationic surfactants (amine salts, quaternary ammonium salts), and amphoteric surfactants (carboxylic acid) Amphoteric surfactants, sulfate ester amphoteric surfactants, sulfonic acid amphoteric surfactants, phosphate ester amphoteric surfactants), nonionic surfactants (ether nonionic surfactants, etherester nonionic surfactants) Ionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, Polymer surfactants, surfactants containing titanium and silicon, and fluorocarbon surfactants).

As vitamins, retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A) in the vitamin A group, thiamine hydrochloride, thiamine sulfate (vitamin B1) in the vitamin B group, Riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group, ascorbic acid and its derivatives, vitamin D group Ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotaxerol, vitamin E group, tocopherol and its derivatives, ubiquinones, 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, arginine , Ornithine, histidine and the like, and their sulfates, phosphates, nitrates, citrates, and amino acid derivatives such as pyrrolidone carboxylic acid.

As a whitening agent, ascorbic acid or its derivative, sulfur, placenta hydrolyzate, ellagic acid or its derivative, kojic acid or its derivative, glucosamine or its derivative, arbutin or its derivative, hydroxycinnamic acid or its derivative, glutathione, arnica Extracts, Ogon extract, Sohakuhi extract, Psychoextract, Boufu extract, Mannentake mycelium culture or extract thereof, Shinanoki extract, peach leaf extract, Agetsu extract, Kuddin extract, Jiyu extract, Touki extract, Yokunin extract, Oyster leaf extract, Daio extract , Peanut extract, hamamelis extract, maroni extract, hypericum extract, oil-soluble licorice extract and the like.

Examples of humectants include hyaluronic acid, polyglutamic acid, serine, glycine, threonine, alanine, collagen, hydrolyzed collagen, hydronectin, fibronectin, keratin, elastin, royal jelly, chondroitin sulfate heparin, glycerophospholipid, glyceroglycolipid, sphingolipids , Glycosphingolipids, linoleic acid or esters thereof, eicosapentaenoic acid or esters thereof, pectin, fermented product of bifidobacteria, lactic acid fermented product, yeast extract, litchi mycelium culture or extract thereof, wheat germ oil, avocado Oil, rice germ oil, jojoba oil, soybean phospholipid, γ-oryzanol, below oil extract, yokuinin extract, jiou extract, daisy extract, diatom extract, kidachi aloe extract, burdock extract, mannen roeki , Arnica extract, wheat bran, and the like.

As a hair restorer, pentadecanoic acid glyceride, coleus extract, gentian extract, matsukasa extract, royal jelly extract, kumazasa extract, t-flavanone, 6-benzylaminopurine, assembly extract, carpronium chloride, minoxidil, finasteride, adenosine, nicotinamide, Mulberry root extract, dirt extract, 5-aminolevulinic acid, and the like.

Examples of extracts or extracts of animals or plants and crude drugs include Asenyaku (Asenyaku), Ashitaba, Acerola, Altea, Arnica, Avocado, Amacha (Amcha), Aloe, Aloe vera, Nettle, Ginkgo (Ginkgo leaf, Ginkgo), Fennel ( Fencil), Turmeric (Utsukin), Usubasaishin (spicy), Plum (Umebai), Vladimir, Owaurushi, Noibara (real fruit), Hiokikoshi (extended life grass), Ougi (Yoki), Koganebana (Ougon), Yamazakura (cherry bark) ), Yellowfin (Huangkashio), Spinach (Yellow), Panax ginseng (Ginseng), Hypericum perforatum (Brother's cut grass), Odorikosou, Holland garashi, Orange, Itohimemegi (Enshi), Utsubogusa (Summer hay), Tsurukudami (Hanshu crow), Endju ), Mugwort (Gai leaf), gajutsu (shajutsu), kuzu (kakkon), kanokoso (kichi) Roots), chamomile, black-necked cucumber cucumber (urorone), sagebrush (inchinko), licorice (licorice), coltsfoot (poor winter flowers, succulent winter leaves), raspberry, kiwi fruit, kikyo (bellflower), chrysanthemum (chrysanthemum flower) (Azumimi), Fruits of Citrus Plants (Kikomi), Tachibana (Tachibana Skin), Cucumber, Udo or Shishudo (Qanggai, Independent), Apricot (Apricot Jin), Wolfberry (Skin Bone, Ginkgo Biloba, Tulberry Leaf), Clara ( Bittersweet), camphor tree, kumazasa, grapefruit fruit, Nikkei (cinnamon bark), kaygai (caygai), ibisgusa (decided child), malva asagao or morning glory (ken beef), safflower (safflower), gobishi (fivefold), comfrey, copaiba, Gardenia, gentian, honoki (thick), hinata nokozuchi (cow knees), goshyu (goshuyu), burdock, kostrum (five) Ajiko), rice, rice bran, wheat, mishimasaiko (saiko), saffron, sabonso, hawthorn (yamazako), sansho (sansho), salvia, sanshin ginseng (seven carrots), shiitake (shiitake mushroom), jiou (Jihuang), Sikunshi (Kimiko), Murasaki (Shikon), Shiso (Shiyoha, Shisoko), Oyster (Kakidai), Peony (Shakuyaku), Psyllium (Kuramaeko, Komaegusa), Ginger (Ginger), Shobu (Irises), Scutellaria spp. (Sadako), Spiraea edulis, Birch, Honeysuckle (Golden and silver flowers, Shinobi winter), Ivy ivy, Achillea millefolium, Aesculus elder, Azuki (red azuki bean), Elderberry (bone grafted tree), Zeniaoi, Senkyu , Assembly (this medicine), mulberry (mulberry bark, mulberry leaf), jujube (large jujube), soybean, taranoki, chikusetsu carrot (bamboo ginseng), hanasuge ( Mother), Waremokou (Jiyu), Dokudami (Juyaku), Fuyushinatsukutaketake (Cyceps sinensis), Pepper, Physalis (Torone), Tachijakoso, Ryokucha (green tea), Kocha (tea), Chouzi (claw), Eunshu Mandarin orange (Chen skin), Camellia, Camellia, Pepper (Pan pepper), Touki (Toki), Tokinsenka, Daidai (Orange skin), Waremokou (Jiyu), Maize (Nanban hair), Eucommia (Tonaka, Tonakaha), Tomato, Nanten (Southern Tenmi), Garlic (Large San), Barley (Malt), Hakusen (White Moss), Janohige (Wheat Gate), Parsley, Batata, Mint (Light Load), Hamamelis, Rose, Loquat Leaves (Loquat) Leaf), matsuhodo (bukuryo), grape or its leaves, loofah, bodaiju, button (peony), hop, maikai (my rose flower), pine needle, maroni D, Mannenrou, Mukuroji, Melissa, Meliloth, Bokeh (Gourd), Sprouts, Peaches (Peach, Peach), Hiogi (Shiboshi), Areca (Betel wax), Mehajiki (Emperor grass), Cornflower, Yukinoshita (Tiger ear grass), Bayberry (Yumebuki), Yashabushi (Yari), Adlay (Yokuinin), Mokogomigi, Yamagomimugi, Lavender, Apple fruit, Mannentake (Lingzhi), Lemon fruit, Forsythia (rengi), Astragalus, Gennoshoko (Old grass), Hashidokoro ( Roto root), chicken tosaka, bovine / human placenta extract, swine / cattle stomach, duodenum, or intestinal extract or its degradation product, water-soluble collagen, water-soluble collagen derivative, collagen hydrolyzate, elastin, elastin Hydrolysate, water-soluble elastin derivative, silk protein, silk protein hydrolyzate, cow Sphere protein degradation product and the like.

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 fermentation of skim milk powder, and the like.

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

As inorganic pigments, silicic anhydride, 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, Bengala, black iron oxide, gunjou, chromium oxide, chromium hydroxide, carbon black, calamine and the like.

Examples of the ultraviolet absorber 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 a pyrone derivative, a nucleic acid derivative, an allantoin derivative, a nicotinic acid derivative, a vitamin B6 derivative, oxybenzone, benzophenone, guaiazulene, shikonin, baicalin, baicalein, and berberine.

Astringents include lactic acid, tartaric acid, succinic acid, citric acid, allantoin, zinc chloride, zinc sulfate, zinc oxide, calamine, zinc p-phenolsulfonate, aluminum potassium sulfate, resorcinol, ferric chloride, tannic acid, etc. No.

As antioxidants, ascorbic acid and its salts, stearic acid esters, tocopherol and its ester derivatives, nordihydroguaselethenoic acid, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), parahydroxyanisole, propyl gallate , Sesamol, sesamolin, gossypol and the like.

Examples of anti-inflammatory agents include ictamole, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d- or dl-camphor, hydrocortisone, guaiazulene, camazulene, chlorpheniramine maleate, glycyrrhizinate and salts thereof. Glycyrrhetinic acid and salts thereof.

(4) Examples of the disinfectant / disinfectant include acrinol, sulfur, benzalkonium chloride, benzethonium chloride, methylrosaniline chloride, cresol, calcium gluconate, chlorhexidine gluconate, sulfamine, mercurochrome, lactoferrin or a hydrolyzate thereof.

As a hair preparation, selenium disulfide, alkylisoquinolinium bromide, zinc pyrithione, biphenamine, thiantol, castal tincture, show tincture, capsicum tincture, quinine hydrochloride, strong aqueous ammonia, potassium bromate, sodium bromate, thioate Glycolic acid and the like.

Examples of flavors include natural animal flavors such as musk, civet, castorium, ambergris, anise essential oil, angelica essential oil, Iran essential oil, iris essential oil, fennel essential oil, orange essential oil, cananga essential oil, caraway essential oil, cardamom essential oil, guaiac wood essential oil, cumin Essential oil, black letter essential oil, cinnamon essential oil, cinnamon essential oil, geranium essential oil, copaiba balsam essential oil, coriandel essential oil, perilla essential oil, cedarwood essential oil, citronella essential oil, jasmine essential oil, ginger grass essential oil, cedar essential oil, spearmint essential oil, western mint essential oil, large Fengxiang essential oil, tuberose essential oil, clove essential oil, orange flower essential oil, winter green essential oil, Torubal sam essential oil, baturi essential oil, rose essential oil, palmarosa essential oil, cypress essential oil, hiba essential oil, sandalwood essential oil, petitgren essential oil, bay essential oil, vetiver essential oil, bergamot 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 mint essential oil and other vegetable flavors, etc. Synthetic fragrances and the like can be mentioned.

Examples of pigments and colorants include red cabbage pigment, red rice pigment, madder pigment, anato pigment, squid pigment, turmeric pigment, orange pigment, krill pigment, persimmon pigment, caramel, gold, silver, gardenia pigment, corn pigment, onion pigment , Tamarind pigment, spirulina pigment, whole buckwheat pigment, cherry pigment, laver pigment, hibiscus pigment, grape juice pigment, marigold pigment, purple potato pigment, purple yam pigment, lac pigment, rutin and the like.

Examples of the sweetener include sugar, sweet tea, fructose, arabinose, galactose, xylose, mannose, maltose, honey, glucose, miraculin, monelin and the like.

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

Others, hormones, sequestering agents, pH adjusters, chelating agents, preservatives / anti-binders, cooling agents, stabilizers, emulsifiers, animal / vegetable proteins and their degradation products, animal / vegetable polysaccharides and their Degradation products, animal and plant glycoproteins and their degradation products, blood flow enhancers, anti-inflammatory agents / antiallergic agents, cell activators, keratolytic agents, wound healing agents, foaming agents, thickening agents, oral agents, Deodorants and deodorants, bitters, seasonings, enzymes and the like.

The dosage form of the present invention is arbitrary, and can be in the form of an ampule, capsule, powder, granule, pill, tablet, solid, liquid, gel, foam, emulsion, cream, ointment, sheet. And quasi-drugs such as mousse, skin and hair cosmetics, bath preparations, foods and drinks, and pharmaceuticals.

Specifically, cosmetics and quasi-drugs include, for example, medicinal preparations for internal and external use, lotions, emulsions, creams, ointments, lotions, oils, basic cosmetics such as packs, facial cleansers and skin cleansers, Shampoos, rinses, hair treatments, hair creams, pomades, hair sprays, hair styling products, perm agents, hair tonics, hair cosmetics such as hair dyes, hair growth and hair restoration, foundations, white powder, whitening, lipsticks, blushers, eye shadows, Makeup cosmetics such as eyeliner, mascara, eyebrows and eyelashes, finishing cosmetics such as beautiful nails, perfumes, bath agents, other toothpastes, mouth fresheners / gargles, liquid odor and deodorants, Sanitary goods, sanitary cotton, wet tissue, and the like.

Foods and drinks include, for example, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, drinks such as lactic acid drinks, ice cream, ice sorbet, shaved ice, etc., buckwheat, udon, harusame, gyoza skin, sweet potato skin, Chinese Noodles such as noodles, instant noodles, candy, candy, gum, chocolate, tablets, snacks, biscuits, jellies, jams, creams, baked goods, baked goods, crabs, salmon, clams, tuna, sardines, shrimp, Fish products such as bonito, mackerel, whale, oyster, saury, squid, red mussel, scallop, abalone, sea urchin, salmon roe, tocobushi, fish and livestock processed foods such as kamaboko, ham, sausage, milk powder, processed milk, fermented milk, etc. Products, salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc. Processed fats, sauces, seasonings such as sauces, curry, stew, oyakodon, porridge, porridge, porridge, Chinese rice bowl, katsudon, tendon, unadon, hayashi rice, oden, marbodough, gyudon, meat sauce, egg soup, omelet, Examples include retort pouch foods such as gyoza, shomai, hamburger and meatballs, various forms of health and nutritional supplements, health foods, tablets, capsules, drinks, troches and the like.

抗 The antioxidant of the present invention is suitably applied to humans, but can be applied to animals other than humans as long as the respective effects can be expected.

Although the aspect of the antioxidant of the present invention is not particularly limited, for example, face wash, cleanser, lotion (eg, whitening lotion), cream (eg, vanishing cream, cold cream), milky lotion, gel, beauty serum , Pack (for example, jelly-like peel-off type, paste-type wipe-off type, powdery rinse-out type), face mask, cleansing, foundation, lipstick, lip balm, lip gloss, lip liner, blush, makeup base, shaving lotion, sunscreen, After sun lotion, deodorant lotion, body lotion (including hand care lotion and foot care lotion), body oil and the like.

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.

水 The aqueous extract of bark tree bark used in the following examples was prepared by the method disclosed in JP-A-2018-58783.

Example 1: Evaluation of the effect of enhancing the expression of a target gene in human normal neonatal epidermal keratinocytes The effect of increasing the expression of a target gene was evaluated by the following test method using the extract of Neem tree as a test sample. The target gene in this example means SOD, PRX, TXN, TRD, GRD and GXN.

A normal human neonatal keratinocyte (NHEK) is seeded on a T75 flask coated with collagen by Collagen Coating Solution (manufactured by Toyobo Co., Ltd.), and a medium for growing normal human keratinocyte (HuMedia-KG2) is added. And cultured at 37 ° C., 10% CO ₂ . The medium was replaced as appropriate, and the culture was continued until the confluence reached 80% or more. After removing the medium and rinsing with PBS, the cells were collected by trypsinization. Cells were seeded in a 96-well plate at 1 × 10 ⁴ cells / well and cultured at 37 ° C., 10% CO _{2 for} 1 day.
The medium was removed, the test sample was replaced with a medium containing a predetermined concentration, and incubated at 37 ° C., 10% CO _{2 for} 72 hours. After the culture, 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 composition of the reaction solution was in accordance with the package insert. However, the reaction volume was 20 μl / well, and the cDNA volume 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 primers used were the nucleotide sequences shown in SEQ ID NOs: 1 to 6. The instrument used was 7500 \ Fast \ Real-Time \ PCR \ System (Applied Biosystems). The expression level of the target gene was normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal standard (SEQ ID NOs: 13 to 14). The relative expression was calculated by dividing the normalized expression level of each target gene by the expression level of the negative control gene not containing the test sample.

The obtained results are shown in Figs. As can be seen from FIGS. 1 to 3, it was confirmed that the gene expression level of the antioxidant-related enzyme and / or the redox-related factor was improved in NHEK as compared with the negative control to which the Neem tree extract was not added. Was. From the present results, it has been clarified that the extract of Neem tree has an effect of enhancing gene expression of antioxidant-related enzymes and / or redox-related factors in epidermal cells and improving antioxidant ability in a living body.

Example 2: Evaluation of the effect of enhancing expression of the target gene in human normal neonatal fibroblasts T-flasks were inoculated with human normal neonatal fibroblasts (NHDF), and D-MEM (+ 10% FBS) was added. At 37 ° C., 5% CO ₂ . The medium was replaced as appropriate, and the culture was continued until the confluence reached 80% or more. After removing the medium and rinsing with PBS, the cells were collected by trypsinization. Cells were seeded in a 96-well plate at 1 × 10 ⁴ cells / well and cultured at 37 ° C., 5% CO _{2 for} 1 day.
The medium was removed, the test sample was replaced with a medium containing a predetermined concentration, and incubated at 37 ° C., 5% CO _{2 for} 72 hours. After the culture, 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 composition of the reaction solution was in accordance with the package insert. However, the reaction volume was 20 μl / well, and the cDNA volume 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. As the primers, the nucleotide sequences shown in SEQ ID NOs: 1 to 12 were used. The instrument used was 7500 \ Fast \ Real-Time \ PCR \ System (Applied Biosystems). The expression level of the target gene was normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal standard (SEQ ID NOs: 13 to 14). The relative expression was calculated by dividing the normalized expression level of each target gene by the expression level of the negative control gene not containing the test sample.

The obtained results are shown in FIGS. As can be seen from FIGS. 4 to 9, it was confirmed that in NHDF, the gene expression levels of antioxidant-related enzymes and redox-related factors were improved as compared to the negative control without the addition of the Neem tree extract. From the present results, it has been clarified that the extract of Neem tree has an effect of enhancing gene expression of antioxidant-related enzymes and / or redox-related factors in epidermal cells and improving antioxidant ability in a living body.

Example 3 Evaluation of Reducing Activity of Reactive Oxygen in Human Normal Neonatal Fibroblasts T75 flasks were inoculated with human normal neonatal fibroblasts (NHDF), and D-MEM (+ 10% FBS) was added thereto. ° C., and cultured in 5% CO _2. The medium was replaced as appropriate, and the culture was continued until the confluence reached 80% or more. After removing the medium and rinsing with PBS, the cells were collected by trypsinization. The cells were seeded in a 96-well plate at 3 × 10 ⁴ cells / well and cultured at 37 ° C., 5% CO _{2 for} 1 day.
After removing the culture and washing with HBSS, the cells were replaced with 20 μM H ₂ DCFDA (2 ′, 7′-dichlorodifluorofluorescein diacetate) and incubated at 37 ° C., 5% CO ₂ (dark place) for 30 minutes. After removing H ₂ DCFDA and washing with HBSS, the test sample and HBSS containing hydrogen peroxide at a predetermined concentration were replaced, and the mixture was further incubated at 37 ° C. and 5% CO ₂ (in a dark place). The fluorescence intensity was measured by (ARVO MX) at Ex / Em = 485 nm / 535 nm.

The results obtained are shown in FIG. As can be seen from FIG. 10, the effect of reducing the active oxygen in cells induced by oxidative stress due to hydrogen peroxide in NHDF was confirmed. This is because the gene expression of antioxidant-related enzymes and / or redox-related factors is enhanced in addition to the antioxidant effect contained in the extract of Alnus elegans, so that the ability of the living body to scavenge active oxygen is improved and the activity in the living body is improved. It is thought that it led to the reduction of oxygen.

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

(4) The test sample was applied to the face of the subject, and cells were obtained from the beard in the application area and evaluated. Specifically, before application of the test sample and 3 hours after application, the beard in the application area was removed from the subject, and hair follicle cells were collected. The cells were stored by immersion in RNAlater (manufactured by Qiagen) and stored at 4 ° C. until RNA extraction. After completion of collection of the hair follicle cells, total RNA was extracted from the hair follicle cells using RNeasy plus micro kit (manufactured by Qiagen) according to the attached protocol. For the extracted total RNA, cDNA was prepared according to the attached protocol using ReverseTra @ Ace (registered trademark) qPCR @ RT @ Master @ Mix (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 composition of the reaction solution was in accordance with the package insert. However, the reaction solution volume was 20 μl / well, and the cDNA volume 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 primers used were the nucleotide sequences shown in SEQ ID NOs: 7 and 8. The instrument used was 7500 \ Fast \ Real-Time \ PCR \ System (Applied Biosystems). The expression level of the target gene was normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal standard (SEQ ID NOs: 13 to 14). The relative expression was calculated by dividing the standardized expression level of each target gene by the expression level of the gene in the negative control before application of the test sample.

The results obtained are shown in FIG. As can be seen from FIG. 11, in the cDNA obtained from the hair follicle cells, it was confirmed that the PRX gene expression level was improved 3 hours after the application, as compared to the negative control before the application of the Alnus extract. Was. From these results, it is clear that the extract of Alnus has the effect of enhancing 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 the living body. became.

Example 5: Formulation of external preparation for skin The following shows a formulation example of the external preparation for skin in the present invention. All of these preparations are expected to have an effect of enhancing the expression of antioxidant-related enzyme genes caused by the Alnus serrata extract, and are effective as antioxidants.

Lotion According to the following composition, lotion was produced by a conventional method.
・ Purified water: 89.80 g
・ Glycerin: 3.00 g
・ Phenoxyethanol ・・・ 0.20g
-Butylene glycol: 5.00 g
・ Pentylene glycol ・・・ 1.00g
・ Neem tree extract ・・・ 1.00g

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

Cream According to the following composition, a cream was produced by a conventional method.
・ Purified water: 58.50 g
・ Butylene glycol ・・・ 10.00g
・ Glycerin: 5.00 g
・ Phenoxyethanol ・・・ 0.20g
・ Ethylhexyl glycerin: 0.20 g
・ Squalane ・・・ 10.00g
・ Olive oil ・・・ 10.00g
・ Behenyl alcohol: 2.50 g
・ Polyglyceryl pentastearate-10: 1.90 g
・ Na stearoyl lactate ・・・ 0.60g
・ Cetyl palmitate ・・・ 1.00g
・ Neem extract: 0.10 g

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

Claims

An antioxidant containing a Neem tree extract.
The antioxidant according to claim 1, wherein the extract of Alnus is an aqueous extract from a plant of an Alnus plant.
3. The antioxidant according to claim 1, wherein the extract of Alnus has an effect of enhancing gene expression of an antioxidant-related enzyme and / or a redox-related factor. 4.
The antioxidant-related enzyme and / or redox-related factor is a group consisting of superoxide dismutase (SOD), peroxiredoxin (PRX), thioredoxin (TXN), thioredoxin reductase (TRD), glutathione reductase (GRD), and glutaredoxin (GXN). The antioxidant according to any one of claims 1 to 3, which is at least one or more selected from the group consisting of:
The antioxidant according to any one of claims 1 to 4, wherein the extract of Alnus has an activity of reducing intracellular active oxygen.
An external preparation for skin comprising the antioxidant according to any one of claims 1 to 5.
The external preparation for skin according to claim 6, wherein the extract of Alnus serrata is contained at a concentration of 0.00001 to 5.0% by weight.