Antioxidant composition

Abstract

The present invention addresses the problem of providing an antioxidant composition having durable antioxidant activity.　The present invention provides: an antioxidant composition containing, as an active ingredient, a light-irradiated product of a grape pomace extract or a new compound contained in said light-irradiated product; and a method for producing a durable antioxidant material, the method including a step for pressing a grape raw material in a wine production process to obtain a grape pomace extract including grape skin and seeds, and a step for irradiating the obtained grape pomace extract with light.

Description

antioxidant composition

[0001] The present invention relates to antioxidant compositions having sustained antioxidant activity.

[0002] The amount of reactive oxygen species produced in the body is balanced by the amount of reactive oxygen species reduced by reactive oxygen scavenging enzymes, so the amount of reactive oxygen species in the body is kept constant. However, it is known that the amount of reactive oxygen species in the body increases due to the influence of living environments such as aging, stress, poor diet, and poor lifestyle. Typical types of reactive oxygen species produced in the body are hydroxyl radicals, superoxide, and singlet oxygen, with hydroxyl radicals being particularly toxic. Excessive production of reactive oxygen species in the body can cause accelerated aging, lifestyle-related diseases such as arteriosclerosis and hypertension, cancer, and a weakened immune system.

[0003] Therefore, antioxidants, which have the ability to remove excess active oxygen, are attracting attention as effective ingredients for preventing lifestyle-related diseases and anti-aging. Representative examples of antioxidants include polyphenols, such as anthocyanins, catechins, isoflavones, sesamin, sesaminol, and theaflavin.

[0004] It is well known that wine contains a large amount of polyphenols, and the grape press residue generated during wine production also contains various useful substances, including polyphenols, and its effective use has been investigated. The present inventors have reported that this grape wine processing residue has a significantly higher hydroxyl radical scavenging activity, an indicator of antioxidant activity, than currently available raw materials such as grape seed extract and catechin (Non-Patent Document 1).

[0005] However, due to the principle of oxidation-reduction, antioxidant ingredients generally lose their activity once their antioxidant purpose is achieved. Therefore, materials with sustained antioxidant activity are desired.

[0006] Tsukada M, Nakashima T, Kamachi T, Niwano Y. PLoS One 2016, Jun 24;11(6):e0158197. Prooxidative Potential of Photo-Irradiated Aqueous Extracts of Grape Pomace, a Recyclable Resource from Winemaking Process.

[0007] In view of the above circumstances, an object of the present invention is to provide an antioxidant composition having sustained antioxidant activity.

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the inventors have discovered that by irradiating grape pomace, which has high antioxidant activity, with light in a specific wavelength range, it is possible to further enhance and sustain the antioxidant activity. Furthermore, they have succeeded in identifying a novel compound that is the active substance from the light-irradiated grape pomace, leading to the completion of the present invention.

[0009] That is, the present invention encompasses the following inventions: (1) An antioxidant composition containing, as an active ingredient, a light-irradiated product of grape pomace. (2) The antioxidant composition according to (1), wherein the wavelength of the light irradiation is 200 to 700 nm. (3) The antioxidant composition according to (1) or (2), wherein the grape pomace is press lees left after grape pressing, which is a waste product in wine production. (4) An antioxidant composition containing, as an active ingredient, a compound represented by the following general formula (I): (In the formula, the bonds represented by the solid and dotted lines in (a) to (g) are single or double bonds. When (a) and (c) are double bonds and (b) and (d) are single bonds, R １ is an oxygen atom, R ２ represents a hydroxy group, and when (a) and (c) are single bonds and (b) and (d) are double bonds, R １ is a hydroxy group, R ２ represents an oxygen atom, R ３ , R ４ , and R ７each independently represents a hydrogen atom or a hydroxy group, and when (e) and (g) are single bonds and (f) is a double bond, R ５ and R ６ indicates a hydroxy group, and when (e) and (g) are double bonds and (f) is a single bond, R ５ and R ６ represents an oxygen atom, R ８ and R ９ each independently represents a hydrogen atom or a hydroxy group, or R ８ and R ９ (5) The antioxidant composition according to (4), wherein the compound represented by general formula (I) is a compound represented by the following structural formula (1), (2), or (3): (6) The antioxidant composition according to (1) or (4), wherein the antioxidant composition is a cosmetic, a pharmaceutical, or a food or beverage. (7) The antioxidant composition according to (1) or (4), wherein the antioxidant composition is a hygiene product, a cleaning agent, a textile product, a food packaging material, a filter product, or a paper product. (8) A method for producing a sustainable antioxidant material, comprising the steps of: squeezing grape raw materials in a wine production process to obtain grape pomace containing grape skins and seeds; and subjecting the obtained grape pomace to a light irradiation treatment. (9) The production method according to (8), wherein the grape pomace is subjected to an extraction treatment before the light irradiation treatment. (10) A novel compound represented by the following general formula (I): (In the formula, the bonds represented by the solid and dotted lines in (a) to (g) are single or double bonds. When (a) and (c) are double bonds and (b) and (d) are single bonds, R １ is an oxygen atom, R ２ represents a hydroxy group, and when (a) and (c) are single bonds and (b) and (d) are double bonds, R １ is a hydroxy group, R ２ represents an oxygen atom, R ３ , R ４ , and R ７ each independently represents a hydrogen atom or a hydroxy group, and when (e) and (g) are single bonds and (f) is a double bond, R ５ and R ６indicates a hydroxy group, and when (e) and (g) are double bonds and (f) is a single bond, R ５ and R ６ represents an oxygen atom, R ８ and R ９ each independently represents a hydrogen atom or a hydroxy group, or R ８ and R ９ (11) The novel compound according to (10), wherein the compound represented by the general formula (I) is a compound represented by the following structural formula (1), (2), or (3): (12) A method for producing the novel compound according to (10) or (11), characterized in that the method comprises subjecting grape pomace to a light irradiation treatment and then a solvent extraction treatment, or subjecting grape pomace to a solvent extraction treatment and then a light irradiation treatment, and separating and purifying the compound from the treated product.

[0010] This application claims priority to Japanese Patent Application No. 2024-003399, filed on January 12, 2024, and includes the contents described in the specification of that patent application.

[0011] According to the present invention, an antioxidant composition having sustained antioxidant activity and a method for producing a sustained antioxidant material are provided. The antioxidant composition of the present invention is effective in improving and preventing various diseases and pathological conditions associated with reactive oxygen species.

[0012] The hydroxyl radical (.OH) scavenging ability of grape pomace (without light irradiation treatment, after 72 hours of light irradiation (302 nm) treatment) is shown. The superoxide (O ２－) scavenging ability. Figure 3-1 shows the results of LC-MS analysis of epigallocatechin (EGC) before and after 72 hours of light irradiation (302 nm). Figure 3-2 shows the results of LC-MS analysis of grape press pomace (GPE) before and after 72 hours of light irradiation (302 nm). Figure 4-1 shows the results of LC-MS analysis of catechin (Cat) before and after 72 hours of light irradiation (302 nm). Figure 4-2 shows the results of LC-MS analysis of epicatechin (EC) before and after 72 hours of light irradiation (302 nm). Figure 5 shows the condition of sunburned skin in mice in the UVB non-irradiated / normal diet group (control), UVB non-irradiated / GPE-containing diet group, UVB irradiated / normal diet group, and UVB irradiated / GPE-containing diet group. Figure 6 shows the time course of melanin levels in sunburned skin of mice in the following groups: C: non-UVB irradiated / normal diet group (control, n = 4); T1: non-UVB irradiated / GPE-containing diet group (n = 4); T2: UVB irradiated / normal diet group (n = 6); and T3: UVB irradiated / GPE-containing diet group (n = 6) (aa: P < 0.01 (vs. C); b: P < 0.05 (vs. T1); bb: P < 0.01 (vs. T1); cc: P < 0.01 (vs. T2); mean ± standard deviation; Tukey-Kramer test). Figure 7 shows the time course of redness in sunburned skin of mice in the C: non-UVB-irradiated / normal diet group (control, n = 4), T1: non-UVB-irradiated / GPE-containing diet group (n = 4), T2: UVB-irradiated / normal diet group (n = 6), and T3: UVB-irradiated / GPE-containing diet group (n = 6) (a: P < 0.05 (vs. C), aa: P < 0.01 (vs. C), b: P < 0.05 (vs. T1), bb: P < 0.01 (vs. T1), cc: P < 0.01 (vs. T2), mean ± standard deviation; Tukey-Kramer test). Figure 8 shows the results of the d-ROM test in mice from the C: non-UVB-irradiated / normal diet group (control, n = 2), T1: non-UVB-irradiated / GPE-containing diet group (n = 2), T2: UVB-irradiated / normal diet group (n = 3), and T3: UVB-irradiated / GPE-containing diet group (n = 4). Figure 9 shows the results of the d-ROM test in healthy rats from the normal diet group (control), 1% GPE-containing diet group, and 2% GPE-containing diet group (*: P < 0.05).Figure 10 shows macroscopic findings of hind limb joints in healthy rats on a normal diet (control), rheumatoid arthritis model rats on a normal diet (group AA), and rheumatoid arthritis model rats on a 2% GPE-containing diet (group AA+GPE). Figure 11 shows CT image findings of hind limb joints in healthy rats on a normal diet (control), rheumatoid arthritis model rats on a normal diet (group AA), and rheumatoid arthritis model rats on a 2% GPE-containing diet (group AA+GPE). Figure 12 shows the results of the von Frey test for healthy rats fed a normal diet (control, n = 7), rheumatoid arthritis model rats fed a normal diet (AA group, n = 7), and rheumatoid arthritis model rats fed a 2% GPE-containing diet (AA + GPE group, n = 7) (*: P < 0.05, **: P < 0.01, mean ± standard deviation; Tukey-Kramer test). Figure 13 shows the results of the challenge test for social isolation stress model rats fed a normal diet (control, n = 7), social isolation stress model rats fed a 1% GPE-containing diet (Stress + 1% GPE group, n = 7), and social isolation stress model rats fed a 2% GPE-containing diet (Stress + 2% GPE group, n = 7) (*: P < 0.05, **: P < 0.01, mean ± standard deviation; Tukey's post-hoc test).

[0013] The present invention will be described in detail below. 1. Antioxidant Composition The antioxidant composition of a first aspect of the present invention contains, as an active ingredient, a light-irradiated product of grape pomace. Here, "grape pomace" refers to the pomace that remains after grape pressing, which is a waste product in the wine production process. However, it may also include the sediment that settles to the bottom of wine tanks after grape pressing.

[0014] In the present invention, the grape variety used as the raw material for the grape press residue is not particularly limited, and may be a white wine grape variety or a red wine grape variety. Examples of white wine grape varieties include Neo Muscat, Treppiano, Niagara, Kerner, Alexandria, Buffalo, Chardonnay, Semillon, Riesling, Sauvignon Blanc, Muller-Thurgau, Verdelet, Koshu, Traminer, and Delaware. Examples of red wine grape varieties include Merlot, Cabernet Sauvignon, Cabernet Franc, Pinot Noir, Syrah, Alicanto, Nebbiolo, Muscat Bailey A, and Black Queen.

[0015] In the present invention, the wavelength of the light irradiated on the grape pomace is, for example, 200 to 700 nm, preferably 280 to 400 nm. The illuminance of the light irradiation is, for example, 0.01 to 100 mW / cm. ２ , preferably 0.1 to 50 mW / cm ２ The light irradiation time can be, for example, 0.5 to 100 hours, preferably 10 to 24 hours.

[0016] The light-irradiated grape pomace can be used as is, or after drying, pulverization, etc., in the form of a slurry, fine particles, granules, or powder. However, for ease of handling when used in a product, it is preferable to subject it to extraction and use an extract in which grape-derived polyphenol components are extracted. Here, examples of grape-derived polyphenol components include epicatechin (EC), catechin (Cat), epigallocatechin (EGC), epigallocatechin gallate (EGCg), procyanidin B2 (dimer), procyanidin C1 (trimer), etc. In addition, extraction may be performed on the grape pomace before light irradiation treatment.

[0017] The extraction method is not particularly limited, and examples thereof include continuous extraction and immersion extraction. Furthermore, a heated extraction method, room temperature extraction, or cold extraction may be used. Examples of solvents used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water (or hot water) and lower alcohols are preferred. These solvents may be used alone or in combination. Furthermore, a solvent whose pH has been adjusted by adding an acid or alkali to the above-mentioned extraction solvent may also be used.

[0018] The amount of extraction solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, the amount of the raw material to be extracted (dry weight), but for convenience of operations such as concentration and isolation after extraction, it is preferable that the amount be 100 times or less. The extraction temperature and time depend on the type of target plant and solvent used, but examples include 10 to 100°C, preferably 30 to 90°C, and 1 minute to 24 hours, preferably 1 to 10 hours.

[0019] The extract may be used as it is in the form of an extracted solution, or, if necessary, may be subjected to treatment such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon or the like, deodorization, ethanol precipitation, etc., within a range that does not affect the effect of the extract. Furthermore, the extracted solution may be subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0020] The antioxidant composition according to the second aspect of the present invention contains a compound represented by the following general formula (I) as an active ingredient. (In the formula, the bonds represented by the solid lines and dotted lines in (a) to (g) are single bonds or double bonds. When (a) and (c) are double bonds and (b) and (d) are single bonds, R １is an oxygen atom, R ２ represents a hydroxy group, and when (a) and (c) are single bonds and (b) and (d) are double bonds, R １ is a hydroxy group, R ２ represents an oxygen atom, R ３ , R ４ , and R ７ each independently represents a hydrogen atom or a hydroxy group, and when (e) and (g) are single bonds and (f) is a double bond, R ５ and R ６ indicates a hydroxy group, and when (e) and (g) are double bonds and (f) is a single bond, R ５ and R ６ represents an oxygen atom, R ８ and R ９ each independently represents a hydrogen atom or a hydroxy group, or R ８ and R ９ together form a single bond.)

[0021] More specifically, examples of the compound represented by the general formula (I) include compounds represented by the following structural formulas (1) to (3) (referred to as compounds 1 to 3, respectively).

[0022]

[0023]

[0024]

[0025] The antioxidant composition according to the second aspect of the present invention contains, as an active ingredient, at least one selected from the group consisting of compounds 1, 2, and 3. Compounds 1 to 3 may be contained alone or in combination of two or more.

[0026] Compounds 1 to 3 can be separated and purified from a light-irradiated grape pomace. Specifically, after obtaining an extract from the light-irradiated grape pomace by the above-mentioned method, or after subjecting a grape pomace extract to light irradiation, the extract is separated from the residue by a conventional method such as filtration or centrifugation to solid-liquid separation. Active fractions can then be isolated from the resulting extract by solvent fractionation using one or more organic solvents such as methanol, ethanol, propanol, butanol, dichloromethane, chloroform, ethyl acetate, toluene, hexane, benzene, and acetone. Furthermore, if necessary, the active fractions can be purified using one or a combination of two or more suitable separation and purification means such as alumina column chromatography, silica gel chromatography, ODS column chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high-performance liquid chromatography.

[0027] The separation and identification of compounds 1 to 3 from the light-irradiated grape pomace can be carried out specifically according to the description in Example 1(4).

[0028] As described above, Compounds 1 to 3 of the present invention can be isolated and purified from light-irradiated grape pomace. However, the method for producing the compounds is not limited to this. The compounds may be synthesized based on known chemical synthesis methods, or they may be produced by subjecting raw materials obtained from natural products other than grapes to a reaction or other treatment.

[0029] The light-irradiated grape pomace and Compounds 1 to 3 have extremely high and long-lasting antioxidant activity, and therefore can be incorporated as active ingredients into antioxidant compositions either alone or in combination with appropriate additives within a range that does not impair the effects of the present invention.

[0030] In the present invention, "antioxidant activity" refers to the activity of scavenging active oxygen and radicals in the body through at least one of an active oxygen scavenging action and a radical scavenging action. Here, active oxygen includes superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen, etc. Furthermore, radical means a molecule or atom having one or more unpaired electrons, and includes superoxide, hydroxyl radical, DPPH (diphenylpicrylhydrazide), lipid peroxide, etc.

[0031] The form of the antioxidant composition is not particularly limited, and examples thereof include cosmetics, pharmaceuticals, and foods and beverages. Since the active ingredient of the antioxidant composition of the present invention is derived from a natural product, it is highly safe and has no side effects, and therefore, when used as cosmetics, pharmaceuticals, or foods and beverages, it can be continuously ingested or administered to mammals such as humans, mice, rats, rabbits, dogs, and cats.

[0032] Since the skin is directly exposed to environmental factors such as ultraviolet rays, it is an organ in which reactive oxygen species are easily generated, and therefore, an increase in the concentration of reactive oxygen species and the accompanying generation of lipid peroxides are likely to occur, which in turn are likely to cause disorders such as the generation of melanin pigments, age spots, fine wrinkles, etc. Therefore, the antioxidant composition of the present invention, which has excellent antioxidant activity, is preferably provided in the form of a cosmetic product that removes melanin accumulated due to ultraviolet rays, suppresses redness caused by ultraviolet rays, and prevents and / or improves pigmentation (age spots), wrinkles, rough skin, etc.

[0033] The cosmetic formulation may be any of an aqueous solution, solubilized solution, emulsion, powder, powder dispersion, oil solution, gel, ointment, aerosol, water-oil two-layer system, or water-oil-powder three-layer system. The cosmetic can be produced according to methods known in the art by appropriately blending various ingredients, additives, bases, etc. commonly used in topical skin preparations, selected according to their type, together with the light-irradiated grape pomace. The cosmetic may be in any form, such as liquid, emulsion, cream, gel, paste, or spray.

[0034] Examples of types of cosmetics include lotions, emulsions, gels, serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, powders, body lotions, hair shampoos, and hair growth agents.

[0035] When the antioxidant composition of the present invention is used as a pharmaceutical, it can be mixed with pharmacologically and pharmaceutically acceptable additives and formulated into various preparations suitable for application to affected areas. The pharmacologically and pharmaceutically acceptable additives may include, depending on the dosage form and intended use, pharmaceutical bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonicity agents, pH adjusters, propellants, colorants, sweeteners, flavoring agents, flavoring agents, and flavoring agents. These additives may be added as appropriate to prepare various dosage forms suitable for oral or parenteral systemic or local administration by various known methods. When the pharmaceutical of the present invention is provided in the above-mentioned forms, it can be prepared by methods commonly used by those skilled in the art, such as the methods set forth in the General Provisions for Preparations [2], each article of the Pharmaceuticals section of the Japanese Pharmacopoeia.

[0036] The pharmaceutical form may be, but is not limited to, oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs; parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drip infusions, suppositories, transdermal absorption preparations, transmucosal absorption preparations, and patches. The pharmaceuticals of the present invention may also be prepared as dried products that are reconstituted for use, and injectable preparations are provided in unit-dose ampoules or multi-dose containers. When used to treat, improve, or prevent pigmentation (age spots), the pharmaceuticals of the present invention are suitable for topical use, such as ointments, creams, gels, liquids, patches, foams, sprays, and aerosols.

[0037] When used as a pharmaceutical, the antioxidant composition of the present invention is effective in treating, ameliorating, and preventing diseases and pathologies caused by reactive oxygen species due to oxidative stress, including, but not limited to, arteriosclerosis, cancer, skin aging (e.g., age spots and wrinkles), skin diseases (e.g., atopic dermatitis and photosensitive dermatitis), rheumatoid arthritis, mental disorders (e.g., schizophrenia and autism), dementia, eye diseases (e.g., cataracts, retinitis pigmentosa and age-related macular degeneration), cranial nervous system diseases (e.g., Parkinson's disease and Alzheimer's disease), respiratory system diseases (e.g., bronchitis), circulatory system diseases (e.g., ischemic arrhythmia, myocardial infarction, cerebral infarction, hypertension), digestive system diseases (e.g., gastric ulcer, colitis, fatty liver), and diabetes.

[0038] The amount used or administered of the cosmetic or pharmaceutical product of the present invention can be determined appropriately depending on its type and form, and the age, sex, weight, and severity of symptoms of the subject. For example, when orally administered to an adult, the dose of light-irradiated grape pomace is in the range of 0.1 to 1000 mg / day, preferably 1 to 500 mg / day, and more preferably 5 to 300 mg / day, once to several times a day. In some cases, a dose less than the above-mentioned dosage range is sufficient, and in other cases, it may be necessary to administer an amount exceeding the range.

[0039] When the light-irradiated grape pomace is incorporated into the above-mentioned cosmetics or pharmaceuticals, its content is not particularly limited, but is usually in the range of 0.001 to 99 wt % (w / w), preferably 0.01 to 90 wt % (w / w), and more preferably 10 to 85 wt % (w / w), calculated as dry solids relative to the total weight of the formulation (composition), and it is preferable that the daily dosage be controlled so that the above-mentioned daily intake amount for an adult can be achieved. Furthermore, the method of adding the active ingredient in the formulation may be either added in advance or during production, and may be selected appropriately taking into account workability.

[0040] Furthermore, the light-irradiated grape pomace or compounds 1 to 3 can also be incorporated into foods and beverages. In the present invention, the term "foods and beverages" refers to not only general foods and beverages, but also foods other than pharmaceuticals that can be consumed for the purpose of maintaining or improving health, such as health foods, functional foods, foods with health claims, or foods for special dietary uses. Health foods include foods offered under names such as dietary supplements, health supplements, and supplements. Health claims are defined by the Food Sanitation Act or the Food Promotion Act, and include foods for specified health uses and foods with health claims that can claim specific health benefits, nutritional component functions, or disease risk reduction. The form of the foods and beverages may be any form suitable for consumption, such as solid, liquid, granular, powdered, capsule-shaped, creamy, or paste-shaped.

[0041] Types of food and beverages include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock foods, oils and fats and processed oil and fat foods, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated concentrates and powders for adjusting such beverages.

[0042] The food and drink products of the present invention may be appropriately blended with commonly used additives depending on the type of food and drink product. Any additives that are acceptable from the standpoint of food hygiene can be used, including, for example, sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and antiseptics.

[0043] The amount of the light-irradiated grape pomace compound to be incorporated into the food or beverage of the present invention may be any amount that can exert an antioxidant effect, and may be appropriately set taking into consideration the general intake amount of the target food or beverage, the form of the food or beverage, efficacy / effect, taste, preference, cost, etc.

[0044] Other forms of the antioxidant composition include hygiene products (nonwoven masks, disposable diapers, wet towels, etc.), detergents (clothing detergents, kitchen detergents, bathroom detergents, household detergents, etc.), textile products (sheets, towels, underwear, etc.), food packaging materials (freshness-keeping sheets, freshness-keeping bags, etc.), filter products (air conditioner filters, air purifier filters, vacuum cleaner filters, ventilation fan filters, etc.), paper products (tissue paper, toilet paper, packaging paper, etc.), etc. For example, when used as a nonwoven fabric product having antioxidant and active oxygen removal effects, it can be produced by applying an aqueous solution of light-irradiated grape pomace to a nonwoven fabric and drying it.

[0045] 2. Method for Producing an Antioxidant Material The light-irradiated grape pomace, which is the active ingredient of the antioxidant composition, can be used as a long-lasting antioxidant material. The antioxidant material of the present invention can be produced by a method including a step of squeezing grape raw materials in a wine production process to obtain grape pomace containing grape skins and seeds, and a step of irradiating the obtained grape pomace with light.

[0046] The step of obtaining grape pomace is carried out by recovering the residue (including skins and seeds) remaining after squeezing grapes in a typical wine production process. The step of subjecting the obtained grape pomace to light irradiation may be carried out under the aforementioned light irradiation conditions. Furthermore, the method may include a step of subjecting the grape pomace to extraction under the aforementioned extraction conditions prior to the light irradiation treatment.

[0047] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0048] Example 1 (1) Production of Light-Irradiated Grape Pomace Immediately after squeezing, grape pomace (varieties 1 to 8) was collected, dried, and pulverized. 10 mL of solvent (water) was added to 1 g of the resulting dried powder, followed by extraction at room temperature for 1 hour and centrifugal filtration at 4,000 rpm for 15 minutes. The resulting extract was irradiated with 302 nm light for 72 hours.

[0049] (2) Antioxidant activity of grape pomace before and after light irradiation. The hydroxyl radical (.OH) scavenging ability and superoxide radical (O) scavenging ability of the extract obtained in (1) before and after light irradiation were ２－ ・) Extinction ability was investigated.

[0050] In the hydroxyl radical (.OH) scavenging ability test, hydroxyl radicals were artificially produced by the Fenton reaction, and the amount of hydroxyl radicals scavenged when a 10-fold diluted sample of each sample was added was measured, and the scavenging rate was calculated. ２－ The scavenging ability test used the principle of the HPX-XOD generation system to measure the amount of superoxide radicals scavenged when a 100-fold diluted version of each sample was added. Both tests were measured using an electron spin resonance (ESR) instrument.

[0051] The hydroxyl radical scavenging ability increased from 20-46% before light irradiation to 32-51% after light irradiation. In all varieties, the activity increased after light irradiation (Figure 1).

[0052] The superoxide scavenging ability increased from 67-75% before light irradiation to 70-80% after light irradiation. In all varieties, the activity increased after light irradiation (Figure 2).

[0053] (3) Component Analysis by LC / MS The samples used were the grape pomace (GPE), epigallocatechin (EGC), catechin (Cat), and epicatechin (EC). Light irradiation was performed under the same conditions as in (1), and LC / MS analysis was performed on the samples before and after light irradiation. A Xevo G2 Q Tof (Waters) system was used for analysis. Samples were prepared by centrifugation at 19,600 g, and 4 μL of the supernatant was subjected to analysis. A Waters Symmetry C18 (length 150 mm, inner diameter 2.1 mm, particle size 3.5 μm) was used as the analytical column, and a linear gradient method of a 0.1% formic acid aqueous solution based on 0.1% formic acid acetonitrile was used as the mobile phase. Q-Time of Day (Tof) measurements using positive ion electrospray ionization (ESI) showed a new peak at around 0.89 minutes in the photoirradiated GPE sample (Figure 3-2), but no new peak was detected in the photoirradiated epigallocatechin (EGC) sample (Figure 3-1). Furthermore, judging from the new peaks detected in the photoirradiated catechin (Cat) and epicatechin (EC) samples (Figures 4-1 and 4-2), it was estimated that photoirradiated GPE contains multiple components, including at least catechin and epicatechin, as well as new components.

[0054] (4) Structural analysis of the new component (extraction and separation of compounds from light-irradiated grape pomace (GPE)) (4-1) Analysis method Since epicatechin is found in a new peak (around 0.89 minutes in LC / MS) that is presumed to contain the new component, a sample containing the target compound was separated from the epicatechin preparation using HPLC.

[0055] A 1 mM aqueous solution of (-)-epicatechin was irradiated with 302 nm UV light for 24 hours and then freeze-dried. The freeze-dried product was dissolved and the target compound was separated by reverse-phase HPLC using an ODS column. The mobile phase was a 5% (v / v) acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution at a flow rate of 8 mL / min. The column used was a TSK-gel 80TS (inner diameter: 20 mm, length: 250 mm).

[0056] To determine the structure of the target compound, IR spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance (NMR) were performed using the following equipment and conditions. 1 H-NMR spectrum, 13 In addition to C-NMR spectra, data were collected from two-dimensional spectra [HH COSY, HSQC (Heteronuclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Correlation), and INADEQUATE (Incredible Natural Abundance Double Quantum Transfer Experiment)].

[0057] <IR spectrum> Apparatus: FT / IR-4X1 type A (JASCO) Measurement method: FT / IR-ATR method_Attenuated Total Reflection (total reflection measurement method) Measurement conditions: Measurement range 4000.6 cm -1 From 399.193cm -1 , resolution 4cm -1 , 16 times of integration, sample chamber temperature 293K

[0058] <Mass spectrometry (MS)> Instrument: JMS-DART-TOF-MS T100LP Ion source: DART Mass analyzer: TOF Measurement conditions: Ion source temperature 250°C, DART gas He

[0059] <Magnetic resonance analysis (NMR)> Apparatus: JNM-ECZ800MHz ( 13 C-NMR measurement only) and JNM-ECZ500MHz Measurement solvent: DMSO-d6

[0060] (4-2) Analysis results <IR spectrum> 3190cm -1 Alcohol or phenolic hydroxyl group (OH) near 1644cm -1 The carbonyl group (C=O) stretching absorption was observed near the nucleus, indicating the presence of alcohol or phenolic hydroxyl groups and carbonyl groups in the sample.

[0061] <Mass spectrometry (MS)> MS:m / z [M+H]+ , calc for [C 15 H 12 O6] + =289.07;found 289.07 MS:m / z [M+H] + , calc for [C 15 H 12 O ７ ] + =305.07;found 305.07

[0062] <Nuclear magnetic resonance (NMR)> 1 H-NMR (500MHz, DMSO-d6) δ 6.87(s, 1H), 6.19(s, 1H), 5.21(d, J=1.5Hz, 1H), 5.17(d, J=1Hz, 1H), 4.14(d, J=8Hz, 1H), 3.97(ddd, J=12, 8, 4.5Hz 1H), 2.05(dd, J=13.5, 10.5Hz, 1H), 1.95(dd, J=13.5, 4.5Hz, 1H) 13 C-NMR (200MHz, DMSO-d6) δ 170-190, 170-190, 170-190, 145.1, 144.8, 132.1, 124.7, 114.7, 112.9, 99.8, 98.5, 72.5, 69.0, 51.2, 36.9

[0063] 1 H-NMR, 13 The results obtained by the C-NMR, HH COSY, HSQC, and HMBC measurements are summarized in Table 1 below.

[0064]

[0065] From the above results, it was determined that the compounds contained in the light-irradiated grape pomace were Compound 1 (molecular weight 289.07, molecular formula C 15 H 12 O6), compound 2 (molecular weight 289.07, molecular formula C 15 H 12 O6), compound 3 (molecular weight 305.07, molecular formula C 15 H 12 O7).

[0066] ​

[0067] Example 2: Effect test on sunburn model mice Sunburn model mice were prepared by inducing inflammatory redness and melanin production by irradiating HRM-2 hairless mice with UVB light, and a study was conducted to determine whether differences in inflammatory redness and melanin pigmentation appearing on the skin surface were observed between a group that ingested grape pomace (hereinafter referred to as "GPE") and a group that did not. In this test, the extract of grape pomace from grape variety A produced in Example 1 (without light irradiation) was used as "GPE."

[0068] The test was performed according to a previous report (Biol. Pharm. Bull. 2017 40, 1381-1388, Cancer Epidemiol Biomarkers Prev. 2011 Aug; 20(8): 1622-8.), and the UVB irradiation conditions were set to an illuminance that would not cause rapid and significant skin inflammation in mice (wavelength: 302 nm, illuminance: 38 mJ / cm ２ UVB irradiation was performed six times every other day.

[0069] The UVB-irradiated mice were fed a diet containing 3% grape pomace powder (GPE powder) or regular diet starting 4 weeks before the start of UVB irradiation. Melanin levels and redness, which represent skin color, were measured using a skin colorimeter (Delphin Technologies).

[0070] The results are shown in Figures 5 to 7. UVB-irradiated mice fed a GPE-containing diet (UVB-irradiated / GPE-containing diet group) showed significantly reduced sunburn-induced inflammation and darkening compared with mice fed a standard diet (UVB-irradiated / standard diet group) (Figure 5). A significant difference in melanin levels was observed between unirradiated and irradiated mice on day 8 of UVB irradiation, and by day 12, the UVB-irradiated / GPE-containing diet group showed a significant reduction in melanin levels compared with the UVB-irradiated / standard diet group (Figure 6). A significant difference in redness was observed between unirradiated and irradiated mice from day 2 of UVB irradiation, and by day 6, the UVB-irradiated / GPE-containing diet group showed a significant reduction in redness compared with the UVB-irradiated / standard diet group, suggesting that the reduction in redness persisted until day 10 (Figure 7). Note that the degree of redness tended to decrease relatively after day 8, when melanin levels increased.

[0071] Furthermore, on day 14 after the start of UVB irradiation, blood hydroperoxide levels, a marker of oxidative stress, were measured using the d-ROM test. In the UVB-irradiated / GPE-containing diet group, the increase in oxidative stress due to UVB irradiation was suppressed (Figure 8).

[0072] Example 3 Effect Test on Rheumatoid Arthritis Model Rats were prepared by administering 0.1 mL of Freund's complete adjuvant to the hind leg joints of male Wistar rats, and it was examined whether there were any differences in the irregularities of the joint surface and pain threshold due to rheumatoid arthritis between the grape pomace intake group and the non-intake group.

[0073] First, the antioxidant activity of grape pomace was investigated in vivo using healthy rats. Male Wistar rats were divided into three groups: a control group, a 1% GPE-containing group, and a 2% GPE-containing group. The GPE-containing group was given ad libitum GPE mixed with powdered feed at a ratio of 1% or 2%. Plasma hydroperoxide concentrations, a marker, were measured using the d-ROM test one, two, and three weeks after the start of feeding.

[0074] The measurement results are shown in Figure 9. In the normal diet group (control), the plasma oxidative stress level (Diacron-Reactive Oxygen Metabolites: dROM value) was significantly elevated, but in the 2% GPE-containing diet group, no elevation was observed. These results confirmed that grape pomace has antioxidant properties in vivo.

[0075] Next, the effect on a rheumatoid arthritis model was examined. The results of comparing the macroscopic findings of the hind limb joints for three groups: healthy rats fed a normal diet (control), rheumatoid arthritis model rats fed a normal diet (AA group), and rheumatoid arthritis model rats fed a diet containing 2% GPE (AA + GPE group) are shown in Figure 10. Compared to the control, the AA group showed significant swelling and redness that spread from around the hind limb joints to the toes. In contrast, the AA + GPE group showed swelling of the hind limb joints, but the degree of swelling was milder than that of the AA group.

[0076] In addition, CT scans of the hindlimb joints were performed for the three groups, and the results of comparing the image findings are shown in Figure 11. In the control group, smooth cortical bone and well-arranged tarsal bones were observed, while in the AA group, irregularities in the articular surface, osteophyte formation, and the presence of loose bodies were observed. These findings strongly suggested that the AA group had developed clear arthritis in the hindlimb joints, resulting in bone and cartilage destruction. In contrast, in the AA + GPE group, although mild joint irregularities were observed, the progression of bone destruction was clearly suppressed compared to the AA group.

[0077] Furthermore, the results of pain evaluation using the von Frey test for the above three groups are shown in Figure 12. The pain threshold decreased in the AA group, but this decrease was significantly suppressed in the AA+GPE group.

[0078] These test results suggest that grape pomace is effective in alleviating the symptoms of rheumatoid arthritis, and that this effect is due to the antioxidant properties of grape pomace.

[0079] [Example 4] Effect test on social isolation stress model rats Psychiatric disorders such as schizophrenia and autism have been reported to be caused by oxidative stress. Therefore, the effectiveness of grape pomace for treating psychiatric disorders was examined.

[0080] Social isolation stress model rats were prepared as a psychiatric model by rearing them in isolation for 28 days. At the same time as the start of the isolation rearing, they were given normal food, food containing 1% GPE, or food containing 2% GPE. After 28 days of rearing, another rat was introduced into the cage as an intruder. The time (seconds) spent by the three groups of social isolation stress model rats to attack the newly introduced rat was counted within 10 minutes of being placed in the cage.

[0081] The results are shown in Figure 13. The average attack time for socially stressed rats on the normal diet (control) was 250 seconds, whereas the attack time for socially stressed rats on the 1% GPE-containing diet (Sress + 1% GPE group) and socially stressed rats on the 2% GPE-containing diet (Sress + 2% GPE group) was approximately 150 seconds and approximately 70 seconds, respectively, indicating a grape pomace concentration-dependent decrease in attack time.

[0082] Example 5: Antioxidant Activity of Novel Compounds Among the novel compounds contained in the photoirradiated grape pomace whose structure was determined in Example 1, Compound 1 was evaluated for its antioxidant activity, as its hydroxyl radical (·OH) scavenging ability. Epigallocatechin gallate (EGCG), which has been highly evaluated for its antioxidant activity, was used as a comparative compound. The hydroxyl radical (·OH) scavenging ability test was performed as in Example 1 by generating hydroxyl radicals via the Fenton reaction, measuring the amount of hydroxyl radicals scavenged by the sample, and calculating the scavenging rate. Specifically, the absorbance of the well containing solvent alone (no Compound 1 added) was defined as 100% radical intensity, and the radical intensity of the well containing Compound 1 (10-fold diluted) was expressed as a relative percentage (%). The 50% inhibitory concentration (IC50) of hydroxyl radical (·OH) was calculated from this value. The results are shown in Table 3.

[0083]

[0084] As shown in Table 3, epigallocatechin gallate (EGCG) had a 50% inhibitory concentration (IC ５０ ) value was 720 μg / mL, whereas the IC ５０ The value was 14.8 μg / mL, indicating approximately 50 times higher antioxidant activity.

[0085] Furthermore, the hydroxyl radical scavenging rate of compound 1 before light irradiation was 50%, but after light irradiation it increased to 60%. On the other hand, the hydroxyl radical scavenging rate of EGCG before light irradiation was 50%, but after light irradiation it decreased to 28% (Table 4). Therefore, it was proven that the hydroxyl radical scavenging rate of compound (1) is sustained.

[0086]

[0087] The antioxidant composition of the present invention has sustained antioxidant activity in vivo or in vitro. Therefore, the present invention can be used in the fields of manufacturing cosmetics, pharmaceuticals, foods, etc. for treating, ameliorating, and preventing diseases and pathologies caused by reactive oxygen species due to oxidative stress. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An antioxidant composition containing, as an active ingredient, a photo-irradiated product of grape pomace.

2. The antioxidant composition according to claim 1, wherein the wavelength of the photo-irradiation is 200 to 700 nm.

3. The antioxidant composition according to claim 1 or 2, wherein the grape pomace is the pomace after grape pressing, which is a waste in wine production.

4. An antioxidant composition containing, as an active ingredient, a compound represented by the following general formula (I). (In the formula, the bonds represented by solid and dotted lines of (a) to (g) are single bonds or double bonds. When (a) and (c) are double bonds and (b) and (d) are single bonds, R １ is an oxygen atom, R ２ represents a hydroxy group. When (a) and (c) are single bonds and (b) and (d) are double bonds, R １ is a hydroxy group, R ２ represents an oxygen atom. R ３ , R ４ , and R ７ each independently represent a hydrogen atom or a hydroxy group. When (e) and (g) are single bonds and (f) is a double bond, R ５ and R ６ represent hydroxy groups. When (e) and (g) are double bonds and (f) is a single bond, R ５ and R ６ represent oxygen atoms. R ８ and R ９ each independently represent a hydrogen atom or a hydroxy group, or R ８ and R ９ together form a single bond.) 5. The antioxidant composition according to claim 4, wherein the compound represented by the general formula (I) is a compound represented by the following structural formula (1), (2) or (3).

6. The antioxidant composition according to claim 1 or 4, wherein the antioxidant composition is a cosmetic, a pharmaceutical, or a food or drink product.

7. The antioxidant composition according to claim 1 or 4, wherein the antioxidant composition is a hygiene product, a detergent, a textile product, a food packaging material, a filter product, or a paper product.

8. A method for producing a sustainable antioxidant material, comprising a step of pressing grape raw materials in a wine production process to obtain grape pomace containing grape skins and seeds, and a step of performing a photo-irradiation treatment on the obtained grape pomace.

9. The production method according to claim 8, wherein an extraction treatment of the grape pomace is performed before the photo-irradiation treatment.

10. A novel compound represented by the following general formula (I). (In the formula, the bonds represented by the solid and dotted lines of (a) to (g) are single bonds or double bonds. When (a) and (c) are double bonds and (b) and (d) are single bonds, R １ represents an oxygen atom, and R ２ represents a hydroxy group. When (a) and (c) are single bonds and (b) and (d) are double bonds, R １ represents a hydroxy group, and R ２ represents an oxygen atom. R ３ , R ４ , and R ７ each independently represent a hydrogen atom or a hydroxy group. When (e) and (g) are single bonds and (f) is a double bond, R ５ and R ６ represent hydroxy groups. When (e) and (g) are double bonds and (f) is a single bond, R ５ and R ６ represent oxygen atoms. R ８ and R ９ each independently represent a hydrogen atom or a hydroxy group, or R ８ and R ９ together form a single bond.) 11. The novel compound according to claim 10, wherein the compound represented by the general formula (I) is a compound represented by the following structural formula (1), (2) or (3).

12. A method for producing a novel compound, which is the production method according to claim 10 or 11, characterized in that after performing a photo-irradiation treatment on grape pomace, a solvent extraction treatment is performed, or after performing a solvent extraction treatment on grape pomace, a photo-irradiation treatment is performed, and the compound is separated and purified from the treated product.

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