Antioxidant for skin, Anti-wrinkle agent, Anti-oxidizing food for skin or Anti-aging beverage for skin, Anti-aging medicine for skin, Anti-oxidizing medicine for skin, and Anti-oxidizing cosmetic for skin

Abstract

This antioxidant for skin contains nicotinamide mononucleotide, docosahexaenoic acid and eicosapentaenoic acid.

Description

Antioxidants for skin, anti-wrinkle agents, antioxidant foods or anti-aging beverages for skin, anti-aging drugs for skin, antioxidant drugs for skin, and antioxidant cosmetics for skin.

[0001] This invention relates to antioxidants for skin, antioxidant foods or beverages for skin, antioxidant pharmaceuticals for skin, and antioxidant cosmetics for skin.

[0002] The three major aging reactions in living organisms are glycation, oxidation, and chronic inflammation (which can also be called vascular aging, such as arteriosclerosis). Furthermore, the three major causes of aging are obesity, decreased immune function efficiency, and the accumulation of senescent cells. Senescent cells accumulate reactive oxygen species, and this accumulation further accelerates cellular aging, so the function of eliminating these reactive oxygen species is considered important.

[0003] In recent years, nicotinamide mononucleotide (hereinafter sometimes referred to as NMN) has attracted attention. NMN is a vitamin B ３ A derivative of niacin, which in the body is converted into nicotinamide adenine dinucleotide (oxidized form) (NAD ＋ It is converted into a molecule called NAD. ＋ Because it activates sirtuin genes, also known as anti-aging genes or longevity genes, it is expected to have an anti-aging effect. Studies have also been conducted on combining NMN with other substances. For example, Patent Document 1 proposes a hepatocyte cell activator that combines NMN with other substances including naturally derived calcined zeolite powder and a food additive reducing agent.

[0004] Patent No. 7610823

[0005] While Patent Document 1 concerns hepatocytes, there is a need for a method that is effective against other cells and tissues.

[0006] The object of the present invention is to provide a skin antioxidant that has antioxidant activity on the skin, an antioxidant food or beverage for the skin using this skin antioxidant, an antioxidant pharmaceutical, and an antioxidant cosmetic for the skin.

[0007] The inventors of the present invention discovered that the above objective can be achieved by combining NMN with a predetermined substance, and thus completed the present invention.

[0008] In other words, according to the present invention: (1) a skin antioxidant comprising nicotinamide mononucleotide, docosahexaenoic acid and eicosapentaenoic acid; (2) the skin antioxidant according to (1) further comprising 5-aminolevulinic acid; (3) the skin antioxidant according to (1) or (2) comprising 80 to 120 parts by weight of eicosapentaenoic acid per 100 parts by weight of docosahexaenoic acid; (4) the skin antioxidant according to (3) comprising 100 parts by weight of eicosapentaenoic acid per 100 parts by weight of docosahexaenoic acid; (5) an antioxidant food or antioxidant beverage for the skin comprising the skin antioxidant according to (1) or (2); (6) an antioxidant pharmaceutical for the skin comprising the skin antioxidant according to (1) or (2); (7) A skin antioxidant cosmetic comprising the skin antioxidant described in (1) or (2) is provided.

[0009] According to the present invention, there are skin anti-aging agents having an anti-aging effect on the skin, antioxidant foods or beverages for the skin using this skin antioxidant, antioxidant pharmaceuticals and antioxidant cosmetics for the skin.

[0010] This graph shows the measurement results of ORAC in the examples. This graph shows the measurement results of Superoxide (SOD) in relation to hydrogen peroxide in the examples. This graph shows the results of the study on anti-glycation effect in the examples. This graph shows the results of the study on anti-wrinkle and moisturizing effects in the examples.

[0011] The following describes the anti-aging agent for skin and the method for producing the same according to the present invention.

[0012] The anti-aging agent for skin of the present invention comprises nicotinamide mononucleotide, docosahexaenoic acid (hereinafter sometimes referred to as "DHA"), and eicosapentaenoic acid (hereinafter sometimes referred to as "EPA").

[0013] (NMN) NMN has α-type and β-type optical isomers, and either α-type or β-type may be used, but β-type is preferred. Furthermore, NMN may be derived from natural products or artificially synthesized. If derived from natural products, extraction is preferably carried out by known extraction methods.

[0014] (DHA) The DHA used in this invention is a type of omega-3 fatty acid, which is mainly found in abundance in marine foods.

[0015] DHA can be obtained from marine fish oil and algae. For fish oil, it is particularly preferable to use fish oil from oily fish such as sardines, mackerel, and saury. Specifically, DHA can be obtained from fish oil and algae by pressing or solvent extraction, followed by purification and concentration. In the manufacturing process, to obtain a high concentration of DHA, steps such as concentration by molecular distillation or esterification followed by separation and concentration may be included.

[0016] (EPA) EPA is a type of omega-3 fatty acid, and is particularly abundant in marine foods. EPA can be obtained from marine fish oil and algae. As for fish oil, it is preferable to use fish oil from oily fish such as sardines, mackerel, and saury. Specifically, it can be obtained from fish oil or algae by pressing or solvent extraction, followed by purification and concentration. In addition, in order to obtain high concentrations of EPA, the manufacturing process may include steps such as concentration by molecular distillation or esterification followed by separation and concentration.

[0017] For the DHA and EPA used in the present invention, LDR-DHA / EPA Premier® is preferred, which contains EPA in an amount of 80 to 120 parts by weight, more preferably 90 to 110 parts by weight, and particularly preferably 100 parts by weight, of DHA per 100 parts by weight. In LDR-DHA / EPA Premier®, the ratio of DHA to EPA is approximately equal, which is a unique ratio not seen elsewhere. In LDR-DHA / EPA Premier, DHA and EPA are each preferably contained in an amount of 10 to 50% by weight, more preferably 20 to 40% by weight, and even more preferably 25 to 35% by weight.

[0018] (5-ALA) In addition to the above-mentioned NMN, DHA, and EPA, the anti-aging agent for skin of the present invention may further contain 5-aminolevulinic acid (hereinafter sometimes referred to as "5-ALA"). 5-ALA may be produced by chemical synthesis or by microbial fermentation.

[0019] (Anti-aging agent for skin) The anti-aging agent for skin of the present invention contains NMN, DHA, and EPA, and optionally contains 5-ALA. The mixing ratio is not particularly limited, but it is preferable to mix 1 to 10,000 parts by weight of DHA per 100 parts by weight of NMN. It is also preferable to mix 1 to 10,000 parts by weight of EPA per 100 parts by weight of NMN. Furthermore, when 5-ALA is included, it is preferable to mix 1 to 10,000 parts by weight of 5-ALA per 100 parts by weight of NMN. The above components may be mixed by known methods. As mentioned above, it is preferable to use LDR-DHA / EPA Premier®, which contains approximately equal amounts of DHA and EPA.

[0020] The anti-aging agent for skin of the present invention may contain other ingredients, provided they do not inhibit the effects of the present invention. Other ingredients include resveratrol, L-cystine, vitamin C, pineapple extract (containing ceramide), red orange extract, pasenol, GABA, coenzyme Q10, lactic acid bacteria powder, odorless garlic powder, alpha-lipoic acid, ginger powder, vitamin A, vitamin D, vitamin E, biotin, and vitamin B １ Vitamin B ２ Vitamin B ６ Vitamin B １２ Examples include niacin, pantothenic acid, folic acid, calcium, magnesium, iron, manganese, copper, selenium, chromium, molybdenum, zinc, valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, histidine, glycine, arginine, glutamic acid, alanine, aspartic acid, proline, serine, and tyrosine.

[0021] The anti-aging agent for skin of the present invention exhibits excellent antioxidant effects in the skin. Furthermore, it also exhibits excellent anti-glycation, anti-wrinkle, and moisturizing effects in the skin. In other words, it demonstrates superior anti-aging effects on the skin.

[0022] (Anti-aging foods or beverages for the skin) The anti-aging agent for the skin of the present invention can be incorporated into anti-aging foods or beverages for the skin. Examples of anti-aging foods for the skin include bread, noodles, confectionery, processed meat products, processed seafood products, frozen foods, jellies, ice cream, dairy products, and various seasonings. In addition to general foods, it can also be incorporated into foods for specified health uses, quasi-drugs, health foods, and supplements. Examples of anti-aging beverages for the skin include soft drinks, dairy drinks, alcoholic beverages, tea, black tea beverages, coffee, fruit juices, carbonated drinks, mineral water, and fruit and vegetable beverages.

[0023] Furthermore, when manufacturing foods or beverages containing the anti-aging agent for skin of the present invention, additives such as sweeteners, colorants, preservatives, thickeners, stabilizers, gelling agents, antioxidants, color fixatives, bleaching agents, emulsifiers, leavening agents, acidulants, glazing agents, and flavorings; solvents; and oils may be added as needed, to the extent that they do not interfere with the effects of the present invention. These additives may be used individually or in combination of two or more types.

[0024] The proportion of the anti-aging agent for skin of the present invention incorporated into the above-mentioned anti-aging food or beverage for skin can be appropriately adjusted according to the intended use, but the proportion of the ingredient formulation incorporated into the above-mentioned food or beverage is preferably 0.01 to 20% by weight, more preferably 0.01 to 15% by weight, and even more preferably 0.1 to 10% by weight.

[0025] Furthermore, the anti-aging food or beverage for skin of the present invention may be in the same form as the oral formulations such as tablets, capsules, and syrups used in the anti-aging pharmaceuticals for skin described later.

[0026] (Anti-aging drugs for the skin) Furthermore, the anti-aging agent for the skin of the present invention can be incorporated into anti-aging drugs for the skin. Examples of anti-aging drugs for the skin include preventive drugs and therapeutic drugs.

[0027] When incorporated into anti-aging drugs for skin use, the active ingredient may be used alone or mixed with generally pharmaceutically acceptable excipients to form a formulation. Regarding the form of administration, there are no particular restrictions, and the appropriate form can be selected as appropriate depending on the purpose of treatment or prevention. Examples of administration methods include oral formulations such as tablets, granules, capsules, pills, powders, liquids, suspensions, emulsions, syrups, elixirs, and extracts, or parenteral formulations such as injections, liquids, suppositories, ointments, patches, poultices, and lotions.

[0028] Furthermore, in the case of tablets, granules, pills, capsules, and powders, additives such as excipients, binders, disintegrants, and lubricants may be included. Examples of excipients include starch, carboxymethylcellulose, sucrose, dextrin, and corn starch.

[0029] Examples of binders include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carmellose sodium, ethylcellulose, carboxymethyl ethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl methacrylate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, refined sucrose, and macrogol.

[0030] Examples of disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, partially pregelatinized starch, hydroxypropyl starch, carboxymethyl starch sodium, and tragacanth.

[0031] Examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrated silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dried aluminum hydroxide gel, talc, magnesium aluminometasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid esters, waxes, hydrogenated vegetable oils, and polyethylene glycol.

[0032] Furthermore, in the case of liquid preparations, syrups, suspensions, emulsions, and elixirs, in addition to commonly used inert diluents such as water and vegetable oil, colorants, flavoring agents, and fragrances may be included as additives.

[0033] Furthermore, in the case of injectable preparations, additives such as suspensions, emulsions, and solvents for use may be included. In the case of ointments and suppositories, additives such as fats, fatty oils, lanolin, petrolatum, paraffin, waxes, resins, plastics, bases, glycols, higher alcohols, water, emulsifiers, and suspending agents may be included. In the case of poultices, additives such as glycerin, water, water-soluble polymers, and superabsorbent polymers may be included. In the case of lotions, additives such as solvents, emulsifiers, and suspending agents may be included.

[0034] Furthermore, the anti-aging agent for skin of the present invention can also be incorporated into cosmetics. Examples of cosmetics include lotions, emulsions, facial washes, cleansers, serums, creams, foundations, eyebrow products, mascaras, eyeshadows, eyeliners, lipsticks, lip glosses, blushes, face powders, and nail polishes. The cosmetic can also be in the form of a liquid, cream, solid, stick, or powder.

[0035] The present invention will be described below with reference to examples, but the present invention is not limited thereto. In these examples, parts and percentages are based on weight unless otherwise specified.

[0036] (Example 1: ORAC Measurement) The antioxidant capacity of the sample was measured using the ORAC (Oxygen Radical Absorbance Capacity) method with Trolox as the standard antioxidant. The measurement was performed using the ORAC Activity Assay Kit. The sample was as follows. The sample was prepared so that the main component was several tens of μg / mL and serially diluted before use.

[0037] Sample 1: DHA and EPA (blended in a 1:1 weight ratio) Sample 2: NMN Sample 3: 5-ALA Sample 4: DHA, EPA, NMN, and 5-ALA (DHA and EPA are blended in a 1:1 weight ratio) Sample 5: DHA, EPA, and NMN (DHA and EPA are blended in a 1:1 weight ratio)

[0038] The results are shown in Figure 1. The values ​​on the horizontal axis of "ORAC" in Figure 1 indicate the dilution ratio. As shown in Figure 1, sample 4 showed a high ORAC value.

[0039] (Example 2: Measurement of Superoxide (SOD) in response to hydrogen peroxide) Human dermal fibroblasts were incubated for 2 hours in a culture medium containing 0.2 mM hydrogen peroxide, then washed off with PBS and replaced with normal culture medium to continue culturing. Materials 1 to 5 described above were added after the addition of hydrogen peroxide, and co-cultured for 4 hours. After that, the cells or culture medium were collected.

[0040] Subsequently, superoxide (SOD) levels were measured using the SOD Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). After culturing the treated cells for 24 hours, Lysis buffer was added, homogenized, and centrifuged. Then, 10 μL of each sample was mixed with 200 μL of xanthine oxidase at room temperature. After 30 minutes, the absorbance at 450 nm was measured.

[0041] The results are shown in Figure 2. In Figures 2 and 3, the numerical values on the horizontal axis represent the number of serial dilutions performed, 1 / 5<n indicates that 5-fold dilution was performed n times (n is an integer from 1 to 7), and "1" indicates the sample before serial dilution.

[0042] As shown in Figure 2, SOD scavenging or inhibitory effects were observed in dermal fibroblasts in Samples 4 and 5

[0043] (Example 3: Examination of anti-glycation effect) (Preparation of AGE) AGE was obtained by dissolving bovine serum albumin (25 mg / mL) in DL-glyceraldehyde and culturing in 0.1 M phosphate buffered saline (pH 7.4; sometimes referred to as "PBS" in this example) at 37°C for one week. Human dermal fibroblasts (Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin (C-12300, PromoCell)) were cultured at 37°C in 5% CO ２ incubator. Next, human dermal fibroblasts were treated with AGE (100 μg / mL), and the above-mentioned Samples 1 to 5 serially diluted (1-fold, 1 / 10-fold, 1 / 100-fold and 1 / 1000-fold) were added respectively, followed by holding at 37°C for 4 hours.

[0044] Total RNA was extracted from the cells using Trizol reagent (Ambion), and mRNA levels of AGE receptors (CD-36, AGE-R1, and AGE-R3) were measured using qRT-PCR (one-step quantitative reverse transcription-polymerase chain reaction). The results are shown in Figure 3. CD-36, AGE-R1, and AGE-R3 are AGE receptors involved in the degradation and removal of AGEs. The qRT-PCR method will be described in detail later in the section "qRT-PCR Method". As shown in Figure 3, elevated mRNA levels of CD-36, AGE-R1, and AGE-R3 were observed in AGE-glycation stress cells at high concentrations of DHA & EPA (Sample 1). In addition, all samples showed a tendency to increase CD-36 and AGE-R1 levels.

[0045] (Example 4: Investigation of anti-wrinkle and moisturizing effects) Human dermal fibroblasts were incubated for 2 hours in a culture medium containing 0.2 mM hydrogen peroxide, then washed with PBS, and cultured in normal medium. Samples 1 to 4 were added after hydrogen peroxide was added to each sample and co-cultured for 4 hours. After that, the cells or culture medium were collected. Total RNA was extracted from the cells using Trizol reagent (Ambion), and the mRNA level of hyaluronic acid was measured by qRT-PCR. The results are shown in Figure 4(a).

[0046] Furthermore, using a predetermined culture medium, samples 1 to 4, each serially diluted (1x, 1 / 10x, 1 / 100x, and 1 / 1000x), were added to stem cells (human mesenchymal stem cells) and cultured in an incubator at 37°C. Exosomes were then extracted from the supernatant of the culture medium containing the stem cells using an ultracentrifuge. The extracted exosomes were added to dermal fibroblasts and incubated for 4 hours.

[0047] Furthermore, human skin fibroblasts (Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin (C-12300, PromoCell)) were cultured at 37°C under 5% CO ２ in an incubator using a dedicated medium, Fibroblast Growth Medium (C-23010, PromoCell).

[0048] Then, total RNA was extracted from the cells using Trizol reagent (Ambion), and the mRNA levels of collagen, elastin and hyaluronic acid were measured by qRT-PCR. The results are shown in Figure 4(b) for collagen, Figure 4(c) for elastin, and Figure 4(d) for hyaluronic acid, respectively.

[0049] As shown in Figure 4, under H2O2 oxidative stress, an increase in hyaluronic acid synthase mRNA level was observed in Sample 1 (DHA&EPA). Furthermore, when exosomes obtained by stimulating stem cells with each sample were added to dermal fibroblasts, the mRNA levels of collagen and elastin increased. In addition, the mRNA level of hyaluronic acid synthase increased in all samples. This is considered to be caused by the secretion of exosomes different from those normally secreted. That is, it is considered that these results suggest anti-wrinkle effects and moisturizing effects in skin cells.

[0050] (qRT-PCR method) The qRT-PCR method used for measuring the aforementioned AGE receptor, collagen, elastin and hyaluronic acid is described below. For the qRT-PCR method, 500 ng of the obtained total RNA was used as a PCR template, and One step RT-PCR, which enables cDNA synthesis by reverse transcription (RT reaction) and quantitative PCR in a single tube, was performed using the Luna Universal One-Step qRT-PCR Kit.

[0051] A Takara Thermal Cycler Dice Real Time System II PCR instrument was used. For one reaction system, 10 μL of Luna Universal One-Step Reaction Mix (2x), 1 μL of Luna WarmStart RT Enzyme Mix (20x), 0.8 μL of Forward primer (10 μM), 0.8 μL of Reverse primer, and Total RNA as the PCR template were prepared by adding 20 μL of Nuclease-free Water. The reaction was carried out under conditions of 30 seconds per cycle at 95°C, followed by 50 cycles of 5 seconds at 95°C and 30 seconds at 60°C.

Claims

1. A skin antioxidant comprising nicotinamide mononucleotide, docosahexaenoic acid, and eicosapentaenoic acid.

2. The skin antioxidant according to claim 1, further comprising 5-aminolevulinic acid.

3. The skin antioxidant according to claim 1 or 2, comprising 80 to 120 parts by weight of eicosapentaenoic acid per 100 parts by weight of docosahexaenoic acid.

4. The skin antioxidant according to claim 3, comprising 100 parts by weight of eicosapentaenoic acid per 100 parts by weight of docosahexaenoic acid.

5. A skin antioxidant food or skin antioxidant beverage comprising the skin anti-aging agent described in claim 1 or 2.

6. A skin antioxidant pharmaceutical comprising the skin anti-aging agent described in claim 1 or 2.

7. A skin antioxidant cosmetic comprising the skin anti-aging agent described in claim 1 or 2.

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