Anti-wrinkle agent and moisturizer for skin cells

Abstract

The present invention provides an anti-aging agent for the skin that has an anti-aging effect on the skin. [Solution] The solution includes a stem cell-derived exosome obtained in the presence of nicotinamide mononucleotide, docosahexaenoic acid, eicosapentaenoic acid, and 5-aminolevulinic acid.

Description

TECHNICAL FIELD

[0001] The present invention relates to an anti-aging agent for skin, an anti-wrinkle agent, an anti-aging food for skin or an anti-aging beverage for skin, an anti-aging pharmaceutical for skin, an anti-aging cosmetic for skin, and a method for producing the anti-aging agent for skin.

BACKGROUND ART

[0002] The three major aging reactions in living organisms include glycation reaction, oxidation reaction, and chronic inflammation (which can also be referred to as vascular aging such as arteriosclerosis). In addition, the three major causes of aging include obesity, reduced efficiency of immune function, and accumulation of senescent cells. Senescent cells accumulate reactive oxygen species, and the accumulation of reactive oxygen species further accelerates cellular aging, so the function of eliminating them is highly valued.

[0003] In recent years, nicotinamide mononucleotide (hereinafter sometimes referred to as NMN) has attracted attention. NMN is a derivative of vitamin B3 (niacin), and in the body it is converted into nicotinamide adenine dinucleotide (oxidized form) (NAD + ) molecule. NAD + activates sirtuin genes, which are also called anti-aging genes or longevity genes, and thus is expected to have an anti-aging effect. Studies on combining NMN with other substances have also been conducted. For example, Patent Document 1 proposes a cell activator for hepatocytes by combining NMN with other substances including naturally derived calcined zeolite powder and a reducing agent for food additives.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0004]

PATENT DOCUMENT 1

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[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 an anti-wrinkle agent and a moisturizer that are effective against skin cells. [Means for solving the problem]

[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) Anti-wrinkle agents in skin cells, including stem cell-derived exosomes obtained in the presence of nicotinamide mononucleotide, docosahexaenoic acid, eicosapentaenoic acid, and 5-aminolevulinic acid. (2) Moisturizing agent for skin cells containing stem cell-derived exosomes obtained in the presence of nicotinamide mononucleotide, docosahexaenoic acid, eicosapentaenoic acid, and 5-aminolevulinic acid It will be provided. [Effects of the Invention]

[0009] According to the present invention, an anti-wrinkle agent and a moisturizer that are effective against skin cells are provided. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the ORAC measurement results in the example. [Figure 2] This graph shows the measurement results of superoxide (SOD) in relation to hydrogen peroxide in the examples. [Figure 3] This graph shows the results of the study on the anti-glycation effect in the examples. [Figure 4] This graph shows the results of the study on the anti-wrinkle and moisturizing effects in the examples. [Modes for carrying out the invention]

[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- and β-optical isomers, and either the α- or β-form can be used, but the β-form is preferred. Furthermore, NMN may be derived from natural products or artificially synthesized. If it is derived from natural products, it is preferable to perform extraction using 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, it can be obtained by extracting from fish oil or algae using pressing or solvent extraction methods, followed by purification and concentration. Furthermore, in order to obtain a high concentration of DHA, the manufacturing process may include steps such as concentration by molecular distillation or esterification followed by further separation and concentration.

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

[0017] Regarding DHA and EPA used in the present invention, LDR-DHA·EPA PREMIA (registered trademark), in which EPA is preferably blended 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 relative to 100 parts by weight of DHA, is suitable. In LDR-DHA·EPA PREMIA (registered trademark), the blending ratio of DHA and EPA is approximately equal, which is a blending ratio not found elsewhere. In LDR-DHA·EPA PREMIA, DHA and EPA are each contained in an amount of preferably 10 to 50% by weight, more preferably 20 to 40% by weight, and even more preferably 25 to 35% by weight.

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

[0019] (Anti-aging agent for skin) The anti-aging agent for skin of the present invention comprises NMN, DHA and EPA, and further contains 5-ALA as necessary. The blending ratio is not particularly limited, but it is preferable to blend DHA in an amount of 1 to 10000 parts by weight relative to 100 parts by weight of NMN. Moreover, it is preferable to blend EPA in an amount of 1 to 10000 parts by weight relative to 100 parts by weight of NMN. When 5-ALA is blended, it is preferable to blend 5-ALA in an amount of 1 to 10000 parts by weight relative to 100 parts by weight of NMN. The above components may be mixed by a known method. As described above, as DHA and EPA, it is preferable to use LDR-DHA·EPA PREMIA (registered trademark) in which these are blended in approximately equal amounts.

[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, vitamin B1, vitamin B2, vitamin B6, and vitamin B1. 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 skin of the present invention can be incorporated into anti-aging foods or beverages for skin. Examples of anti-aging foods for 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 skin include soft drinks, dairy beverages, 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 depending on 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 skin) Furthermore, the anti-aging agent for skin of the present invention can be incorporated into anti-aging pharmaceuticals for skin. Examples of anti-aging pharmaceuticals for 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. [Examples]

[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: Measurement of ORAC) The antioxidant capacity of the samples was measured using the ORAC (Oxygen Radical Absorbance Capacity) method with Trolox as the standard antioxidant. The measurements were performed using the ORAC Activity Assay Kit. The samples are as follows. The samples were prepared so that the main component was present in concentrations of several tens of μg / mL, and serially diluted.

[0037] Sample 1: DHA and EPA (combined 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 in a weight ratio of 1:1) Sample 5: DHA, EPA, and NMN (DHA and EPA are combined in a weight ratio of 1:1)

[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 relation to hydrogen peroxide) 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 standard medium. Materials 1-5 were added after the addition of hydrogen peroxide, and co-cultured for 4 hours. Afterward, 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 dilution cycles, 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 was performed.

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

[0043] (Example 3: Examination of anti-glycation effect) (Preparation of AGEs) AGEs were obtained by culturing bovine serum-derived albumin (25 mg / mL) in phosphate-buffered saline (pH 7.4; sometimes referred to as "PBS" in this Example) dissolved with DL-glyceraldehyde to a concentration of 0.1 M at 37°C for 1 week. Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin (C-12300, PromoCell) were cultured in a dedicated medium, Fibroblast Growth Medium (C-23010, PromoCell), at 37°C in a 5% CO2 incubator. Next, human skin fibroblasts were treated with AGEs (100 μg / mL), and the above serially diluted Samples 1 to 5 (1-fold, 1 / 10-fold, 1 / 100-fold and 1 / 1000-fold) were added respectively, and the mixture was held at 37°C for 4 hours.

[0044] Then, total RNA was extracted from the cells using Trizol reagent (Ambion), and the mRNA levels of AGE receptors (CD-36, AGE-R1 and AGE-R3) were measured by the qRT-PCR (one-step quantitative reverse transcription-polymerase chain reaction) method. 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-glycated stress cells at high concentrations of DHA & EPA (Sample 1). Furthermore, all samples showed a tendency to increase CD-36 and AGE-R1 levels.

[0045] (Example 4: Examination of anti-wrinkle and moisturizing effects) Human dermal fibroblasts were incubated for 2 hours in a culture medium containing 0.2 mM hydrogen peroxide. After the initial rinse, the culture was washed with PBS and replaced with standard culture medium to continue. Samples 1-4 were added to each medium after the addition of hydrogen peroxide, and co-cultured for 4 hours. After that, the cells or culture medium were collected. Total RNA was then 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] The dermal fibroblasts used were human dermal fibroblasts (NHDF), juvenile foreskin (C-12300, PromoCell), cultured in a dedicated medium, Fibroblast Growth Medium (C-23010, PromoCell), at 37°C in a 5% CO2 incubator.

[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 for collagen are shown in Figure 4(b), for elastin in Figure 4(c), and for hyaluronic acid in Figure 4(d).

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

[0050] (qRT-PCR method) The qRT-PCR method used for measuring AGE receptors, collagen, elastin, and hyaluronic acid, as described above, is explained. In the qRT-PCR method, 500 ng of obtained total RNA was used as a PCR template, and a one-step RT-PCR was performed using the Luna Universal One-Step qRT-PCR Kit, which allows for cDNA synthesis by reverse transcription (RT reaction) and quantitative PCR to be performed in a single test tube.

[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. An anti-wrinkle agent for skin cells, comprising stem cell-derived exosomes obtained in the presence of nicotinamide mononucleotide, docosahexaenoic acid, eicosapentaenoic acid, and 5-aminolevulinic acid.

2. A moisturizer for skin cells containing stem cell-derived exosomes obtained in the presence of nicotinamide mononucleotide, docosahexaenoic acid, eicosapentaenoic acid, and 5-aminolevulinic acid.

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