β-NMN and proteoglycan-containing oral composition

Abstract

Provided is an oral composition containing β-nicotinamide mononucleotide (β-NMN) and a proteoglycan as active ingredients. The composition can be used for improving joint function as well as skin function.

Description

Oral composition containing β-NMN and proteoglycan

[0001] The present invention relates to an oral composition containing β-nicotinamide mononucleotide (β-NMN) and a glycoprotein called proteoglycan.

[0002] β-Nicotinamide mononucleotide (β-NMN) is an intermediate metabolite of β-nicotinamide adenine dinucleotide (NAD) (Patent Document 1, Non-Patent Document 1). Administering β-NMN to the body directly induces NAD biosynthesis and increases NAD concentration in tissues (Non-Patent Document 2).

[0003] Proteoglycans are a type of glycoconjugate, consisting of a core protein bound to glycosaminoglycans (acidic mucopolysaccharides). Proteoglycans are a major component of the extracellular matrix, present in skin, cartilage, bone, blood vessel walls, etc., and are involved in cell proliferation and adhesion together with collagen, laminin, fibronectin, etc. (Non-Patent Documents 3 and 4). Proteoglycans, along with chondroitin sulfate, are the main components of articular cartilage and are thought to be important for preventing joint disorders in the elderly.

[0004] International Publication No. 2014 / 146044

[0005] Liana R. Stein et al. The dynamic regulation of NAD metabolism in mitochondria. Trends in Endocrinology and Metabolism. 2012, Vol. 23, No. 9J. Yoshino et al. Nicotinamide Mononucleotide, a Key NAD+ Intermediate, Treats the Pathophysiology of Diet- and Age-Induced Diabetes in Mice. Cell Metab. 2011, 14(4), P. 528-536.CBNM. de Aguiar et al. Glycosaminoglycans mediate C6 glioma cell adhesion to extracellular matrix components and alter cell proliferation and cell migration. BMC Cell Biol 6:31, 2005.H. Munakata et al. Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor. Glycobiology 9:1023-1027, 1999.

[0006] The problems to be solved by the present invention include providing an oral composition that can protect cartilage and improve joint function, and providing an oral composition that can improve skin function.

[0007] The present inventors have discovered that the combined use of β-NMN and proteoglycan enhances the effect of improving joint function or skin function due to the synergistic effect of β-NMN and proteoglycan, and have completed the present invention.

[0008] Specifically, (1) an oral composition containing β-nicotinamide mononucleotide (β-NMN) and proteoglycan as active ingredients, (2) the composition of (1) for improving joint function, (3) the composition of (1) or (2) in which the ratio of β-NMN to proteoglycan is 10:90 to 90:10, (4) any of the compositions of (1) to (3) which is a pharmaceutical composition, (5) the composition of (4) for the prevention or treatment of osteoarthritis, rheumatoid arthritis, spondylosis osteoarthritis, intervertebral disc herniation, chondrodysplasia, articular cartilage damage due to trauma, articular disc damage, acute septic arthritis, tuberculous arthritis, syphilitic arthritis, rheumatic fever or systemic lupus erythematosus, or for cartilage repair in fractures or transplants, (6) any of the compositions of (1) to (3) which is a food composition, (7) The composition according to (1) for improving skin functions (for example, promoting skin fibroblast proliferation, type I collagen production, and / or elastin production).

[0009] According to the present invention, the effects of β-NMN and proteoglycan promote the differentiation and metabolism of chondrocytes, thereby exhibiting, for example, an effect of improving joint function and / or skin function.

[0010] This is a graph showing cartilage growth upon addition of β-NMN, proteoglycan, and a mixture of β-NMN and proteoglycan. Left: Addition of β-NMN only. Right: Comparison between addition of proteoglycan only and a mixture of β-NMN and proteoglycan. *, $, #: p<0.05. **, $$, ##: p<0.005. ***, $$$, ###: p<0.001.

[0011] In one aspect, the present invention provides an oral composition comprising β-NMN and a proteoglycan.

[0012] In this embodiment, β-NMN (nicotinamide mononucleotide) is represented by the following formula (I), and exists as optical isomers in α- and β-forms, but the present invention is directed to β-form nicotinamide mononucleotide. NAD is not absorbed into the body when orally ingested, but β-NMN is absorbed into the body, directly induces NAD biosynthesis, and can increase NAD concentrations in blood and tissues.

[0013]

[0014] In this embodiment, the method for obtaining β-NMN is not particularly limited, and a generally available product (e.g., commercially available β-NMN) may be used, or β-NMN may be prepared by a method described in a publication (e.g., WO 2019 / 181961). The β-NMN may be a crude product or a purified product. For example, any composition containing β-NMN, such as β-NMN contained in yeast extract, may be used.

[0015] In this embodiment, the proteoglycan used in combination with β-NMN is a glycoprotein in which sugar chains called glycosaminoglycans (hereinafter referred to as GAG), such as chondroitin sulfate and dermatan sulfate, are covalently bonded to a core protein. Proteoglycans are one of the main components of the extracellular matrix and are widely distributed throughout the body, including in skin and cartilage. GAG chains have a long, unbranched, linear structure. They are negatively charged due to the presence of numerous sulfate and carboxyl groups, and the GAG ​​chains assume an extended shape due to their electrical repulsive force. Furthermore, proteoglycans can retain large amounts of water due to the water affinity of sugars. The numerous GAG chains contained in proteoglycans flexibly retain water like a sponge, while also fulfilling functions unique to cartilage, such as elasticity and shock resistance.

[0016] The core protein of proteoglycans has the property of binding to various molecules in the matrix. In the case of cartilage proteoglycans, they have binding domains at the N-terminus that bind to hyaluronic acid and link proteins, and they can bind to these substances and even associate with each other in the same molecule. The C-terminus contains lectin-like domains and EGF-like domains, which bind to various other molecules. This property allows proteoglycans to build structures that are suited to each tissue.

[0017] Among proteoglycans, chondroitin sulfate-type proteoglycans are proteoglycans in which glycosaminoglycans, each of which has chondroitin sulfate as a constituent, are covalently bound to a core protein.

[0018] The salmon nasal cartilage-derived proteoglycan is a proteoglycan extracted from salmon nasal cartilage. Here, the salmon is, for example, a fish belonging to the genus Oncorhynchus, and preferably, salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of cell proliferation (e.g., more efficient cultivation of desired cells).

[0019] The method for obtaining the proteoglycan contained in the composition of this embodiment is not particularly limited, and a generally available proteoglycan (e.g., a commercially available proteoglycan) may be used, or the proteoglycan may be prepared by a method described in, for example, Japanese Patent No. 6317053 or Japanese Patent No. 7295572. The proteoglycan may be a crude product or a purified product.

[0020] The ratio of β-NMN to proteoglycan in the composition of this embodiment is arbitrary, and may be 1:99 to 99:1, for example, 10:90 to 90:10, 15:85 to 85:15, 20:80 to 80:20, 25:75 to 75:25, 30:70 to 70:30, 35:65 to 65:35, 40:60 to 60:40, etc., and preferably 80:20 to 50:50.

[0021] The β-NMN content in the composition of this embodiment is optional and may be 10 μg to 2000 mg, for example, 50 μg to 1000 mg, 100 μg to 500 mg, 200 μg to 250 mg, etc. The daily intake of β-NMN can be adjusted appropriately depending on the intake form, purpose of use, age, body weight, etc., but is generally adjusted to be in the range of 10 mg to 1000 mg.

[0022] The proteoglycan content in the composition of this embodiment is optional and may be 10 μg to 2000 mg, for example, 50 μg to 1000 mg, 100 μg to 500 mg, 200 μg to 250 mg, etc. The daily intake of proteoglycan can be adjusted as appropriate depending on the intake form, purpose of use, age, body weight, etc., but generally, the daily intake of proteoglycan for an adult human is preferably adjusted to be in the range of 1 mg to 100 mg.

[0023] In this embodiment, the amounts and ratios of β-NMN and proteoglycan to be mixed can be adjusted as needed to achieve the intake amounts described above.

[0024] In one embodiment, the composition of this aspect is a pharmaceutical composition. The pharmaceutical composition may be a mixture of β-NMN and proteoglycan formulated according to conventional methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and may contain pharmaceutically acceptable carriers and additives. Examples of suitable carriers include, but are not limited to, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Other commonly used carriers may also be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, maltitol, sorbitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, dextrin, and inorganic salts.

[0025] The method of administration of the pharmaceutical composition of this embodiment is not particularly limited, and examples thereof include oral administration, injection, transdermal administration, etc., with oral administration being preferred. The form of the pharmaceutical composition is not particularly limited, and examples thereof include tablets, powders, granules, pills, suspensions, emulsions, infusions / decoctions, capsules, syrups, liquids, elixirs, extracts, tinctures, and liquid extracts.

[0026] The subjects to which the pharmaceutical composition is administered include humans, animals including humans, and animals not including humans, such as, but not limited to, humans, mice, rats, monkeys, pigs, cows, horses, goats, sheep, donkeys, dogs, cats, rabbits, hamsters, guinea pigs, chickens, and other livestock, poultry, and pets.

[0027] The pharmaceutical composition may be administered three times a day, twice a day, or once a day. For example, the pharmaceutical composition may be administered to a subject with or before or after each meal, or once a day, for example, with or before or after breakfast, lunch, or dinner. The pharmaceutical composition may also be administered between meals.

[0028] In some embodiments, the composition of this aspect is a food composition, more specifically, a functional food, dietary supplement, or supplement. The food composition may contain the carriers and additives listed above for the pharmaceutical composition. The form of the food composition is not particularly limited, and examples include concentrated liquids, powders, granules, tablets, gums, candies, capsules, pastes, jellies, drinks, etc.

[0029] Examples of other ingredients that can be contained in the food composition include seasonings such as sugar, salt, soy sauce, miso, and vinegar; sweeteners such as honey, maple syrup, oligosaccharides, and high-intensity sweeteners; acidulants such as citric acid, malic acid, tartaric acid, and acetic acid; bittering agents such as caffeine and naringin; spices such as oregano, pepper, cinnamon, ginger, turmeric, chili pepper, basil, paprika, and wasabi; oils such as salad oil, sesame oil, safflower oil, sunflower oil, rapeseed oil, fish oil, beef tallow, and lard; flavor oils; extracts such as yeast extract, seafood extract, beef extract, pork extract, and chicken extract; fruit juice; vegetable juice; vitamins; minerals; sugars; oligosaccharides; polysaccharides; dietary fiber; amino acids; peptides; fatty acids; coenzymes; flavorings; emulsifiers; thickeners; and colorants.

[0030] The food composition may be taken three times a day, twice a day, or once a day. In some embodiments, the food composition may be taken as a functional food, for example, with or before or after each meal, or once a day, for example, with or before or after breakfast, lunch, or dinner. It may also be taken between meals.

[0031] The composition of this embodiment can be used to improve joint function. The improvement of joint function of the present invention refers to the treatment, improvement, or prevention of cartilage damage. Furthermore, the composition of the present invention can be used as a chondrocyte differentiation promoter, chondrocyte proliferation promoter, or cartilage matrix production promoter that is excellent in the action of promoting differentiation into chondrocytes, the action of promoting proliferation of chondrocytes, or the action of promoting cartilage matrix production.

[0032] Furthermore, the pharmaceutical composition of this embodiment can be used for the prevention or treatment of, for example, osteoarthritis, rheumatoid arthritis, spondylosis osteoarthritis, intervertebral disc herniation, chondrodysplasia, articular cartilage damage due to trauma, articular disc damage, acute septic arthritis, tuberculous arthritis, syphilitic arthritis, rheumatic fever or systemic lupus erythematosus, or for cartilage repair in bone fractures or transplants.

[0033] The composition of this embodiment can be used to improve skin function. The improvement of skin function in the present invention refers to the effects of promoting skin fibroblast proliferation, type I collagen production, and / or elastin production. More specifically, the composition of this embodiment can be used as a skin fibroblast proliferation promoter, type I collagen production promoter, and / or elastin production promoter. For example, the composition of this embodiment can be used as a skin moisturizer, wrinkle reduction agent, sagging reduction agent, or rough skin reduction agent. In some embodiments, the composition that can be used to improve skin function is, for example, a food composition, a pharmaceutical composition, or the like.

[0034] A composition that can be used to improve skin function can have a long-lasting effect, for example, 24 to 144 hours, 48 ​​to 120 hours, or 72 to 96 hours.

[0035] In one aspect, the present invention provides a method for producing the above-mentioned composition. The production method of this aspect is not particularly limited and can be a general production method. A general production method used to produce pharmaceutical compositions or food compositions can be appropriately selected depending on the form of the composition. For example, if the food composition is in the form of a powder, it can be produced by uniformly kneading β-NMN, proteoglycan, and other ingredients that are added as needed. The amount of each ingredient to be added can be determined appropriately. The conditions for the raw materials, amounts to be added, etc. used in this production method are as described above.

[0036] In one aspect, the present invention provides use of β-NMN and proteoglycan for the manufacture of a pharmaceutical or food composition for improving joint function. The raw materials, conditions, amounts, etc., used in this aspect are as described above.

[0037] In one aspect, the present invention provides a method for improving joint function by administering or ingesting the pharmaceutical or food composition to a subject. The raw materials, form, amount of formulation, subject to be administered or ingested, administration or intake amount, target disease, etc. in the method of this aspect are as described above.

[0038] In one aspect, the present invention provides a method for enhancing the joint function-improving effect of proteoglycan using β-NMN. The raw materials, form, amount, subjects for administration or intake, and amount to be administered or taken in the method of this aspect are as described above.

[0039] In one aspect, the present invention provides use of β-NMN and proteoglycan for the manufacture of a pharmaceutical or food composition for improving skin function. The raw materials, conditions, amounts, etc., used in this aspect are as described above.

[0040] In one aspect, the present invention provides a method for improving skin function by administering or ingesting the above pharmaceutical or food composition to a subject. The raw materials, form, amount of formulation, subject to be administered or ingested, administration or intake amount, target disease, etc. in the method of this aspect are as described above.

[0041] In one aspect, the present invention provides a method for enhancing the skin function improving effect of proteoglycan using β-NMN. The raw materials, form, amount, subjects for administration or intake, and amount to be administered or taken in the method of this aspect are as described above.

[0042] The present invention will be specifically described below, but the present invention is not limited thereto.

[0043] (Experiment 1: Confirmation of proliferation ability of mouse-derived chondroprogenitor cell line) Mouse-derived chondroprogenitor cell line ATDC5 cells (obtained from the RIKEN BioResource Research Center) were placed at 2 × 10 in a microplate containing 0.1 mL of Dulbecco's modified Eagle's medium (DMEM) / Ham's F-12 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). ３ The cells were seeded at 1000 cells / well and incubated in 5% CO ２ The cells were pre-cultured at 37°C for 24 hours. After culturing, the cells were washed once with PBS(-) by standard methods, and serum-free medium containing β-NMN, salmon nasal cartilage-derived proteoglycan, and β-NMN + proteoglycan at the concentrations shown in Table 1 was added, followed by culturing for 72 hours. After culturing, cell proliferation was confirmed by the CCK-8 method. The results are shown in Table 1 and Figure 1.

[0044]

[0045] As shown in Table 1 and Figure 1, the addition of β-NMN alone had little effect on the proliferation of chondroprogenitor cell lines, while the addition of proteoglycan alone was confirmed to induce proliferation of chondroprogenitor cell lines in a concentration-dependent manner. A combination of β-NMN and proteoglycan was confirmed to synergistically enhance cell proliferation of ATDC5 cells.

[0046] (Experiment 2: Confirmation of cell proliferation ability of normal human dermal fibroblasts) This confirmation was carried out using normal human dermal fibroblasts (KURABO, KF-4009, derived from a newborn).

[0047] (Experimental Method) For the preparation of normal human skin fibroblasts, DMEM medium containing 5% FBS (hereinafter, DMEM medium may be referred to as "DMEM", Fujifilm Wako Pure Chemical Industries, Ltd., 041-29775) was used for pre-culture, and DMEM containing 0.1% FBS was used in this experiment. ２ The cells were cultured at 37°C.

[0048] Normal human dermal fibroblasts were cultured in DMEM medium containing 5% FBS at a concentration of 2 × 10 ３ The cells were seeded onto a 96-well plate at a cell number of 1000 cells / well. ２ The mixture was cultured at 37°C for 24 hours.

[0049] After this culture, the medium in the well plate was replaced with DMEM medium containing 0.1% FBS. After this replacement, the following two patterns of addition (two groups) were carried out. The NMN and PG used in Experiment 2 were the same as those used in Experiment 1. - Control group: A group to which the NMN and PG were not added. - NMN10 and PG10 addition group: A group to which β-NMN was added at a final concentration of 10 μg / mL and proteoglycan derived from salmon nasal cartilage was added at a final concentration of 10 μg / mL.

[0050] After this addition, the cells were incubated in 5% CO ２ The cells were cultured at 37°C for 72 hours. After this culture, the cells from each group were collected. The collected cells were analyzed using CellTiter-Glo. ＴＭ Intracellular ATP was measured using a Luminescent Cell Viability Assay kit (Promega, G7571) according to the kit's instructions (the collected cells were dissolved in the kit's solution, and the ATP in the lysate was reacted with luciferase (luciferin → oxyluciferin)). The luminescence value of intracellular ATP obtained in the test medium without sample was used as a control (100% reference value, average value), and the cell proliferation ability of each addition group was confirmed. The confirmed results (average value for each group) are shown in Table 2. A significance test was performed using JMP statistical software.

[0051]

[0052] The results shown in Table 2 are as follows: Comparing the control group with the NMN10 and PG10 added group, the values ​​in the NMN10 and PG10 added group were higher (the cell proliferation ability was high).

[0053] (Experiment 3: Confirmation of type I collagen gene expression in normal human skin fibroblasts) This confirmation was carried out using the RT-PCR method.

[0054] (1) Cell Preparation Normal human dermal fibroblasts were cultured in DMEM medium containing 5% FBS at a concentration of 4 × 10 ４ The cells were seeded onto a 24-well plate at a cell number of 1000 cells / well. ２ The cells were cultured for 24 hours at 37°C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.25% FBS. After this replacement, the cells were cultured under 5% CO ２ The cells were cultured for 24 hours at 37° C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.25% FBS.

[0055] (2) Experimental Groups The following experimental groups were prepared for the replaced cells. (Experimental Groups) Control group: A group to which the NMN and PG were not added. NMN1 and PG100 addition group: A group to which β-NMN was added to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL. NMN10 and PG100 addition group: A group to which β-NMN was added to a final concentration of 10 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL.

[0056] (3) Confirmation of Type I Collagen Gene Expression (qRT-PCR) After preparing the experimental groups, the mice were incubated in a 5% CO atmosphere. ２The cells were cultured at 37°C for 24 hours. mRNA was purified from the cultured cells. The mRNA was purified using RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Thereafter, qRT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 3 below, and changes in expression levels were analyzed by relative quantification. The qRT-PCR was performed using TB Green EX TaqII Master Mix (Takara Bio Inc.). The qRT-PCR reaction was performed using LightCycler 96 (Roche). Furthermore, the amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0057]

[0058] These results are shown in Table 4 below. Table 4 below shows values ​​when the value of the control group (change in expression level by the relative quantification) is set to 100. In addition, in Table 4 below, the mark * indicates a significant difference (p<0.05) compared to the value of the control group calculated using JMP statistical software. It was confirmed that the addition of NMN and PG improved the expression of type I collagen genes.

[0059]

[0060] (Experiment 4: Confirmation of elastin gene expression in normal human skin fibroblasts) This confirmation was carried out using the RT-PCR method.

[0061] (1) Cell Preparation Normal human dermal fibroblasts were cultured in DMEM medium containing 5% FBS at a concentration of 4 × 10 ４ The cells were seeded onto a 24-well plate at a cell number of 1000 cells / well. ２ The cells were cultured for 24 hours at 37°C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.25% FBS. After this replacement, the cells were cultured under 5% CO ２The cells were cultured for 24 hours at 37° C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.25% FBS.

[0062] (2) Experimental Groups The following experimental groups were prepared for the replaced cells. (Experimental Groups) Control group: A group to which the NMN and PG were not added. NMN1 and PG100 addition group: A group to which β-NMN was added to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL. NMN10 and PG100 addition group: A group to which β-NMN was added to a final concentration of 10 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL.

[0063] (3) Confirmation of elastin gene expression (qRT-PCR) After preparing the experimental groups, the mice were incubated in a 5% CO ２ The cells were cultured at 37°C for 24 hours. mRNA was purified from the cultured cells. The mRNA was purified using RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, a reverse transcription reaction was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Thereafter, qRT-PCR was performed using the primer pairs corresponding to each target factor shown in Table 5 below, and changes in expression levels were analyzed by relative quantification. The qRT-PCR was performed using TB Green EX TaqII Master Mix (Takara Bio Inc.). The qRT-PCR reaction was performed using LightCycler 96 (Roche). Furthermore, the amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0064]

[0065] These results are shown in Table 6 below. Table 6 below shows values ​​when the value of the control group (change in expression level by the relative quantification) is set to 100. In addition, in Table 6 below, the mark * indicates a significant difference (p<0.05) compared to the value of the control group calculated using JMP statistical software. It was confirmed that the addition of NMN and PG improved elastin gene expression.

[0066]

[0067] (Experiment 5: Confirmation of sustained type I collagen gene expression in normal human dermal fibroblasts after long-term culture) This confirmation was carried out using the RT-PCR method.

[0068] (1) Cell Preparation Normal human dermal fibroblasts (KURABO, KF-4109, adult-derived) were cultured at 4 × 10 ４ The cells were seeded onto a 24-well plate at a cell number of 1000 cells / well. ２ The cells were cultured for 24 hours at 37°C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.5% FBS. After this replacement, the cells were cultured under 5% CO ２ The cells were cultured for 24 hours at 37° C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.5% FBS.

[0069] The following experimental groups were prepared for the replaced cells. (2) Experimental Groups - Control group (1): A group cultured for 24 hours without the addition of NMN and PG. - PG100-added group (1): A group cultured for 24 hours after adding salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. - Control group (2): A group cultured for 72 hours without the addition of NMN and PG. - PG100-added group (2): A group cultured for 72 hours after adding salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. - NMN1 and PG100-added group: A group cultured for 72 hours after adding β-NMN to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. NMN10 and PG100 addition group: A group to which β-NMN was added to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL, and cultured for 72 hours.

[0070] (3) Confirmation of Type I Collagen Gene Expression (qRT-PCR) After preparing the experimental groups, the mice were incubated in a 5% CO atmosphere. ２The cells were cultured at 37°C for 24 and 72 hours. mRNA was purified from the cultured cells. The mRNA was purified using RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription reaction was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Then, qRT-PCR was performed using primer pairs corresponding to each target factor shown in Table 3, and changes in expression levels were analyzed by relative quantification. The qRT-PCR was performed using TB Green EX TaqII Master Mix (Takara Bio Inc.). The qRT-PCR reaction was performed using LightCycler 96 (Roche). Furthermore, the amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0071] These results are shown in Table 7 below. Table 7 below shows values ​​when the value of the control group (change in expression level by the relative quantification) is set to 100. In Table 7 below, * indicates a significant difference (p<0.05) compared to the value of the control group, calculated using JMP statistical software. The addition of PG was confirmed to improve type I collagen gene expression after 24 hours of culture, but no significant increase was observed after 72 hours. On the other hand, the addition of NMN was confirmed to significantly increase type I collagen gene expression compared to the control group even after 72 hours of culture, and these gene expressions were significantly higher than those in the group to which PG alone was added after 72 hours (significant difference compared to the PG100-added group (2); # indicates p<0.05, ## indicates p<0.01). This confirmed that the type I collagen gene expression induced by the addition of PG is continuously improved by the addition of NMN.

[0072]

[0073] (Experiment 6: Confirmation of sustained elastin gene expression in normal human skin fibroblasts after long-term culture) This confirmation was carried out using the RT-PCR method.

[0074] (1) Cell Preparation Normal human dermal fibroblasts (KURABO, KF-4109, adult-derived) were cultured at 4 × 10 ４ The cells were seeded onto a 24-well plate at a cell number of 1000 cells / well. ２ The cells were cultured for 24 hours at 37°C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.5% FBS. After this replacement, the cells were cultured under 5% CO ２ The cells were cultured for 24 hours at 37° C. After this culture, the medium in the well plate was replaced with DMEM medium containing 0.5% FBS.

[0075] The following experimental groups were prepared for the replaced cells. (2) Experimental Groups - Control group (1): A group cultured for 24 hours without the addition of NMN and PG. - PG100-added group (1): A group cultured for 24 hours after adding salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. - Control group (2): A group cultured for 72 hours without the addition of NMN and PG. - PG100-added group (2): A group cultured for 72 hours after adding salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. - NMN1 and PG100-added group: A group cultured for 72 hours after adding β-NMN to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan to a final concentration of 100 μg / mL. NMN10 and PG100 addition group: A group to which β-NMN was added to a final concentration of 1 μg / mL and salmon nasal cartilage-derived proteoglycan was added to a final concentration of 100 μg / mL, and cultured for 72 hours.

[0076] (3) Confirmation of elastin gene expression (qRT-PCR) After preparing the experimental groups, the mice were incubated in a 5% CO ２The cells were cultured at 37°C for 24 and 72 hours. mRNA was purified from the cultured cells. The mRNA was purified using RNeasy Mini Kit (QIAGEN). Using the purified mRNA as a template, reverse transcription reaction was performed using PrimeScript RT master Mix (Takara Bio Inc.) to synthesize cDNA. Then, qRT-PCR was performed using primer pairs corresponding to each target factor shown in Table 5, and changes in expression levels were analyzed by relative quantification. The qRT-PCR was performed using TB Green EX TaqII Master Mix (Takara Bio Inc.). The qRT-PCR reaction was performed using LightCycler 96 (Roche). Furthermore, the amplification results of RPS18 (ribosomal protein S18) were used as a reference for the analysis of relative abundance changes.

[0077] These results are shown in Table 8 below. Table 8 below shows values ​​when the value of the control group (change in expression level by relative quantification) is set to 100. In Table 8 below, * indicates a significant difference (p<0.05) compared to the value of the control group, calculated using JMP statistical software. The addition of PG was confirmed to improve elastin gene expression after 24 hours of culture, but no significant increase was observed after 72 hours. On the other hand, the addition of NMN was confirmed to significantly improve elastin gene expression compared to the control group even after 72 hours of culture, and these gene expressions were significantly higher than those in the group to which PG alone was added after 72 hours (significant difference compared to the PG100-added group (2); # indicates p<0.05, ## indicates p<0.01). This confirmed that the addition of NMN continuously improves elastin gene expression induced by the addition of PG.

[0078]

[0079] The present invention provides an oral composition that can improve joint function or skin function when orally ingested. The oral composition can be used as a pharmaceutical composition or a food composition.

Claims

1. An oral composition comprising β-nicotinamide mononucleotide (β-NMN) and proteoglycan as active ingredients.

2. The composition according to claim 1 for improving joint function.

3. The composition according to claim 1, wherein the ratio of β-NMN to proteoglycan is 10:90 to 90:

10.

4. The composition according to any one of claims 1 to 3, which is a pharmaceutical composition.

5. The composition according to claim 4 for the prevention or treatment of osteoarthritis, chronic rheumatoid arthritis, osteoarthritis of the spine, herniated disc, chondrodysplasia, traumatic articular cartilage damage, articular disc damage, acute suppurative arthritis, tuberculous arthritis, syphilitic arthritis, rheumatic fever or systemic lupus erythematosus, or for cartilage repair in fractures or transplants.

6. The composition according to any one of claims 1 to 3, which is a food composition.

7. The composition according to claim 1 for improving skin function.

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