Blood osteocalcin secretion promoter

Abstract

The present invention relates to a blood osteocalcin secretion promoter containing fucosylated chondroitin sulfate.

Description

Blood osteocalcin secretion promoter

[0001] This invention relates to an agent that promotes the secretion of osteocalcin in the blood. Specifically, this invention relates to an agent that promotes the secretion of osteocalcin in the blood containing fucosylated chondroitin sulfate. Furthermore, this invention also relates to a composition for the prevention or treatment of symptoms or diseases, or a food or pharmaceutical composition containing fucosylated chondroitin sulfate.

[0002] Fucosylated chondroitin sulfate, a fucosylated polysaccharide, is a sulfated polysaccharide specifically present in the body wall of sea cucumbers and is known to have various functions. The physiologically active functions of fucosylated chondroitin sulfate include anti-obesity effects through regulation of the gut microbiota in vivo (Non-Patent Literature 1), improvement of atherosclerosis in vivo (Non-Patent Literature 2), anticoagulant effects (Non-Patent Literature 3), anticancer effects in vivo such as reduction of metastatic lesions of cancer cells and inhibition of neutrophil recruitment in induced peritonitis (Non-Patent Literature 4), and antiviral effects in vivo that prevent HIV from entering host cells (Non-Patent Literature 5).

[0003] Furthermore, osteocalcin, a physiologically active hormone (Non-Patent Literature 6, 7), is attracting attention as a blood biomarker that predicts decreased activity in the elderly (Non-Patent Literature 8).

[0004] Zhenjun Zhu et al., Sulfated Polysaccharide from Sea Cucumber and its Depolymerized Derivative Prevent Obesity in Association with Modification of Gut Microbiota in High-Fat Diet-Fed Mice. Mol Nutr Food Res. 2018 Dec;62(23):e1800446.Nian Wu et al., Sulfation pattern of fucose branches affects the anti-hyperlipidemic activities of fucosylated chondroitin sulfate. Carbohydr Polym. 2016 Aug 20:147:1-7. doi: 10.1016 / j.carbpol.2016.03.013. Epub 2016 Mar 8.Qinying Li et al., A novel structural fucosylated chondroitin sulfate from Holothuria Mexicana and its effects on growth factors binding and anticoagulation. Carbohydr Polym. 2018 Feb 1:181:1160-1168. doi: 10.1016 / j.carbpol.2017.10.100. Epub 2017 Nov 2.Lubor Borsig et al., Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment. J Biol Chem. 2007 May 18;282(20):14984-91. doi: 10.1074 / jbc.M610560200.Wu Lian et al., Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope. Biochim Biophys Acta. 2013 Oct;1830(10):4681-91. doi: 10.1016 / j.bbagen.2013.06.003.Cell Physiol. 2008 Dec;217(3):769-77. doi: 10.1002 / jcp.21557.Valerie Le Doan et al., Osteocalcin and glucose metabolism: assessment of human studies. Med Sci (Paris). 2017 Apr;33(4):417-422. doi: 10.1051 / medsci / 20173304012.Martha Cristina Diaz-Franco et al., Osteocalcin-GPRC6A: An update of its clinical and biological multi-organic interactions (Review). Mol Med Rep. 2019 Jan;19(1):15-22. doi: 10.3892 / mmr.2018.9627. Epub 2018 Nov 5.Tomohiko Urano et al., Low serum osteocalcin concentration is associated with incident type 2 diabetes mellitus in Japanese women. J Bone Miner Metab. 2018 Jul;36(4):470-477. doi: 10.1007 / s00774-017-0857-0. Epub 2017 Aug 1.

[0005] Therefore, the present invention aims to provide a novel agent for promoting the secretion of osteocalcin in the blood.

[0006] The inventors of this invention have for the first time discovered that fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood.

[0007] In other words, the present invention encompasses the following inventions: [1] A blood osteocalcin secretion promoter comprising fucosylated chondroitin sulfate. [2] The blood osteocalcin secretion promoter according to [1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [3] The blood osteocalcin secretion promoter according to [1] or [2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [4] A composition for the prevention or treatment of symptoms or diseases comprising fucosylated chondroitin sulfate, The aforementioned symptoms or diseases include sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function due to muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function due to decreased insulin secretion, decreased physical function due to decreased hormone levels, decreased physical function due to worsening of the gut environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy Roffey-Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia Type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 11, Dentatorubral-pallidoluysian atrophy, Gluten ataxia, Neuronal ceroid lipofuscinosis, Infant neuronal ceroid lipofuscinosis, Late infant neuronal ceroid lipofuscinosis, Juvenile neuronal ceroid lipofuscinosis, Adult-onset neuronal ceroid lipofuscinosis, Alzheimer's disease, Early-onset Alzheimer's disease, Late-onset Alzheimer's disease,A composition selected from the group consisting of familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications. [5] The composition according to [4], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [6] The composition according to [4] or [5], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [7] The composition according to any one of [4] to [6], wherein the composition for the prevention or treatment of symptoms or diseases is a food or pharmaceutical composition. [8] A composition for the prevention or treatment of symptoms or diseases, comprising a blood osteocalcin secretion promoter according to any one of [1] to [3], The aforementioned symptoms or diseases include sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function due to muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function due to decreased insulin secretion, decreased physical function due to decreased hormone levels, decreased physical function due to worsening of the gut environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, and primary progressive multiple sclerosis. Myasthenia gravis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy,Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinal Myocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's A composition selected from the group consisting of Luzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin-resistant diseases, sleep apnea, hypertension, renal disease, hypertensive diseases, inflammatory diseases, and knee joint complications. [9] A composition for the prevention or treatment of symptoms or diseases, comprising fucosylated chondroitin sulfate, wherein fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis,Primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy Fee, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infantile neuronal ceroid Lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, A composition selected from the group consisting of juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin-resistant diseases, sleep apnea, hypertension, renal disease, hypertensive diseases, inflammatory diseases, and knee joint complications.

[10] A composition comprising fucosylated chondroitin sulfate, wherein fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood.Food or pharmaceutical composition.

[11] For the prevention or treatment of symptoms or diseases, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid loop Drug-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy Oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca,A food or pharmaceutical composition according to

[10] , selected from the group consisting of dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications.

[12] A blood osteocalcin secretion promoter according to any one of [1] to [3] for use in non-therapeutic or therapeutic methods. The fucosylated chondroitin sulfate contained in the food or pharmaceutical composition according to

[10] or

[11] may be the fucosylated chondroitin sulfate according to any one of [1] to [3].

[0008] The present invention also encompasses the following inventions: [A1] A method for preventing or treating a symptom or disease, comprising administering an effective amount of fucosylated chondroitin sulfate to a subject in need thereof, wherein the symptom or disease is sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus Drug-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy strophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis Fustinism, adult-onset neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome,A method selected from the group consisting of spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications. [A2] The method according to [A1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [A3] The method according to [A1] or [A2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [A4] The method according to any one of [A1] to [A3], wherein the fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood.

[0009] [B1] Use of fucosylated chondroitin sulfate in the manufacture of a blood osteocalcin secretion promoter. [B2] The use according to [B1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [B3] The use according to [B1] or [B2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [B4] The use according to [B1] to [B3], wherein the blood osteocalcin secretion promoter is a food or a pharmaceutical composition. [B5] The use according to [B1] to [B4], wherein the blood osteocalcin secretion promoter is for use in healthy or unhealthy individuals.

[0010] [C1] Use of fucosylated chondroitin sulfate for the prevention or treatment of symptoms or diseases, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, new Lupus in newborns, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharynx Type muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss,Uses selected from the group consisting of concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications. [C2] The use according to [C1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [C3] The use according to [C1] or [C2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [C4] The method according to any one of [C1] to [C3], wherein the fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood.

[0011] [D1] Fucosylated chondroitin sulfate for use in the prevention or treatment of symptoms or diseases, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus Neonatal lupus, diabetes, type 1 diabetes, type 2 diabetes, early-onset adult diabetes, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, Oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss,Fucosylated chondroitin sulfate selected from the group consisting of hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications. [D2] The fucosylated chondroitin sulfate according to [D1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [D3] The fucosylated chondroitin sulfate according to [D1] or [D2], wherein fucose is bonded to the hydroxyl group at position 3 of glucuronic acid in chondroitin. [D4] The fucosylated chondroitin sulfate according to any one of [D1] to [D3] that promotes the secretion of osteocalcin in the blood.

[0012] [E1] Non-therapeutic use of fucosylated chondroitin sulfate for promoting the secretion of osteocalcin in the blood. [E2] The non-therapeutic use according to [E1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [E3] The non-therapeutic use according to [E1] or [E2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin.

[0013] [F1] A method for promoting the secretion of osteocalcin in the blood, comprising administering a composition containing fucosylated chondroitin sulfate to a subject. [F2] The method according to [F1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [F3] The method according to [F1] or [F2], wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin. [F4] The method according to any one of [F1] to [F3], wherein the composition is a food or pharmaceutical composition. [F5] The method according to any one of [F1] to [F3], for the purpose of treating a subject or not for the purpose of treating a subject.

[0014] [G1] Fucosylated chondroitin sulfate for use in a non-therapeutic method for promoting the secretion of osteocalcin in the blood. [G2] The fucosylated chondroitin sulfate according to [G1], wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less. [G3] The fucosylated chondroitin sulfate according to [G1] or [G2], wherein fucose is bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin.

[0015] In the present invention, the invention may be therapeutic or non-therapeutic. That is, it may be an invention of a therapeutic or non-therapeutic method, an invention of a therapeutic or non-therapeutic use, or an invention of a therapeutic or non-therapeutic product.

[0016] In any of the inventions [1] to

[12] , [A1] to [A4], [B1] to [B5], [C1] to [C4], [D1] to [D4], [E1] to [E3], [F1] to [F5], and [G1] to [G3], the configuration of the invention may be selected from any combination of preferred embodiments described below as embodiments. Preferred embodiments include more preferred embodiments and even more preferred embodiments.

[0017] According to the present invention, a novel agent for promoting the secretion of osteocalcin in the blood can be provided.

[0018] The results for osteocalcin concentration in osteoblast culture supernatant are shown. Fluorescence microscopy images of myoblasts stained by immunofluorescence are shown. The results for measuring the area and diameter of myotubes are shown. Sequence IDs 1-6 are shown.

[0019] The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

[0020] The blood osteocalcin secretion promoter of this embodiment contains fucosylated chondroitin sulfate.

[0021] <Fucosylated Chondroitin Sulfate> Fucosylated chondroitin sulfate is a polysaccharide having a structure in which a sulfate group and sulfated fucose are bonded to the hydroxyl group of chondroitin. In this specification, chondroitin means a sugar chain having a disaccharide composed of glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) as constituent units. That is, chondroitin is a sugar chain having a structure in which the disaccharides GlcA and GalNAc are alternately repeated via β(1-3) and β(1-4) glycosidic bonds. Furthermore, sugar chains in which GalNAc is bonded to GlcA at the end of the repeating structure, or sugar chains in which GlcA is bonded to GalNAc at the end of the repeating structure, are also included in chondroitin. The GlcA and GalNAc that constitute chondroitin are generally D-type, but L-type GlcA or GalNAc may also be included. Furthermore, the reducing end of chondroitin is preferably GalNAc. Chondroitin is not particularly limited as long as it has a structure of two or more sugars, but it is preferably chondroitin with 3 to 15 sugars, more preferably chondroitin with 3 to 9 sugars, and even more preferably chondroitin with 5 to 7 sugars. The molecular weight of chondroitin is not particularly limited, but varies from 500 to 300,000 Da depending on the chain length, preferably 500 to 100,000 Da, more preferably 500 to 10,000 Da, and even more preferably 1,000 to 3,000 Da. Chondroitin sulfate has a structure in which a sulfate group is bonded to the hydroxyl group of chondroitin, and the sulfate group can be bonded to the hydroxyl groups at the 4th and / or 6th positions of GalNAc. Both the 4th and 6th hydroxyl groups may be sulfated, or only one of them may be sulfated.

[0022] Fucosylated chondroitin is a sugar chain having a structure in which one or more fucose molecules are bonded to the hydroxyl group of chondroitin.

[0023] The number of fucose molecules in fucosylated chondroitin is not particularly limited. The number of fucose molecules in fucosylated chondroitin can be determined according to the number of hydroxyl groups to which fucose can be bound in chondroitin. When there is one fucose molecule in fucosylated chondroitin, it may be bound to GlcA or GalNAc, and it is preferable that the fucose is bound to GlcA. When there are two or more fucose molecules in fucosylated chondroitin, multiple fucose molecules may be bound to one GlcA or GalNAc, or multiple fucose molecules may be bound to multiple GlcA or GalNAc, but it is preferable that one fucose molecule is bound to one GlcA or GalNAc. Furthermore, if fucosylated chondroitin contains two or more fucose molecules, one or more fucose molecules may be bound to each of several adjacent GlcA and / or GalNAc molecules, or one or more fucose molecules may be bound to each of several non-adjacent GlcA and / or GalNAc molecules. It is preferable that one or more fucose molecules are bound to each of several non-adjacent GlcA and / or GalNAc molecules, and it is more preferable that one or more fucose molecules are bound to each of several non-adjacent GlcA molecules. Below is a schematic diagram of a heptasaccharide fucosylated chondroitin in which one fucose molecule is bound to each of several non-adjacent GlcA molecules. In this specification, when "X sugar" (where X is any integer) is used, X sugar does not contain fucose. Specifically, when we refer to heptasaccharide fucosylated chondroitin, it means that the fucosylated chondroitin contains seven sugars other than fucose (for example, GlcA or GalNAc).

[0024]

[0025] The circle (〇) represents GalNAc, the square (□) represents GlcA, and the triangle (△) represents fucose. The GalNAc at the far right represents the reducing end of the sugar chain.

[0026] The mode of fucose bonding in fucosylated chondroitin is not particularly limited and may be α-linked and / or β-linked, and may be 1,2-linked, 1,3-linked, 1,4-linked and / or 1,6-linked. When fucose is bonded to GlcA, 1,3-linked bonding is preferred. Chondroitin sulfate or fucosylated chondroitin sulfate is a polysaccharide having a structure in which one or more sulfate groups are bonded to the hydroxyl group of chondroitin or fucosylated chondroitin. The number of sulfate groups contained in chondroitin sulfate or fucosylated chondroitin sulfate is not particularly limited. The number of sulfate groups contained in chondroitin sulfate or fucosylated chondroitin sulfate can be determined according to the number of hydroxyl groups to which the sulfate groups can be bonded in chondroitin or fucosylated chondroitin. In chondroitin sulfate, sulfate groups can be attached to the hydroxyl groups at position 1 (reducing end), position 3 (non-reducing end), position 4, position 6 of GalNAc, position 1 (reducing end), position 2, position 3, and position 4 (non-reducing end) of GlcA. All of these hydroxyl groups may be sulfated, or one or more of them may be sulfated. In fucosylated chondroitin sulfate, sulfate groups can be attached to the hydroxyl groups at position 1 (reducing end), position 3 (non-reducing end), position 4, position 6 of GalNAc, position 1 (reducing end), position 2, position 3, position 4 (non-reducing end) of GlcA, position 2, position 3, position 4 of GlcA, position 2, position 3, and position 4 of fucose. All of these hydroxyl groups may be sulfated, or one or more of these hydroxyl groups may be sulfated. Chondroitin sulfate or fucosylated chondroitin sulfate may also be salts. Chondroitin sulfate or fucosylated chondroitin sulfate is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, organic amine salts such as ammonium salt, triethanolamine salt and triethylamine salt, or basic amino acid salts such as lysine salt and arginine salt.The molecular weight of fucosylated chondroitin sulfate is not particularly limited, but varies from 500 to 300,000 Da depending on the chain length, with 500 to 100,000 Da being preferred, 1,000 to 10,000 Da being more preferred, and 1,500 to 4,000 Da being even more preferred.

[0027] <Method for producing fucosylated chondroitin or fucosylated chondroitin sulfate> In this embodiment, fucosylated chondroitin sulfate may be extracted from living organisms by known methods, for example, by extraction and purification from sea cucumbers (e.g., VIEIRA, Ricardo P.; MULLOY, B.; MOURAO, PA, Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues. Journal of Biological Chemistry, 1991;266.21:13530-13536). In this embodiment, fucosylated chondroitin sulfate may be produced by known chemical synthesis methods, enzymatic methods, microbial fermentation methods, or combinations thereof, for example, from chondroitin, fucosylated chondroitin, or chondroitin sulfate obtained by known production methods or commercially available. Fucosylated chondroitin sulfate can be produced from fucosylated chondroitin, for example, by sulfating fucosylated chondroitin as a substrate. Fucosylated chondroitin sulfate can be produced from chondroitin sulfate, for example, by reacting chondroitin sulfate with an enzyme having fucose transfer activity as a substrate. Alternatively, for example, chondroitin sulfate can be reacted with an enzyme having fucose transfer activity to produce fucosylated chondroitin sulfate, and then sulfated again. Sulfation of the hydroxyl groups at the 2-position, 3-position, and / or 4-position of fucose may occur after the fucose structure is bound to chondroitin or chondroitin sulfate, or the sulfated fucose structure may be bound to chondroitin or chondroitin sulfate. When a sulfated fucose structure is bound to chondroitin or chondroitin sulfate, specifically when fucosylation is performed using chondroitin or chondroitin sulfate as a substrate, a compound in which GDP-fucose has been pre-sulfated can be used as the substrate.

[0028] The fucose transfer activity in fucosylation of chondroitin or chondroitin sulfate refers to the activity of transferring fucose to chondroitin or chondroitin sulfate using GDP-fucose as a substrate to produce a fucosylated polysaccharide. Specifically, the fucose transfer activity to chondroitin refers to the activity of transferring fucose to chondroitin using GDP-fucose as a substrate to produce fucosylated chondroitin, and the fucose transfer activity to chondroitin sulfate refers to the activity of transferring fucose to chondroitin sulfate using GDP-fucose as a substrate to produce fucosylated chondroitin sulfate. In addition, fucose is usually transferred to the hydroxyl group of GlcA or GalNAc. As long as fucose can be bound, it may be bound to either the hydroxyl group of GlcA or GalNAc that constitutes chondroitin, but it is preferable for fucose to be bound to the hydroxyl group of GlcA.

[0029] The number of fucose molecules transferred to chondroitin is not particularly limited and may be determined according to the number of hydroxyl groups to which fucose can bind in chondroitin, or it may be adjusted by the amount of GDP-fucose.

[0030] In this embodiment, fucosyltransferase (fucT) may be used as the enzyme having fucose transfer activity. Examples of fucT include fucosyltransferases derived from Helicobacter acinonychis, Helicobacter ailurogastricus, Helicobacter baculiformis, Helicobacter bizzozeronii, Helicobacter cetorum MIT 99-5656, Helicobacter cynogastricus, Helicobacter felis, Helicobacter heilmannii, Helicobacter labacensis, Helicobacter mehlei, Helicobacter salomonis, Helicobacter sp.219-2, and Helicobacter Examples include fucosyltransferase derived from sp.L8, fucosyltransferase derived from Helicobacter sp.NHP19-003, fucosyltransferase derived from Helicobacter suis, fucosyltransferase derived from Helicobacter vulpis, or fucosyltransferase derived from Fusobacterium mortiferum.

[0031] In this embodiment, the method for producing fucosylated chondroitin or fucosylated chondroitin sulfate refers to a method for producing fucosylated chondroitin or fucosylated chondroitin sulfate by using an enzyme having fucose transfer activity to chondroitin or chondroitin sulfate, and transferring fucose from GDP-fucose using chondroitin or chondroitin sulfate as a substrate. For example, a method for producing fucosylated chondroitin or fucosylated chondroitin sulfate can be given by using the above-mentioned enzyme having fucose transfer activity, and transferring fucose from GDP-fucose using chondroitin or chondroitin sulfate as a substrate. The above-mentioned production method includes using a microorganism having an enzyme having fucose transfer activity to chondroitin or chondroitin sulfate. Fucosylated chondroitin or fucosylated chondroitin sulfate may be produced in the microorganism, or the microorganism may be used as an enzyme preparation to produce fucosylated chondroitin or fucosylated chondroitin sulfate. A method for producing fucosylated chondroitin or fucosylated chondroitin sulfate using the microorganism in question is sometimes called the fermentation method. A method for producing fucosylated chondroitin or fucosylated chondroitin sulfate using the microorganism as an enzyme preparation is sometimes called the enzymatic method. The microorganism used in producing fucosylated chondroitin or fucosylated chondroitin sulfate is a microorganism that has an enzyme with fucose transfer activity to chondroitin or chondroitin sulfate. A microorganism that has an enzyme with fucose transfer activity to chondroitin or chondroitin sulfate may be a microorganism that originally possesses a protein with fucose transfer activity to chondroitin or chondroitin sulfate. If the reference strain used is a microorganism that does not originally possess a protein with fucose transfer activity to chondroitin or chondroitin sulfate, it may be a genetically modified microorganism in which the protein with fucose transfer activity to chondroitin or chondroitin sulfate has been artificially enhanced.This may be a genetically modified microorganism that originally possesses a protein having fucose transfer activity to chondroitin or chondroitin sulfate, and in which the protein having fucose transfer activity to chondroitin or chondroitin sulfate is further enhanced. Furthermore, it is possible to obtain an enzyme preparation characterized by containing an enzyme having fucose transfer activity to chondroitin or chondroitin sulfate, in the form of a culture of the above-mentioned microorganism or a processed product of the culture. Fucosylated chondroitin or fucosylated chondroitin sulfate can also be produced by using this enzyme preparation.

[0032] Regarding methods for producing fucosylated chondroitin or fucosylated chondroitin sulfate, an example of a method for producing fucosylated chondroitin is described below. 1. Method for producing fucosylated chondroitin by fermentation One method for producing fucosylated chondroitin is a method for producing fucosylated chondroitin by fermentation, which includes culturing the above-mentioned microorganisms in a culture medium to produce fucosylated chondroitin in the culture. The microorganism used when producing fucosylated chondroitin by fermentation is a microorganism that has an enzyme having fucose transfer activity to chondroitin and produces fucosylated chondroitin. The microorganism may be one in which the activity of the protein having fucose transfer activity to chondroitin is enhanced compared to the parent strain, and the productivity of fucosylated chondroitin is improved. The microorganism having an enzyme having fucose transfer activity to chondroitin may be a microorganism that originally has an enzyme having fucose transfer activity to chondroitin. If the reference strain used is a microorganism that does not originally possess an enzyme with fucose transfer activity to chondroitin, a genetically modified microorganism that has been artificially conferred with an enzyme with fucose transfer activity to chondroitin may be used. Alternatively, a genetically modified microorganism that originally possesses a protein with fucose transfer activity to chondroitin may be used in which the enzyme with fucose transfer activity to chondroitin has been further enhanced.

[0033] In the method for producing fucosylated chondroitin by fermentation, it is preferable that the genetically modified microorganism used is one that has the ability to produce GDP-fucose and / or chondroitin. If the microorganism used does not originally have the ability to produce GDP-fucose and / or chondroitin, this ability may be conferred upon it. Furthermore, if the microorganism used originally has the ability to produce GDP-fucose and / or chondroitin, this ability may be further enhanced.

[0034] In a method for producing fucosylated chondroitin by fermentation, if the genetically modified microorganism used does not have the ability to produce GDP-fucose, GDP-fucose may be added to the culture medium during cultivation as a substrate for fucosylated chondroitin, or GTP or mannose, which are substrates of GDP-fucose, may be added to the culture medium. Alternatively, in a method for producing fucosylated chondroitin by fermentation, if the genetically modified microorganism used does not have the ability to produce GDP-fucose, instead of adding GDP-fucose to the culture medium during cultivation, GDP-fucose may be supplied to the genetically modified microorganism by culturing a microorganism capable of producing GDP-fucose simultaneously with the genetically modified microorganism. Furthermore, in order to enhance the supply of GTP, a substrate of GDP-fucose, a microorganism capable of producing GTP may be cultured simultaneously. Examples of microorganisms capable of producing GTP include known microorganisms in which the expression of enzymes involved in the GTP biosynthesis pathway has been enhanced by various genetic manipulations (e.g., Biotechnol Bioeng. 2019 Sep:3).

[0035] In the method for producing fucosylated chondroitin by fermentation, if the genetically modified microorganism used does not have the ability to produce chondroitin, chondroitin may be added to the culture medium during cultivation as a substrate for fucosylated chondroitin, or UDP-glucuronic acid and / or UDP-N-acetylgalactosamine, which are substrates of chondroitin, may be added to the culture medium. Alternatively, in the method for producing fucosylated chondroitin by fermentation, if the genetically modified microorganism used does not have the ability to produce chondroitin, instead of adding chondroitin to the culture medium during cultivation, chondroitin may be supplied to the genetically modified microorganism by culturing a microorganism capable of producing chondroitin simultaneously with the genetically modified microorganism in this embodiment. The method for culturing the genetically modified microorganism can be carried out according to the usual methods used for culturing microorganisms.

[0036] As a culture medium for genetically modified microorganisms, either a natural medium or a synthetic medium may be used, as long as it contains a carbon source, nitrogen source, and / or inorganic salts that the genetically modified microorganisms can utilize, and is capable of efficiently culturing the genetically modified microorganisms.

[0037] As a carbon source, any material that can be utilized by genetically modified microorganisms is acceptable, such as glucose, fructose, sucrose, molasses containing these, sugars such as starch or starch hydrolysates, organic acids such as acetic acid or propionic acid, or alcohols such as glycerol, ethanol or propanol. As a nitrogen source, examples include inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate or ammonium phosphate, ammonium salts of organic acids, other nitrogen-containing compounds, or peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysates, soybean meal, soybean meal hydrolysates, various fermentation cells or their digests. As an inorganic salt, examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, or calcium carbonate.

[0038] Cultivation is preferably carried out under aerobic conditions such as shaking culture or deep aeration agitation culture. The cultivation temperature is usually 15 to 40 °C, and the cultivation time is usually 5 hours to 7 days. The pH of the culture broth during cultivation is usually maintained at 3.0 to 9.0. The pH adjustment is carried out using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, or ammonia.

[0039] Also, if necessary during cultivation, an antibiotic such as ampicillin or tetracycline may be added to the medium. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added to the medium. For example, when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacrylic acid or the like may be added to the medium.

[0040] By the above cultivation, fucosylated chondroitin is produced and accumulated in the culture, and fucosylated chondroitin can be produced by collecting fucosylated chondroitin from the culture.

[0041] Analysis of the obtained fucosylated chondroitin can be carried out by ordinary analysis methods using sugar chains such as ion exchange chromatography or gel filtration chromatography. Collection or purification of fucosylated chondroitin from the culture or a treated product of the culture can be carried out by ordinary methods using filtration, activated carbon, and / or ion exchange resin. When fucosylated chondroitin accumulates in the cells, for example, the cells are disrupted by ultrasonic treatment or chemical treatment, the cells are removed by centrifugation, and fucosylated chondroitin can be collected from the supernatant obtained by using activated carbon and / or ion exchange resin.

[0042] 2. Method for producing fucosylated chondroitin using GDP-fucose and chondroitin as substrates As a method for producing fucosylated chondroitin, there is also a method for producing fucosylated chondroitin using GDP-fucose and chondroitin as substrates.

[0043] Specifically, the method for producing fucosylated chondroitin by an enzymatic method includes an enzyme source containing an enzyme having fucose transfer activity with respect to chondroitin produced by a cell-free protein synthesis system, or a culture of a microorganism having the ability to produce an enzyme having fucose transfer activity with respect to chondroitin or a processed product of the culture as an enzyme source, and a substrate or the enzyme source is allowed to exist in an aqueous medium, and fucosylated chondroitin is produced in the aqueous medium. The production method may include, for example, producing fucosylated chondroitin in an aqueous medium, accumulating it, and collecting or purifying fucosylated chondroitin from the aqueous medium.

[0044] GDP-fucose and chondroitin may be of any origin as long as they can be substrates for the enzymes possessed by genetically modified microorganisms, but a culture of a microorganism having the ability to produce GDP-fucose or chondroitin or a processed product of the culture may be used as it is, or GDP-fucose and chondroitin collected from the culture or the processed product of the culture may be used.

[0045] Examples of the processed product of the culture include a concentrate of the culture, a dried product of the culture, cells obtained by centrifuging the culture, a dried product of the cells, a freeze-dried product of the cells, a surfactant-treated product of the cells, an ultrasonic-treated product of the cells, a mechanically milled product of the cells, a solvent-treated product of the cells, an enzyme-treated product of the cells, a protein fraction of the cells, or an immobilized product of the cells or an isolated and purified enzyme extracted from the cells. Among them, the solvent-treated product of the cells and the isolated and purified enzyme extracted from the cells are preferable.

[0046] Examples of solvent-treated bacterial cells include those treated with organic solvents such as xylene. For example, treating bacterial cells with xylene allows for the incorporation of GDP-fucose and chondroitin into the bacterial cells without completely disrupting them, while also maintaining the fucosyltransferase in the bacterial cells.

[0047] Examples of aqueous media include water, phosphates, carbonates, acetates, borates, citrates, buffers such as Tris, alcohols such as methanol or ethanol, esters such as ethyl acetate, ketones such as acetone, or amides such as acetamide. Additionally, for example, the culture medium of microorganisms used as an enzyme preparation can also be used as an aqueous medium.

[0048] The method for analyzing, collecting, or purifying fucosylated chondroitin produced in an aqueous medium is the same as in 1. Method for producing fucosylated chondroitin by fermentation.

[0049] The above-described manufacturing method can be used as a method for industrially producing fucosylated chondroitin. Furthermore, the fucosylated chondroitin obtained by the above-described manufacturing method can be used to produce fucosylated chondroitin sulfate, which can be used as a pharmaceutical or food product. Fucosylated chondroitin sulfate can be produced by sulfating the fucosylated chondroitin obtained by the above-described manufacturing method as a substrate using known chemical or enzymatic reactions (e.g., Garg, G Hari et al., Effect of Fully Sulfated Glycosaminoglycans on Pulmonary Artery Smooth Muscle Cell Proliferation. Archives of Biochemistry and Biophysics. 1999 November 15;371(2):228-233.). In addition, fucosylated chondroitin sulfate can be analyzed, collected, or purified by the same method as the analysis, collection, or purification method for fucosylated chondroitin described above.

[0050] <Promotion of Osteocalcin Secretion> In this embodiment, promotion of osteocalcin secretion means activating or stimulating the secretion of osteocalcin and relatively increasing the total amount of osteocalcin in the secreted substance. Specifically, it means that by using the blood osteocalcin secretion promoter of the present invention, the amount of osteocalcin in the blood increases by 10% or more, preferably 15% or more, more preferably 20% or more, and even more preferably 50% or more compared to before use.

[0051] <Osteocalcin> Osteocalcin is a calcium-binding protein derived from osteoblasts, formed from 49 amino acids. Osteocalcin is known to have functions such as improving blood glucose levels, reducing body fat, improving fatty liver, improving brain function, increasing muscle mass (e.g., increased expression of mTOR or S6K1), strengthening muscles, maintaining muscle mass during exercise, promoting myokine (e.g., IL-6) secretion, promoting energy metabolism, promoting insulin sensitivity, and promoting glucose uptake, lowering blood pressure, inducing testosterone biosynthesis in testicular cells, and promoting myogenic differentiation in vitro. Non-carboxyl osteocalcin (GluOC), which contains three glutamic acid residues (Glu) per molecule, is converted to carboxyl osteocalcin (GlaOC) by vitamin K-dependent carboxylase, which γ-carboxylates the glutamic acid. GlaOC is mostly incorporated into the bone matrix and binds to calcium ions, participating in bone formation (e.g., calcium deposition), while some is released into the bloodstream. GluOC has a weak affinity for the bone matrix and is released into the bloodstream. Therefore, the blood GluOC / GlaOC ratio can be used as an indicator of bone metabolism.

[0052] <Compositions for the Prevention or Treatment of Symptoms or Diseases Containing Fucosylated Chondroitin Sulfate> In this embodiment, compositions for the prevention or treatment of symptoms or diseases, containing fucosylated chondroitin sulfate, are provided. In this specification, prevention usually means preventing the onset or progression of symptoms or diseases, and does not include treatment. Treatment means the cure, improvement, or suppression of the worsening of symptoms or diseases. That is, fucosylated chondroitin sulfate may be used as an active ingredient for therapeutic purposes or for non-therapeutic purposes. Unless otherwise specified, the respective effects related to the prevention or treatment of symptoms or diseases may be obtained for therapeutic purposes or for non-therapeutic purposes. Therapeutic purposes may include, for example, medical procedures (e.g., acts of performing surgery, treatment, or diagnosis on a person), and more specifically, may include procedures on the human body through treatment. Non-therapeutic purposes may include, for example, concepts that do not include medical procedures, and more specifically, may include procedures on the human body through treatment. Non-therapeutic purposes include, for example, health promotion or cosmetic purposes.

[0053] Osteocalcin, through interaction with GPRC6A, a type of G protein-coupled receptor, (1) triggers the secretion of hormones such as insulin and adiponectin, and is activated by phospholipase C (PLC) IP3-Ca 2+(2) It is known to activate the Mek-Erk cascade, which activates the adenylyl cyclase-cAMP-PKA pathway. Osteocalcin is also known to have effects such as promoting lipid metabolism, adiponectin secretion, suppression of pro-inflammatory cytokine production (e.g., IL-10), insulin secretion, β-cell proliferation, recovery from fatty liver, increased growth hormone receptor and IGF-1 via testosterone, suppression of luteinizing hormone (LH) secretion in the pituitary gland, increased expression of growth hormone (GH) via testosterone, contribution to spatial learning and memory processing, glucose consumption in muscles, improved exercise performance, gastrin secretion in the intestines, intestinal GLP-1 secretion, spermatogenesis, testosterone secretion, conversion of fibroblasts to myofibroblasts, and vasodilation. Symptoms or diseases include, for example, sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function due to muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function due to decreased insulin secretion, decreased physical function due to decreased hormone levels, decreased physical function due to worsening of the gut environment, traumatic brain injury (TBI), multiple sclerosis (MS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), progressive relapsing multiple sclerosis (PRMS), lupus, systemic lupus erythematosus (SLE), discoid lupus, drug-induced lupus (dil), neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, and early-onset adult-onset diabetes mellitus (MO). DY: MODY1, MODY2, MODY3, MODY4, MODY5, MODY6, MODY7, MODY8, MODY9, MODY10, MODY11), Non-alcoholic steatohepatitis (NASH), Myasthenia gravis (MG), Ocular myasthenia gravis, Congenital myasthenia gravis, Generalized myasthenia gravis, Rheumatoid arthritis (RA), Graves' disease, Guillain-Barré syndrome (GBS), Muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Myotonic muscular dystrophy, Congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, Facioscapulohumeral muscular dystrophy, Limb-girdle muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, Amyotrophic lateral sclerosis (ALS),Ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 11 (SCA11), dentatorubral-pallidoluysian atrophy (DRPLA), gluten ataxia, neuronal ceroid lipofuscinosis (NCL), Infant Neuronal Ceroid Lipofuscinosis (INCL), Late Infant Neuronal Ceroid Lipofuscinosis (LINCL), Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Adult Neuronal Ceroid Lipofuscinosis (ANCL), Alzheimer's Disease, Early-Onset Alzheimer's Disease, Late-Onset Alzheimer's Disease, Familial Alzheimer's Disease (FAD), Optic Neuritis (ON), Leber Hereditary Optic Neuropathy (LHON), Autism Spectrum Disorder This may include conditions such as athletic dysplasia (ASD), Asperger's syndrome, pervasive developmental disorder (PDD), childhood disintegrative disorder (CDD), autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy (SMA; types I, II, III, and IV), noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance, sleep apnea, hypertension, kidney disease, hypertensive diseases, inflammatory diseases, or knee joint complications. The symptoms or diseases are preferably sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, or decreased physical function associated with deterioration of the intestinal environment.

[0054] Sarcopenia is a condition or disease characterized by a progressive and generalized decline in skeletal muscle mass and strength. Sarcopenia includes primary (age-related) sarcopenia, which has no apparent cause other than aging, and secondary sarcopenia, which has one or more apparent causes other than aging. Secondary sarcopenia can be caused by reduced physical activity, disease (e.g., associated with severe organ failure, inflammatory diseases, malignancies, endocrine disorders, etc.), and nutrient deficiencies. Frailty refers to a state of increased vulnerability to a variety of health problems, based on various age-related functional declines (reduced reserve capacity). Frailty is also commonly used as a concept that includes physical and / or mental problems.

[0055] The composition in this embodiment can exert a preventive or therapeutic effect on symptoms or diseases when administered or ingested directly or indirectly to the site where symptoms or diseases occur or are likely to occur.

[0056] The composition in this embodiment may be for food or pharmaceutical use. For example, it can be in a form suitable for the intended use, such as for food or pharmaceutical purposes. It is not particularly limited, but can be in a form suitable for the intended use, such as solid, liquid, granular, powder, capsule, cream, paste, or jelly. For example, when forming capsules, it may be coated with one or more films as needed. These forms can be produced using known additives. It is not particularly limited, but known excipients, binders, diluents, disintegrants, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, and / or preservatives may be used individually or in combination as needed.

[0057] The food composition is not particularly limited, but examples include food and beverages, health foods, functional foods, nutritional supplements, supplements, health functional foods, foods for specified health uses, nutritional functional foods, or foods with functional claims. Examples of food composition include foods, beverages, seasonings, and food additives. Specific examples of foods include confectionery, dairy products, bread, noodles, rice, soups, or prepared foods. Specific examples of beverages include milk beverages, soft drinks, fruit juices, carbonated drinks, sports drinks, nutritional drinks, or alcoholic beverages. The food composition may optionally contain known colorants, flavorings, sweeteners, bittering agents, acidulants, preservatives, antioxidants, pH adjusters, and / or thickening stabilizers. The food composition may be provided after heat sterilization, or in a sealed form in a bag or container. The food composition according to this embodiment (including feed for animals other than humans in general) may be provided or sold as food or beverages labeled with an intended use related to the preventive or therapeutic effect of symptoms or diseases (for example, health use, functional use, enteral nutrition use, special use, nutritional function use, health function use, or nutritional supplement use). The food composition according to this embodiment may be provided or sold as food or beverages labeled with an intended target. Labeling includes all actions to inform consumers of the above-mentioned uses, and any expression that can cause consumers to recall or infer the above-mentioned uses constitutes labeling, regardless of the purpose, content, object, and medium of the labeling. In particular, labeling may be carried out in an expression that allows consumers to directly recognize the above-mentioned uses. Examples of indications include transferring, delivering, displaying for transfer or delivery, or importing a product or packaging of a food composition according to this embodiment that has the above-mentioned uses described on it; or displaying or distributing an advertisement, price list, or transaction document relating to the product that includes the above-mentioned uses; or providing information containing such information by electromagnetic means (such as the Internet).Examples of labeling include labeling on packaging, containers, catalogs, brochures, promotional materials at sales sites (such as POP displays), and other documents. Examples of labeling include labeling as health foods, functional foods, enteral nutrition foods, foods for special dietary uses, health functional foods (such as foods for specified health uses, foods with nutritional function claims, and foods with functional claims), or nutritional supplements. Preferably, the labeling may be approved by the government or other authorities (for example, labeling approved under various systems established by the government and carried out in accordance with such approval). Examples of labeling approved by the government or other authorities include labeling approved by the Consumer Affairs Agency of Japan. In the case of food and beverage compositions, examples of labeling approved by the Consumer Affairs Agency of Japan include labeling approved under the health functional food system (such as foods for specified health uses, foods with nutritional function claims, and foods with functional claims) and similar systems. Specific examples of labeling approved by the Consumer Affairs Agency of Japan include labeling as a food for specified health uses, labeling as a conditionally approved food for specified health uses, labeling that it affects the structure or function of the body, labeling that it reduces the risk of disease, or labeling that it has scientifically proven functionality. More specifically, the labels approved by the Consumer Affairs Agency of Japan include labels for Foods for Specified Health Uses (especially labels for health purposes) as defined in the Cabinet Office Ordinance concerning the Permission for Special Use Labeling under the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009), and similar labels.

[0058] The food composition in this embodiment may be labeled with a functional claim such as, for example, "Helps suppress the decline in muscle mass and muscle strength," "Helps improve walking ability," "Maintains muscle mass and muscle strength," "Supports the maintenance of muscle mass and muscle strength," "Maintains walking ability that declines with age," "Helps maintain muscles that decline with age," or a functional claim with the same meaning.

[0059] The composition in this embodiment may contain other active ingredients as long as they do not impair its effect. Furthermore, the amount of fucosylated chondroitin sulfate in the composition in this embodiment can be appropriately selected within a range that provides the effects of the present invention. The composition in this embodiment is administered to or ingested by animals in general, including humans. The dosage or intake may be appropriately set according to the route of administration or intake, as well as various circumstances such as the age, sex, weight, and symptoms or diseases of the recipient or recipient. In this embodiment, the recipient of the composition may be a healthy person or a person with the symptoms or diseases described above. In this embodiment, methods of administering or ingesting the composition include oral, rectal, parenteral (intravenous, intramuscular, subcutaneous, or transdermal absorption, etc.), intracisional, intravaginal, intraperitoneal, intravesical, or topical administration or inhalation (injection, infusion, powder, ointment, gel, cream, oral, or nasal spray, etc.). These dosage forms include, for example, oral preparations such as tablets, capsules, granules, fine granules, powders, liquids, syrups, chewables, and lozenges, as well as ointments, gels, creams, injections, sublingual preparations, inhalants, suppositories, topical applications, patches, eye drops, and nasal drops. The composition of the present invention may be administered alone or in combination with other components, such as pharmaceuticals or pharmaceutical compositions, foods or food compositions, or components contained therein. In this embodiment, the single dose or intake of the composition may be determined within a range that does not impair its effect, and is appropriately determined according to the symptoms or disease of the subject being administered or ingested, the animal species, age, weight, sex, or body length. For example, when the subject is a human, the daily dose or intake of fucosylated chondroitin sulfate administered to an adult may be adjusted in the range of, for example, 1 mg to 10,000 mg, more preferably 100 mg to 3,000 mg. In this embodiment, "adult" means males and females aged 15 years or older. However, the compositions according to this embodiment are not limited to those administered or ingested by adults, but may also be administered or ingested by children under 15 years of age.When administering to or ingesting children, the dosage may be reduced to 1 / 2 or 2 / 3 of the adult daily dose or intake, depending on the age group. The same applies to components other than fucosylated chondroitin sulfate. In this embodiment, the number of times the composition is administered or ingested is not particularly limited, and may be, for example, once or more, three or more, five or more, or seven or more times per week. Administration or ingestion may be once a day, or the daily dose or intake may be divided into multiple doses. In this embodiment, the duration of administration or ingestion of the composition is not particularly limited, and may be, for example, one or more days, three or more days, one or more weeks, four or more weeks, eight or more weeks, or twelve or more weeks.

[0060] Examples of the present invention are shown below, but the present invention is not limited to these examples.

[0061] [Test Example 1] Fucosylated Chondroitin Production (1) Creation of fucT-expressing strain Using the expression vector pET28a (Novagen) containing the T7 promoter as a template, a PCR reaction was performed using oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 as a primer set to obtain fragment 1. Escherichia coli having a plasmid for gene expression encoding fucT consisting of the amino acid sequence represented by SEQ ID NOs: 3 was created by the following method. Using the DNA of SEQ ID NOs: 4 listed in the "Template" column of the table below as a template, PCR was performed using DNA consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6 as a primer set to obtain each amplified DNA fragment. The artificially synthesized gene of SEQ ID NOs: 4 was synthesized by optimizing the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 3 for Escherichia coli using Eurofins Genomics' artificial gene synthesis service. Primer 5 has a 15-residue nucleotide sequence from the 3' end of pET28a fragment 1 added to the 5' end of the region containing the start codon of the fucT gene. Primer 6 is formed by adding a nucleotide sequence complementary to the 5' end of the nucleotide sequence complementary to the sequence containing the stop codon of the fucT gene, to the nucleotide sequence complementary to 15 residues from the 5' end of pET28a fragment 1.

[0062] The amplification product obtained by the PCR described above and pET28a fragment 1 were reacted with each other using an In-fusion Kit (Takara Bio Inc.) at 50°C for 15 minutes, and the expression plasmid pET28a-FmfucT2 was obtained by ligating them with the expression vector pET28a. Escherichia coli BL21(DE3) was transformed with the expression plasmid obtained above to obtain BL21(DE3) / pET28a-FmfucT2.

[0063] (2) Fucosylated chondroitin production by stationary cell reaction using fucT-expressing strain The BL21(DE3) / pET28a-FmfucT2 obtained in (1) was inoculated into a test tube containing 2 mL of LB medium with 100 mg / L kanamycin and cultured with shaking at 30°C for 16 hours. The culture solution was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of LB medium with 100 mg / L kanamycin and cultured at 30°C for 5 hours. IPTG was then added to a final concentration of 0.1 mM, and the culture was further cultured at 15°C for 24 hours. The culture solution was centrifuged to obtain wet cells. Xylene was added to the wet cells to a final concentration of 10 mL / L, and membrane treatment was performed at 30°C at 850 rpm for 30 minutes. A 0.2 mL reaction solution consisting of 100 g / L of each type of wet bacterial cell, 50 mM Tris-HCl (pH 7.5), 2.5 mM pentasaccharide-non-sulfated chondroitin (CS-0, GalNAc-GlcA-GalNAc-GlcA-GalNAc) (hereinafter, "-0" means non-sulfated), 10 mM GDP-Fucose, and 10 mM magnesium chloride was placed in an Eppendorf tube, and the reaction was carried out for 24 hours under conditions of 37°C and 850 rpm. The reaction was carried out the required number of times to obtain a sufficient amount for use in cell testing. The obtained reaction solution was centrifuged, the supernatant was collected, and the supernatant was freeze-dried to obtain a powder containing fucosylated chondroitin. (3) Production of fucosylated chondroitin sulfate The sulfation of the fucosylated chondroitin obtained in (2) was carried out according to the sulfation of glycosaminoglycans as previously reported (Garg, G Hari et al., Effect of Fully Sulfated Glycosaminoglycans on Pulmonary Artery Smooth Muscle Cell Profitation. Archives of Biochemistry and Biophysics. 1999 November 15;371(2):228-233.), and a powder containing fucosylated chondroitin sulfate was obtained. The resulting powder containing fucosylated chondroitin sulfate was dissolved in water and then subjected to Bio-Gel P-2 (Bio-rad) for fractionation. The fractionated fractions were spotted onto a TLC plate and colored with 5% sulfuric acid ethanol.The relevant fraction was recovered and freeze-dried to obtain fucosylated chondroitin sulfate (FucCS, pentasaccharide).

[0064] [Test Example 2] Cell Culture (1) Osteoblast Culture In this test, MC3T3-E1 Subclone 4 (hereinafter, MC3T3) (ATCC, CRL-2593) was used as the preosteoblast. MC3T3 was cultured using Minimum Essential Medium α (MEMα; Gibco, A1049001) containing 10% Fetal Bovine Serum (FBS; Gibco, 10270106) and 1% penicillin-streptomycin (p / s; Fujifilm Wako Pure Chemical Industries, 168-23191) as the growth medium. MC3T3 was peeled off using a 0.05% Trypsin-EDTA solution (Thermo Fisher Scientific, 25300054) at a concentration density of 80-90%, resulting in 3.0 × 10⁻⁶ ４ cells / cm ２ Seeds were seeded and subcultured. MC3T3 cells with 20-30 subculturings were used in the experiment. When inducing differentiation of MC3T3 into osteoblasts, 1.0 × 10⁶ cells were placed on a 24-well plate. ５ cells / cm ２ The cells were seeded in this manner, and when the cells reached confluence, the culture medium was switched to differentiation medium (the growth medium of this study with Osteoblast-Inducer Reagent (for animal cell) (TAKARA, MK430) added). On day 23 of differentiation induction, the culture supernatant was collected, and the supernatant obtained by centrifugation (15,000 rpm, 4°C, 10 min) was used as a preparation sample for osteocalcin measurement and addition to myoblasts. 24 hours before collecting the culture supernatant, PBS (control reagent, Control) (Thermo Fisher Scientific, 10010023) or fucosylated chondroitin sulfate (FucCS, pentasaccharide) obtained in [Test Example 1] dissolved in PBS was added to a final concentration of 100 μg / mL. All cultures are performed at 37°C and 5% CO2. ２ It was carried out inside the incubator.

[0065] In addition, osteoblast culture was performed in the same manner as above, except that chondroitin (CS-0, 5-sugar), chondroitin sulfate (CS, 5-sugar), or fucosylated chondroitin (FucCS-0, 5-sugar) was used instead of PBS or FucCS.

[0066] (2) Myoblast culture In this study, C2C12 (RIKEN Cell Bank, RCB0987) was used as myoblasts. C2C12 was cultured using Dulbecco's Modified Eagle Medium (DMEM; WAKO, 044-29765) containing 10% FBS (Thermo, A5256701) and 1% p / s (Wako, 168-23191) as the growth medium. C2C12 was detached using a 0.05% Trypsin-EDTA solution (Wako, 204-16935) at a confluence density of 80-90%, seeded at a confluence density of 10-15%, and subcultured. C2C12 with passage numbers 5-20 was used in the test. When inducing the differentiation of C2C12 into myotube cells, it was seeded in a 24-well plate at 2.5 x 10 ４ cells / cm ２ and when the cells reached confluence, the medium was switched to a differentiation medium (DMEM containing 2% Horse Serum (HS; Thermo, 16050130) and 1% p / s). The culture supernatant of osteoblasts was centrifuged (140,000 g, 4 °C, 30 minutes) using a centrifugal filter unit with a 10,000 NMWL (Merck millipore, UFC501008), followed by centrifugation (140,000 g, 4 °C, 60 minutes) using a centrifugal filter unit with a 3000 NMWL (Merck millipore, UFC500308), and then adjusted to 3000-10,000 Da by reverse centrifugation of the filter side. The prepared sample was added on the 3rd day of myoblast differentiation, and myotube formation was evaluated on the 6th day of differentiation. All cultures were performed in an incubator at 37 °C and 5% CO ２ .

[0067] [Test Example 3] The concentration of osteocalcin (ng / mL) in the culture supernatant of osteoblasts obtained in Test Example 2 (1) was measured using commercially available ELISA kits: Mouse Gla-Osteocalcin High Sensitive EIA Kit (TAKARA, MK127) and Mouse Glu-Osteocalcin High Sensitive EIA Kit (TAKARA, MK129). The amount of osteocalcin was calculated as the total amount of Gla-type osteocalcin and Glu-type osteocalcin. Each data point is shown as mean ± standard error. Significant difference testing was performed using Dunnett's test with SPSS software for comparisons between groups, and a p < 0.05 result was considered statistically significant. The results are shown in Figure 1. Figure 1 shows that the addition of 100 μg / mL of FucCS significantly increased the osteocalcin concentration in the culture supernatant compared to the control, CS-0, CS, and FucCS-0.

[0068] [Test Example 4] Myoblasts cultured in 24-well plates in the myoblast culture obtained in Test Example 2 (2) were fixed with the same volume of 4% paraformaldehyde (Wako, 163-20145) as the culture medium for 20 minutes, and then washed three times with phosphate-buffered saline (PBST) containing 0.1% Triton X100 (Wako, 160-24751). After that, the cells were blocked / permeabilized at room temperature for 1 hour with PBST containing 3% donkey serum (Sigma, D9663-10ML, Lot. SLCJ7887), and then the myosin heavy chain-specific primary antibody Mouse Anti-myosin4 (Thermo Fisher, 14-6503-82) was added and incubated overnight at 4°C. After washing the cells three times with PBST, a mixture of the corresponding secondary antibody Donkey anti-Mouse Alexa Flor 594 (Thermo Fisher, A-21203) or Donkey anti-Mouse Alexa Flor 488 (Thermo Fisher, A-21202), nuclear staining DAPI (Wako, 340-07971), and PBST was added, and the mixture was allowed to stand at room temperature for 2 hours. The cells were then washed three times with PBST, and fluorescence images were acquired using a fluorescence microscope (Leica DMI4000 B). The area and thickness of myotubes stained by immunofluorescence and imaged were measured using the image analysis software MetaMorph. Stained images were taken four times per well, and the 10 myotubes with the largest area were selected from these images. The average of their measurements was used as the well value. Each data point is shown as mean ± standard error. Significant difference testing was performed using Dunnett's test with SPSS software for comparisons between groups, and a p < 0.05 was considered statistically significant. Figure 2 shows the results of fluorescence microscopy confirmation of myoblasts stained by immunofluorescence. Figure 3 shows the results of measuring the area and thickness of myotubes. From Figure 3, it was shown that adding osteoblast culture supernatant containing 100 μg / mL of FucCS to myoblasts significantly increased the area and length of myotubes compared to the control group.

[0069] Although the present invention has been described in detail above with reference to specific embodiments, the present invention is not limited thereto. Additions, omissions, substitutions, and other modifications to the configuration are possible without departing from the spirit of the present invention.

Claims

1. A blood osteocalcin secretion promoter containing fucosylated chondroitin sulfate.

2. The blood osteocalcin secretion promoter according to claim 1, wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less.

3. The fucosylated chondroitin sulfate is a blood osteocalcin secretion promoter according to claim 1, wherein fucose is bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin.

4. A composition for the prevention or treatment of symptoms or diseases, comprising fucosylated chondroitin sulfate, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus, and new Lupus in newborns, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharynx Type muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss,A composition selected from the group consisting of concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive diseases, inflammatory diseases, and knee joint complications.

5. The composition according to claim 4, wherein the molecular weight of the fucosylated chondroitin sulfate is 4000 Da or less.

6. The composition according to claim 4, wherein the fucosylated chondroitin sulfate has fucose bonded to the 3-position hydroxyl group of glucuronic acid in chondroitin.

7. The composition according to any one of claims 4 to 6, wherein the composition for preventing or treating the symptoms or disease is a food or pharmaceutical composition.

8. A composition for the prevention or treatment of symptoms or diseases, comprising a blood osteocalcin secretion promoter according to any one of claims 1 to 3, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug-induced lupus Lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy Rophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss,A composition selected from the group consisting of drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications.

9. A composition for the prevention or treatment of symptoms or diseases, comprising fucosylated chondroitin sulfate, wherein fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood, wherein the symptoms or diseases are sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug Substance-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy Distal muscular dystrophy, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, Adult-onset ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy,A composition selected from the group consisting of noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma, Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance disease, sleep apnea disease, hypertension, renal disease, hypertensive disease, inflammatory disease, and knee joint complications.

10. A food or pharmaceutical composition comprising fucosylated chondroitin sulfate, wherein fucosylated chondroitin sulfate promotes the secretion of osteocalcin in the blood.

11. For the prevention or treatment of symptoms or diseases, the symptoms or diseases being sarcopenia, frailty, muscle loss, muscle atrophy, decreased physical function associated with muscle loss and / or muscle atrophy, obesity, sarcopenic obesity, fat accumulation in muscle tissue, dynapenia, decreased physical function associated with decreased insulin secretion, decreased physical function associated with decreased hormones, decreased physical function associated with deterioration of the intestinal environment, traumatic brain injury, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, lupus, systemic lupus erythematosus, discoid lupus, drug Substance-induced lupus, neonatal lupus, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, early-onset adult-onset diabetes mellitus, non-alcoholic fatty liver disease, myasthenia gravis, ocular myasthenia gravis, congenital myasthenia gravis, generalized myasthenia gravis, rheumatoid arthritis, Graves' disease, Guillain-Barré syndrome, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, Emery-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy Muscular dystrophy, amyotrophic lateral sclerosis, ataxia, Friedreich's ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 11, dentatorubral-pallidoluysian atrophy, gluten ataxia, neuronal ceroid lipofuscinosis, infant neuronal ceroid lipofuscinosis, late infant neuronal ceroid lipofuscinosis, juvenile neuronal ceroid lipofuscinosis, adult neuronal ceroid lipofuscinosis, Alzheimer's disease, early-onset Alzheimer's disease, late-onset Alzheimer's disease, familial Alzheimer's disease, optic neuritis, Leber hereditary optic neuropathy, autism spectrum disorder, Asperger's syndrome, pervasive developmental disorder, childhood disintegrative disorder, autism, Parkinson's disease, idiopathic Parkinson's disease, vascular parkinsonism, Lewy body dementia, hereditary Parkinson's disease, drug-induced parkinsonism, juvenile Parkinson's disease, atypical parkinsonism, Wolfram syndrome, spinal muscular atrophy, noise-induced hearing loss, age-related hearing loss, drug-induced hearing loss, hereditary hearing loss, concussion, keratoconjunctivitis sicca, dry eye disease, glaucoma,A food or pharmaceutical composition according to claim 10, selected from the group consisting of Sjögren's syndrome, rheumatoid arthritis, Walcott-Larrisson syndrome, mitochondrial disease, metabolic syndrome, autoimmune disorders, insulin resistance, sleep apnea, hypertension, renal disease, hypertensive diseases, inflammatory diseases, and knee joint complications.

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