Composition for preventing or treating muscle diseases comprising probiotic metabolite as active ingredient

Abstract

A probiotic metabolite of the present invention was found to protect muscle cells and promote differentiation of muscle fibers. In addition, the probiotic metabolite was found to increase muscle mass and muscle function, inhibit the expression of muscle degradation-related factors, and increase the expression of muscle synthesis-and regeneration-related factors in a dexamethasone-induced muscular atrophy or sarcopenia animal model. In addition, the probiotic metabolite was found to increase muscle function, protect against damage to muscle tissue, inhibit the expression of factors related to muscle degradation, and increase the expression of factors related to muscle synthesis and regeneration in a fasting-induced muscular atrophy or sarcopenia animal model.

Description

Composition for preventing or treating muscle disease containing probiotic metabolites as active ingredients

[0001] The present invention relates to a composition for preventing or treating muscle disease, comprising a probiotic metabolite as an active ingredient.

[0002] Prebiotics, probiotics, and postbiotics are relatively new terms used to describe a variety of substances that confer health and nutritional benefits to animals. Typically, the term prebiotic refers to substances that stimulate the growth or activity of bacteria in the digestive tract of animals, leading to beneficial health effects. Prebiotics can be selectively fermented ingredients that induce specific changes in both the composition and activity of the gastrointestinal microflora, conferring health benefits to the host. Probiotics generally refer to microorganisms that contribute to the intestinal microbial balance and, consequently, to maintaining health. Many species of lactic acid bacteria (LAB), such as Lactobacillus and Bifidobacterium, are commonly considered probiotics, but some species of Bacillus and some yeasts have also been identified as suitable candidates. Postbiotics refer to non-viable bacterial products or metabolic by-products derived from probiotic organisms that possess biological activity in the host.

[0003] The use of probiotics to improve animal health and nutrition has been shown to be effective in a variety of diseases and health conditions. Furthermore, prebiotics and postbiotics offer potential alternatives or adjunctive therapies to the use of live microorganisms. There is growing understanding of the role of prebiotics, probiotics, and postbiotics in modulating immune responses, specifically in regulating the expression of cytokines that control inflammatory responses at both local and systemic levels. For example, the consumption of probiotic bacteria can potentially stabilize the immunological barrier in the gut mucosa by reducing the production of local proinflammatory cytokines. Furthermore, alterations in the characteristics of the indigenous microbiota by probiotic therapy have been shown to reverse some immunological disturbances in human conditions such as Crohn's disease, food allergy, and atopic eczema.

[0004] Meanwhile, muscle atrophy can be caused by a variety of factors, including the absence of mechanical stimulation, starvation, and cancer. Muscle atrophy can be defined as the loss of muscle tissue resulting from disuse, disease of the muscle itself, or damage to the nerves that control it. In general, disuse can lead to a significant loss of muscle strength, which can gradually progress to muscle atrophy. Furthermore, individuals living in environments without gravity can also experience muscle weakness due to decreased calcium and muscle strength. Muscle atrophy due to disease of the muscle itself includes myasthenia gravis, muscular dystrophy (progressive muscular dystrophy, myotonic dystrophy, Duchenne, Becker, limb-girdle, facioscapulohumeral), and inflammation that occurs in the muscle itself, and muscle atrophy due to damage to the nerves that control the muscle includes spinal muscular amyotrophy (Berardnig-Hoffmann type, Kugelberg-Welander disease), amyotrophic lateral sclerosis (ALS): Lou Gehrig's disease, and spinobular muscular atrophy (Kennedy's disease). For example, muscle degeneration can inevitably progress due to conditions such as spaceflight or disability, even if exercise or other countermeasures are continuously applied. Therefore, research is needed on other countermeasures that can overcome disability conditions, either as an alternative to mechanical stimulation or as an addition to mechanical stimulation. An appropriate approach to such countermeasures is to utilize functional biomaterials derived from natural products that can help maintain muscle mass even under conditions that induce atrophy of muscle fiber proteins.

[0005] Sarcopenia refers to a condition in which skeletal muscle mass and function are reduced. Sarcopenia can be caused by a variety of factors, including aging, hormonal imbalances, nutritional deficiencies, lack of physical activity, inflammation, and degenerative diseases. Aging and sex hormone deficiencies are known to be the primary causes. Advances in medical technology and the development of various treatments have led to increased life expectancy worldwide, leading to a growing aging population. Consequently, the demand for sarcopenia treatment is expected to continue to grow.

[0006] In patients with sarcopenia, the number of myoblasts decreases due to impaired recruitment, activation, or proliferation of satellite cells, which are stem cells of muscle cells, and the proliferation and differentiation of myoblasts decreases. As a result, the muscles of patients with sarcopenia show symptoms of decreased muscle function due to a decrease in the size and number of muscle fibers at the histological level.

[0007] Exercise, protein, and calorie supplementation are known to help with sarcopenia. However, they are not particularly effective in the elderly, who account for the majority of sarcopenia patients. Therefore, treatments for sarcopenia are urgently needed. However, currently, treatments that demonstrate a direct effect on improving muscle loss and increasing muscle mass are still in the clinical trial stage, and no drug has received final FDA approval.

[0008] Accordingly, the inventors of the present invention isolated useful metabolites from probiotic strains and confirmed that the isolated compounds have effects of increasing muscle mass and inhibiting muscle atrophy, thereby completing the present invention.

[0009] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease, which comprises a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] [Chemical Formula 1]

[0011]

[0012] Another object of the present invention is to provide a food composition for preventing or improving muscle disease, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0013] Another object of the present invention is to provide a pharmaceutical composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a food composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0015] Another object of the present invention is to provide a pharmaceutical composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Another object of the present invention is to provide a food composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0017] Another object of the present invention is to provide a pharmaceutical composition for improving muscle function, which comprises a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0018] Another object of the present invention is to provide a food composition for improving muscle function, which comprises a compound represented by the above chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

[0019] Another object of the present invention is to provide a method for treating muscle disease, comprising the step of administering to a subject a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0020] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, which comprises a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] [Chemical Formula 1]

[0022]

[0023] In addition, the present invention provides a food composition for preventing or improving muscle disease, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0024] In addition, the present invention provides a pharmaceutical composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0025] In addition, the present invention provides a food composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0026] In addition, the present invention provides a pharmaceutical composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0027] In addition, the present invention provides a food composition for increasing muscle mass or promoting muscle production, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0028] In addition, the present invention provides a pharmaceutical composition for improving muscle function, which comprises a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] In addition, the present invention provides a food composition for improving muscle function, which comprises a compound represented by the above chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

[0030] In addition, the present invention provides a method for treating muscle disease, comprising the step of administering to a subject a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0031] The probiotic metabolites of the present invention were confirmed to protect muscle cells and promote muscle fiber differentiation. Furthermore, in an animal model of dexamethasone-induced muscle atrophy or sarcopenia, they were confirmed to increase muscle mass and muscle function, suppress the expression of factors associated with muscle breakdown, and increase the expression of factors associated with muscle synthesis and regeneration. Furthermore, in an animal model of fasting-induced muscle atrophy or sarcopenia, they were confirmed to increase muscle function, protect against muscle tissue damage, suppress the expression of factors associated with muscle breakdown, and increase the expression of factors associated with muscle synthesis and regeneration, and thus can be usefully utilized in related industries.

[0032] Figure 1 is a schematic diagram of the production and experimental process of a dexamethasone-induced muscle atrophy or sarcopenia animal model.

[0033] Figure 2 is a schematic diagram of the production and experimental process of a fasting-induced muscle atrophy or sarcopenia animal model.

[0034] Figure 3 is a diagram confirming the MGO-AGEs crushing ability according to the treatment of the probiotic metabolite of the present invention.

[0035] Figure 4 is a diagram confirming the cell viability and cell proliferation ability of muscle cells according to the probiotic metabolite of the present invention and dexamethasone treatment.

[0036] A: Confirmation of cytotoxicity of metabolites

[0037] B: Confirmation of protection against dexamethasone toxicity

[0038] C: Confirmation of cell proliferation induction by metabolites

[0039] D: Confirmation of protection against dexamethasone toxicity

[0040] Figure 5 is a diagram showing the formation of myotubes confirmed by Jenner-Giemsa staining according to the probiotic metabolite of the present invention and dexamethasone treatment (A: staining result, B: quantification of myotube diameter).

[0041] Figure 6 is a diagram showing the expression of muscle breakdown proteins analyzed by Western blot according to the probiotic metabolites of the present invention and dexamethasone treatment (A: Western blot analysis, B: quantification of analysis results).

[0042] Figure 7 is a diagram showing the expression of muscle production and growth proteins according to the probiotic metabolites of the present invention and dexamethasone treatment, analyzed by Western blot (A: Western blot analysis, B: quantification of analysis results).

[0043] Figure 8 is a diagram showing the quantification of body weight changes and food intake according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: confirmation of body weight change, B: quantification of body weight change, C: quantification of food intake).

[0044] Figure 9 shows the quantification of muscle mass by muscle according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease.

[0045] Figure 10 is a diagram showing muscle function confirmed according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: running time quantification, B: speed quantification, C: grip strength quantification).

[0046] Figure 11 is a diagram showing the results of micro-CT analysis of calf thickness according to administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: micro-CT results, B: calf area quantification, C: fibula and tibia distance quantification).

[0047] Figure 12 is a Western blot analysis of the expression of muscle degradation proteins in tissues following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: Western blot analysis, B: quantification of analysis results).

[0048] Figure 13 is a diagram showing the expression of Atrogin-1 in tissues analyzed by immunohistochemical staining according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: staining results, B: quantification of staining results).

[0049] Figure 14 is a Western blot analysis of the expression of fibrotic factors in tissues following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: Western blot analysis, B: quantification of analysis results).

[0050] Figure 15 is a diagram showing the collagen accumulation confirmed by Sirius red staining following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: staining result, B: quantification of staining result).

[0051] Figure 16 is a Western blot analysis of the expression of myogenic factors according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: Western blot analysis, B: quantification of analysis results).

[0052] Figure 17 is a diagram showing the expression of MyH confirmed by immunohistochemical staining according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: staining result, B: quantification of staining result).

[0053] Figure 18 is a diagram showing the muscle fiber size confirmed by H&E staining according to the administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: staining result, B: quantification of staining result).

[0054] Figure 19 is a Western blot analysis of the activation of the muscle synthesis pathway following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease (A: Western blot analysis, B: quantification of analysis results).

[0055] Figure 20 is a diagram showing the results of multivariate statistical analysis of the metabolic differences between groups in blood and gastrocnemius muscle tissue according to the administration of probiotic metabolites in a dexamethasone-induced muscle atrophy or sarcopenia animal model.

[0056] Figure 21 is a diagram analyzing the pattern of metabolite content and changed metabolic pathways between groups in blood and gastrocnemius muscle tissue according to the administration of probiotic metabolites in an animal model of dexamethasone-induced muscular atrophy or sarcopenia (A. Comparison of metabolite content between groups, B. Changed major metabolic pathways).

[0057] Figure 22 is a diagram confirming the effect of improving muscle strength by administering probiotic metabolites to a fasting-induced muscle disease animal model.

[0058] Figure 23 is a diagram showing the muscle fiber protection effect confirmed by administration of probiotic metabolites to a fasting-induced muscle disease animal model using H&E staining.

[0059] Figure 24 is a diagram showing the activity of creatine kinase, an indicator of muscle damage in the blood, following administration of probiotic metabolites to a fasting-induced muscle disease animal model.

[0060] Figure 25 is a diagram showing the concentration of β-hydroxybutanoic acid, an indicator of muscle damage in the blood, following administration of probiotic metabolites to a fasting-induced muscle disease animal model.

[0061] Figure 26 is a diagram confirming the correlation between fasting and body composition.

[0062] Figure 27 is a Western blot analysis of the expression of muscle protein degradation factors following administration of probiotic metabolites to a fasting-induced muscle disease animal model (A: Western blot analysis, B: quantification of analysis results).

[0063] Figure 28 is a Western blot analysis of protein expression of the muscle degradation pathway following administration of probiotic metabolites to a fasting-induced muscle disease animal model (A: Western blot analysis, B: quantification of analysis results).

[0064] Figure 29 is a Western blot analysis of protein expression of muscle synthesis and growth pathways following administration of probiotic metabolites to a fasting-induced muscle disease animal model (A: Western blot analysis, B: quantification of analysis results).

[0065] Figure 30 is a Western blot analysis of the expression of cell death factors following administration of probiotic metabolites to a fasting-induced muscle disease animal model (A: Western blot analysis, B: quantification of analysis results).

[0066] Figure 31 is a Western blot analysis of the expression of inflammatory and oxidative stress factors following administration of probiotic metabolites to a fasting-induced muscle disease animal model.

[0067] A: Western blot results for inflammatory factors

[0068] B: Quantification of inflammatory factor expression

[0069] C: Western blot results of oxidative stress factors

[0070] D: Quantification of oxidative stress factor expression

[0071] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0072] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0073] The present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0074]

[0075] The compound of the above chemical formula 1 of the present invention may be named 2-hydroxyisovaleric acid and may be a compound with CAS number 17407-56-6.

[0076] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0077] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0078] The term "pharmaceutically acceptable salts" means those salts which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, and the like, and which are proportional to a reasonable advantage / disadvantage ratio. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0079] Salts derived from suitable bases are made of alkali metals, alkaline earth metals, ammonium and N + (C 1-4 alkyl) tetra-salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0080] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0081] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0082] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0083] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0084] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0085] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0086] According to one embodiment of the present invention, the compound may increase muscle mass, and the increase in muscle mass may be an increase in muscle canal diameter or muscle thickness.

[0087] According to one embodiment of the present invention, the compound may increase muscle function.

[0088] According to one embodiment of the present invention, the compound may suppress the expression of a muscle degradation factor, and the muscle degradation factor may be a protein selected from the group consisting of muscle atrophy F-box (MAFbx / Atrogin-1), muscle-specific RING finger protein 1 (MuRF1), forkhead box O3 (FoxO3a), and glucocorticoid receptor (GR).

[0089] According to one embodiment of the present invention, the compound may increase the expression of a muscle growth factor, and the muscle growth factor may be myoblast determination protein 1 (MyoD), myogenin, or myosin heavy chain (MyH).

[0090] According to one embodiment of the present invention, the compound may increase phosphorylation or expression of a muscle synthesis pathway protein, and the muscle synthesis pathway protein may be a protein selected from the group consisting of mammalian target of rapamycin (mTOR), AKT Serine / Threonine Kinase 1 (AKT), insulin like growth factor 1 receptor (IGF-1R), and NAD-dependent deacetylase sirtuin 2 (SIRT2).

[0091] According to one embodiment of the present invention, the compound may inhibit phosphorylation of a muscle degradation pathway, and the muscle degradation pathway may be an extracellular signal-mediated kinase (ERK1 / 2).

[0092] According to one embodiment of the present invention, the compound may reduce the LC3(1A / 1B-light chain 3)B-II / LC3B-I ratio of the muscle degradation pathway.

[0093] According to one embodiment of the present invention, the compound may inhibit fibrosis of muscle tissue, and the inhibition of fibrosis may be by inhibiting Mothers against decapentaplegic homolog 7 (Smad7) in muscle tissue, and may be by inhibiting collagen accumulation in muscle tissue.

[0094] According to one embodiment of the present invention, the compound may reduce a muscle damage indicator in the blood, and the muscle damage indicator may be creatine kinase or β-hydroxybutyrate.

[0095] According to one embodiment of the present invention, the compound may regulate the expression of a metabolite related to muscle synthesis.

[0096] According to one embodiment of the present invention, regulating the expression of the muscle synthesis-related metabolite may be by increasing the amount of a metabolite selected from the group consisting of glycine, tyrosine, sucrose, pyruvate, thymine, ethylene glycol, and 1,2-propanediol in serum, and by increasing the amount of 12-S-HETE or LPE 18:3 in muscle.

[0097] According to one embodiment of the present invention, regulating the expression of the muscle synthesis-related metabolites may be by reducing the amount of lactose, mannitol, sorbitol, 2-hydroxybutyric acid, fumaric acid, and uric acid in serum, and by reducing the amount of glycine or mannitol in muscle.

[0098] According to one embodiment of the present invention, the muscle disease may be a disease selected from the group consisting of muscular atrophy, myopathy, muscular degeneration, myasthenia, muscular injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia, and is preferably muscular atrophy or sarcopenia, but is not limited thereto.

[0099] In addition, the present invention provides a food composition for preventing or improving muscle disease, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0100] The term "improvement" as used herein means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0101] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0102] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0103] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0104] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating muscle diseases. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0105] In addition, the present invention provides a pharmaceutical composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0106] In addition, the present invention provides a food composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0107] In addition, the present invention provides a pharmaceutical composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0108] In addition, the present invention provides a food composition for increasing muscle mass or promoting muscle production, which comprises a compound represented by the above chemical formula 1 or a food-wise acceptable salt thereof as an active ingredient.

[0109] In addition, the present invention provides a pharmaceutical composition for improving muscle function, comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0110] In addition, the present invention provides a food composition for improving muscle function, which comprises a compound represented by the above chemical formula 1 or a food-related acceptable salt thereof as an active ingredient.

[0111] In addition, the present invention provides a method for treating muscle disease, comprising the step of administering to a subject a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0112] The treatment method of the present invention comprises administering to a subject a therapeutically effective amount of the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof. It is preferred that the specific therapeutically effective amount for a specific subject be applied differently depending on various factors including the type and degree of the response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health condition, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration of each active ingredient should be such that the content of each active ingredient is not excessively high and side effects are not caused. Therefore, it is preferred that the effective amount of a composition suitable for the purpose of the present invention be determined in consideration of the aforementioned matters.

[0113] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0114] The compound of chemical formula 1 of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are conceivable, and for example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0115] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0116] <Preparation Example 1> Preparation of metabolites derived from probiotic strains

[0117] The compound of chemical formula 1, a metabolite derived from the probiotic strain of the present invention, was identified through strain metabolite profiling. Thereafter, in order to utilize the compound of chemical formula 1 in an experiment to improve muscle disease, the compound of chemical formula 1 below, a probiotic metabolite (ProM), was purchased from Sigma Aldrich.

[0118] [Chemical Formula 1]

[0119]

[0120] <Experimental Example 1> Preparation for confirmation of improvement in dexamethasone-induced muscle disease by probiotic metabolites.

[0121] <1-1> Reagent preparation

[0122] In order to confirm the effect of the probiotic metabolite of the present invention on improving dexamethasone (DEX)-induced muscle disease, reagents were prepared. In animal experiments, oxymetholone (OXY; Sigma-Aldrich, USA) was used as a positive control. In cell analysis, curcumin (CU; Sigma-Aldrich, USA) was used as a positive control, and the probiotic metabolite and all compounds were dissolved in distilled water and used. Dexamethasone (DEX; Sigma-Aldrich, USA) was used as a negative control that reduces muscle mass, inducing muscle atrophy and sarcopenia. In animal experiments, it was dissolved in 4% DMSO, 1% tween-20, and 95% saline, and in cell experiments, it was dissolved in 10% DMSO.

[0123] <1-2> Analysis of MGO-AGEs fragmentation ability

[0124] To evaluate the degrading activity of MGO-AGEs (methylglyoxal-derived advanced glycation endproducts), a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay was performed. Specifically, MGO-AGEs, prepared by reacting methylglyoxal (MGO) with bovine serum albumin (BSA) at 37°C for 7 days, were mixed with the compound of formula 1 (100, 200, or 400 μM) and dexamethasone (100, 200, or 400 μM) at a concentration of 1 mg / mL, homogenized, and reacted for 24 hours. Afterwards, 0.1% TNBSA and 34% NaHCO were added, and reacted for 2 hours. The reaction was then terminated by adding 10% SDS and 1 N HCl, and the AGE degradation products were quantified by measuring the absorbance at 340 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

[0125] <1-3> Cell culture and drug treatment

[0126] C2C12 cells (ATCC, USA), a mouse myoblast cell line, were cultured in Dulbecco's Modified Eagle Medium (DMEM, Welgene, Korea) supplemented with 10% fetal bovine serum (Welgene) and 1% penicillin / streptomycin at 5% CO2 and 37°C. To induce myotube differentiation of myoblasts, 2.5 × 10 cells were seeded in 6-well plates. 5The cells were seeded at a cell density of 10 and cultured for 2 days. After the cells reached 90% or more confluence, the medium was replaced with differentiation DMEM containing 2% horse serum, and the compound of chemical formula 1 or 1 μM curcumin (CU), a positive control, was treated during the differentiation process for 6 days. On the 6th day of differentiation, the C2C12 myotubes were reacted with 50 μM DEX for 24 hours.

[0127] <1-4> Cell viability and proliferation analysis

[0128] Cell viability was analyzed using the MTT assay, and C2C12 cells were seeded at 1 × 10 in a 96-well plate. 4 The cells were seeded at a concentration of 100 μL and cultured under conditions of 5% CO2 and 37°C. When the cells were more than 80% confluent, the cells were treated with the compound of formula 1 and cultured in serum-free medium for an additional 24 hours. After that, 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg / ml, Sigma-Aldrich) was added and reacted for 2 hours. The resulting formazan product was dissolved in DMSO and the absorbance was measured at 540 nm using a microplate reader (BioTek, Winooski, VT, USA).

[0129] In addition, proliferation analysis was evaluated using BrdU analysis, and cell proliferation was measured using BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, USA) on cells treated with the compound of chemical formula 1 and cultured for 24 hours in the same manner as above.

[0130] <1-5> Jenner-Giemsa staining and root canal confirmation

[0131] The length, width, and area of ​​the root canals were measured by staining differentiated C2C12 cells with Jenner-Giemsa. After Jenner-Giemsa staining, the root canals were washed twice with cold PBS and fixed with 4% paraformaldehyde. The root canal images were then examined using an optical microscope (Olympus, Tokyo, Japan). The root canal diameters were measured from the images of each group, and the average values ​​were quantified using Image J software.

[0132] <1-6> Preparation of animal models

[0133] Seven-week-old C57BL / 6N mice were purchased from Orient Bio (Republic of Korea) and acclimated for 1 week before use in the experiment. Mice were housed at 23 ± 1°C with a 12-h light / dark cycle and were provided unlimited access to water and a standard laboratory diet. All animal experiments were performed in accordance with the ethical guidelines established by the Laboratory Animal Research Center, College of Pharmacy, Gachon University, Seongnam, Republic of Korea, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Gachon University (GU1-2023-IA0032-00).

[0134] The habituated mice were divided into the following groups: a vehicle group (CON group) administered with a solvent; a DEX group administered with dexamethasone; a positive control group (OXY) treated with 50 mg / kg of oxymetholone and dexamethasone; a group treated with 5 mg / kg of the compound of formula 1 and dexamethasone (ProM 5); and a group treated with 20 mg / kg of the compound of formula 1 and dexamethasone (ProM 20). The compound of formula 1 and oxymetholone were administered orally daily for 14 days, and dexamethasone was injected subcutaneously daily at a concentration of 20 mg / kg to induce muscle atrophy. The specific drug administration schedule and classification of the experimental groups are shown in Fig. 1.

[0135] <1-7> Measurement of body weight, food intake, and muscle mass

[0136] Throughout the animal model experiments, body weight and food intake were monitored daily. At the end of the experiment, mice were humanely sacrificed, and the quadriceps femoris (QD), gastrocnemius (GCM), plantaris (PLA), extensor digitorum longus (EDL), and soleus (SOL) muscles were isolated and weighed. Muscle tissues were then frozen in liquid nitrogen and stored and fixed at -80°C.

[0137] <1-8> Treadmill and grip strength test

[0138] To measure muscle strength, treadmill and grip strength tests were performed. Specifically, the treadmill test was performed on days 7 and 14 after sample administration using an 8-lane treadmill with a motivation grid (JD-A-22, Republic of Korea). The treadmill test involved placing mice on a flat treadmill at a speed of 10 m / min for 3 min, increasing the speed to 2 m / min every 2 min. The running time (seconds) and speed (meters per minute) until exhaustion (maximal exercise) were recorded and compared between each group.

[0139] Grip strength of mice was measured using a grip dynamometer (BIO-G53, BIOSEB, USA). The grip strength test involved allowing mice to grasp a wire mesh with their forelimbs, and the grip strength value obtained when force was applied momentarily to the tail was recorded. Grip strength was measured twice, on days 7 and 14 after inducing muscle atrophy and after sample administration. Each group of mice was tested three times. The recorded grip strength values ​​were finally quantified by dividing the value by body weight (g / g).

[0140] <1-9> micro CT

[0141] The thighs obtained from sacrificed mice were fixed in 10% neutral buffered formalin and photographed using a Micro-CT scanner (SkyScan1276, Bruker, Belgium) to measure the width and radius of the thigh. The analysis conditions are as shown in Table 1 below. Using the CTAn program (Bruker microCT), the width and radius of the thigh were measured at the midpoint, with the total length being from the thigh head to the beginning of the fibula bone based on the tibia bone. The width measurement refers to the measurement of the muscle width in the cut plane, and the radius measurement refers to the length of the line segment from the location corresponding to the shank to the center of the fibula bone. The Micro CT Scan conditions are shown in Table 1 below.

[0142] MicroCT scan conditionsResolution2 KSource Voltage40 kVSource Current200 μAImage Pixels18 μmExposure Times650 msRotation Step2 °Rotation in Degree180 °Average Frames2Filter1 mm AlScan Duration3 m: 28 s

[0143] <1-10> Muscle histological analysis

[0144] Quadriceps femoris (QD) and gastrocnemius (GCM) muscles obtained from sacrificed mice were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 2.5 μm. The cross-sectional area of ​​each muscle tissue was measured using hematoxylin and eosin staining (H&E staining; Sigma-Aldrich, USA). The degree of collagen fibrosis was analyzed using Picro-Sirius Red (Sigma-Aldrich, USA) staining. Both staining processes included deparaffinization, rehydration, dehydration, dexylene, and mounting using DPX mounting agent (Sigma-Aldrich, USA).

[0145] <1-11> Western blot analysis

[0146] QD tissue (30 mg) or C2C12 myotubes were homogenized using RIPA buffer containing a protease / phosphatase inhibitor cocktail. The homogenate was then centrifuged at 12,000 rpm for 1 h at 4°C, and protein (30 μg) was separated by SDS-PAGE to determine expression. The separated proteins were then transferred to a nitrocellulose membrane and reacted with primary antibodies of muscle atrophy F-box (MAFbx / Atrogin-1), Muscle-specific RING finger protein 1 (MuRF1), Glucocorticoid Receptor (GR), Myoblast Determination protein 1 (MyoD), Myogenin, Myosin heavy chain (MyH), The mammalian target of rapamycin (mTOR), p-mTOR, FoxO3a (Forkhead box O3), p-FoxO3a, AKT Serine / Threonine Kinase 1 (AKT), p-AKT, Mothers against decapentaplegic homolog 7 (Smad7), insulin-like growth factor 1 receptor (IGF-1R), p-IGF-1R, and NAD-dependent deacetylase sirtuin2 (SIRT2) and GAPDH, which are proteins related to muscle atrophy, at 4°C for 18 hours. Afterwards, the membrane was washed with TBST, and HRP-conjugated secondary antibody was added and reacted at room temperature for 1 hour. After the reaction was completed, the membrane was visualized and analyzed using the ChemiDoc XRS+ imaging system (Bio-Rad, USA).

[0147] <1-12> Immunohistochemical analysis

[0148] QD tissues sectioned at 4 μm were deparaffinized with xylene, rehydrated in 70–100% ethanol, and treated with an endogenous peroxidase blocker. The sections were then washed with phosphate-buffered saline (PBS) and reacted with primary antibodies, MAFbx / Atrogin-1 and MyHC, at 4°C. The sections were then washed with PBS and incubated with biotinylated anti-rabbit and anti-mouse IgG antibodies for 1 h, followed by incubation with an avidin-biotin horseradish peroxidase complex. The optical density of MAFbx / Atrogin-1 and MyHC immunoreactivity in the QD tissues was then analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0149] <1-13> Analysis of metabolite changes in blood and muscle tissue

[0150] Metabolomic analysis was performed on serum and QD tissue obtained from sacrificed mice. 80 μL of serum was homogenized with 1 mL of 100% methanol and incubated at -20°C for 2 hours to precipitate proteins. The homogenate was centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was dried using a speed vacuum. The dried extract was re-dissolved in 0.3 mL of 50% methanol and used for instrumental analysis. Furthermore, 25 mg of QD tissue was homogenized with 1 mL of a mixed solvent (methanol:water:chloroform = 2.5:1:1). The homogenate was centrifuged at 10,000 g for 5 minutes at 4°C, and the supernatant was transferred to a 2-mL tube, where 0.5 mL of water was added and mixed. After centrifugation of the mixture using the same method, 1 mL of the polar layer (upper part) and 0.2 mL of the non-polar layer (lower part) were obtained from the separated mixture and dried using a speed vacuum. The dried extracts of the polar and non-polar layers of the muscle were redissolved using 0.2 mL of 50% and 100% methanol, respectively, and then filtered through a 0.22 μm syringe before being used for instrumental analysis.

[0151] LC-MS analysis was performed on the nonpolar layer extracts of serum and muscle. 0.1 mL of the solution was placed in a container and analyzed using UHPLC-Orbitrap-MS / MS. A Phenomenes KINETEX C18 column was used as the stationary phase, and water (A) with 0.1% formic acid and acetonitrile (B) with 0.1% formic acid were used as the mobile phase to separate the substances. In addition, GC-MS analysis was performed on the polar layer extracts of serum and muscle. After redrying the re-dissolved solution, derivatization (oximation, silylation) was performed. An Rtx-5MS column was used as the stationary phase, and the substances were separated through helium and temperature control.

[0152] <1-14> Statistical analysis

[0153] All collected data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., USA) and expressed as mean ± standard error of the mean (SEM). All results were analyzed using one-way analysis of variance (ANOVA) to compare means across multiple groups followed by Tukey's post hoc test or two-way analysis of variance (ANOVA), and statistical significance was set at p<0.05. In addition, the collected data for metabolite analysis were subjected to multivariate statistical analysis using Simca-P+ software (version 13; Umetric, Umea, Sweden). Differential metabolites were selected based on variable importance in projection (VIP>1.0) obtained from the PLS-DA model, and then metabolic pathways were analyzed using Metabonanalyst (https: / www.metaboanalyst.ca / ).

[0154] <Experimental Example 2> Preparation for confirmation of improvement in fasting-induced muscle disease by probiotic dead cells and metabolites.

[0155] <2-1> Animal model and experimental design

[0156] To confirm the effect of the probiotic metabolite of the present invention on improving fasting-induced muscular dystrophy or sarcopenia, an animal model in which fasting-induced sarcopenia was induced was created. Specifically, 10-week-old male C57BL / 6J mice (Laon Bio, Republic of Korea) were habituated in individual cages under conditions of 22 ± 1℃, 50 ± 5% humidity, and a 12-hour light / dark cycle, with free access to water and food. All experimental procedures related to the animal model were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHSASP-23-183).

[0157] After 7 days of acclimation, mice were divided into the following groups: a normal control group (FED or CON group) continuously supplied with distilled water and diet; a control group fed only distilled water and fasted for 48 hours (FAST group); a WF group administered 50 mg / kg of a wild-type probiotic strain extract; an LProM group administered 5 mg / kg of the compound of formula 1 and fasted for 48 hours; and an HProM group administered 20 mg / kg of the compound of formula 1 and fasted for 48 hours. Mice, except for the FED group, received a 7-day preconditioning regimen using oral gavage, followed by a 48-hour fasting period designed to induce muscle atrophy. All groups were fed AIN-93G feed (Dooyeol Biotech, Korea) during the treatment period. To ensure uniform experimental conditions for both fed and fasted mice, none of the mice in each group were fed food until 5 hours before the start of the dark condition, after which all mice were fed food, and the FED group was maintained on this condition until the end of the experiment. The fasted mice were fed food for 2 hours and then food was withheld for 48 hours, and the mice in each group were provided with drinking water ad libitum. The body weights of the mice were measured before the start of the fasting period and at 24 and 48 hours after the start of the fasting period. The specific drug administration schedule and classification of the experimental groups are shown in Fig. 2.

[0158] <2-2> Evaluation of evil power

[0159] Grip strength was evaluated using a grip strength measuring device (Grip test package GS3 (25N), Harvard Apparatus, Holliston, MA, USA) in the same manner as in Experimental Example 1-8 above, and was performed before and after fasting.

[0160] <2-3> Blood ketone measurement and body composition evaluation

[0161] After 48 hours of fasting, blood glucose and ketone levels, including β-hydroxybutyrate, were measured using a blood glucose and ketone meter (FreeStyle Optium, Abbott Laboratories, Australia).

[0162] Body composition was assessed using Dual-energy X-ray Absorptiometry (DXA; InAlyzer, Korea) after a 48-h fast. After ketamine and xylazine anesthesia, each mouse was placed on a scanner bed with its tail and limbs extended away from the body. Lean body mass and fat mass were then quantified using the manufacturer's software.

[0163]

[0164] <2-4> Plasma creatine kinase activity analysis

[0165] Plasma creatine kinase activity was analyzed using a colorimetric assay kit (ECPK-100, BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions.

[0166] <2-5> Histological analysis

[0167] At the end of the experiment, gastrocnemius muscle tissue was obtained from mice humanely sacrificed, fixed in 10% formalin, and embedded in paraffin. The tissue was then sectioned at 4 μm and stained with hematoxylin and eosin (H&E). The stained tissue was then examined under a light microscope (Nikon ECLIPSE Ci, Konan, Tokyo, Japan), and the average percentage of muscle fiber area was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

[0168] <2-6> Western blot analysis

[0169] Proteins were extracted from the gastrocnemius muscle tissue, and the expression of Myostatin, p-ERK1 / 2, extracellular signal-mediated kinase (ERK1 / 2), FOXO3a, MuRF-1, MAFbx / Atrogin-1, p-AMPKα, AMPKα, p-mTOR, mTOR, LC3B, Bax, Bcl-2, CuZnSOD, Nrf2, and PCNA and α-tubulin, which are proteins related to muscle atrophy, were analyzed by Western blot, in the same manner as in Experimental Example 1-11.

[0170] <2-7> Statistical analysis

[0171] As in Experimental Examples 1-13 above, all numerical data were expressed as mean ± standard deviation (SD). To evaluate the significance of differences between experimental groups, one-way analysis of variance (ANOVA) was performed, followed by Duncan's multiple range test using SPSS software (version 28 for Windows, SPSS Inc., IL, USA). Statistical significance was set at p<0.05.

[0172] <Example 1> Confirmation of improvement in dexamethasone-induced muscle atrophy by probiotic metabolites.

[0173] <1-1> Confirmation of MGO-AGEs crushing ability

[0174] Methylglyoxal-derived advanced glycation end products (MGO-AGEs) are compounds produced through metabolic reactions with various biomolecules, and have recently been reported to have negative effects on skeletal muscle, reducing exercise capacity and promoting muscle atrophy. Therefore, it was confirmed whether the compound of chemical formula 1 of the present invention decomposes MGO-AGEs. As a result, as shown in Fig. 3, compared to the CON group, the DEX group did not exhibit MGO-AGE decomposition ability, but the group treated with the compound of chemical formula 1 showed excellent MGO-AGE decomposition ability in a concentration-dependent manner.

[0175] <1-2> Confirmation of the effect on myoblast toxicity and proliferation

[0176] In order to confirm whether the compound of chemical formula 1 of the present invention has a protective effect on myoblasts, cell viability and proliferation ability were evaluated. As a result, as shown in Fig. 4A, it was confirmed that the compound of chemical formula 1 of the present invention had no cytotoxicity, and it was confirmed that cell viability was significantly reduced in the DEX group. In addition, when the compound of chemical formula 1 and DEX were treated together, cell death induced by DEX was inhibited, confirming that the compound of chemical formula 1 of the present invention has a protective effect on myoblasts (Fig. 4B). In addition, when treated with the compound of chemical formula 1, cell proliferation significantly increased at a concentration of 20 μM (Fig. 4C), and while cell proliferation decreased in the group treated with DEX alone, cell proliferation did not decrease in the group treated together with the compound of chemical formula 1 (Fig. 4D), confirming that the probiotic metabolite of the present invention protects myoblasts and promotes proliferation.

[0177] <1-3> Confirmation of the effect of probiotic metabolites on root canal formation and size

[0178] It was confirmed whether the compound of chemical formula 1 of the present invention affects myotube formation. As a result, as shown in Fig. 5, it was confirmed that when exposed to DEX after myotube differentiation, the diameter of the myotube decreased, inducing myotube atrophy. In the group pretreated with the compound of chemical formula 1 and curcumin, a positive control, the diameter of the myotube increased, confirming that it protected against inhibition of myotube formation caused by exposure to DEX.

[0179] <1-4> Confirmation of muscle breakdown protein expression

[0180] It was confirmed whether the compound of chemical formula 1 of the present invention regulates the expression of muscle degradation proteins. The expression levels of Atrogin-1 and MuRF1, muscle-specific ubiquitin ligases that play an important role in protein degradation during muscle wasting, were quantified, and as a result, as shown in Fig. 6, in the group treated with DEX, the expression of MuRF-1 and Atrogin-1 significantly increased compared to the CON group, but in the group treated with the compound of chemical formula 1, the expression of MuRF-1 and Atrogin-1, which had increased in a concentration-dependent manner, was confirmed to decrease.

[0181] <1-5> Confirmation of muscle production and growth protein expression

[0182] It was confirmed that the compound of chemical formula 1 of the present invention increases the expression of proteins related to muscle production and growth. Proteins of the MyoD and Myogenin pathways are known as proteins for muscle differentiation, and MyH is known as a marker of muscle differentiation. As a result, as shown in Fig. 7, the expression of MyoD was decreased in the group treated with DEX compared to the CON group, but it was confirmed that the decreased expression of MyoD was significantly increased when treated with the compound of chemical formula 1. In addition, Myogenin showed a tendency to increase with treatment with chemical formula 1, and it was confirmed that the expression level of MyH was significantly increased with treatment with chemical formula 1.

[0183] In addition, in the muscle protein synthesis pathway, the protein expression of the AKT-mTOR pathway, known to promote protein synthesis and inhibit protein degradation, was confirmed. As a result, the phosphorylation of mTOR and AKT was decreased in the DEX group compared to the CON group, but the phosphorylation of mTOR and AKT was significantly increased in the group treated with the compound of formula 1. Therefore, it was confirmed that the probiotic metabolite of the present invention increases muscle protein synthesis and inhibits muscle atrophy induced by DEX by activating the expression of MyoD and HyH and the mTOR-AKT pathway.

[0184] <1-6> Confirmation of increased muscle mass

[0185] It was confirmed whether the probiotic metabolite of chemical formula 1 increases muscle mass in an animal model. As a result, as shown in FIGS. 8A and 8B, in mice administered DEX, the body weight was significantly reduced compared to the CON group, and it was confirmed that the body weight loss was affected by muscle mass. However, in the group administered the compound of chemical formula 1, there was no significant difference in body weight compared to the CON group despite DEX treatment, and there was no significant difference in the food amount of the mice in each group (FIG. 8C). In addition, the compound of chemical formula 1 increased the muscle mass of the quadriceps femoris, gastrocnemius, and plantaris muscle in DEX-induced muscular dystrophy, and no significant difference was confirmed in the extensor digitorum longus and soleus muscles (FIG. 9), confirming that the compound of chemical formula 1 of the present invention has various effects on muscle protection depending on the type of muscle. Therefore, it was confirmed that the probiotic metabolite of the present invention increases muscle mass in muscles that exercise a lot, even when a muscle atrophy-inducing substance such as DEX is administered.

[0186] <1-7> Muscle function check

[0187] It was confirmed whether the compound of chemical formula 1 increases muscle function. Specifically, physical activity was confirmed by a treadmill and grip strength test, and as a result, as shown in Fig. 10, in mice administered DEX, muscle function was confirmed to decrease, and running speed and grip strength were confirmed to decrease. However, in mice administered the compound of chemical formula 1 of the present invention, running speed and grip strength were significantly increased, and in particular, it was confirmed that running speed and grip strength were significantly increased on the 14th day after the start of the experiment compared to the 7th day, confirming that the probiotic metabolite of the present invention can increase muscle function even in a state of muscle wasting.

[0188] <1-8> Confirmation of increased calf thickness

[0189] Lower extremity sarcopenia accompanied by a decrease in calf circumference is known to be associated with a higher mortality rate even in patients with sarcopenia. Therefore, in order to determine whether the probiotic metabolite of the present invention protects lower extremity muscles from muscle atrophy, the volume of the calf muscles was measured by micro-CT analysis. As a result, as shown in Fig. 11A, in mice administered DEX, calf muscle atrophy was induced, resulting in a significant decrease in muscle mass. However, in the group administered the compound of chemical formula 1 of the present invention, the decreased muscle mass was increased, and it was confirmed that the cross-sectional area of ​​the calf muscle was increased by 1.61 to 3.4 mm² (Fig. 11B). In addition, for accurate measurement, the distance between the fibula and the tibia was measured, and as a result, in the group administered the compound of chemical formula 1, the distance between the fibula and the tibia was significantly increased compared to the DEX group (Fig. 11C), confirming that the lower extremity muscles were protected.

[0190] <1-9> Confirmation of inhibition of quadriceps muscle protein breakdown

[0191] In the quadriceps femoris, the effect on muscle breakdown proteins was confirmed. As a result, as shown in Figure 12, in mice administered DEX, MuRF-1 and Atrogin-1 were significantly increased in quadriceps femoris tissue. However, in mice administered the compound of Chemical Formula 1, the increased MuRF-1 and Atrogin-1 were significantly reduced.

[0192] In addition, FoxO3a, a major transcriptional regulator of muscle atrophy, was significantly increased in the DEX group compared to the CON group, but it was confirmed that the expression of FoxO3a was significantly reduced in the group treated with the compound of chemical formula 1.

[0193] In addition, as a result of confirming the expression of the glucocorticoid receptor (GR), the expression of GR was significantly increased in the DEX group compared to the CON group, but in the group administered the compound of chemical formula 1, the increased expression of GR was significantly reduced.

[0194] In the results of quantitative analysis of the expression of Atrogin-1 in quadriceps femoris tissue using immunohistochemical staining, it was confirmed that the expression of Atrogin-1 significantly increased in the DEX group, but decreased in the group administered the compound of chemical formula 1, confirming that the probiotic metabolite of the present invention inhibits proteins of the muscle degradation pathway (Fig. 13).

[0195] <1-10> Confirmation of the effect on muscle fibrosis in quadriceps femoris tissue

[0196] In the quadriceps femoris, the effects of probiotic metabolites on fibrosis-related factors were examined, confirming their effects on abnormal tissue repair and collagen accumulation. The Smad7 protein is known to be a key regulator of muscle fibrosis, and Smad7 is also known to increase muscle differentiation. As shown in Figure 14, the DEX group showed a significant decrease in Smad7 expression, but the group treated with the compound of Chemical Formula 1 showed a marked increase in the decreased expression of Smad7.

[0197] In addition, when collagen accumulation was confirmed by Sirius red staining, it was confirmed that the collagen content in the muscle tissue increased in the DEX group, but in the group treated with the compound of chemical formula 1, the collagen content in the muscle tissue was significantly reduced (Fig. 15).

[0198] <1-11> Confirmation of increased myogenic factors and muscle fiber size in quadriceps femoris tissue

[0199] The effects of probiotic metabolites on the development of quadriceps femoris were evaluated. In muscle formation, MyoD and Myogenin regulate differentiation, and MyH is an essential factor in the maturation and contraction of muscle fibers. In the DEX group, MyoD and Myogenin protein expression was significantly reduced compared to the CON group, and when the compound of formula 1 was administered, the reduced MyoD and Myogenin protein expression significantly increased (Fig. 16). In addition, when the expression of MyH was confirmed in tissues by immunohistochemical staining, MyH expression was reduced in the DEX group, but increased by the administration of the compound of formula 1 (Fig. 17).

[0200] The quadriceps femoris tissue was stained with H&E and histologically evaluated. As a result, the size of the muscle fibers was significantly reduced in the DEX group, but in the group treated with the compound of chemical formula 1, the size of the reduced muscle fibers was confirmed to increase in a concentration-dependent manner (Fig. 18).

[0201] <1-12> Confirmation of activation of muscle synthesis pathway in quadriceps femoris tissue

[0202] In the muscle protein synthesis pathway, the IGF1R-AKT-mTOR and SIRT2-AKT pathways are mainly activated, and it was confirmed that the phosphorylation of IGF-1R, AKT, and mTOR decreased in the group treated with DEX compared to the CON group. However, in the group administered the compound of formula 1, it was confirmed that the decreased phosphorylation of IGF1R, AKT, and mTOR increased in a concentration-dependent manner. In addition, the expression of SIRT2 was significantly decreased in the DEX group, but the compound of formula 1 increased the decreased expression of SIRT2 in a concentration-dependent manner (Fig. 19), confirming that the probiotic metabolite of the present invention improves sarcopenia induced by DEX by activating the muscle protein synthesis pathway.

[0203] <1-13> Confirmation of changes in metabolic pathways of metabolites in blood and muscle tissue

[0204] We confirmed the changes in metabolites in blood and QD muscle tissue due to probiotic metabolites. As a result, as shown in Fig. 20, both tissues showed a clear distinction between mice that were not administered DEX (CON group) and mice administered DEX (NC group), and the groups administered probiotic metabolites (ProM_Low, ProM_High) showed a clear distinction from the NC group. Based on the PLS-DA model of Fig. 20, metabolites showing differences between groups were selected and the content patterns between groups were compared. As a result, many substances in the amino acid, fatty acid, and organic acid series were similar in the Con and ProM treatment groups compared to the NC group in both tissues, confirming that the change in metabolite content due to DEX treatment was alleviated (Fig. 21A). Next, metabolic pathway analysis was performed on the selected metabolites. As the substances in the blood pyruvate metabolism, TCA cycle, and muscle galactose, sucrose, and starch metabolism were identified as the major metabolites that were changed, it was assumed that ProM administration may have contributed to the alleviation of DEX-induced inhibition of glycolysis. In addition, as the "glycine, serine, threonine metabolism" and "taurine and hypotaurine metabolism" in muscle were major changes, it is thought that there may be a correlation with the alleviation of DEX-induced muscle proteolysis. It was confirmed that metabolites in the "glycoxylate and dicarboxylate metabolism" were mainly changed in relation to the alleviation of muscle myogenesis inhibition by ProM administration (Fig. 21B). Additionally, among the metabolites that were changed by DEX treatment with a fold change >1.5 or <0.5, those that showed a recovery pattern were suggested as indicator substances for ProM administration (Table 2).

[0205] [Correction pursuant to Rule 91, July 8, 2025]

[0206] <Example 2> Confirmation of improvement in fasting-induced muscular dystrophy or sarcopenia by probiotic metabolites

[0207] <2-1> Confirmation of weight, body composition, and organ weight control effects

[0208] In fasting-induced muscular dystrophy or sarcopenia, the improving effect of probiotic metabolites was confirmed. Specifically, the body weight of mice significantly decreased 24 hours after fasting and further decreased at 48 hours. In the group treated with the compound of formula 1 before fasting, there was no significant effect on body weight loss. Body composition analysis results also showed that treatment with the compound of formula 1 did not significantly affect the fasting-induced decrease in lean body mass and body fat mass. Although gastrocnemius muscle weight and total muscle weight significantly decreased after fasting, the weights of the quadriceps femoris, EDL, TA, and soleus muscles were not affected by 48 hours of fasting, and fasting decreased the weights of all types of adipose tissue. However, pretreatment with the compound of formula 1 did not alleviate the loss of muscle and adipose tissue. Brain weight was not affected by the fasting period, and liver weight was significantly reduced in fasted mice, but pretreatment with the compound of formula 1 did not increase the decrease in liver weight. There was no significant difference in the amount of food consumed in each group of mice (Table 3).

[0209] [Correction pursuant to Rule 91, July 8, 2025]

[0210] <2-2> Confirmation of inhibition of fasting-induced muscle strength loss

[0211] It was confirmed that the probiotic metabolite of the chemical formula 1 of the present invention protects against fasting-induced muscle strength loss. As a result, as shown in Figure 22, grip strength was significantly reduced in fasting-induced muscular dystrophy mice, but pretreatment with the compound of chemical formula 1 significantly increased the reduced grip strength, confirming that the probiotic metabolite increases muscle strength.

[0212] <2-3> Confirm muscle fiber protection

[0213] We confirmed whether the probiotic metabolite of the present invention protects against fasting-induced muscle fiber loss. As a result, as shown in Figure 23, in mice subjected to 48-hour fasting to induce muscle atrophy, the area of ​​muscle fibers decreased. However, in the group pretreated with the compound of Chemical Formula 1, the decreased area of ​​muscle fibers significantly increased, confirming that the probiotic metabolite suppresses muscle loss.

[0214] <2-4> Checking muscle damage indicators in blood

[0215] We confirmed whether the probiotic metabolites of the present invention modulate plasma creatine kinase activity, an indicator of muscle damage. As a result, as shown in Figure 24, plasma creatine kinase activity significantly increased in fasting-induced mice, but in the group treated with the compound of Chemical Formula 1, plasma creatine kinase activity decreased in a concentration-dependent manner, confirming a decrease in plasma metabolites associated with muscle loss.

[0216] In addition, as a result of checking the concentration of β-hydroxybutanoic acid, a blood ketone, when pretreated with the compound of chemical formula 1 before fasting, there was no significant change in the concentration of β-hydroxybutanoic acid compared to the control group, but in fasted mice, the concentration of β-hydroxybutanoic acid in the blood was confirmed to have significantly increased (Fig. 25), confirming that the probiotic metabolite stabilizes the blood metabolite related to muscle loss.

[0217] <2-5> Correlation Analysis between Fasting and Body Composition

[0218] The above results showed a significant positive correlation between muscle strength and body weight and total adipose tissue mass, regardless of drug treatment. A significant positive correlation was also observed between total muscle mass and total fat mass. Furthermore, there was no correlation between blood β-hydroxybutanoic acid levels and muscle strength (Figure 26).

[0219] <2-6> Confirmation of myostatin signaling and muscle protein degradation factor expression

[0220] In the group administered the compound of chemical formula 1, the expression of myostatin protein was significantly reduced, and the expression of p-ERK1 / 2, a phosphorylated extracellular signal-mediated kinase, and the ratio of p-ERK1 / 2 and ERK1 / 2 were significantly reduced by treatment with the compound of chemical formula 1, confirming that the probiotic metabolite reduces the expression of muscle protein degradation factors (Fig. 27).

[0221] In addition, fasting significantly increased the expression of E3 ubiquitin ligases, MuRF-1, and MAFbx / Atrogin-1, but in the group administered the compound of chemical formula 1, the expression of MuRF-1 decreased similarly to the CON group, and the expression of Atrogin-1 significantly decreased, confirming that probiotic-derived metabolites reduce the expression of proteins in the pathway related to muscle degradation (Fig. 28).

[0222] In addition, in mice induced with fasting, p-mTOR expression and the ratio of p-mTOR and mTOR were significantly reduced, but in the group administered the compound of formula 1, the reduced p-mTOR expression and the ratio of p-mTOR / mTOR were significantly increased, and 1A / 1B-light chain 3 (LC3) protein expression and the ratio of LC3B-II / LC3B-I were increased in fasted mice, but in the group administered the compound of formula 1, LC3 protein expression and the ratio of LC3B-II / LC3B-I were significantly reduced, confirming that postbiotic metabolites inhibit muscle protein degradation (Fig. 29).

[0223] There was no significant difference between the groups in Bcl2 and BAX proteins related to apoptosis (Fig. 30), and there was no significant difference between the groups in the expression of factors related to inflammation and oxidative stress (Fig. 31).

[0224] Therefore, it was confirmed that the probiotic metabolite of the present invention protects muscle cells and promotes muscle fiber differentiation. In addition, it was confirmed that in an animal model of dexamethasone-induced muscle atrophy or sarcopenia, it increases muscle mass and muscle function, suppresses the expression of factors related to muscle breakdown, and increases the expression of factors related to muscle synthesis and regeneration. In addition, it was confirmed that the probiotic metabolite contributes to the alleviation of muscle atrophy by regulating the metabolism of sugars, amino acids, and fatty acids changed by dexamethasone. In addition, in an animal model of fasting-induced muscle atrophy or sarcopenia, it was confirmed that it increases muscle function, protects muscle tissue damage, suppresses the expression of factors related to muscle breakdown, and increases the expression of factors related to muscle synthesis and regeneration.

Claims

1. A pharmaceutical composition for the prevention or treatment of muscle disease, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 2. In paragraph 1, A composition wherein the compound increases muscle mass.

3. In paragraph 2, A composition that increases the muscle mass by increasing the diameter of the muscle canal or muscle thickness.

4. In paragraph 1, A composition wherein the compound increases muscle function.

5. In paragraph 1, A composition wherein the compound inhibits the expression of muscle decomposition factors.

6. In paragraph 5, A composition wherein the muscle degradation factor is a protein selected from the group consisting of muscle atrophy F-box (MAFbx / Atrogin-1), muscle-specific RING finger protein 1 (Muscle-specific RING finger protein 1, MuRF1), forkhead box (Forkhead box O3, FoxO3a), and glucocorticoid receptor (GR).

7. In paragraph 1, A composition wherein the compound increases the expression of a muscle growth factor.

8. In paragraph 7, A composition wherein the muscle growth factor is myoblast determination protein 1 (MyoD), myogenin or myosin heavy chain (MyH).

9. In paragraph 1, A composition wherein the compound increases phosphorylation or expression of a muscle synthesis pathway protein.

10. In paragraph 9, A composition wherein the muscle synthesis pathway protein is a protein selected from the group consisting of mammalian target of rapamycin (mTOR), AKT Serine / Threonine Kinase 1 (AKT), insulin like growth factor 1 receptor (IGF-1R), and NAD-dependent deacetylase sirtuin 2 (SIRT2).

11. In paragraph 1, A composition wherein the compound inhibits phosphorylation of the muscle degradation pathway.

12. In paragraph 11, A composition wherein the above muscle decomposition pathway is an extracellular signal-mediated kinase (ERK1 / 2).

13. In paragraph 1, A composition wherein the compound reduces the LC3(1A / 1B-light chain 3)B-II / LC3B-I ratio of the muscle degradation pathway.

14. In paragraph 1, A composition wherein the compound inhibits fibrosis of muscle tissue.

15. In paragraph 14, A composition that inhibits the above fibrosis by inhibiting Mothers against decapentaplegic homolog 7 (Smad7) in muscle tissue.

16. In paragraph 14, A composition that inhibits the above fibrosis by inhibiting collagen accumulation in muscle tissue.

17. In paragraph 1, A composition wherein the compound reduces muscle damage indicators in the blood.

18. In paragraph 17, A composition wherein the above muscle damage indicator is creatine kinase or β-hydroxybutyrate.

19. In paragraph 1, A composition wherein the compound regulates the expression of metabolites related to muscle synthesis.

20. In paragraph 19, A composition for regulating the expression of the above muscle synthesis-related metabolites by increasing the amount of a metabolite selected from the group consisting of glycine, tyrosine, sucrose, pyruvate, thymine, ethylene glycol, and 1,2-propanediol in serum.

21. In paragraph 19, A composition that regulates the expression of the above muscle synthesis-related metabolites by increasing the amount of 12-S-HETE or LPE 18:3 in the muscle.

22. In paragraph 19, A composition that regulates the expression of the above muscle synthesis-related metabolites by reducing the amounts of lactose, mannitol, sorbitol, 2-hydroxybutyric acid, fumaric acid and uric acid in the serum.

23. In paragraph 19, A composition that regulates the expression of the above muscle synthesis related metabolites by reducing the amount of glycine or mannitol in the muscle.

24. In paragraph 1, A composition, wherein the muscle disease is a disease selected from the group consisting of muscular atrophy, myopathy, muscular dystrophy, myasthenia, muscular injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia.

25. A food composition for preventing or improving muscle disease, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof as an active ingredient: [Chemical Formula 1] 26. A pharmaceutical composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 27. A food composition for promoting muscle differentiation, muscle regeneration or muscle strengthening, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof as an active ingredient: [Chemical Formula 1] 28. A pharmaceutical composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 29. A food composition for increasing muscle mass or promoting muscle production, comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof as an active ingredient: [Chemical Formula 1] 30. A pharmaceutical composition for improving muscle function comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] 31. A food composition for improving muscle function comprising a compound represented by the following chemical formula 1 or a food-based acceptable salt thereof as an active ingredient: [Chemical Formula 1] 32. A method for treating muscle disease, comprising the step of administering to a subject a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1]

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