WO2025244466A1 - Composition for preventing or treating muscle diseases comprising probiotic metabolite as active ingredient - Google Patents

Abstract

It was confirmed that probiotic metabolites of the present invention protect muscle cells and promote differentiation of muscle fibers. In addition, it was confirmed in an animal model of muscle atrophy or sarcopenia induced by dexamethasone that probiotic metabolites increased muscle mass and muscle function, inhibited expression of factors associated with muscle degradation, and increased expression of factors associated with muscle synthesis and regeneration. It was confirmed that probiotic metabolites contribute to alleviation of muscle atrophy by regulating sugar, amino acid, and fatty acid metabolism altered by dexamethasone.

Description

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

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

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 bacterial balance and, consequently, to maintaining health. Many species of lactic acid bacteria (LAB), such as Lactobacillus and Bifidobacterium , are commonly considered probiotics, although 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.

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.

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.

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.

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.

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.

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.

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.

[Chemical Formula 1]

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.

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.

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.

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.

Another object of the present invention is to provide 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.

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.

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-wise acceptable salt thereof as an active ingredient.

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.

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.

[Chemical Formula 1]

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.

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.

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.

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.

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.

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.

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-wise acceptable salt thereof as an active ingredient.

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.

The probiotic metabolite of the present invention was confirmed to protect muscle cells and promote muscle fiber differentiation. Furthermore, in an animal model of dexamethasone-induced muscle atrophy or sarcopenia, it was 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. The probiotic metabolite contributes to the alleviation of muscle atrophy by modulating dexamethasone-induced sugar, amino acid, and fatty acid metabolism, and thus can be usefully utilized in related industries.

Figure 1 is a diagram illustrating the method for producing a sarcopenia animal model of the present invention, the drug administration schedule, and the experimental group.

Figure 2 is a diagram confirming the MGO-AGEs crushing ability of the probiotic metabolite of the present invention.

Figure 3 is a diagram confirming the cytotoxicity and cytoprotective effects of the probiotic metabolite of the present invention (A: cytotoxicity confirmed, B: muscle cell protection confirmed).

Figure 4 is a diagram confirming the increase in myotube formation of the probiotic metabolite of the present invention and the positive control group using Jenner-Giemsa staining.

Figure 5 is a diagram confirming the protective effect of probiotic metabolites on myotube formation in dexamethasone-induced myotube formation inhibition.

Figure 6 is a Western blot analysis of the expression of muscle synthesis factors in a cell model in which dexamethasone-induced muscle atrophy was induced.

A: Western blot results

B: Quantification of MyoD expression

C: Quantification of Myogenin expression

D: Quantification of MyHC expression

Figure 7 is a diagram quantifying the weight loss following administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia (A: confirmation of weight change, B: quantification of weight change, C: quantification of food intake).

Figure 8 is a diagram quantifying muscle weight according to administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia.

Figure 9 is a diagram showing an increase in quadriceps femoris tissue density confirmed by H&E staining following administration of probiotic metabolites in an animal model of dexamethasone-induced muscular atrophy or sarcopenia (A: staining result, B: quantification of staining result).

Figure 10 is a diagram showing the accumulation of collagen in the quadriceps femoris muscle following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease, as confirmed by Sirius red staining.

Figure 11 is a diagram showing an increase in gastrocnemius muscle tissue density confirmed by H&E staining following administration of probiotic metabolites in an animal model of dexamethasone-induced muscular atrophy or sarcopenia (A: staining result, B: quantification of staining result).

Figure 12 is a diagram showing the accumulation of collagen in the gastrocnemius muscle following administration of probiotic metabolites to an animal model of dexamethasone-induced muscle disease, as confirmed by Sirius red staining.

Figure 13 is a diagram showing the quantification of intestinal length according to the administration of probiotic metabolites in an animal model of dexamethasone-induced muscular atrophy or sarcopenia (A: intestinal length quantification, B: intestinal length/body weight quantification).

Figure 14 is a diagram quantifying the results of a treadmill test according to the administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia (A and C: results at week 1, B and D: results at week 2).

Figure 15 shows the quantification of grip strength according to the administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia (A: 1st week results, B: 2nd week results).

Figure 16 is a diagram showing the results of analyzing the calf thickness according to the administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia using Micro-CT (A: micro-CT results, B: calf area quantification, C: fibula and tibial distance quantification).

Figure 17 is a Western blot analysis of the expression of muscle decomposition or muscle synthesis factors in the gastrocnemius muscle tissue following administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia.

Figure 18 is a Western blot analysis of the expression of muscle breakdown or muscle synthesis factors in quadriceps femoris tissue following administration of probiotic metabolites in a dexamethasone-induced muscle atrophy or sarcopenia animal model.

Figure 19 is a Western blot analysis of the expression of muscle decomposition or muscle synthesis factors in the extensor digitorum longus muscle tissue following administration of probiotic metabolites in an animal model of dexamethasone-induced muscle atrophy or sarcopenia.

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.

Figure 21 is a diagram analyzing the pattern of metabolite content and altered 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 dystrophy or sarcopenia (A. Comparison of metabolite content between groups, B. Altered major metabolic pathways).

Figure 22 is a diagram confirming the MGO-AGEs decomposition ability according to the form and amino acid composition of probiotic metabolites.

Figure 23 is a diagram showing the expression of muscle decomposition factors according to the form and amino acid composition of probiotic metabolites using Western blot (A: Western blot results, B: protein expression quantification).

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.

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.

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.

The compound of the above chemical formula 1 of the present invention may be named gamma-glutamylmethionine (GM) and may be a compound with CAS number 17663-87-5.

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.

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.

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.

Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium, and N ⁺ ( _C1-4 alkyl) ₄ 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.

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.

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.

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.

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.

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.

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.

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.

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

According to one embodiment of the present invention, the compound may inhibit the expression of a muscle degradation factor, and the muscle degradation factor may be a glucocorticoid receptor (GR), muscle atrophy F-box (MAFbx/Atrogin-1), or muscle-specific RING finger protein 1 (MuRF1).

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).

According to one embodiment of the present invention, the compound may increase intestine length.

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

According to one embodiment of the present invention, regulating the muscle synthesis-related metabolites may be by increasing the amount of a factor selected from the group consisting of glycine, tyrosine, sucrose, pyruvic acid, glyoxylic acid, 3-deoxytetronic acid, octadecanedioic acid, thymine, 1,2-propanediol, and ethylene glycol in plasma, and by increasing the amount of 12-S-HETE or LPE 18:3 in muscle.

According to one embodiment of the present invention, regulating the muscle synthesis-related metabolites may be by reducing the amount of a factor selected from the group consisting of threonine, lactose, mannitol, sorbitol, fumaric acid, 2-hydroxybutric acid, 15,16-DiHODE, dodecanedioic acid, uric acid, and indol-3-propanoic acid in plasma, and may be by reducing the amount of gluconic acid in muscle.

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.

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.

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.

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.

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.

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.

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 capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, 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 an excipient, a binder, a disintegrant, etc., using a conventionally known method, and, if necessary, can be coated with white sugar or another coating agent, or the surface can be coated with a material such as starch or talc. The granular health functional food can be manufactured into a granular form by mixing a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

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.

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.

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.

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.

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.

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-wise acceptable salt thereof as an active ingredient.

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.

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 above-mentioned matters.

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.

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.

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.

<Preparation Example 1> Preparation of metabolites derived from probiotic strains

The compound of chemical formula 1, which is 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 (γ-Glutamyl-methionine, γ-GM), which is a probiotic metabolite (ProM), was purchased from Peptron through a request for synthesis.

[Chemical Formula 1]

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

<1-1> Analysis of MGO-AGEs fragmentation activity

To evaluate the degradation 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 (10, 100, or 200 μM) at a concentration of 1 mg/mL, homogenized, and reacted for 24 hours. Then, 0.1% TNBSA and 4% NaHCO ₃ 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).

<1-2> Cell culture and drug treatment

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, cells were seeded at a cell density of 2.5 × ¹⁰⁵ in 6-well plates and cultured for 2 days. When the cells reached 90% confluence, the medium was replaced with differentiation DMEM containing 2% horse serum, and the cells were treated with the compound of formula 1 or 1 and 5 μM curcumin (CU), as a positive control, during the differentiation process for 6 days. On the 6th day of differentiation, C2C12 myotubes were treated with 50 μM DEX for 24 h.

<1-3> Cell viability

Cell viability was analyzed using the MTT assay. C2C12 cells were seeded in 96-well plates at a concentration of 1 × 10 ⁴ cells and cultured under 5% CO ₂ and 37°C conditions. When the cells were more than 80% confluent, the cells were treated with the compound of formula 1 at a concentration of 0.01, 0.1, 1, 5, 10, or 20 μM 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).

<1-4> Jenner-Giemsa staining and root canal confirmation

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 observed using an optical microscope (Olympus, Tokyo, Japan).

<1-5> Preparation of animal models

Eight-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).

The habituated mice were divided into the following groups: a vehicle group (CON group) administered with a solvent; a DEX group (NC group) administered with dexamethasone at a concentration of 20 mg/kg; a positive control group (OXY, PC group) treated with 50 mg/kg of oxymetholone and 20 mg/kg of dexamethasone; a group treated with 5 mg/kg of the compound of formula 1 and 20 mg/kg of dexamethasone (ProM 5); and a group treated with 20 mg/kg of the compound of formula 1 and 20 mg/kg of 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.

<1-6> Measurement of body weight, food intake, and muscle mass

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 gastrocnemius (GCM), quadriceps femoris (QD), extensor digitorum longus (EDL), plantaris (PLA), and soleus (SOL) muscles were isolated and weighed. Muscle tissues were then frozen in liquid nitrogen and stored and fixed at -80°C.

<1-7> Treadmill and grip strength test

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.

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).

<1-8> micro CT

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 of the midline of the fibula bone from the position corresponding to the shank.

MicroCT scan conditions Resolution 2 K Source Voltage 40 kV Source Current 200 μA Image Pixels 18 μm Exposure Times 650 ms Rotation Step 2 ° Rotation in Degree 180 ° Average Frames 2 Filter 1 mm Al Scan Duration 3 m : 28 s

<1-9> Muscle histological analysis

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).

<1-10> Western blot analysis

QD tissue (30 mg) or C2C12 myotubes were homogenized in RIPA buffer containing a protease/phosphatase inhibitor cocktail. The homogenate was centrifuged at 12,000 rpm for 1 h at 4°C, and protein (30 μg) was separated by SDS-PAGE to measure expression. The separated proteins were then transferred to nitrocellulose membranes and incubated with primary antibodies against muscle atrophy F-box (MAFbx/Atrogin-1), Muscle-specific RING finger protein 1 (MuRF1), Myoblast Determination protein 1 (MyoD), Myogenin, Myosin heavy chain (MyH), and GAPDH, which are proteins associated with muscle atrophy, at 4°C for 18 h. The membranes were then washed with TBST, and HRP-conjugated secondary antibodies were added and incubated at room temperature for 1 h. After the reaction was completed, the membrane was visualized and analyzed using a ChemiDoc XRS+ imaging system (Bio-Rad, USA).

<1-11> Analysis of metabolite changes in blood and muscle tissue

Metabolomic analysis was performed on serum and GCM 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 GCM 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.

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.

<1-12> Statistical analysis

All collected activity-related 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), and different 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/) .

<Example 1> Confirmation of improvement in dexamethasone-induced muscle atrophy by probiotic metabolites (in vitro)

<1-1> Confirmation of MGO-AGE crushing ability

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. Accordingly, the crushing ability of MGO-AGE of chemical formula 1 of the present invention was confirmed. As a result, as shown in Fig. 2, it was confirmed that when the compound of chemical formula 1 was treated, the degree of MGO-AGE crushing increased in a concentration-dependent manner, and thus the ratio of free amines increased.

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

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. 3A, 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 was treated on myoblasts induced by DEX, DEX-induced cell death was inhibited, confirming that the compound of chemical formula 1 of the present invention has a protective effect on myoblasts (Fig. 3B).

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

It was confirmed whether the compound of chemical formula 1 of the present invention affects myotube formation. As a result, as shown in Fig. 4, it was confirmed that myotube formation was promoted in the curcumin (Cu group), which is the positive control group, in the differentiation medium. In addition, it was confirmed that myotube formation increased in a concentration-dependent manner in the group treated with the compound of chemical formula 1 of the present invention (Fig. 4).

In addition, it was confirmed that when exposed to DEX after root canal differentiation, the formation of root canals decreased, inducing muscle atrophy. In the group pretreated with curcumin, which is a positive control, it was confirmed that the formation of root canals increased (Fig. 5). In the group treated with the compound of chemical formula 1 of the present invention, it was confirmed that muscle atrophy caused by DEX was suppressed in a concentration-dependent manner and muscle canal formation increased (Fig. 5), confirming that the compound of chemical formula 1 of the present invention protects against the inhibition of root canal formation caused by DEX exposure.

<1-4> Confirmation of expression of muscle breakdown proteins and muscle building proteins

Proteins of the MyoD and Myogenin pathways are known as proteins for muscle differentiation, and MyH is known as a marker of muscle differentiation. Therefore, the expression of muscle differentiation proteins according to treatment with the compound of chemical formula 1 of the present invention was quantified. As a result, as shown in Fig. 6, the expression of MyoD, Myogenin, and MyH related to muscle synthesis and differentiation was confirmed. Compared to the group treated with DEX, when the compound of chemical formula 1 was treated, the expression of MyoD and Myogenin was confirmed to increase in a concentration-dependent manner. In addition, it was confirmed that MyH tended to increase with treatment with chemical formula 1.

<Example 2> Confirmation of improvement in dexamethasone-induced muscle atrophy by probiotic metabolites (in vivo)

<2-1> Increased muscle mass and muscle tissue confirmation

We confirmed whether the probiotic metabolite of Chemical Formula 1 increases muscle mass in an animal model. As a result, as shown in Figures 7A and 7B, in mice administered DEX (NC group), body weight was significantly reduced compared to the CON group, and it was confirmed that the 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 even after DEX treatment, and there was no significant difference in the food intake of the mice in each group (Figure 7C). In addition, the compound of chemical formula 1 increased the muscle mass of the gastrocnemius (GCM), quadriceps femoris (QD), extensor digitorum longus (EDL), and plantaris (PLA) in DEX-induced muscular dystrophy, while no significant difference was observed in the soleus (SOL) (Fig. 8), 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 the muscle mass of muscles with high exercise volume even when a muscular dystrophy-inducing substance such as DEX is administered.

In addition, in order to confirm the protection of muscle tissue, the quadriceps femoris (QD) and gastrocnemius (GCM) tissues were stained with H&E staining, and as a result, in the NC group treated with DEX, the density of muscle fibers in the tissues decreased compared to the CON group, but in the group treated with the compound of chemical formula 1 of the present invention, the density of the decreased muscle fibers increased in a concentration-dependent manner (Figs. 9 to 11). In addition, as a result of confirming the degree of collagen accumulation in the quadriceps femoris (QD) and gastrocnemius (GCM) tissues using Sirius red staining, in the NC group treated with DEX, the degree of collagen accumulation increased compared to the CON group, whereas in the group treated with the compound of chemical formula 1 of the present invention, the degree of collagen accumulation decreased in a concentration-dependent manner similar to the PC group (Fig. 12).

In addition, intestinal tissues were obtained from each group of sacrificed mice, and intestinal length was measured to confirm whether intestinal muscle damage was induced by DEX. As a result, in the NC group administered with DEX, the intestinal length was reduced compared to the CON group, but in the group treated with the compound of chemical formula 1 of the present invention, the reduced intestinal length was confirmed to have significantly increased (Fig. 13).

<2-2> Muscle function check

It was confirmed whether the compound of chemical formula 1 increases muscle function. Specifically, physical activity was confirmed using a treadmill and grip strength test, and as a result, as shown in FIGS. 14 and 15, in mice administered DEX, muscle function was decreased, and it was confirmed that the running time, speed, and grip strength decreased in the second week of the experiment. However, in mice administered the compound of chemical formula 1 of the present invention, it was confirmed that the running time, speed, and grip strength significantly increased, confirming that the probiotic metabolite of the present invention can increase muscle function even in a state of muscle wasting.

<2-3> Confirmation of increased calf thickness

Sarcopenia in the lower extremities, 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 using micro-CT analysis. As a result, as shown in Figure 16A, 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 muscles was increased in a concentration-dependent manner (Figure 16B). In addition, for accurate measurement, the distance between the fibula and the tibia was measured, and as a result, it was confirmed that the group administered the compound of Chemical Formula 1 increased the distance between the fibula and the tibia in a concentration-dependent manner compared to the DEX group (Figure 16C), protecting the lower extremity muscles.

<2-4> Confirmation of regulation of muscle breakdown protein and myogenic factor expression

We investigated the effects of probiotic metabolites on the regulation of muscle breakdown protein and myogenic factor expression. In muscle formation, MyoD and Myogenin regulate differentiation, while MyH is an essential factor for muscle fiber maturation and contraction.

In the gastrocnemius (GCM), the expression of muscle degradation proteins and myogenic factors was confirmed by Western blot. The expression of muscle degradation proteins, Atrogin-1 and MuRF-1, was significantly increased in the NC group administered DEX compared to the CON group. However, when the compound of chemical formula 1 of the present invention was administered, the increased expression of Atrogin-1 and MuRF-1 was confirmed to decrease. In addition, the expression of muscle synthesis factors, MyoD and Myogenin, was decreased by the administration of DEX. However, when the compound of chemical formula 1 was treated, the decreased expression of MyoD and Myogenin was confirmed to increase significantly (Fig. 17).

In addition, in the quadriceps femoris (QD), the expression of muscle degradation proteins Atrogin-1 and MuRF-1 was significantly increased in the NC group administered DEX compared to the CON group, but it was confirmed that the increased expression of Atrogin-1 and MuRF-1 was significantly reduced when the compound of chemical formula 1 of the present invention was administered. The expression of muscle synthesis factors MyoD and Myogenin was decreased by the administration of DEX, but a tendency to increase was confirmed when the compound of chemical formula 1 was treated (Fig. 18).

In the extensor digitorum longus (EDL) muscle, the expression of muscle degradation proteins, Atrogin-1 and MuRF-1, was significantly increased in the NC group administered DEX compared to the CON group, but it was confirmed that the increased expression of Atrogin-1 and MuRF-1 was significantly reduced when the compound of chemical formula 1 of the present invention was administered. In addition, the expression of muscle synthesis factors, MyoD and Myogenin, was decreased by the administration of DEX, but it was confirmed that the decreased expression of MyoD and Myogenin was increased when the compound of chemical formula 1 was treated (Fig. 19).

<2-5> Confirmation of changes in metabolic pathways of metabolites in blood and muscle tissue

We confirmed the changes in metabolites in blood and GCM muscle tissue by 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 and confirmed that there were metabolic differences. Based on the PLS-DA model of Fig. 20, metabolites showing differences between groups were selected and the content patterns between groups were compared. In both tissues, many substances in the amino acid, fatty acid, and organic acid series showed similar increase/decrease patterns in the Con and ProM treatment groups compared to the NC group, confirming that the metabolic changes induced by DEX treatment were alleviated. 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 inhibition of glycolysis and the alleviation of glucose metabolism disorders caused by DEX. In addition, since the "glycine, serine, threonine metabolism" and "taurine and hypotaurine metabolism" in the muscle were major changes, it is thought that there may be a correlation with the alleviation of muscle proteolysis caused by DEX. It was confirmed that the metabolites in the "glycoxylate and dicarboxylate metabolism" were mainly changed in relation to the alleviation of muscle myogenesis inhibition caused by ProM administration (Fig. 21). 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 caused by ProM administration (Table 2).

<Example 3> Comparison of effects according to the type of probiotic metabolite

<3-1> Confirmation of MGO-AGE crushing ability

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. Accordingly, in the present invention, the MGO-AGEs decomposition ability was confirmed according to the structure and amino acid composition of the probiotic metabolite. As a result, as described in Fig. 22, among the GM (Glutamyl-methionine) of the present invention, it was confirmed that γ-GM had a significantly increased free amine ratio compared to alpha or beta GM. In addition, as a result of comparing the effects in dipeptides composed of other amino acids after γ-Glutamyl, it was confirmed that among the metabolites treated at 200 μM (high concentration), the ratio of free amines was significantly increased in γ-GM, confirming that among the probiotic metabolites, γ-GM has an excellent ability to break down MGO-AGEs.

<3-2> Confirmation of regulation of proteins related to muscle loss in DEX-treated myotubes

We examined whether DEX and probiotic metabolites regulate the expression of muscle-degrading enzymes during the myotube differentiation process for 7 days. As a result, as shown in Fig. 23, DEX treatment was found to increase the activity of the glucocorticoid receptor (GR) and to increase the expression of Atrogin-1 and MuRF1, a type of E3 ligase that is a biomarker of muscle atrophy. However, in the group treated with probiotic metabolites, similar to the positive control group, curcumin, it was confirmed to suppress GR activity and regulate the expression of Atrogin-1 and MuRF.

Therefore, the probiotic metabolite of the present invention was confirmed to protect muscle cells and promote muscle fiber differentiation. Furthermore, in an animal model of dexamethasone-induced muscle atrophy or sarcopenia, it was confirmed to increase muscle mass and muscle function, suppress the expression of factors related to muscle breakdown, and increase the expression of factors related to muscle synthesis and regeneration. It was confirmed that the probiotic metabolite contributed to the alleviation of muscle atrophy by regulating the metabolism of sugars, amino acids, and fatty acids altered by dexamethasone.

Claims (23)

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: [Chemical Formula 1] In paragraph 1, A composition wherein the compound increases muscle mass. In the second paragraph, A composition that increases the muscle mass by increasing the diameter of the muscle canal or muscle thickness. In paragraph 1, A composition wherein the compound increases muscle function. In paragraph 1, A composition wherein the compound inhibits the expression of muscle decomposition factors. In paragraph 5, A composition wherein the muscle decomposition factor is a glucocorticoid receptor (GR) muscle atrophy F-box (MAFbx/Atrogin-1) or a muscle-specific RING finger protein (Muscle-specific RING finger protein 1, MuRF1). In paragraph 1, A composition wherein the compound increases the expression of a muscle growth factor. In paragraph 7, A composition wherein the muscle growth factor is myoblast determination protein 1 (MyoD), myogenin or myosin heavy chain (MyH). In paragraph 1, A composition wherein the compound increases intestine length. In paragraph 1, A composition wherein the compound regulates metabolites related to muscle synthesis. In paragraph 10, A composition that regulates the above muscle synthesis-related metabolites by increasing the amount of a factor selected from the group consisting of glycine, tyrosine, sucrose, pyruvic acid, glyoxylic acid, 3-deoxytetronic acid, octadecanedioic acid, thymine, 1,2-propanediol and ethylene glycol in plasma. In paragraph 10, A composition that regulates the above muscle synthesis related metabolites by increasing the amount of 12-S-HETE or LPE 18:3 in the muscle. In paragraph 10, A composition for regulating the above muscle synthesis-related metabolites, wherein the amount of a factor selected from the group consisting of threonine, lactose, mannitol, sorbitol, fumaric acid, 2-hydroxybutric acid, 15,16-DiHODE, dodecanedioic acid, uric acid, and indol-3-propanoic acid in plasma is reduced. In paragraph 10, A composition that regulates the above muscle synthesis-related metabolites by reducing the amount of gluconic acid in the muscles. 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. 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] 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] 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] 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] 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] 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] A food composition for improving muscle function, comprising a compound represented by the following chemical formula 1 or a food-related acceptable salt thereof as an active ingredient: [Chemical Formula 1] 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]