Composition for preventing or treating muscle diseases containing inotodiol

Abstract

The present invention relates to a composition for preventing, improving, or treating muscle diseases, which contains an inotodiol compound, and the composition increases muscle mass through the effects of enhancing the self-renewal of muscle stem cells and promoting the differentiation of myoblasts, promotes the regeneration of muscle fibers, and enhances muscle strength by inducing mitochondrial functional activity, thereby having preventive or therapeutic effects against various muscle diseases, and includes an effect of increasing athletic ability by strengthening muscle strength.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing, ameliorating or treating a muscular disease, comprising inotodiol. [Background technology]

[0002] Muscles play an important role in bodily functions such as energy metabolism and athletic performance, and can be damaged or weakened by a variety of factors, including sarcopenia due to aging, muscle atrophy due to nutritional imbalance or lack of exercise, other diseases such as other cancers, and aging.

[0003] Sarcopenia, a major muscle-damaging disease, is a disease in which muscle strength decreases as skeletal muscle mass decreases with aging. The most notable feature of sarcopenia is the loss of muscle mass, and the type of muscle fiber may change. While type 1 and type 2 muscle fibers decrease in a similar proportion with aging, the thickness of type 1 muscle fibers decreases more significantly in sarcopenic patients. Such sarcopenia has been reported to cause muscle weakness and functional impairment in the elderly (Roubenoff R., Can. J. Appl. Physiol. 26, 78-89, 2001).

[0004] Muscle atrophy is induced by malnutrition or long-term disuse of muscles, and occurs when the balance between normal protein synthesis and breakdown is disrupted, causing proteins in the muscles to break down.

[0005] Various treatment methods are being developed to fundamentally treat such muscle diseases, and in particular, treatment methods using mechanisms that promote muscle regeneration or promote differentiation of muscle cells from stem cells to strengthen muscles have been proposed. These methods can fundamentally treat muscle diseases, so research into various therapeutic substances that can make this possible is currently necessary.

[0006] The present inventors have conducted research into various compounds that have the effect of improving muscle diseases through the above-mentioned effects, and have thus completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors have discovered that inotodiol promotes the differentiation of myoblasts, has the effects of muscle regeneration and increasing muscle mass, improves muscle energy metabolism and strengthens muscle strength, and is particularly effective in treating muscle diseases such as muscular atrophy, and have completed the present invention.

[0008] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a muscular disease, comprising inotodiol or a pharma- ceutically acceptable salt thereof.

[0009] Another object of the present invention is to provide a health functional food composition for preventing or improving a muscular disease, which contains inotodiol or a dietarily acceptable salt thereof.

[0010] It is still another object of the present invention to provide an animal feed composition for preventing or improving a muscle disease, which comprises an inotodiol compound or a salt thereof.

[0011] Yet another object of the present invention is to provide a composition for promoting muscle regeneration capacity or increasing muscle mass by enhancing the self-renewal capacity of muscle stem cells and the differentiation capacity of myoblasts, comprising an inotodiol compound.

[0012] A further object of the present invention is to provide a composition for improving muscle function, comprising an inotodiol compound.

[0013] It is still another object of the present invention to provide a muscle strengthening composition comprising inotodiol.

[0014] Yet another object of the present invention is to provide a composition for improving muscle fibrosis, comprising inotodiol.

[0015] It is yet another object of the present invention to provide a composition for improving athletic performance, comprising inotodiol.

[0016] It is yet another object of the present invention to provide a method for preventing, ameliorating or treating a muscle disorder by administering inotodiol or a pharma- ceutically acceptable salt thereof to an individual.

[0017] It is still another object of the present invention to provide use of inotodiol or a pharma- ceutical acceptable salt thereof for the prevention, amelioration or treatment of a muscular disease. [Means for solving the problem]

[0018] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a muscular disease, comprising inotodiol or a pharma- ceutically acceptable salt thereof.

[0019] In order to achieve another object of the present invention, the present invention provides a functional health food composition for preventing or improving a muscle disease, comprising inotodiol or a dietarily acceptable salt thereof.

[0020] In order to achieve still another object of the present invention, the present invention provides an animal feed composition for preventing or improving a muscle disease, which comprises an inotodiol compound or a salt thereof.

[0021] In order to achieve yet another object of the present invention, the present invention provides a composition for promoting muscle regeneration or increasing muscle mass by enhancing the self-renewal of muscle stem cells or enhancing the muscle differentiation ability of myoblasts, comprising an inotodiol compound.

[0022] In order to achieve still another object of the present invention, the present invention provides a composition for improving muscle function, comprising an inotodiol compound.

[0023] In order to achieve still another object of the present invention, the present invention provides a composition for strengthening muscle, comprising inotodiol.

[0024] In order to achieve still another object of the present invention, the present invention provides a composition for improving muscle fibrosis, comprising inotodiol.

[0025] In order to achieve yet another object of the present invention, there is provided a composition for improving athletic performance, comprising inotodiol.

[0026] In order to achieve yet another object of the present invention, the present invention provides a method for preventing, ameliorating or treating a muscular disease by administering inotodiol or a pharma- ceutically acceptable salt thereof to an individual.

[0027] In order to achieve still another object of the present invention, the present invention provides use of inotodiol or a pharma- ceutical acceptable salt thereof for preventing, improving or treating a muscular disease. Effect of the Invention

[0028] The present invention relates to a composition for preventing, improving or treating muscle diseases, which contains an inotodiol compound. The composition increases muscle mass and muscle fiber regeneration by promoting myoblast differentiation, and enhances muscle strength by inducing activation of mitochondrial function, thereby having therapeutic effects on various muscle diseases, and can be used as a pharmaceutical or health functional food for the same. [Brief description of the drawings]

[0029] [Figure 1a-1b] Figures 1a and 1b show the degree of differentiation of mouse myoblasts (C2C12) by administration of different concentrations of inotropic diol. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 through One-way ANOVA test. [Diagram 2]Figure 2 shows the concentration-dependent increase in PGC-1α luciferase activity in myoblasts (C2C12) treated with inotropic dihydrochloride (IDH). Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using One-way ANOVA test. [Diagram 3] Figure 3 shows the results of qRT-PCR to confirm the mRNA expression level of PGC-1α in myoblasts (C2C12) treated with inotropic dihydrochloride. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 by Student t-test. [Figure 4] FIG. 4 shows the results of treating myoblasts (C2C12) with various concentrations of inotropic dihydrochloride and observing the expression of mitochondrial activity markers in muscle. [Diagram 5] Figure 5 shows the effect of inotropic dihydrochloride on increasing muscle mass (TA, EDL, SOL, GAS) in the hind limbs of mice in a mouse model with muscle damage induced by CTX. Figure 5a shows the change in body weight, and Figure 5b shows the change in weight of each muscle in the hind limbs. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student's t-test. [Figure 6a-6c] Figures 6a-6c show the effects of inotropic dihydrochloride on muscle fiber regeneration and increased muscle fiber cross-sectional area (CSA) in a mouse model with muscle damage induced by CTX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student's t-test. [Figure 7] Figure 7 shows the results of comparing the relative mRNA expression rate by muscle fiber type when indomethacin was administered to mice with muscle damage induced by CTX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student t-test. [Figure 8]Figure 8 shows the effect of increasing grip strength when inotoxin was administered to a mouse model with induced muscle damage. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student t-test. [Figure 9] Figure 9 shows the blood glucose reduction by administration of inotropic dihydrochloride in a mouse model with induced muscle damage. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student t-test. [Figure 10-12] 10 to 12 show the process and results of a BrdU analysis experiment to confirm the proliferation of muscle stem cells when indomethacin was administered after inducing muscle damage with CTX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student's t-test. [Figure 13] Figure 13 shows the results of comparing the relative mRNA expression levels of genes involved in the cell cycle when indomethacin was administered after muscle damage was induced by CTX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student's t-test. [Figure 14] Figure 14 shows the results of confirming the effect of inotropic acid administration on the expression of genes involved in muscle growth after muscle damage was induced by CTX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student t-test. [Figure 15] 15a and 15b show the results of observing the effect of inotodiol administration on improving muscle fibrosis after muscle damage was induced by CTX. [Figure 16-17] 16 and 17 show the results of observing changes in muscle strength and exercise performance when inotodiol was administered to a high-fat diet mouse model. [Figure 18-19]Figures 18 and 19 show the changes in muscle, muscle fiber size, and myotube cross-sectional area size by administration of inotropic dihydrochloride in a mouse model with muscle atrophy induced by DEX. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 through One-way ANOVA test. [Figure 20] Figure 20 shows the results of measuring muscle mass and muscle strength changes after long-term administration of inotropic dihydrochloride to normal mice for 2 months. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 using Student's t-test. [Figure 21] Figure 21 shows the results of examining the expression levels of PGC1a and mitochondrial activity genes to confirm mitochondrial activity in muscle of normal mice. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 through Student t-test. [Figure 22-23] 22 and 23 show the results of observing the changes in muscle and fat weights relative to the changes in body weight in normal mice. Statistics are expressed as *p<0.05, **p<0.01, and ***p<0.001 through Two-way ANOVA or Student's t-test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention relates to a pharmaceutical composition for preventing or treating a muscular disease, comprising inotodiol or a pharma-ceutical acceptable salt thereof.

[0031] The present invention relates to a functional health food composition for preventing or improving a muscular disease, which contains inotodiol or a food-logically acceptable salt thereof.

[0032] The present invention relates to an animal feed composition for preventing or ameliorating a muscular disease, which comprises an inotodiol compound or a salt thereof.

[0033] The present invention relates to a composition containing an inotodiol compound for enhancing the self-renewal of muscle stem cells, promoting muscle regeneration capacity by enhancing the muscle differentiation capacity of myoblasts, or for increasing muscle mass.

[0034] The present invention relates to a composition for improving muscle function, which contains an inotodiol compound.

[0035] The present invention relates to a muscle strengthening composition containing inotodiol.

[0036] The present invention relates to a method for preventing, ameliorating or treating a muscle disorder by administering inotodiol or a pharma- ceutically acceptable salt thereof to an individual.

[0037] The present invention relates to use of inotodiol or a pharma- ceutical acceptable salt thereof for the prevention, amelioration or treatment of a muscular disease.

[0038] The present invention will be described in detail below.

[0039] As one aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating muscle diseases, comprising the inotodiol and a pharma- ceutical acceptable salt thereof. Inotodiol is known to be a component contained in large amounts in Chagatake mushrooms, and is known to have anti-cancer, immune-enhancing, and other effects. The inotodiol of the present invention may be extracted from Chagatake mushrooms or may be chemically synthesized, and is represented by the following chemical formula:

[0040] [ka]

[0041] In one embodiment of the present invention, the inotodiol of the present invention has an effect of preventing or treating muscle diseases caused by muscle function decline, muscle loss, muscle atrophy, muscle wasting or muscle degeneration. The term "muscle disease" refers to a state in which muscles are damaged or lost due to aging or disease, resulting in weakened muscle strength, and this can be caused by a variety of reasons, including genetic predisposition, age-related diseases such as hypertension, impaired glucose tolerance, diabetes, obesity, dyslipidemia, atherosclerosis or cardiovascular disease, chronic diseases such as cancer, autoimmune diseases, infectious diseases, AIDS, chronic inflammatory diseases, arthritis, malnutrition, kidney diseases, chronic obstructive pulmonary disease, emphysema, rickets, chronic lower spinal pain, peripheral nerve damage, central nerve damage and chemical damage, fractures, trauma, etc., or loss of movement due to long-term bed rest, aging, etc.

[0042] The muscle disease may be at least one muscle disease selected from the group consisting of, but not limited to, atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, and senile sarcopenia, and specifically may be a disease such as senile muscular atrophy, muscle disease due to cancer and chronic disease, or muscle atrophy due to disuse of muscle, and more specifically may include senile muscular atrophy or cancer-related muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, senile sarcopenia, and muscle wasting disease. In particular, in the present invention, the muscle disease may be at least one muscle disease selected from the group consisting of muscular atrophy due to disuse, sarcopenia due to aging, and muscular dystrophy (muscular degeneration and atrophy).

[0043] In one embodiment of the present invention, the preventive, therapeutic or ameliorative effect on the muscle disease may be due to the promotion of myoblast differentiation. In one embodiment of the present invention, it was confirmed that inotodiol promotes myoblast differentiation. The "myoblast differentiation" refers to the process in which mononuclear myoblasts fuse to form multinuclear myotubes, and cells in the differentiation stage of forming myotubes may be classified using markers such as Pax7-, MyoD+, and Myogenin. In cells in the early differentiation stage of forming myotubes, the expression of myogenic transcription factors such as Myosin D (Myo D) increases, and in the middle stage, myogenin increases. In the late stage when differentiation is almost complete, the expression of myosin heavy chain (MHC) increases. In one embodiment of the present invention, it was confirmed that when myoblasts were treated with inotodiol, the expression of MHC, a differentiation marker of myoblasts, increased (Figure 1).

[0044] In addition, in the present invention, the preventive, therapeutic or ameliorative effect on the muscle disease may be due to the enhancement of mitochondrial activity in muscle. Through the activation of mitochondria in muscle, muscle growth and muscle endurance can be improved, thereby improving muscle strength. Furthermore, the enhancement of mitochondrial activity in muscle has the effect of increasing PGC-1α expression or increasing the expression of MHC type IIb+ muscle fibers in muscle. In one embodiment of the present invention, it was experimentally confirmed that the activity and expression of PGC-1α, a marker related to mitochondrial activity, increased when myoblasts were treated with inotodiol (FIGS. 2 and 3).

[0045] In addition, as described above, by increasing the activity of mitochondria in the muscles, muscle metabolism can be promoted, resulting in a blood sugar lowering effect (Figure 9).

[0046] Therefore, due to the effects as described above, the inotodiol compound of the present invention can regenerate damaged muscles in mice and have preventive, therapeutic or ameliorative effects against muscular diseases.

[0047] In one embodiment of the present invention, the effect of administering inotodiol to a mouse model of muscle injury (CTX-injury) was examined. As a result, no change in body weight was observed compared to the control group (vehicle administration group), but it was confirmed that the muscle mass of the hind-limb increased relatively (Figure 5). When the degree of regeneration of muscles and muscle fibers damaged by CTX (Cardiotoxin) was evaluated based on muscle fiber size, it was confirmed that the muscle fiber cross-sectional area (CSA) of the inotodiol administration group increased by an average of 89.4% compared to the control group (Figure 6).

[0048] The inotodiol compound of the present invention has a muscle strengthening effect. By strengthening muscle strength, the ability to perform exercise can be improved, and in this case, "ability to perform exercise" refers to the ability to perform exercise using muscle strength. The muscle strength may be improved by increasing muscle mass, muscle endurance, and oxidative muscle mass, improving muscle recovery and energy balance in muscle, and may also be promoted by reducing fatigue substances in muscle, etc., but the inotodiol compound of the present invention has an effect of promoting muscle strength through the effects of particularly enhancing muscle self-regeneration, muscle cell differentiation, increasing muscle mass, and muscle regeneration.

[0049] In relation to the above effects, in one embodiment of the present invention, it was confirmed that the actual exercise capacity of a mouse model (CTX-injury) with muscle injury was increased in a group administered with inotodiol compared to a control group (vehicle administration group) through a grip test.

[0050] The composition of the present invention may be used for various purposes such as pharmaceuticals, functional health foods, functional foods, animal feed, cell culture medium compositions, etc., and has the effect of promoting myoblast differentiation or enhancing mitochondrial activity in muscles.

[0051] As used herein, the term "prevention" refers to any action that can suppress or delay the onset of a muscular disorder by administering the pharmaceutical composition according to the present invention.

[0052] As used herein, the term "treatment" refers to any action in which symptoms are ameliorated or beneficially altered by administration of the pharmaceutical composition according to the present invention.

[0053] The pharmaceutical composition of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injections by a conventional method, and may further include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may be further included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0054] For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract or compound with at least one excipient, such as at least menthol, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to water and liquid paraffin, which are commonly used simple diluents.

[0055] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin, etc. may be used.

[0056] The pharmaceutical composition of the present invention may be administered orally or parenterally (by intravenous injection, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage varies depending on the condition and weight of the patient, the degree of the disease, the drug form, the administration route and time, and may be selected as an appropriate form by those skilled in the art.

[0057] The pharmaceutical composition of the present invention is administered in a pharma- ceutical effective amount. In the present invention, the term "pharma-ceutical effective amount" means a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease, and the criteria may be determined based on the patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, combined ingredients, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. Taking all of the above factors into consideration, the dosage may be determined at a level that minimizes side effects, which may be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition varies depending on the patient's age, body weight, severity, sex, etc., and may be generally 0.001 to 150 mg per kg body weight, more preferably 0.01 to 100 mg, administered daily or every other day, 1 to 3 times a day. However, this is merely an example, and the dosage may be set differently as necessary.

[0058] In addition, the composition of the present invention may be a food or a functional health food, and in particular, the term "functional health food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body based on Act No. 6727 on Functional Health Foods, and "functional" refers to intake for the purpose of regulating nutrients for the structure and function of the human body or obtaining beneficial effects for health purposes such as physiological actions.

[0059] The food or health functional food of the present invention can be manufactured and processed as pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or as health functional foods such as tea bags, infused tea, beverages, candies, jellies, gums, etc., for the purpose of preventing and improving muscle diseases.

[0060] The food or health functional food composition of the present invention may be used as a food additive, or may be manufactured alone or in combination with other ingredients. It may also contain nutrients, vitamins, electrolytes, flavorings, colorants and enhancers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. The above ingredients may be used alone or in combination, and may be used in combination in appropriate amounts.

[0061] In another embodiment, the present invention relates to a feed or feed additive composition comprising an inotodiol compound.

[0062] In the present invention, the term "feed" refers to a substance that supplies organic or inorganic nutrients necessary to maintain the life of an animal. The feed includes nutrients such as energy, protein, lipids, vitamins, and minerals required by animals such as livestock, and includes, but is not limited to, vegetable feed such as grains, roots and fruits, food processing by-products, algae, fibers, oils and fats, starches, butterbur, and grain by-products, and animal feed such as proteins, minerals, oils and fats, minerals, oils and fats, and single-cell proteins.

[0063] In the present invention, the term "feed additive" refers to a substance added to feed to improve the productivity and health of an animal, and may further include, but is not limited to, amino acid preparations, vitamin preparations, enzyme preparations, flavoring agents, silicate preparations, buffering agents, extractants, oligosaccharides, etc. for growth promotion, disease prevention, etc. The feed or feed additive composition of the present invention has an effect of promoting myoblast differentiation, muscle regeneration, or muscle strengthening in an animal, and further has an effect of preventing, treating, or improving muscle-related diseases in an animal.

[0064] In still another aspect, the present invention relates to a composition for enhancing muscle self-regeneration, promoting muscle differentiation, muscle regeneration or increasing muscle mass, and further to a composition for strengthening muscle strength and improving muscle function, comprising an inotodiol compound.

[0065] Inotodiol of the present invention has an effect of promoting muscle self-renewal, and the term "promotion of muscle self-renewal" refers to the ability of muscle stem cells to maintain their numbers without completely differentiating into myoblasts. In other words, by promoting muscle self-renewal, muscle stem cells can maintain a certain number, which means that they have a sustained muscle regeneration ability even in the case of exogenous damage caused by external impact or muscle damage caused by endogenous factors including chronic inflammation, ultimately maintaining muscle mass.

[0066] In addition, the composition of the present invention has an effect of "promoting myoblast differentiation", and "myoblast differentiation" refers to the process in which mononuclear myoblasts fuse to form multinucleated myotubes, and refers to inducing this process. Cells in the differentiation stage that form myotubes can be identified by the presence or absence of markers such as Pax7-, MyoD+, and MyoG+. By promoting myoblast differentiation, muscle cells increase, which ultimately leads to increased muscle mass in the individual.

[0067] In addition, the composition of the present invention has a "muscle regeneration" effect, and the "muscle regeneration" means the ability of damaged muscles to recover normally. In one embodiment of the present invention, it was confirmed through an experiment that muscle tissue acutely damaged by injection of a neurotoxin, Cardiotoxin, recovers faster through administration of Inotoxin. The muscle regeneration may be an effect of promoting differentiation of muscle cells to increase the absolute amount of muscle cells, or an effect of increasing the diameter of individual myotubes.

[0068] The present invention also relates to a composition for strengthening muscles, comprising inotodiol. "Strengthening muscles" refers to strengthening physical performance, strengthening maximum endurance, increasing muscle mass, enhancing muscle recovery, reducing muscle fatigue, improving energy balance, or a combination of these effects. As described above, the composition of the present invention can increase muscle mass and increase overall muscle mass through its ability to differentiate myoblasts into muscle cells, and can enhance maximum endurance by promoting muscle regeneration, thereby strengthening physical performance and reducing muscle fatigue. In addition, since muscle cells can be rapidly replaced, muscle damage can be rapidly healed.

[0069] The inotodiol compound of the present invention has an effect of improving muscle function, and "improvement of muscle function" refers to the ability to exert force by muscle contraction, including muscle strength, which is the ability of muscles to exert maximum contractile force to overcome resistance, muscle endurance, which is the ability of muscles to repeat contraction and relaxation for a given weight for a long time or how many times, and explosive power, which is the ability to exert strong force within a short period of time. Such muscle function is proportional to muscle mass, and "improvement of muscle function" means to improve muscle function. In addition, the inotodiol of the present invention can have an effect of improving muscle function because it improves the energy balance in muscles by promoting the activity of mitochondria in muscles.

[0070] The inotodiol compound of the present invention has an effect of improving muscle fibrosis, and "muscle fibrosis" refers to a symptom in which muscle elasticity is reduced, and is caused by the tendons that compose the muscle becoming stiff and stiff for a long period of time. As shown in one experimental example of the present invention, the effect of improving muscle fibrosis by administering the inotodiol compound was confirmed.

[0071] The composition for enhancing the self-renewal of muscle stem cells, promoting the differentiation ability of myoblasts, muscle regeneration, increasing muscle mass, strengthening muscle, or improving muscle function of the present invention may be prepared in the form of a food composition or food additive, and may be prepared in the form of a health food composition. The food composition is as described above. Therefore, the composition for strengthening muscle of the present invention may be used in the form of an adjuvant for muscle generation and strengthening muscle of the general public, as well as for muscle loss due to aging or disease.

[0072] MODE FOR CARRYING OUT THEINVENTION

[0073] Hereinafter, the present specification will be described in detail with reference to examples in order to specifically explain the present specification. However, the examples according to the present specification may be modified into various other forms, and the scope of the present specification should not be interpreted as being limited to the examples described below. The examples of the present specification are provided to more completely explain the present specification to those having average knowledge in the art.

[0074] Example 1. Cultivation and differentiation induction of myoblast cell line C2C12 C2Cl2 is a myoblast cell line derived from a C3H mouse and is widely used in muscle cell differentiation research. The C2C12 cells were cultured in a general cell culture medium and a differentiation medium, respectively. The normal cell culture medium (GM, growth media) was DMEM supplemented with 10% fetal bovine serum, and the differentiation medium (DM, differentiation media) was DMEM supplemented with 2% horse serum.

[0075] The cells were dispensed into cell culture medium (GM) and cultured for 24 hours, after which the cells were treated with DMSO and inotropic diol (0.1, 0.5, 1.0, and 10 μM) at different concentrations in differentiation medium (DM) to induce differentiation for 2 to 3 days.

[0076] Example 2. CTX injury mouse model Ten 5-month-old male C57BL / 6 mice were used as experimental animals. The experimental animals were divided into a control group not administered inotodiol and an experimental group administered inotodiol, with five mice of similar weight. Inotodiol was dissolved in 80% saline, 10% PEG400, and 10% EtOH at 300 μg / kg and orally administered to the experimental group for 7 days. Then, 10 μM cardiotoxin (CTX) was directly injected into the muscle at 2 μl per gram of body weight to induce muscle damage, and inotodiol (300 μg / kg) was orally administered for 21 days.

[0077] Example 3. Muscle atrophy (DEX) model The experimental animals were 20 male C57BL / 6 mice, 16 weeks old, grouped into groups of 10 with similar body weights, and one group was administered 20 mg / kg dexamethasone to induce muscle atrophy.

[0078] Experimental example 1. Confirmation of myoblast differentiation enhancement effect C2C12 cells induced to differentiate in Example 1 were washed with 1X PBS, fixed with 3.7% paraformaldehyde at room temperature, and reacted with permeabilization buffer at room temperature. Non-specific antibody binding was inhibited by reacting with PBST (blocking buffer) containing 3% BSA and PBS containing 0.1% Tween 20. Primary antibody against myosin heavy chain (MHC) was added at room temperature after dilution at 1:500 in blocking buffer. Secondary antibody was added at room temperature after dilution at 1:5000 in blocking buffer, and reacted at room temperature. After applying mounting solution and fixing, the results were analyzed by taking a photograph under a fluorescent microscope.

[0079] As a result, in the case of C2C12 cells treated with various concentrations of inotoxin, immunostaining images on the second day of differentiation confirmed that the formation of multinucleated myotubes increased in an inotoxin concentration-dependent manner (Figure 1 (top)).

[0080] C2C12 cells induced to differentiate in Example 1 were obtained and dissolved by adding lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X, proteinase inhibitor), and the cell lysis sample was quantified, and the same amount of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and washed with TTBS (0.03% Tween 20, Tris 2.42 g, NaCl 9 g, pH 7.41 / L). A differentiation marker MHC (myosin heavy chain) primary antibody was added at 1:500 in TTBS containing 5% BSA, and reacted overnight at 4°C. Then, a secondary antibody was added at 1:5000 in TTBS containing 5% skim milk, and reacted at room temperature, and ECL (Enhanced Chemiluminescent solution, pierce) was added. The membrane was then exposed to an X-ray film to confirm the amount of protein.

[0081] As a result, as shown in Figure 1 (bottom), it was confirmed that the expression level of MHC, a differentiation marker, increased in a concentration-dependent manner in C2C12 cells treated with inotodiol at various concentrations compared to the control group (DMSO-treated group). Taking the above results together, it was confirmed that inotodiol promotes the differentiation of myoblasts.

[0082] Experimental example 2. PGC-1α reporter assay 1×10 per well in a 96-well culture plate 4The cells were dispensed at 1000 cells / well, and immediately transfected with PGC-1α (DNA) and plasmid at a ratio of [150ng / 10μl] + [0.3μl / 10μl] per well. After 24 hours, the differentiation medium was treated with inotropic diol at different concentrations (0, 0.1, 0.5, and 1.0μM) to induce differentiation for 24 hours. After 24 hours of drug treatment, the cells were dissolved in 30μl of passive lysis buffer (Promega) per well, and the dissolved cell solution was transferred to a luminometer read plate. The same amount of luciferin, a substrate of luciferase, was added to cause an enzyme reaction and the luminescence level was measured. The measured values ​​were standardized for each well and plate, and the measured values ​​in wells treated with DMSO, the solvent for the compound, were used as the control group and expressed as a relative ratio. The level of luciferase expression in the PGC-1α reporter cell line depends on the stimulation of the PGC-1α promoter. That is, when a compound is a PGC-1α expression inducer, it stimulates the PGC-1α promoter to increase luciferase expression, and when a compound is a PGC-1α expression inhibitor, it decreases luciferase expression. The degree of expression was measured by adding a luciferase substrate and measuring the luminescence that appeared, thereby measuring the degree of activation or inhibition of PGC-1α transcription by inotodiol.

[0083] As a result, it was confirmed that the luciferase activity against PGC-1α in the group treated with inotodiol increased in an inotodiol concentration-dependent manner, as shown in Figure 2. Furthermore, the luciferase activity against PGC-1α was higher than that of 0.5 mM AICAR, which was used as a positive control.

[0084] Experimental example 3. Analysis of PGC-1α mRNA expression by qRT-PCR C2C12 cells induced to differentiate in Example 1 were dissolved in Easy-spin Total RNA Extraction Kit, chloroform was added, and centrifugation was performed. After separating the supernatant, the same volume of isopropanol was added for binding. After centrifugation, the cells were washed with 70% EtOH mixed with DEPC water, and 20 to 50 μl of DEPC solution was added to quantify the RNA concentration. RNA was reverse transcribed with PrimeScript cDNA synthesis kit to synthesize cDNA. qRT-PCR was performed to analyze gene expression using a Real-Time PCR system using SYBR Premix EX Taq. The fold change in gene expression was normalized for the expression of the ribosomal gene 18s rRNA.

[0085] As a result, it was confirmed that the mRNA expression for PGC-1α was significantly increased in the inotodiol-treated group compared to the control group, as shown in Figure 3. Taken together, it was confirmed that inotodiol increases both the enzyme activity and expression of PGC-1α during the differentiation of myoblasts.

[0086] Experimental Example 4. Mitochondrial activity enhancement effect Differentiation of myoblasts C2C12 was induced in the same manner as in Example 1, and the expression characteristics of antibody T-OxPHOS, which is a mixture of ATP5A, MTCO1, and NDUF88, was confirmed by immunoblotting to confirm mitochondrial activity. Regarding each protein contained in the T-OxPHOS antibody, ATP5A is a component protein of ATP synthase and contributes to ATP synthesis in mitochondria, MTCO1 is involved in the oxidase activity of cytochrome-c, a component of the electron transport chain in the inner mitochondrial membrane, and NDUF 88 encodes a subunit of NADH:ubiquinone oxidoratesase (complex 1), the first enzyme complex of the mitochondrial electron transport chain.

[0087] As a result, it was confirmed that the expression levels of all of the above proteins increased in proportion to the concentration of inotoxin, as shown in Figure 4. This result proves that inotoxin enhances mitochondrial activity in muscle.

[0088] Experimental Example 5. Improvement of muscle function in mouse model of muscle injury (CTX-administered) 5-1. Confirmation of muscle mass increase effect As in Example 2, the weight and hindlimb muscle weights were measured 21 days after administration of inotodiol to the CTX muscle injury mouse model. As a result, as shown in FIG. 5a, no change in the weight of the experimental animals was observed when inotodiol was administered to the CTX muscle injury model. However, the weights of the hindlimb muscle tissues of the experimental animals, namely, TA (Tibialis anterior muscle), EDL (Extensor digitorum longus muscle), Sol (Soleus muscle), and Gas (Gastrocnemius muscle), were measured, and it was confirmed that the experimental group administered inotodiol had a 16.0% increase in TA, 18.9% in EDL, 5.2% in Sol, and 9.5% in Gas compared to the control group (FIG. 5b). In particular, the TA muscle mass increased by approximately 16.0%, which means that the regenerative ability of the muscle damaged by CTX was further enhanced by the administration of inotoxin.

[0089] 5-2. Confirmation of muscle regeneration and muscle fiber size increase effects H&E staining (hematoxylin & eosin staining) was performed using TA muscle isolated from the tissue of the experimental animals. Frozen sections fixed in 4% paraformaldehyde were stained with hematoxylin, and then stained with eosine for contrast staining, mounted, and observed under an optical microscope. In addition, to analyze the size of muscle fibers, TA muscle tissue was immunohistochemically stained using a laminin antibody and observed under a fluorescent microscope.

[0090] As a result of H&E staining, it was confirmed that administration of inotodiol to mice with CTX muscle damage showed a significant muscle regeneration effect compared to the control group (Fig. 6a, 6b), as shown in Figure 6. In addition, analysis of the cross-sectional area (CSA, Cross Section Area) of the TA muscle by Laminin staining confirmed that the proportion of muscle fibers with large cross-sectional area in the TA muscle of the inotodiol-treated group increased compared to the control group, and in particular, the cross-sectional area ratio of the entire TA muscle increased by about 89.4% compared to the control group (Fig. 6c).

[0091] 5-3. Changes in skeletal muscle fiber types by administration of inotoxin In a CTX-injected mouse model, changes in skeletal muscle fiber type were observed when inotodiol was administered 21 days after muscle damage.Comparing the relative mRNA expression rates of Myh7 (MHCI), Myh2 (MHCIIa), Myh4 (MHCIIb), and Myh1 (MHCIIx) by muscle type, as shown in Figure 7, there was no significant difference, but the expression level generally tended to increase, and in particular, the expression of Myh2 (MHCIIa), which is a glycated myofiber, tended to increase significantly.

[0092] 5-4. Confirmation of muscle strength increase effect On the 21st day after inducing CTX muscle damage, a grip strength test was performed to confirm the muscle strength of the inotodiol-treated group and the control group. Grip strength tests were performed using a mouse grip strength meter manufactured by BIOSEB. Mice were placed on a wire mesh attached to an instrument panel that can monitor strength, and the strength with which the mice gripped the wire mesh was measured while pulling down on their tails. The average value of five consecutive measurements was used. As a result, it was confirmed that grip strength in the inotodiol-treated group increased by approximately 6.8% compared to the control group (Figure 8).

[0093] 5-6. Blood sugar lowering effect When inotoxin was administered to experimental animals with CTX muscle damage, the change in blood glucose was measured. Improvement of muscle mitochondrial function is closely related to the activation of energy metabolism in muscles, and its concomitant effect can induce a blood glucose lowering effect. When blood glucose of the experimental animals was analyzed on the 21st day after inducing CTX muscle damage, the effect of lowering blood glucose in the inotoxin-administered group compared to the control group was confirmed (Figure 9).

[0094] It can be inferred that the reduction in blood glucose in the inotodiol administration group is the result of increased glucose consumption in the muscle due to the activity of mitochondria in the muscle. As confirmed in Experimental Examples 2-3, when inotodiol is administered, the activity of mitochondria in the muscle is promoted, so the effect of reducing blood glucose is due to the increase in blood glucose consumption by mitochondria in the muscle. Another reason is that the administration of inotodiol promotes muscle differentiation and regeneration, which leads to an increase in absolute muscle mass, which is thought to be the result of increased glucose consumption due to muscle energy metabolism.

[0095] 5-7. Stem cell proliferation promotion effect by administration of inotodiol An experiment was conducted to confirm the muscle regeneration effect of administering inotodiol to a mouse model in which muscle damage was induced by injection of CTX. Mice were administered inotodiol once a day for 7 days, and then injected with CTX for 3 days to induce muscle damage. We then used BrdU analysis to confirm whether muscle stem cell proliferation was promoted (Figure 10).

[0096] As a result, as shown in Figures 11a and 11b, it was confirmed that the expression level of BrdU was increased in the muscle tissue of mice administered indomethacin, and as shown in Figures 12a and 12b, the expression levels of Pax7 and Ki67 and the expression ratio of Ki67 to Pax7 were increased.

[0097] Furthermore, as shown in FIG. 13, an increase in the relative mRNA expression level of genes involved in the cell cycle was confirmed, and as shown in FIG. 14, an effect of promoting the expression of genes involved in muscle proliferation was confirmed.

[0098] These results confirm that inotoxin promotes muscle stem cell proliferation.

[0099] 5-8. Inhibitory effect of inotoxin on muscle fibrosis We conducted an experiment to confirm the effect of administering inotoxin on muscle fibrosis in a mouse model in which muscle damage was induced by injection of CTX. After injecting CTX for 7 days to induce muscle damage, we administered inotoxin to confirm whether muscle fibrosis could be improved.

[0100] As a result, as shown in Figures 15a and 15b, it was confirmed that fibrosis was improved in muscle tissue administered with inotodiol, and the area in which fibrosis progressed was reduced.

[0101] Experimental Example 6. Effect of high-fat diet on improving athletic performance The effects of administering inotodiol to mice fed a high-fat diet on muscle strength and exercise performance were confirmed. After administering inotodiol to a mouse model fed a high-fat diet, exercise duration and distance traveled were measured through a treadmill experiment. As a result, it was confirmed that the inotodiol-administered group recovered, on average, the exercise performance to the same level as the normal control group (Figure 16). In addition, the results of the grip test confirmed that the high-fat diet model had lower muscle strength than average, but that muscle strength was improved when inotodiol was administered (Figure 17).

[0102] Experimental Example 7. Improvement effect of myotube atrophy in muscle atrophy-induced (DEX) model The myotube atrophy improvement and recovery effect of administration of inotodiol was confirmed for the muscle atrophy mouse model of Example 3. As a result, it was confirmed that the size of the muscle and muscle fiber increased. In addition, as a result of checking through the size of the myotube cross-sectional area, it was confirmed that administration of inotodiol to the DEX-induced myotube atrophy model improved the myotube cross-sectional area to the same level as the normal group (Figures 18 and 19).

[0103] Experimental Example 10. Increased muscle mass and strength in a long-term inotodiol administration model 10-1. Increased muscle mass An experiment was conducted to confirm whether administration of inotodiol to normal mice without induced muscle damage or muscle atrophy would increase muscle mass and strength. Mice were administered inotodiol for two months, and the changes in muscle mass and strength (Grip test) of the hindlimb muscles were measured after administration of inotodiol.

[0104] As a result, as shown in FIG. 20, it was confirmed that the muscle mass in the hind legs was increased compared to the control group, and that muscle strength was also increased.

[0105] 10-2.Enhancing effect of PGC-1a and mitochondria-related genes in muscle When indomethacin was administered for a long period of time to the normal mice, the expression levels of the muscle metabolic marker PGC-1a and mitochondrial-related genes in the muscles were examined. As shown in Figure 21, it was confirmed that the expression levels were increased compared to the control group.

[0106] In addition, the change in fat weight relative to the amount of body weight increase in normal mice was examined. As a result, as shown in Figure 22, it was confirmed that the group administered inotodiol had a relatively small increase in fat weight, and as shown in Figure 23, it was confirmed that the muscle mass relative to body weight was higher than that of the non-administered group.

[0107] In other words, the above results indicate that, not only in cases of muscle disease, but also in normal muscle cells, an increase in muscle mass and mitochondrial expression in the muscle can be increased, resulting in muscle strength improvement effects.

[0108] The present invention has been described above with reference to its preferred embodiment. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined in the claims, not the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. A pharmaceutical composition for preventing or treating a muscular disease, comprising an inotodiol compound or a pharma- ceutical acceptable salt thereof as an active ingredient.

2. A functional health food composition for preventing or improving a muscle disease, comprising an inotodiol compound or a food-logically acceptable salt thereof as an active ingredient.

3. An animal feed composition for preventing or improving a muscle disease, comprising an inotodiol compound or a salt thereof as an active ingredient.

4. The composition according to any one of claims 1 to 3, wherein the muscle disease is selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, muscle wasting disease and sarcopenia.

5. The composition according to any one of claims 1 to 3, wherein the muscle disease is induced by aging, muscle decline, muscle wasting, muscle degeneration, disuse or muscle damage.

6. The composition according to any one of claims 1 to 3, wherein the muscle disease is sarcopenia induced by cancer or aging.

7. The composition according to any one of claims 1 to 3, characterized in that the muscle disease is muscle atrophy induced by disuse of skeletal muscles.

8. The composition according to any one of claims 1 to 3, characterized in that the composition enhances mitochondrial activity in muscle.

9. The composition according to any one of claims 1 to 3, characterized in that the composition lowers blood sugar by promoting metabolism by mitochondria in muscle.

10. A composition for enhancing the self-renewal of muscle stem cells, promoting muscle differentiation, regenerating muscle, or increasing muscle mass, comprising an inotodiol compound.

11. A composition for improving muscle function comprising an inotodiol compound.

12. A composition for strengthening muscles, comprising an inotodiol compound.

13. A composition for improving athletic performance, comprising an inotodiol compound.

14. A composition for improving muscle fibrosis, comprising an inotodiol compound.

15. The composition according to any one of claims 10 to 14, which is used for at least one selected from the group consisting of food, functional food, health functional food, pharmaceuticals, animal feed, and feed additives.

16. The composition according to any one of claims 10 to 14, characterized in that the composition is administered to an injured or aged individual.

17. The composition according to any one of claims 10 to 14, characterized in that the composition is administered to a normal individual.

18. Use of the composition according to claim 1 in the manufacture of a medicament for the prevention, amelioration or treatment of a muscle disease.

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