KR102645399B1 - Composition for improving muscular function, or preventing, ameliorating or treating muscle disease comprising euscaphic acid - Google Patents

Abstract

The present invention relates to a composition for improving muscle function or preventing, ameliorating or treating muscle disease, including Euscapic acid. More specifically, it relates to a composition containing Euscapic acid, which exhibits the activity of inhibiting muscle loss, increasing muscle energy production, and increasing muscle protein synthesis. Provided are food compositions for improving muscle function or preventing or treating muscle diseases, health functional food compositions, and/or pharmaceutical compositions for improving muscle function or preventing or treating muscle diseases.

Description

Composition for improving muscle function or preventing, ameliorating or treating muscle disease comprising Euscapic acid {COMPOSITION FOR IMPROVING MUSCULAR FUNCTION, OR PREVENTING, AMELIORATING OR TREATING MUSCLE DISEASE COMPRISING EUSCAPHIC ACID}

The present invention relates to a composition for improving muscle function or preventing, ameliorating or treating muscle disease, including Euscapic acid. More specifically, it relates to a composition containing Euscapic acid, which exhibits the activity of inhibiting muscle loss, increasing muscle energy production, and increasing muscle protein synthesis. Provided are food compositions for improving muscle function or preventing or treating muscle diseases, health functional food compositions, and/or pharmaceutical compositions for improving muscle function or preventing or treating muscle diseases.

Muscle atrophy is caused by a gradual decrease in muscle mass and refers to muscle weakness and degeneration (Cell, 119(7): 907-910, 2004). Muscle atrophy is promoted by inactivity, oxidative stress, or chronic inflammation and weakens muscle function and exercise capacity (Clinical Nutrition, 26(5): 524-534, 2007).

In this regard, the most important factor determining muscle function is muscle mass, which is maintained by the balance of muscle protein synthesis and breakdown. Muscular dystrophy occurs when protein degradation occurs more than synthesis (The International Journal of Biochemistry and Cell Biology, 37(10): 1985-1996, 2005).

Muscle size is controlled by intracellular signaling pathways that induce anabolism or catabolism within the muscle, and when signaling reactions that induce synthesis rather than decomposition of muscle proteins occur more often. Muscle protein synthesis is increased, which appears as an increase in muscle size (hypertrophy) or an increase in the number of muscle fibers (hyperplasia) due to an increase in muscle protein (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011).

Muscle hypertrophy-inducing factors induce protein synthesis by phosphorylating downstream proteins starting from stimulation of the PI3K (phosphatidylinositol-3 kinase)/Akt pathway within muscle cells. Among these, activation of mTOR (mammalian target of rapamycin) by PI3K/Akt signaling is recognized as a major growth signaling mechanism that integrates various growth signals within cells. Activation of mTOR can contribute to an increase in muscle mass by inducing muscle protein synthesis by activating two downstream targets, 4E-BP1 (4E-binding protein) and p70S6K (phosphorylated 70-kDa ribosomal S6 kinase) (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011; The International Journal of Biochemistry and Cell Biology, 43(9): 1267-1276, 2011).

One of the representative methods for improving muscle function by promoting energy consumption is to generate ATP energy by increasing fatty acid oxidation by mitochondria. It was found that the number and ability of mitochondria that control this are controlled by PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1 alpha) coactivator, and PGC-1α activity is controlled by SIRT1 (sirtuin 1) (EMBO J., 26:1913-1923, 2007, Cell Metab., 1: 361-370, 2005).

When the transcription factor FoxO (forhead box) moves from the cytoplasm into the nucleus, the E3 ubiquitin ligase factors Atrogin-1/MAFbx (muscle atrophy F-box) and MuRF-1 are involved in protein degradation. Increases the expression of (muscle RING-finger protein-1) (Dis. Model. Mech., 6: 25-39, 2013). As their expression level increases, protein breakdown within the muscle is promoted, resulting in a decrease in muscle mass. Therefore, suppressing the expression of Atrogin-1/MAFbx and MuRF-1 reduces the loss of muscle protein amount and maintains normal muscle function.

Differentiation of muscle cells and muscle formation are regulated by various muscle regulatory factors. Among them, MyoD initiates the expression of muscle-specific genes and induces the differentiation of muscle satellite cells into myoblasts. Induction of myogenin expression by MyoD activity is the most important factor in the fusion of myogenic cells and is involved in the formation of myotube cells. Muscle fibers formed through this process form bundles and ultimately form muscles (Zanou N and Gailly P, Cellular and Molecular Life Sciences, 70:4117-4130, 2013).

While research investment is focused on developing treatments related to geriatric diseases due to the rapid increase in the elderly population, the World Health Organization (WHO) established sarcopenia, which is related to skeletal muscle atrophy, as a disease code (ICD-10-CM) in 2017 and provides treatments for it. Although development is actively taking place, there are currently no drugs approved by the U.S. FDA for the treatment of sarcopenia.

Republic of Korea Patent Publication No. 10-2022-0061070 Republic of Korea Patent Publication No. 10-2021-0122125

Against this background, the present inventors confirmed that Euscapic acid regulates various indicators involved in muscle loss, muscle energy production, or muscle protein synthesis, thereby suppressing muscle loss, increasing muscle energy production, or increasing muscle protein synthesis, The present invention has been completed.

Accordingly, the purpose of the present invention is to provide a composition for improving muscle function or preventing, improving, or treating muscle disease using Euscaphic acid.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above-described problems, the present invention provides a food composition for improving muscle function or preventing or ameliorating muscle disease containing Euscaphic acid or a foodologically acceptable salt thereof.

Additionally, the present invention provides a health functional food composition for improving muscle function or preventing or improving muscle disease containing Euscaphic acid or a foodologically acceptable salt thereof.

The present invention also provides a pharmaceutical composition for improving muscle function or preventing or treating muscle disease, containing Euscaphic acid or a pharmaceutically acceptable salt thereof.

According to one embodiment of the present invention, the muscle disease may be a muscle disease caused by decreased muscle function, muscle wasting, or muscle degeneration.

Additionally, the muscle disease may be any one or more selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, and sarcopenia.

In addition, the Euscapic acid, its food-acceptable salt or pharmaceutically-acceptable salt may exhibit any one or more of the following effects i) to iii):

i) effect of inhibiting muscle loss;

ii) the effect of increasing muscle energy production; and

iii) Effect of increasing muscle protein synthesis.

The composition for improving muscle function or preventing, improving or treating muscle disease containing euscapic acid according to the present invention downregulates Atrogin-1 and MuRF-1, E3 ubiquitin ligase factors involved in muscle loss. By upregulating mTOR involved in muscle protein synthesis, PGC-1α involved in energy metabolism, and MyoD and Myogenin involved in muscle differentiation, it suppresses muscle loss, increases muscle energy production, and increases muscle protein synthesis. In addition to improving muscle function, it can be effectively used to prevent, improve, or treat muscle diseases caused by decreased muscle function, muscle wasting, or muscle degeneration.

Figure 1 is a graph showing changes in the mRNA expression levels of muscle loss-related markers (Atrogin-1 and MuRF1) in C2C12 cells, which are muscle stem cells, according to euscapic acid treatment.
Figure 2 shows changes in the mRNA expression levels of muscle protein synthesis-related marker (mTOR), energy metabolism-related marker (PGC-1α), and muscle differentiation-related markers (MyoD and Myogenin) according to euscapic acid treatment in C2C12 cells, which are muscle stem cells. This is the graph shown.

As mentioned above, to date, there are no drugs approved by the U.S. FDA for the treatment of sarcopenia, and therefore, there is still a need to discover substances that can be substantially effective as a treatment for sarcopenia.

Accordingly, the present inventors confirmed that Euscapic acid regulates various indicators involved in muscle loss, muscle energy production, or muscle protein synthesis to inhibit muscle loss, increase muscle energy production, or increase muscle protein synthesis, and that Euscapic acid The present invention has been completed by providing a composition for improving muscle function or preventing, improving or treating muscle disease, including.

Therefore, the first aspect of the present invention is a food composition for improving muscle function or preventing or improving muscle disease containing Euscapic acid or a foodologically acceptable salt thereof and/or a food composition containing Euscapic acid or a foodologically acceptable salt thereof. It relates to a health functional food composition for improving muscle function or preventing or improving muscle disease.

In the food composition of the present invention, Euscaphic acid, which is included as an active ingredient, is a pentacyclic triterpenoid compound having the structure of the following [Formula 1], and is activated by calf DNA polymerase α ( It is known as a DNA polymerase inhibitor that inhibits pol α) and rat DNA polymerase β (pol β) (Murakami C, et al. Novel anti-inflammatory compounds from Rubus sieboldii, triterpenoids, are inhibitors of mammalian DNA polymerases. Biochim Biophys Acta. 2002 Apr 29;1596(2):193-200.), is known to induce apoptosis (Dai W, et al. Euscaphic acid inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by silencing the PI3K /AKT/mTOR signaling pathway. Am J Transl Res. 2019 Apr 15;11(4):2090-2098.).

[Formula 1]

In the food composition of the present invention, the Euscapic acid can be artificially synthesized and used, or a commercially available one can be used. Additionally, it can be purified and used from materials containing euscapic acid or natural sources.

By use of the term “purified,” it is intended to mean that Euscapic acid is in a more concentrated form compared to the form in which it can be obtained from natural sources. Purified ingredients can be concentrated from their natural sources or obtained through chemical synthesis methods.

In the present invention, the term "muscle disease" is preferably a muscle disease caused by decreased muscle function, muscle wasting, or muscle degeneration, and is preferably a disease reported in the art, specifically atony and muscular atrophy. , muscular dystrophy, myasthenia, cachexia, and sarcopenia, but is not limited thereto.

The muscle wasting or degeneration occurs due to genetic factors, acquired factors, aging, etc., and muscle wasting is characterized by gradual loss of muscle mass and weakening and degeneration of muscles, especially skeletal muscles, voluntary muscles, and cardiac muscles.

E3 ubiquitin ligase factors Atrogin-1/MAFbx (Muscle atrophy F-box) and MuRF-1 (Muscle RING-finger protein-1) are involved in protein degradation, and when their expression level increases, intramuscular Protein breakdown is promoted and muscle mass decreases.

Therefore, in a specific embodiment of the present invention, in order to confirm the effect of Euscapic acid on improving muscle function or preventing, improving or treating muscle diseases, muscle stem cells C2C12 were treated with dexamethasone to induce muscle loss and then treated with Euscapic acid. After processing, changes in gene expression levels of Atrogin-1 and MuRF-1, which are markers related to muscle loss, were analyzed. As a result, as seen in Figure 1, the mRNA expression levels of Atrogin-1 and MuRF1 were significantly increased in the dexamethasone-treated group (CTL) compared to the untreated control group (NC), confirming that muscle loss was induced. On the other hand, in all groups treated with Euscapic acid after dexamethasone treatment, the levels of Atrogin-1 and MuRF1 mRNA expression, which were increased by dexamethasone, were significantly reduced by Euscapic acid treatment. Treatment with Euscapic acid reduced the expression levels of these genes in a concentration-dependent manner. Therefore, Euscapic acid according to the present invention can be effectively used in the treatment of muscle diseases caused by decreased muscle function, muscle wasting, or muscle degeneration through its activity in suppressing muscle loss.

It is known that when mTOR protein is phosphorylated and activated, it can induce the activation of proteins involved in muscle protein synthesis and muscle mass increase in the intracellular PI3K/Akt signaling pathway.

In addition, PGC-1α is an important factor that regulates energy metabolism within muscles and increases the amount of energy by controlling fatty acid oxidation and enhancing mitochondrial biosynthesis. In addition, PGC-1α is known to activate transcription factors such as NRF1, TFAM, and Sirt1 that affect mitochondrial proliferation, energy homeostasis regulation, and respiration.

Therefore, in another specific embodiment of the present invention, in order to confirm the effect of Euscapic acid on improving muscle function or preventing, improving or treating muscle diseases, C2C12, a muscle stem cell, is treated with Euscapic acid to obtain a marker related to muscle protein synthesis. Changes in gene expression levels of mTOR and PGC-1α, a marker related to energy metabolism, were analyzed. As a result, as seen in Figure 2, it was confirmed that the mRNA expression levels of mTOR and PGC-1α were increased in the group treated with Euscapic acid compared to the untreated control group (CTL). Treatment with Euscapic acid increased the expression levels of these genes in a concentration-dependent manner. Therefore, Euscapic acid helps form muscle proteins by increasing muscle protein synthesis and helps produce muscle energy by increasing the expression of mitochondria-mediated energy metabolism regulators, which not only improves muscle function but also reduces muscle function decline and muscle wasting. Alternatively, it can be effectively used to treat muscle diseases caused by muscle degeneration.

MyoD is an important protein involved in muscle differentiation, and Myogenin is a muscle cell-specific transcription factor that is involved in muscle development. Increased mRNA expression of this myogenic regulatory factor protein induces muscle cell differentiation and protein synthesis, thereby increasing muscle mass.

Therefore, in another specific embodiment of the present invention, in order to confirm the effect of Euscapic acid on improving muscle function or preventing, improving or treating muscle diseases, C2C12, a muscle stem cell, is treated with Euscapic acid to produce a marker related to muscle differentiation. Changes in gene expression levels of MyoD and Myogenin were confirmed. As a result, as shown in Figure 2, it was confirmed that the mRNA expression levels of MyoD and Myogenin were significantly increased in the group treated with Euscapic acid compared to the untreated control group (CTL). Treatment with Euscapic acid increased the expression levels of these genes in a concentration-dependent manner. Therefore, Euscapic acid ultimately has an effect on the formation of muscle tissue by promoting muscle differentiation, so it can be effectively used in the treatment of muscle diseases caused by decreased muscle function, muscle wasting, or muscle degeneration.

In the present invention, the term “prevention” refers to any action that suppresses or delays the progression of muscle disease by administering the composition of the present invention.

As used herein, the term “improvement” means that the extent and/or undesirable clinical signs of a muscle disease condition are reduced, or the time course of progression is slowed or lengthened.

In the present invention, the term "muscular function" refers to the ability to exert force by contracting muscles, including strength that allows muscles to exert maximum contractile force to overcome resistance; Muscular endurance, which is the ability of a muscle to contract and relax for a given weight or how many times; and agility, which is the ability to exert strong force in a short period of time. The muscle function is proportional to muscle mass, and the term "improving muscle function" means improving the decline in muscle function due to muscle wasting or degeneration in a more positive direction.

In the present invention, the term “health functional food” includes both “functional food” and “health food”.

In the present invention, the term "functional food" is the same as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, and medical effects. It means high food.

In the present invention, the term "health food" refers to food that has a more active health maintenance or promotion effect compared to general food, and health supplement food refers to food for health supplement purposes. In some cases, the terms functional food, health food, and health supplement are used interchangeably. The food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills.

As a specific example of such a functional food, the composition can be used to produce processed food that preserves the characteristics of agricultural products, livestock products, or marine products while modifying them and improving storage properties.

The health functional food composition of the present invention can also be manufactured in the form of nutritional supplements and food additives, and is intended for consumption by humans or animals, including livestock.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , can be manufactured by adding Euscapic acid to vegetable proteins, retort foods, frozen foods, and various seasonings (e.g. soybean paste, soy sauce, sauce, etc.).

In addition, nutritional supplements are not limited to this, but can be manufactured by adding Euscapic acid to capsules, tablets, pills, etc.

In addition, the health functional food is not limited to this, but for example, the Euscapic acid can be prepared in the form of tea, juice, and drinks and consumed by liquefying, granulating, encapsulating, and powdering so that it can be consumed (health beverage). You can. Additionally, in order to use the Euscapic acid in the form of a food additive, it can be prepared and used in powder or concentrate form. In addition, the Euscapic acid can be prepared in the form of a composition by mixing it with known active ingredients known to be effective in improving muscle function and/or preventing, improving or treating muscle diseases.

When the food composition of the present invention is used as a health drink composition, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

Euscapic acid may be contained as an active ingredient in a food composition for improving muscle function and/or preventing or ameliorating muscle disease, and the amount is an amount effective to obtain the above effect, for example, 0.01 to 100% based on the total weight of the entire composition. It is preferable to use % by weight, but it is not particularly limited thereto.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent. In addition, the health food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

A second aspect of the present invention relates to a pharmaceutical composition for improving muscle function or preventing or treating muscle disease, comprising euscapic acid or a pharmaceutically acceptable salt thereof.

In the pharmaceutical composition of the present invention, the description of the active ingredient Euscapic acid and its effects are the same as those described in the first aspect, and therefore the description thereof is omitted.

In the present invention, the term “treatment” refers to any action in which a muscle disease is improved or beneficially changed by administration of the composition of the present invention.

As used herein, the term "pharmaceutically acceptable" refers to something that is physiologically acceptable and does not typically cause allergic reactions or similar reactions when administered to humans, and the salt includes a pharmaceutically acceptable free acid (free acid). Acid addition salts formed by acid are preferred.

The pharmaceutically acceptable salt may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, Malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxy acetic acid, benzenesulfonic acid, p- Contains toluenesulfonic acid or methanesulfonic acid. Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of hydrochloride or acetate, and more preferably in the form of hydrochloride.

In addition, additional possible salt forms include gaba salt, gabapentin salt, pregabalin salt, nicotinate salt, adipate salt, hemimalonate, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt, or aspartate salt. etc.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, suitable oils, saline solutions, aqueous glucose and glycols, and the like. Additionally, stabilizers and preservatives may be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As for other pharmaceutically acceptable carriers, those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and the parenteral administration method is not limited to this, but is intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, and intraperitoneal. , may be administered intranasally, enterally, topically, sublingually, or rectally.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), antioxidants, bacteriostatic agents, and chelating agents (e.g., EDTA). or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, and such solid preparations may contain at least one excipient, for example, in the pharmaceutical composition of the present invention. , starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose , methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose or gelatin can be mixed. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. In the above, 'transdermal administration' means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

For inhalation administration, the compounds used according to the invention may be packaged in pressurized packs or using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

The pharmaceutical composition of the present invention can provide desirable muscle function improvement and/or muscle disease prevention, improvement or treatment effects when it contains an effective amount of euscapic acid. As used herein, the term "effective amount" refers to an amount that produces a greater response compared to a negative control, preferably improving muscle function and/or preventing or ameliorating muscle disease by inhibiting muscle loss, increasing muscle energy production, or increasing muscle protein. Or it refers to an amount sufficient for treatment. The pharmaceutical composition of the present invention may contain 0.01 to 99.9% of Euscapic acid, and the remaining amount may be comprised of a pharmaceutically acceptable carrier. The effective amount of Euscapic acid included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the patient's condition. For example, based on Euscapic acid, it can be administered in one to several divided doses, preferably 0.001 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day. However, the effective dose for the patient is determined by considering various factors such as the administration route and number of treatments of the pharmaceutical composition, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, taking this into account, a person with ordinary knowledge in the art can determine the appropriate effective dosage of the Euscapic acid according to the specific use for improving muscle function and/or preventing, improving or treating muscle disease. You will be able to. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

Additionally, a third aspect of the present invention relates to a quasi-drug composition for improving muscle function and/or preventing or ameliorating muscle disease, including euscapic acid.

In the quasi-drug composition of the present invention, the description of the active ingredient Euscapic acid and its effects are the same as those described in the first aspect, so the description thereof is omitted.

In the present invention, the term "quasi-drug" refers to fibers, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing diseases in humans or animals, which have a weak effect on the human body or do not directly act on the human body, and devices. Or, it is an article that is not a machine or similar, or an agent used for sterilization, insecticide, and similar purposes to prevent infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases in humans or animals. It may refer to articles used that are not instruments, machines, or devices, and articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding articles that are not instruments, machines, or devices. Additionally, the quasi-drugs may include external skin preparations and personal hygiene products. Preferably, it may be a disinfectant cleaner, shower foam, garnish, wet tissue, detergent, soap, hand wash, or ointment, but is not limited thereto.

When using the composition according to the present invention as a quasi-drug additive, the composition can be added as is or used together with other quasi-drugs or quasi-drug ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Identification of markers related to muscle loss following Euscapic acid treatment

1-1. Sample preparation

10 mg of Euscapic acid (BP1819, Biopurify Phytochemicals) was taken and placed in a 10 ml mass flask (volumetric flask), then methanol was added and sonicated for 30 minutes. Afterwards, the solution was filtered through a 0.45 μm membrane filter, diluted to an appropriate concentration, and used in the experiment.

1-2. Preparation of cells

C2C12 cells (ATCC ^® CRL-1772™), which are muscle stem cells, were cultured in DMEM medium containing 10% rFBS, 100U/ml penicillin, and 100ug/ml streptomycin at 37°C under 5% CO₂ conditions. Cells were placed in a 6-well plate at 3 x 10 ⁵ cells/well and cultured for 24 hours.

1-3. Identification of muscle loss-related markers

After 24 hours, the prepared cells were divided into four groups, and the samples were treated as shown in Table 1 in DMEM medium containing 2% HS, 100U/ml penicillin, and 100ug/ml streptomycin, and cultured for 72 hours.

group Sample processing untreated control
(Normal control, NC) - Dexamethasone treatment group (Control, CTL) Dexamethasone 20 uM Dexamethasone + Euscapic acid treatment group (E50 or E100) Dexamethasone 20 uM + Euscapic Acid 50 uM Dexamethasone 20 uM + Euscapic Acid 100 uM

After 72 hours, the medium was removed, washed with PBS, and RNA was extracted using GeneAll Ribospin II 300 scale (Cat No. 314-103). cDNA was synthesized from the extracted RNA using the SensiFAST cDNA synthesis kit (Cat No. BIO65054). Real-time PCR was performed using AccuPower 2X Greenstar qPCR Master mix (Cat No. K-6254). Primers for the Atrogin-1, MuRF1, and GAPDH genes used in PCR were synthesized by Bioneer (Daejeon, Korea), and the relative expression levels of mRNA for each gene were compared based on GAPDH and are shown in Figure 1.

As a result, as seen in Figure 1, the mRNA expression levels of Atrogin-1 and MuRF1 were significantly increased in the dexamethasone-treated group (CTL) compared to the untreated control group (NC), confirming that muscle loss was induced. On the other hand, in all groups treated with Euscapic acid after dexamethasone treatment, the mRNA expression levels of Atrogin-1 and MuRF1, which were increased by dexamethasone, were decreased in a Euscapic acid concentration-dependent manner. Therefore, Euscapic acid can be effectively used in the treatment of muscle diseases caused by decreased muscle function, muscle wasting, or muscle degeneration through its activity in suppressing muscle loss.

Identification of markers related to muscle protein synthesis, energy metabolism, and muscle differentiation following Euscapic acid treatment

2-1. Sample preparation

10 mg of Euscapic acid (BP1819, Biopurify Phytochemicals) was taken and placed in a 10 ml mass flask (volumetric flask), then methanol was added and sonicated for 30 minutes. Afterwards, the solution was filtered through a 0.45 μm membrane filter, diluted to an appropriate concentration, and used in the experiment.

2-2. Preparation of cells

C2C12 cells (ATCC ^® CRL-1772™), which are muscle stem cells, were cultured in DMEM medium containing 10% rFBS, 100U/ml penicillin, and 100ug/ml streptomycin at 37°C under 5% CO₂ conditions. Cells were placed in a 6-well plate at 3 x 10 ⁵ cells/well and cultured for 24 hours.

2-3. Identification of markers related to muscle protein synthesis, energy metabolism and muscle differentiation

After 24 hours, the prepared cells were divided into three groups, and the samples were treated as shown in Table 2 in DMEM medium containing 2% HS, 100U/ml penicillin, and 100ug/ml streptomycin, and then cultured for 24 hours.

group Sample processing untreated control
(Normal control, NC) - Euscapic acid treated group (E50 or E100) Euscapic Acid 50 uM Euscapic Acid 100 uM

After 24 hours, the medium was removed, washed with PBS, and RNA was extracted using GeneAll Ribospin II 300 scale (Cat No. 314-103). cDNA was synthesized from the extracted RNA using the SensiFAST cDNA synthesis kit (Cat No. BIO65054). Real-time PCR was performed using AccuPower 2X Greenstar qPCR Master mix (Cat No. K-6254). Primers for the mTOR, PGC-1α, MyoD, Myogenin, and GAPDH genes used in PCR were synthesized by Bioneer (Daejeon, Korea), and the relative expression level of mRNA for each gene was compared based on GAPDH, as shown in Figure 2. indicated.

As a result, as seen in Figure 2, the mRNA expression levels of mTOR, which is related to muscle protein synthesis, and PGC-1α, which is related to energy metabolism, were significantly increased in a concentration-dependent manner in the group treated with euscapic acid compared to the untreated control group (CTL). And the mRNA expression of MyoD and Myogenin, which are related to muscle differentiation, were also significantly increased in a concentration-dependent manner. Therefore, Euscapic acid can be effectively used in the treatment of muscle diseases caused by decreased muscle function, muscle wasting, or muscle degeneration through its activity in increasing muscle energy production and muscle protein synthesis.

Claims (9)

A food composition for improving skeletal muscle function or preventing or improving musculoskeletal diseases, comprising Euscaphic acid or a foodologically acceptable salt thereof. The food composition for improving skeletal muscle function or preventing or improving musculoskeletal disease according to claim 1, wherein the musculoskeletal disease is a musculoskeletal disease caused by decreased muscle function, muscle wasting, or muscle degeneration. The method of claim 1, wherein the musculoskeletal disease is at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, and sarcopenia. A food composition for improving skeletal muscle function or preventing or improving musculoskeletal diseases. The food composition for improving skeletal muscle function or preventing or improving musculoskeletal diseases according to claim 1, which exhibits one or more of the following effects:
i) effect of inhibiting muscle loss;
ii) the effect of increasing muscle energy production; and
iii) Effect of increasing muscle protein synthesis. According to claim 1, wherein the food composition is a health functional food composition, a food composition for improving skeletal muscle function or preventing or improving musculoskeletal diseases. A pharmaceutical composition for improving skeletal muscle function or preventing or treating musculoskeletal diseases, comprising Euscaphic acid or a pharmaceutically acceptable salt thereof. The pharmaceutical composition for improving skeletal muscle function or preventing or treating musculoskeletal diseases according to claim 6, wherein the musculoskeletal disease is a muscle disease caused by decreased muscle function, muscle wasting, or muscle degeneration. The method of claim 6, wherein the musculoskeletal disease is at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, and sarcopenia. Pharmaceutical composition for improving skeletal muscle function or preventing or treating musculoskeletal diseases. The pharmaceutical composition for improving skeletal muscle function or preventing or treating musculoskeletal diseases according to claim 6, which exhibits one or more of the following effects:
i) effect of inhibiting muscle loss;
ii) the effect of increasing muscle energy production; and
iii) Effect of increasing muscle protein synthesis.