Composition for the prevention or treatment of muscle diseases containing licorice extract or compounds isolated therefrom as an active ingredient

Abstract

The present invention relates to a composition for preventing or treating muscle diseases, and more specifically, provides a pharmaceutical composition and a functional health food composition for preventing or treating muscle diseases, each containing as an active ingredient a licorice extract or a fraction thereof, or a compound isolated therefrom or a pharmaceutically acceptable salt thereof. The licorice extract or a fraction thereof, or the compound or a salt thereof according to the present invention can promote myoblast proliferation, myotube formation, and differentiation into muscle cells. Furthermore, the licorice extract or a fraction thereof, or the compound or a salt thereof has excellent effects in regenerating damaged muscles, and therefore various muscle diseases can be effectively prevented, improved, or treated using the composition.

Description

[Technical Field]

[0001] The present invention relates to a composition for the prevention or treatment of muscle diseases, which contains licorice extract or a compound isolated therefrom as an active ingredient. [Background technology]

[0002] Muscle is a vital component of the human body, a tissue expressed by mesoderm stem cells. Making up about 40% of the body, muscles are supported by bones and ligaments, and consist of bundles of muscle fibers that move with each other, changing cell size to induce contraction. Muscles are classified into skeletal muscles, cardiac muscles, and visceral muscles, each generating force, inducing movement, and protecting bodily organs such as bones, joints, and internal organs. Furthermore, muscles possess regenerative capabilities; when damaged, they can regenerate to their original contractile and relaxed state after being degenerated by satellite cells and their surrounding environment.

[0003] Muscle diseases are caused by congenital genetic or environmental factors, and in recent years, with the aging society and the trend of increased life expectancy, diseases related to muscle loss have also been increasing. Human muscle mass decreases by more than 1% each year from age 40, and by age 80, it has decreased by about 50% of its maximum muscle mass. Muscle loss in old age is recognized as the most important cause of overall decline in physical function. Such muscle diseases are steadily increasing worldwide compared to the past.

[0004] However, muscular diseases are difficult to diagnose accurately because their causes are more diverse than those of other diseases. The symptoms and severity of the disease also vary depending on the type of muscular disease, and many of them are rare diseases whose precise mechanisms have not been clarified. Muscular diseases progress rapidly, and as the disease progresses, patients suffer to the point where they find it difficult to carry out daily activities on their own. However, there are currently very few treatments for the underlying related diseases. [Overview of the project] [Problems that the invention aims to solve]

[0005] An object of the present invention is to provide a composition for preventing or treating muscle diseases, which contains an extract of a natural product or a compound separated therefrom having an excellent effect on treating muscle diseases as an active ingredient.

Means for Solving the Problems

[0006] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating muscle diseases, which contains a licorice extract or a fraction thereof as an active ingredient.

[0007] The present invention provides a health functional food composition for preventing or improving muscle diseases, which contains a licorice extract or a fraction thereof as an active ingredient.

[0008] The present invention provides a pharmaceutical composition for preventing or treating muscle diseases, which contains a compound represented by any one of the following chemical formulas 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] <Chemical Formula 1>

[0010]

Chem.

[0011] <Chemical Formula 2>

[0012]

Chem.

[0013] <Chemical Formula 3>

[0014]

Chem.

[0015] Furthermore, the present invention provides a health functional food composition for preventing or improving muscle diseases, which contains the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Effects of the Invention

[0016] The licorice extract or fraction thereof according to the present invention, compounds isolated therefrom, or pharmaceutically acceptable salts thereof promote the proliferation of myoblasts, the formation of myotubes, and the differentiation of myoblasts into muscle cells. Furthermore, it has an excellent effect in regenerating damaged muscles. Therefore, a pharmaceutical composition or health functional food composition containing the licorice extract or fraction thereof, compounds isolated therefrom, or salts thereof according to the present invention as an active ingredient can effectively prevent, improve, or treat various muscle diseases.

[0017] Furthermore, since the composition utilizes natural products, it has the advantage of having few side effects and being safe. [Brief explanation of the drawing]

[0018] [Figure 1] This diagram schematically illustrates a method for producing licorice extract or its fractions according to one embodiment of the present invention. [Figure 2] The following are observational results of myoblast (C2C12) proliferation and differentiation after treatment with licorice hot water extract: a is an image and graph showing cell proliferation according to the concentration of licorice hot water extract confirmed by the MTT method; b is an image showing the changes after treatment with licorice hot water extract after scratching the cell surface; and c is the result of fusion index, real-time PCR, Western blotting, and immunostaining, which can confirm whether or not myoblasts differentiate into muscle cells. [Figure 3] The following is the result of analyzing the muscles of mice after ingestion of licorice hot water extract: a is a table showing the body weight and muscle loss rate of the mice, b is the Western blot result, c is a graph that quantifies the intensity of the bands obtained from the Western blot, and d is an image showing the changes in protein expression using immunohistochemistry. [Figure 4]The results of determining whether or not myoblast proliferation occurred after treatment with a fraction of licorice extract are shown, where a is an image and graph of the MTT analysis of myoblasts, and b is an image showing the changes after scratching the cell surface and then treating with the fraction. [Figure 5] The images show the results of observing myoblast differentiation after treatment with a fraction of licorice extract, where a is a graph showing the myotube formation image and lysis index, and b and c show the changes in the expression of muscle differentiation / muscle atrophy genes and proteins after the treatment with the fraction. [Figure 6] The chemical structure of the final single substance separated from the ethyl acetate (SiO) fraction according to one embodiment of the present invention is shown. [Figure 7] The following are observation results of myoblast proliferation and differentiation by treatment with a single final substance: a is a graph showing changes in cell proliferation after treatment with 10 final substances isolated according to one embodiment of the present invention; b is a graph showing the myotube formation image and melting index; c is a graph showing changes in cell proliferation after treatment with 3 purchased final substances; and d is a graph showing the myotube formation image and melting index. [Figure 8] The following is an analysis of muscle changes after mice were given liquiritigenin, one of the purchased final substances. Graph a shows body weight, muscle mass, and muscle loss rate; graph b shows H&E staining results; and graph c shows measured muscle diameter (μm). [Modes for carrying out the invention]

[0019] The present invention will be described in detail below.

[0020] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, containing licorice extract or a fraction thereof as an active ingredient.

[0021] Licorice (Glycyrrhiza uralensis Fischer) is a medicinal plant belonging to the legume family (Fabaceae) of the order Rosales, and can be collected from nature, cultivated, or purchased from a commercial source.

[0022] Licorice can be used as is, either as a root or rhizome, or after removing the outer layer. Preferably, dried licorice root can be used.

[0023] In this specification, “extract” means a substance obtained by extracting components of a natural product, regardless of the extraction method, extraction solvent, extracted components, or form of the extract, and may include any substance that can be obtained by processing or treating a substance obtained by extracting components of a natural product using individual methods after extraction.

[0024] The licorice extract may be extracted by conventional methods in the art, such as hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., and these may be performed individually or in combination of two or more methods.

[0025] In the present invention, the licorice extract may be obtained by extracting with water, C1-C4 alcohols, or a mixture thereof. Preferably, the licorice is placed in 15-25 times, more preferably 20 times, distilled water relative to the weight of the licorice, and the extract is obtained by heating at 110-120°C, more preferably 115°C, for 2-4 hours, more preferably 3 hours, and is a hot water extract, but is not limited thereto.

[0026] In the present invention, the fraction of the licorice extract may be obtained by fractionating the licorice extract, preferably the hot water extract of licorice, with one or more solvents selected from the group consisting of dichloromethane, ethyl acetate, and n-butanol, and more preferably by fractionating with ethyl acetate, resulting in an ethyl acetate fraction of the hot water extract of licorice.

[0027] According to one embodiment of the present invention, the hot water extract of licorice was suspended in distilled water, then sequentially partitioned into dichloromethane, ethyl acetate, and n-butanol, and each solution was evaporated to obtain a dichloromethane fraction, an ethyl acetate fraction, and an n-butanol fraction.

[0028] In the present invention, the licorice extract or its fraction can promote the proliferation of myoblasts, the formation of myotubes, and the differentiation of myoblasts into muscle cells. Furthermore, the licorice extract or its fraction can suppress muscle protein breakdown and muscle atrophy, and regenerate damaged muscles.

[0029] The licorice extract or its fraction can increase the expression of one or more genes or proteins selected from the group consisting of MYOG, MYOD, MYL2, and Pax7, which are muscle differentiation or muscle regeneration-related factors, and can decrease the expression of one or more genes or proteins selected from the group consisting of MSTN, MuRF1, Atrogin 1, and Nitrotyrosine, which are muscle protein degradation or muscle atrophy-related factors.

[0030] MYOD initiates the expression of muscle-specific genes, inducing the differentiation of muscle satellite cells into myoblasts. Induction of myogenin (MYOG) expression by MYOD activity is the most important element in myoblast fusion and is involved in the formation of myotubes. Muscle fibers formed through this process bundle together to ultimately form muscle.

[0031] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising a compound represented by any one of the following chemical formulas 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0032] <Chemical formula 1>

[0033] [ka]

[0034] <Chemical formula 2>

[0035] [ka]

[0036] <Chemical formula 3>

[0037] [ka]

[0038] The compounds of chemical formulas 1 to 3 are active ingredients isolated from licorice, and preferably, chemical formula 1 is liquiritigenin, chemical formula 2 is tetrahydroxymethoxychalcone, and chemical formula 3 is licochalcone B.

[0039] The aforementioned compound or salt thereof may be obtained by direct separation or extraction from natural products using methods well known in the art, by chemical synthesis, or by selecting commercially available products, but the method or substance is not particularly limited.

[0040] In the present invention, the compound or salt thereof may be obtained by separating a fraction obtained by fractionating a licorice extract, preferably a licorice methanol extract, extracted with water, a C1-C4 alcohol, or a mixed solvent thereof, with dichloromethane or ethyl acetate, and preferably, by separating a fraction obtained by sequentially partitioning the licorice methanol extract into dichloromethane and ethyl acetate, but is not limited thereto.

[0041] More preferably, the compound or salt thereof may be obtained by reflux extracting the dried roots of licorice with 100% methanol, suspending the methanol extract in distilled water, partitioning it with dichloromethane, removing the dichloromethane from the water fraction, and then partitioning it with ethyl acetate to obtain an ethyl acetate fraction from which it can be separated.

[0042] More specifically, the compound of chemical formula 1 or its salt was subjected to silica gel column chromatography with ethyl acetate fractions hexane-ethyl acetate-methanol (hexane-Â-MeOH, 5.5:1:0.1, 3:1:0.1, v / v), chloroform-acetone-methanol (CHCl3-Acetone-MeOH, 3:1:0.1, v / v), and chloroform-methanol-water (CHCl3-MeOH-H2O, 5:1:0.1, 3:1:0.1, v / v) to obtain five fractions (Fr.E1~E5), of which fraction E2 was subjected to SNAP Ultra Using a C18 cartridge, the mixture was separated by MPLC using a methanol-water (MeOH-Water, 34-75% MeOH, v / v) gradient to obtain 16 fractions (Fr.E2A-E2P). Of these, fraction E2C may be separated using silica gel column chromatography with a hexane-ethyl acetate-methanol (hexane-siRNA-MeOH, 3:1:0.1, v / v) solvent, but is not limited to this method.

[0043] The compound of chemical formula 2 or its salt may be obtained by separating fraction E3 from the five fractions (Fr.E1~E5) separated from the ethyl acetate fraction into 15 fractions (Fr.E3A~E3O) using MPLC with a methanol-water (MeOH-Water, 33~80% MeOH, v / v) gradient using a SNAP Ultra C18 cartridge, and then separating fraction E3E from these using MPLC with a hexane-ethyl acetate-methanol (hexane-siRNA-MeOH, 28~36% siRNA-MeOH, 1:0.1, v / v) gradient using a SNAP KP-SIL cartridge, but is not limited to this method.

[0044] Furthermore, the compound of chemical formula 3 or its salt may be obtained by separating fraction E3F from the 15 fractions separated (Fr.E3A~E3O) into 5 fractions (Fr.E3FA~E3FE) using MPLC with a SNAP KP-SIL cartridge under a gradient of hexane-ethyl acetate-methanol (hexane-siRNA-siRNA-MeOH, 20-34% siRNA-MeOH, 1:0.1, v / v), and then separating fraction E3FB from these using TLC (silica gel 60 F254, hexane-siRNA-siRNA-MeOH, 1:1:0.2, v / v), but is not limited to this.

[0045] The compound can be used in the form of a pharmaceutically or food-safe salt within the range having the same efficacy.

[0046] In this specification, “pharmaceutically or food-safe” means that the composition is not toxic to cells or humans exposed to it.

[0047] The salt may be used in either a pharmaceutically or food-safe basic salt or an acidic salt. The basic salt may be used in either an organic or inorganic basic salt and may be selected from the group consisting of sodium salts, potassium salts, calcium salts, lithium salts, magnesium salts, cesium salts, aminium salts, ammonium salts, triethylaminium salts, and pyridinium salts.

[0048] Acid salts are useful as acid addition salts formed from free acids. Inorganic and organic acids can be used as free acids. Examples of inorganic acids include hydrochloric acid, bromate, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid, and nitric acid. Examples of organic acids include citric acid, acetic acid, maleic acid, malic acid, fumaric acid, glycolic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, gluconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and stearic acid, but are not limited to these, and may include any salts formed from various inorganic and organic acids commonly used in this industry.

[0049] Furthermore, the compound may include any salt, hydrate, solvate, derivative, etc., that can be produced by conventional methods, in addition to pharmaceutically or food-safe salts. Addition salts can be produced by conventional methods, such as by dissolving them in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and then adding an excess amount of organic base or an aqueous solution of inorganic base, followed by precipitation or crystallization. Alternatively, the addition salt can be obtained by evaporating the solvent or excess base from this mixture and then drying it, or by suction filtration of the precipitated salt.

[0050] In the present invention, the compound or a salt thereof can promote the proliferation of myoblasts, the formation of myotubes, and the differentiation of myoblasts into muscle cells. Furthermore, the compound or a salt thereof can suppress muscle protein breakdown and muscle atrophy, and regenerate damaged muscles.

[0051] In the present invention, the muscle disease is a muscle disease resulting from decreased muscle function, muscle wasting, or muscle degeneration, and may be one or more selected from the group consisting of, for example, muscular atrophy, muscular dystrophy, sarcopenia, myopathy, myasthenia, and muscular injury, but is not limited thereto.

[0052] In this specification, “prevention” means any action that suppresses or delays the onset of a muscle disease or at least one symptom thereof by administering a pharmaceutical composition or health functional food composition according to the present invention. It also includes treatment of a subject whose disease is improving in order to prevent or prevent recurrence.

[0053] In this specification, “treatment” means any action that improves or beneficially alters the symptoms of a muscle disease or at least one of its symptoms, such as alleviating, reducing, or eliminating the symptoms of the muscular disease or at least one of its symptoms, by administering the pharmaceutical composition according to the present invention.

[0054] In this specification, “pharmaceutical composition” means a composition administered for a specific purpose, and for the purposes of the present invention, it means a composition administered to prevent or treat a muscle disease or at least one symptom thereof.

[0055] The pharmaceutical compositions according to the present invention can be manufactured by conventional methods in the pharmaceutical field. The pharmaceutical compositions may be compounded with a suitable pharmaceutically acceptable carrier depending on the dosage form, and may further contain, if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The suitable carriers, etc., do not inhibit the activity and properties of the licorice extract or fraction thereof, compounds separated therefrom, or salts thereof according to the present invention, and may be individually selected depending on the administration form and dosage form.

[0056] The pharmaceutical compositions according to the present invention may be applied in any dosage form, and more specifically, they can be used in oral dosage forms, topical preparations, suppositories, and parenteral dosage forms of sterile injection solutions by conventional methods.

[0057] Among the oral dosage forms, the solid dosage forms include tablets, pills, powders, granules, gels, and capsules, and can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. Furthermore, in the case of capsule dosage forms, a liquid carrier such as fatty oil may be included in addition to the substances mentioned above.

[0058] Among the oral dosage forms mentioned above, liquid dosage forms include suspensions, solution-resistant agents, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included.

[0059] The parenteral dosage forms may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Twin 61, cocoa butter, lauric acid butter, and glycerol gelatin. However, the use of any suitable formulation known in the art is not limited thereto.

[0060] Furthermore, the pharmaceutical composition according to the present invention may contain additional calcium or vitamins to enhance its therapeutic effect.

[0061] In the pharmaceutical composition according to the present invention, the pharmaceutical composition can be administered in a pharmaceutically effective amount.

[0062] In this specification, “pharmaceutically effective amount” means an amount that is sufficient to treat a disease without causing side effects, based on a reasonable benefit-to-risk ratio applicable to medical treatment.

[0063] The effective dose level of the pharmaceutical composition may be determined individually based on the intended use, the patient's age, sex, weight and health status, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the method of administration, the time of administration, the route of administration and the elimination ratio, the duration of treatment, the drugs included in the formulation or used concurrently, and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg, can be administered once to several times a day. The above dosage does not limit the scope of the present invention in any respect.

[0064] The pharmaceutical composition according to the present invention can be administered to any animal that may develop muscle disease, and such animals may include, for example, primates including humans, as well as livestock such as cattle, pigs, horses, and dogs.

[0065] The pharmaceutical composition according to the present invention can be administered via an appropriate route of administration depending on the formulation, and can be administered orally or parenterally in any way that reaches the target tissue. The method of administration is not particularly limited and can be administered by conventional methods such as oral, rectal or intravenous, intramuscular, topical application to the skin, intra-respiratory inhalation, intrauterine dura mater, or intravascular (intracere-broventricular) injection.

[0066] The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of muscle diseases, or in combination with surgery or other drug therapies.

[0067] The present invention provides a health functional food composition for preventing or improving muscle diseases, which contains licorice extract or a fraction thereof as an active ingredient.

[0068] Preferably, the licorice extract may be a hot water extract of licorice, and the fraction of the licorice extract may be an ethyl acetate fraction of the hot water extract of licorice.

[0069] The licorice extract or its fraction can promote the proliferation of myoblasts, the formation of myotubes, and the differentiation of myoblasts into muscle cells. Furthermore, the licorice extract or its fraction can suppress muscle protein breakdown and muscle atrophy, regenerate damaged muscles, and can be used as a health functional food composition for the prevention or improvement of muscle diseases.

[0070] The characteristics corresponding to this are as described above.

[0071] The present invention provides a health functional food composition for preventing or improving muscle diseases, which contains a compound represented by any one of the following chemical formulas 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0072] <Chemical formula 1>

[0073] [ka]

[0074] <Chemical formula 2>

[0075] [ka]

[0076] <Chemical formula 3>

[0077] [ka]

[0078] Preferably, the compound or salt thereof may be separated from a fraction obtained by fractionating a licorice methanol extract with dichloromethane or ethyl acetate, and may include liquiritigenin, tetrahydroxymethoxychalcone, or licochalcone B.

[0079] The aforementioned compound or its salt can promote the proliferation of myoblasts, the formation of myotubes, and the differentiation of myoblasts into muscle cells. Furthermore, the aforementioned compound or its salt can suppress muscle protein breakdown and muscle atrophy, regenerate damaged muscles, and can be used as a health functional food composition for the prevention or improvement of muscle diseases.

[0080] The characteristics corresponding to this are as described above.

[0081] In this specification, “improvement” means any action that, by ingesting the health functional food composition according to the present invention, alleviates, reduces, or eliminates a muscle disease or at least one of its symptoms, thereby improving or beneficially altering the symptoms.

[0082] In this specification, “health functional foods” includes foods manufactured and processed using raw materials and components that have functional properties useful to the human body as defined in Act No. 6727 concerning Health Functional Foods, and refers to foods with high medical and therapeutic effects that are processed to efficiently demonstrate biological regulatory functions such as prevention of muscle diseases, biological defense, immunity, and recovery, for the purposes of the present invention, in addition to providing nutrition.

[0083] The health functional food according to the present invention can be manufactured as a powder, granules, tablets, capsules, syrup, or beverage for the purpose of preventing or improving muscle diseases. There are no restrictions on the form that the health functional food can take, and it may be formulated in the same manner as the pharmaceutical composition and used as a functional food, or it may be added to various foods.

[0084] The aforementioned health functional foods may include any food in the ordinary sense. For example, beverages and drinks, fruits and their processed products (canned fruit, jam, etc.), fish, meats and their processed products (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.), and any functional foods in the ordinary sense. Foods used as animal feed may also be included.

[0085] The functional food composition according to the present invention may be manufactured further by including food-grade, food-acceptable food additives and other appropriate auxiliary components commonly used in the industry. Unless otherwise specified, suitability as a food additive can be determined according to the standards and criteria for the item in question, based on the general principles and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety. Examples of items listed in the aforementioned Food Additives Code include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar dye preparations.

[0086] The aforementioned other auxiliary components may further contain, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, coloring agents, pectinic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents. In particular, the natural carbohydrates can include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.

[0087] The effective volume of the licorice extract or its fraction, the compound separated therefrom, or its salt contained in the health functional food according to the present invention may be appropriately adjusted depending on the intended use, such as the prevention or improvement of muscle diseases.

[0088] The aforementioned health functional food composition has the advantage of being made from food ingredients, having no side effects that may occur with long-term use of general medicines, and being highly portable, making it suitable for consumption as an adjunct for preventing or improving muscle diseases.

[0089] The present invention provides a reagent composition for myoblast proliferation containing licorice extract or a fraction thereof as an active ingredient.

[0090] The present invention provides a reagent composition for promoting the differentiation of myoblasts into muscle cells, which contains licorice extract or a fraction thereof as an active ingredient.

[0091] The present invention provides a reagent composition for myoblast proliferation containing a compound represented by any one of the following chemical formulas 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0092] <Chemical formula 1>

[0093] [ka]

[0094] <Chemical formula 2>

[0095] [ka]

[0096] <Chemical formula 3>

[0097] [ka]

[0098] The present invention provides a reagent composition for promoting the differentiation of myoblasts into muscle cells, which contains the above compound or a salt thereof as an active ingredient.

[0099] Furthermore, the present invention provides a method for myoblast proliferation that includes the step of treating an animal other than a human with licorice extract or a fraction thereof.

[0100] The present invention provides a method for promoting the differentiation of myoblasts into muscle cells, which includes the step of treating a non-human animal with licorice extract or a fraction thereof.

[0101] The present invention provides a method for myoblast proliferation that includes the step of treating an animal other than a human with a compound represented by any one of the following chemical formulas 1 to 3 or a pharmaceutically acceptable salt thereof.

[0102] <Chemical formula 1>

[0103] [ka]

[0104] <Chemical formula 2>

[0105] [ka]

[0106] <Chemical formula 3>

[0107] [ka]

[0108] The present invention provides a method for promoting the differentiation of myoblasts into muscle cells, comprising the step of treating an animal other than a human with the compound or a salt thereof.

[0109] The characteristics corresponding to this are as described above. [Modes for carrying out the invention]

[0110] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. The examples of the present invention are provided to give a more complete explanation of the present invention to a person of average knowledge in the industry.

[0111] <Example 1> Preparation of licorice extract and fractions 1.Material preparation The dried roots of licorice (Glycyrrhiza uralensis Fischer) were purchased from dried rhizomes at the Gwangmyeong Herb Center. All plant specimens are stored at the KM Application Center Herb Bank of the Korea Institute of Oriental Medicine.

[0112] 2. Production of licorice hot water extract To prepare licorice hot water extract, 50.0 g of dried licorice pieces were placed in 1,000 mL of distilled water and heated at 115°C for 3 hours to extract the extract. After extraction, the mixture was filtered using standard testing sieves (150 μm) and freeze-dried. The freeze-dried extract powder was dissolved in tertiary distilled water and left at 4°C for 24 hours. After 24 hours, the mixture was centrifuged at 5000 g for 5 minutes, and the supernatant was transferred to a new tube and stored at -20°C.

[0113] 3. Preparation of fractions from licorice hot water extract Figure 1 is a schematic diagram illustrating a method for producing licorice extract or its fractions according to one embodiment of the present invention.

[0114] As shown in Figure 1, the licorice hot water extract (10.0 g) produced in step 2 of Example 1 was suspended in distilled water and then partitioned into dichloromethane (DCM, CH2Cl2), ethyl acetate (siRNA, EA), and n-butanol (BuOH). Next, each solution was evaporated under reduced pressure at 45°C to obtain fractions of dichloromethane (99.6 mg), ethyl acetate (333.0 mg), and n-butanol (1037.0 mg), respectively.

[0115] 4. Extraction and separation of the final substance 4-1. Extraction and Separation Methods 1 H and 1313C NMR spectra were recorded at 600 MHz using a BRUKER AVANCE III HD 600 with tetramethylsilane (TMS) as the internal standard. Medium-pressure liquid chromatography (MPLC) was performed using an Isolera One (Biotage, Uppsala, Sweden) with SNAP KP-SIL and SNAP Ultra C18 cartridges. Column chromatography was performed using silica gel (Kieselgel 60, 70-230 and 230-400 mesh, Merck, Darmstadt, Germany), YMC C18 resin, and thin-layer chromatography (TLC). The samples were washed on silica gel 60 F254 and RP-18 F254S plates (0.25 mm, Merck, Darmstadt, Germany), visualized with UV light (254 and 365 nm), and stained with 10% sulfuric acid (H2SO4).

[0116] 4-2. Extraction and Confirmation of the Final Substance As shown in Figure 1, first, dried licorice roots (Glycyrrhiza uralensis, 4.2 kg) were reflux-extracted three times with 100% methanol (MeOH) (15 L each time). The methanol extract (957.0 g) was suspended in distilled water and then partitioned into dichloromethane (DCM). After removing the dichloromethane solution from the water fraction, the extract was partitioned into ethyl acetate (SiO2). The ethyl acetate solution was then evaporated under reduced pressure at 45°C to obtain the ethyl acetate fraction (137.0 g).

[0117] The ethyl acetate fraction was subjected to silica gel column chromatography with hexane-ethyl acetate-methanol (hexane-Â-MeOH, 5.5:1:0.1, 3:1:0.1, v / v), chloroform-acetone-methanol (CHCl3-Acetone-MeOH, 3:1:0.1, v / v), and chloroform-methanol-water (CHCl3-MeOH-H2O, 5:1:0.1, 3:1:0.1, v / v) to obtain five fractions (Fr.E1~E5) and a semi-crystalline solid (Fr.EC).

[0118] Fraction E2 (4.9 g) was separated by MPLC using a SNAP Ultra C18 cartridge under a methanol-water (MeOH-Water, 34-75% MeOH, v / v) gradient, and 16 fractions (Fr. E2A to E2P) containing compound 1 (23.9 mg) were obtained.

[0119] - Compound 1 (4-hydroxybenzoic acid): White powder, C7H6O3; 1 H NMR (600 MHz, MeOD-d4) δ 7.67 (2H, d, J = 7.2 Hz, H-2, 6), 6.61 (2H, d, J = 7.2 Hz, H-3, 5). 13 C NMR (600 MHz, MeOD-d4) δ 170.1 (C=O), 163.3 (C-4), 132.9 (C-2, 6), 122.7 (C-1), 116.0 (C-3, 5).

[0120] The fraction E2C (243.5 mg) was separated using silica gel column chromatography with hexane-ethyl acetate-methanol (hexane-siRNA-MeOH, 3:1:0.1, v / v) solvent to obtain compound 2 (108.5 mg).

[0121] - Compound 2 (Liquiritigenin): Colorless needle crystals, C 15 H 12 O4; 1 H NMR (600 MHz, MeOD-d4) δ 7.68 (1H, d, J = 8.5 Hz, H-5), 7.28 (2H, d, J = 7.6 Hz, H-2´, 6´), 6.77 (2H, d, J = 7.8 Hz, H-3´, 5´), 6.45 (1H, d, J = 6.5 Hz, H-6), 6.31 (1H, s, H-8), 5.32 (1H, d, J = 12.6 Hz, H-2), 3.00 (1H, t, J = 15.0 Hz, H-3a), 2.64 (1H, d, J = 16.8 Hz, H-3b). 13 C NMR (600 MHz, MeOD-d4) δ 192.1 (C=O), 165.3 (C-7), 164.1 (C-9), 157.5 (C-4´), 129.9 (C-1´), 128.4 (C-5), 127.6 (C-2´, 6´), 114.9 (C-3´, 5´), 113.5 (C-10), 110.3 (C-6), 102.4 (C-8), 79.6 (C-2), 43.5 (C-3).

[0122] Fraction E2D (420.3 mg) was isolated by MPLC using a SNAP KP-SIL cartridge with a gradient of hexane-EtOAc-MeOH (17 - 25% EtOAc-MeOH, 1:0.1, v / v) to afford Compound 3 (51.7 mg) and Compound 4 (15.4 mg).

[0123] - Compound 3 ((R)-(-)-Vestitol): Colorless crystals, C 16 H 16 O4; 1H NMR (600 MHz, Acetone-d6) δ 7.05 (1H, d, J = 7.7 Hz, H-6´), 6.89 (1H, d, J = 7.1 Hz, H-5), 6.50 (1H, s, H-3´), 6.42 (1H, d, J = 6.1 Hz, H-5´), 6.36 (1H, d, J = 5.7 Hz, H-6), 6.28 (1H, s, H-8), 4.23 (1H, d, J = 6.7 Hz, H-2), 3.98 (1H, t, J = 9.5 Hz, H-2), 3.72 (3H, s, -OCH3), 3.47 (1H, m, H-3), 2.99 (1H, m, H-4), 2.82 (1H, d, J = 14.8 Hz, H-4). 13 C NMR (600 MHz, Acetone-d6) δ 160.3 (C-4´), 157.4 (C-7), 156.6 (C-2´), 156.1 (C-9), 131.0 (C-5), 128.7 (C-6´), 120.9 (C-1´), 114.3 (C-10), 108.7 (C-6), 105.6 (C-5´), 103.6 (C-8), 102.4 (C-3´), 70.4 (C-2), 55.3 (-OCH3), 32.6 (C-3), 31.0 (C-4).

[0124] - Compound 4 (Isoliquiritigenin): Yellow crystals, C 15 H 12 O4; 1 H NMR (600 MHz, MeOD-d4) δ 7.90 (1H, d, J = 8.2 Hz, H-6´), 7.73 (1H, d, J = 15.2 Hz, H-β), 7.56 (3H, s, H-2, 6, α), 6.79 (2H, d, J = 8.3 Hz, H-3, 5), 6.36 (1H, dd, J = 8.6, 1.6 Hz, H-5´), 6.24 (1H, s, H-3´). 13C NMR (600 MHz, MeOD-d4) δ 193.5 (C=O), 167.5 (C-4´), 166.3 (C-2´), 161.5 (C-4), 145.6 (C-β), 133.3 (C-6´), 131.8 (C-2, 6), 127.8 (C-1), 118.2 (C-α), 116.9 (C-3, 5), 114.7 (C-1´), 109.1 (C-5´), 103.8 (C- 3´).

[0125] Fraction E2F (95.1 mg) was separated using MPLC with a SNAP KP-SIL cartridge on a hexane-ethyl acetate-methanol (hexane-ÂTED-MeOH, 11-22% ÂTED-MeOH, 1:0.1, v / v) gradient and secured as compound 5 (11.2 mg).

[0126] - Compound 5 (Medicarpin): White amorphous powder, C 16 H 14 O4; 1 H NMR (600 MHz, CDCl3) δ 7.39 (1H, d, J = 7.5 Hz, H-1), 7.13 (1H, d, J = 7.1 Hz, H-7), 6.55 (1H, d, J = 6.0 Hz, H-2), 6.45 (2H, s, H-8, -10), 6.42 (1H, s, H-4), 5.50 (1H, d, J = 5.8 Hz, H-11a), 4.24 (1H, d, J = 5.4 Hz, H-6eq), 3.77 (3H, s, -OCH3), 3.62 (1H, br t, J = 10.4 Hz, H-6ax), 3.54 (1H, d, J = 4.3 Hz, H-6a). 13C NMR (600 MHz, CDCl3) δ 161.2 (C-9), 160.7 (C-10a), 157.1 (C-3), 156.8 (C-4a), 132.3 (C-1), 124.9 (C-7), 119.2 (C-6b), 112.8 (C-11b), 109.9 (C-2), 106.5 (C-8), 103.8 (C-4), 97.0 (C-10), 78.6 (C-11a), 66.6 (C-6), 55.6 (-OCH3), 39.6 (C-6a).

[0127] Fraction E3 (41.6 g) was separated by MPLC using a SNAP Ultra C18 cartridge with a methanol-water (MeOH-Water, 33-80% MeOH, v / v) gradient and secured as 15 fractions (Fr. E3A-E3O). Fraction E3E (140.0 mg) was sequestrated by MPLC using a SNAP KP-SIL cartridge with a hexane-ethyl acetate-methanol (hexane-ÂTED-MeOH, 28-36% ÂTED-MeOH, 1:0.1, v / v) gradient and secured as compound 6 (35.2 mg).

[0128] - Compound 6 (Tetrahydroxymethoxychalcone): Yellow needles, C 16 H 14 O6; 1 H NMR (600 MHz, MeOD-d4) δ 7.89 (1H, d, J = 15.6 Hz, H-β), 7.55 (1H, d, J = 15.6 Hz, H-α), 7.48 (1H, d, J = 6.7 Hz, H-2´), 7.45 (1H, s, H-6´), 7.14 (1H, d, J = 7.9 Hz, H-6), 6.82 (1H, d, J = 7.5 Hz, H-5´), 6.59 (1H, d, J = 7.9 Hz, H-5), 3.78 (3H, s, -OCH3). 13C NMR (600 MHz, MeOD-d4) δ 191.4 (C=O), 152.1 (C-4´), 150.7 (C-4), 149.9 (C-2), 146.5 (C-3´), 140.9 (C-3), 139.6 (C-β), 131.8 (C-1´), 123.4 (C-6´), 121.4 (C-1), 120.5 (C-α), 120.3 (C-6), 116.3 (C-2´), 115.9 (C-5´), 112.7 (C-5), 61.7 (-OCH3).

[0129] Fraction E3F (410.0 mg) was isolated by MPLC using a SNAP KP-SIL cartridge under a hexane-ethyl acetate-methanol (hexane-Â-MeOH, 20-34% Â-MeOH, 1:0.1, v / v) gradient and secured as five fractions (Fr.E3FA~E3FE). Fraction E3FB (17.5 mg) was separated by TLC (silica gel 60 F254, hexane-Â-MeOH, 1:1:0.2, v / v) and secured as compound 7 (5.6 mg).

[0130] - Compound 7 (Licochalcone B): Yellow needles, C 16 H 14 O5; 1 H NMR (600 MHz, MeOD-d4) δ 7.97 - 7.91 (3H, m, H-2´, 6´, β), 7.61 (1H, d, J = 15.7 Hz, H-α), 7.19 (1H, d, J = 7.9 Hz, H-6), 6.85 (2H, d, J = 6.9 Hz, H-3´, 5´), 6.61 (1H, d, J = 7.5 Hz, H-5), 3.81 (3H, s, -OCH3). 13C NMR (600 MHz, MeOD-d4) δ 191.3 (C=O), 163.9 (C-4´), 151.0 (C-4), 149.9 (C-2), 141.0 (C-β), 139.7 (C-3), 132.2 (C-2´, 6´), 131.1 (C-1´), 121.3 (C-1), 120.4 (C-α), 120.3 (C-6), 116.4 (C-3´, 5´), 112.7 (C-5), 61.7 (-OCH3).

[0131] Fraction E5 (20.4 g) was segregated using MPLC with a methanol-water (MeOH-Water, 20-44% MeOH, v / v) gradient using a SNAP Ultra C18 cartridge, and was secured as nine fractions (Fr. E5A-E5I). Fractions E5B-D were combined with a chloroform-methanol-water (CHCl3-MeOH-H2O, 12-20% MeOH-H2O, 1:0.1, v / v) gradient using MPLC with a SNAP KP-SIL cartridge, and secured as five fractions (Fr. E5BA-E5BE). After obtaining a semicrystalline solid from fraction E5BA, it was redistributed with methanol to secure compound 8 (969.0 mg).

[0132] - Compound 8 (Liquiritin): White powder, C 21 H 22 O9; 1 H NMR (600 MHz, DMSO-d6) δ 7.65 (1H, d, J = 7.6 Hz, H-5), 7.44 (2H, s, H-2´, 6´), 7.06 (2H, s, H-3´, 5´), 6.51 (1H, s, H-6), 6.35 (1H, s, H-8), 4.88 (1H, s, H-1´), 3.68 (1H, s, H-6´), 3.10 - 3.50 (6H, m, H-2´, 3´, 4´, 5´, ​​6´, 3), 2.67 (1H, d, J = 15.2 Hz, H-3). 13C NMR (600 MHz, DMSO-d6) δ 190.4 (C=O), 165.1 (C-7), 163.5 (C-9), 157.9 (C-4´), 132.8 (C-1´), 128.8 (C-5), 128.4 (C-2´, 6´), 116.6 (C-3´, 5´), 114.0 (C-10), 111.0 (C-6), 103.0 (C-8), 100.7 (C-1´), 79.1 (C-2), 77.5 (C-5´), 77.0 (C-3´), 73.6 (C-2´), 70.1 (C-4´), 61.1 (C-6´), 43.6 (C-3).

[0133] Fraction E5BB-C was bound and separated with chloroform-methanol-water (CHCl3-MeOH-H2O, 7:1:0.05, 6:1:0.05, and MeOH, v / v) solvent using silica gel column chromatography, and six fractions (Fr.E5BBA~E5BBF) were secured. Fraction E5BBD (220.0 mg) was isolated using MPLC with a SNAP KP-SIL cartridge under a gradient of ethyl acetate-methanol-water (Â-MeOH-H2O, 4~10% MeOH-H2O, 1:0.1, v / v) and secured as compound 9 (113.5 mg).

[0134] - Compound 9 (Liquiritin apioside): Yellow powder, C 26 H 30 O 13 ; 1H NMR (600 MHz, MeOD-d4) δ 7.71 (1H, d, J = 8.3 Hz, H-5), 7.41 (2H, d, J = 6.9 Hz, H-2´, 6´), 7.09 (2H, s, H-3´, 5´), 6.48 (1H, d, J = 6.4 Hz, H-6), 6.34 (1H, s, H-8), 5.44 (1H, br s, H-1´), 5.40 (1H, s, H-2), 4.98 (1H, d, J = 7.0 Hz, H-1´), 3.01 (1H, t, J = 14.5 Hz, H-3), 2.70 (1H, t, J = 16.8 Hz, H-3). 13 C NMR (600 MHz, MeOD-d4) δ 193.2 (C=O), 166.8 (C-7), 165.4 (C-9), 159.1 (C-4´), 134.3 (C-1´), 129.8 (C-5), 128.8 (C-2´, 6´), 117.5 (C-3´, 5´), 115.0 (C-10), 111.8 (C-6), 110.7 (C-1´), 103.8 (C-8), 100.7 (C-1´), 80.7 (C-3´), 80.7 (C-2), 78.6 (C-2´), 78.5 (C-3´), 78.0 (C-2´), 78.0 (C-5´), 75.4 (C-4´), 71.3 (C-4´), 66.0 (C-5´), 62.4 (C-6´), 44.9 (C-3).

[0135] The fraction EC (3.0 g) was separated by MPLC using a SNAP KP-SIL cartridge with a chloroform-methanol-water (CHCl3-MeOH-H2O, 12-16% MeOH-H2O, 1:0.1, 100% acetone, v / v) gradient, and after being set aside as one fraction, it was redispersed with methanol to obtain compound 10 (22.2 mg).

[0136] - Compound 10 (Ononin): White powder, C 22 H 22 O9; 1H NMR (600 MHz, DMSO-d6) δ 8.43 (1H, s, H-2), 8.05 (1H, d, J = 8.5 Hz, H-5), 7.53 (2H, d, J = 7.5 Hz, H-2´, 6´), 7.24 (1H, s, H-8), 7.15 (1H, d, J = 8.1 Hz, H-6), 6.99 (2H, d, J = 7.6 Hz, H-3´, 5´), 5.12 (1H, d, J = 7.8 Hz, H-1´), 3.78 (3H, s, -OCH3), 3.73 (1H, m, C-6´), 3.19 - 3.48 (5H, m, H-2´, 3´, 4', 5', 6'). 13 C NMR (600 MHz, DMSO-d6) δ 175.1 (C-4), 161.9 (C-7), 159.4 (C-4´), 157.5 (C-9), 154.1 (C-2), 130.5 (C-2´, 6´), 127.4 (C-5), 124.4 (C-1´), 123.8 (C-3), 118.9 (C-10), 116.0 (C-6), 114.0 (C-3´, 5´), 103.8 (C-8), 100.4 (C-1´), 77.6 (C-5´), 76.9 (C-3´), 735. (C-2´), 70.7 (C-4´), 61.0 (C-6´), 55.6 (-OCH3).

[0137] <Experimental Example 1> Confirmation of the effects of licorice extract and its fractional treatment on myoblast proliferation, differentiation, and muscle regeneration. 1. Experimental Method 1-1. Observation of growth in C2C12 cell culture and licorice hot water extract, its fractions, or final substance treatment. Mouse myoblast cell line C2C12 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S).

[0138] To verify the effects of licorice hot water extract, its fractions, or the final substance, C2C12 cells (2 × 10⁻¹⁰) were used. 3 Cells were placed in a 12-well cell culture dish and allowed to adhere for 24 hours. Then, the cells were treated with hot water extracts (0, 50, 100, 200 μg / ml), their fractions (25 μg / ml), or the final product (0.5 ng / ml) for 1 day to confirm cell proliferation. The culture medium was changed every two days, and the cells were cultured at 37°C.

[0139] 1-2. Scratch Experiment When C2C12 cells reached 100% growth, a scratch was applied to the cell surface, and the cells were treated with a hot water extract (50 μg / ml) or its fraction (25 μg / ml). After 1 day of culture, the degree of cell recovery was observed.

[0140] 1-3. Verification of myoblast proliferation (MTT method) To verify cell proliferation, the cell culture medium was removed, the cells were washed with DMEM, and 500 μl of MTT reagent (0.5 mg / ml) dissolved in phosphate-buffered saline (PBS) was added to each well. The mixture was left at 37°C for 1 hour. The reaction mixture was removed, and 1000 μl of dimethyl sulfoxide (DMSO) was added to each well. Purple formazan crystals were completely dissolved in DMSO, and the absorbance was measured at 540 nm.

[0141] 1-4. Observation of differentiation of licorice hot water extract, its fractions, and final product treatment. To induce differentiation, when the cells had grown to approximately 70% or more, they were replaced with DMEM (differentiation medium) supplemented with 2% FBS and 1% P / S, treated with hot water extract (100 μg / ml), fraction (25 μg / ml), or final substance (0.25 ng / ml), and then cultured for 2 or 4 days. The medium was changed every two days, and the cells were cultured at 37°C.

[0142] 1-5. Giemsa stain and fusion index The cell culture medium was removed, and the cells were washed with PBS. After washing, a 1:1 volume ratio methanol:PBS reagent was applied and fixed for 2 minutes. Then, a 2:1 volume ratio methanol:PBS reagent was added, and the cells were fixed for another 2 minutes. After 2 minutes, 0.04% Giemsa reagent was added and left for 30 minutes. After 30 minutes, the cells were washed with PBS, observed under a microscope, and three photographs of each cell were taken (300×). From the photographs, the number of fused nuclei in myotubes was counted, and after counting the total number of nuclei in the cells, the percentage value was calculated by dividing the number of fused nuclei by the total number of nuclei in the cells.

[0143] 1-6. RNA extraction and cDNA synthesis TRIzol TM After adding 1 ml of reagent, the cells were pulverized using an ultra-high frequency pulverizer (sonicator). The pulverized sample was centrifuged (12,000 rpm, 10 minutes, 4°C), the supernatant was transferred to a new tube, 200 μl of chloroform was added, and it was left at room temperature for 10 minutes. Then, it was centrifuged again (12,000 rpm, 10 minutes, 4°C) to obtain a clear supernatant.

[0144] Next, 500 μl of isopropanol was added and left for 10 minutes, then centrifuged to obtain an RNA pellet. The RNA pellet was washed with 70% ethanol (ethanol + diethylpyrocarbonate (DEPC) treated distilled water), then completely removed and dried. The dried, clear RNA was added to DEPC-treated distilled water and stored at -80°C. The total RNA amount was measured using Nanodrop, and the 18s and 28s bands were confirmed on a 1.2% agarose gel. cDNA was synthesized with 2 μg of total RNA, random hexamer primers, and reverse transcriptase (25°C: 10 min, 37°C: 120 min, 85°C: 5 min).

[0145] 1-7. Confirmation of gene expression To confirm gene expression, real-time PCR was performed. For real-time gene expression observation, gene expression was analyzed using the Power SYBR Green PCR Master Mix, which contains the SYBR green fluorescent agent (7500 real-time PCR system). PCR primers were designed using Primer 3 software (http: / / frodo.wi.mit.edu) according to nucleotide sequences obtained from NCBI GenBank.

[0146] PCR was performed 40 times, with the reaction cycle consisting of 10 minutes at 95°C, 33 seconds at 95°C, 33 seconds depending on the gene primer temperature (tm), and 33 seconds at 72°C. Gene expression levels were analyzed by analyzing the c(t) values ​​obtained from real-time PCR analysis (fold change 2-△△Ct formula). Gene expression levels of treated cells were calculated after setting the gene expression level of untreated cells to 1. For gene c(t) value analysis, the GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used for normalization.

[0147] The sequences of the PCR primers are shown in Table 1 below.

[0148] [Table 1]

[0149] 1-8. Cell tissue immunostaining method (Immunocytochemistry) The culture medium of the cultured C2C12 cells was removed and washed once with PBS. The washed cells were treated with 4% formaldehyde (Sigma) and fixed for 15 minutes, then washed with PBS and left for 5 minutes with 0.2% Tipton X-100 (Sigma). The cells were washed again with PBS and left for 30 minutes with enhancer solution, then the primary antibody (MYOD, MYOG, myosin light chain 2 (MYL2), Atrogin 1, MuRF1, nitrotyrosine, MSTN, 1:50) was added and incubated at 4°C for 14 hours. The antibody was removed, washed three times with PBS for 10 minutes each, and then cultured with secondary antibody (Alexa Fluor 488 goat anti-rabbit or mouse SFX kit) for 1 hour. After culturing, the antibodies were removed, and the cells were washed with PBS for 10 minutes. The nuclei were then stained with DAP I (4',6-diamidino-2-phenylindol), and protein expression was observed using a fluorescence microscope.

[0150] 1-9. Western blot The culture medium of the cultured C2C12 cells was removed and washed with PBS. After washing the cells, RIPA buffer (Radioimmunoprecipitation assay buffer, Thermo) and a protease inhibitor (Thermo) were added, and the cells were lysed to extract the protein. 40 μg of the extracted protein was subjected to electrophoresis on an 8-10% acrylamide gel and then transferred to a PVDF membrane (Polyvinylidene fluoride membrane, Milipore). This was blocked with 3% skim milk or bovine serum albumin (BSA) at room temperature for 1 hour. Subsequently, primary antibodies (MYOD:1:500, MYOG:1:400, MYL2:1:1000, β-actin:1:1000, MuRF1:1:500, Atrogin 1:1:500, Nitrotyrosine:1:10000, MSTN:1:500, GAPDH:1:1000) diluted in 1% skim milk or BSA were added and reacted at 4°C for at least 16 hours. After 16 hours, the mixture was washed three times with TBST (Tris-Buffered Saline containing Tween20), and the secondary antibody with attached HRP (horseradish peroxidase) was reacted at room temperature for 1 hour. This was then washed three times with TBST, and the mixture was developed with Super Signal West Pico chemiluminescent substrate.

[0151] 1-10. Tissue immunostaining method (Immunohistochemistry) Paraffin-containing muscle tissue was deparaffinized with xylene and ethanol, respectively, and then moistened. To inhibit endogenous peroxidase activity, the tissue was immersed in 0.3% H2OH / methanol. Subsequently, for morphological observation, the tissue was stained with hematoxylin / eosin or reacted with 1% normal goat serum at room temperature for 1 hour to block the reaction with nonspecific antibodies. After that, it was reacted with a primary antibody (1:50) at 4°C for more than 16 hours. After 16 hours, the tissue was washed three times with PBS and reacted with a secondary antibody (1:100) contaminated with HRP (horseradish peroxidase) at room temperature for 1 hour. After detecting protein expression by adding HRP-labeled streptavidin, the tissue was observed under a microscope.

[0152] 1-11. Observation of the muscle regeneration effect of licorice hot water extract diet. To observe the muscle regeneration effect of licorice hot water extract or its final product, liquiritigenin, mice were administered licorice hot water extract or liquiritigenin, and then muscle morphology and regeneration were observed after injection / non-injection of cardiotoxin (CTX) into the muscle. C57BL / 6 mice were administered licorice hot water extract (100 mg / kg) or liquiritigenin (15 mg / kg), and 100 mM cardiotoxin was injected into the gastrocnemius muscle one day later. After cardiotoxin injection, licorice hot water extract or liquiritigenin was administered daily, and muscle tissue was sampled either before or 7 days after cardiotoxin injection. The diameter (μm) was measured using the Image J program from the muscle tissue obtained before or 7 days after cardiotoxin injection.

[0153] 2. Experimental Results 2-1. Confirmation of myoblast proliferation and differentiation by licorice hot water extract treatment. To confirm the proliferation of myoblasts induced by licorice hot water extract treatment, C2C12 cells were treated with the hot water extract at concentrations of 0, 50, 100, and 200 μg / ml for one day, and then cell proliferation was analyzed by the MTT method.

[0154] Figure 2 shows the results of observing the proliferation and differentiation of myoblasts (C2C12) treated with licorice hot water extract. Here, the degree of myoblast proliferation was calculated by setting the value of untreated cells to 100 and then calculating the relative value of cells treated with licorice hot water extract. The degree of differentiation and gene expression was calculated by setting the value of untreated cells to 1 and then calculating the relative value of cells treated with licorice hot water extract. This same method is applied to the experiments described below.

[0155] Referring to Figure 2, image a shows that when cells were treated with 50 and 100 μg / ml of licorice hot water extract, the cells proliferated by approximately 20% compared to cells that were not treated with the extract. Furthermore, image b shows that when C2C12 cells were treated with 50 μg / ml of licorice hot water extract for one day after being scratched, the recovery of the cells treated with the extract was higher than that of the cells that were not treated with the extract.

[0156] Referring to Figure 2c, to confirm the differentiation of C2C12 cells by treatment with licorice hot water extract, cells were treated with 2% FBS differentiation medium and then treated with licorice hot water extract (100 μg / ml) and cultured for 4 days. As a result, myotube formation was observed in the cells treated with the extract, and the expression of genes or proteins such as MYOD, MYOG, and MYL2, which are highly expressed during differentiation into muscle cells, was increased. In addition, the expression of Atrogin 1 and MuRF1 genes or proteins, which are associated with proteolysis and muscle atrophy, was decreased.

[0157] 2-2. Confirmation of muscle regeneration effect by licorice hot water extract treatment. To confirm the muscle regeneration effect of licorice hot water extract treatment, mice were given licorice hot water extract, and then cardiotoxin was injected into the mouse muscles. The degree of regeneration of damaged muscles was then analyzed. Furthermore, to verify the effect of the extract in undamaged muscles, the expression of related proteins was observed after feeding the mice with licorice hot water extract.

[0158] Figure 3 shows the results of an analysis of mouse muscle tissue after ingestion of licorice hot water extract. Referring to this, Table a shows that there was little change in body weight between mice that ingested licorice hot water extract and those that did not, but the rate of muscle loss was lower in the mice that ingested the extract compared to the mice that did not.

[0159] Referring to Figures 3b, c, and d, the expression of Pax7, MYOD, MYOG, and MYL2 proteins, which are highly expressed during muscle regeneration, was increased in the muscles of mice injected with licorice hot water extract / cardiotoxin compared to mice injected with cardiotoxin alone. Furthermore, the expression of MSTN, MuRF1, Atrogin 1, and nitrotyrosine proteins, which are associated with muscle differentiation inhibition, proteolysis, and muscle atrophy, was decreased in the muscles of mice injected with licorice hot water extract / cardiotoxin compared to mice injected with cardiotoxin alone. However, no difference in protein expression due to the intake of licorice hot water extract was observed in the muscles of mice that had not suffered muscle damage.

[0160] 2-3. Confirmation of myoblast proliferation by treatment with licorice extract fractions. To confirm the proliferation of myoblasts induced by treatment with licorice extract fractions, C2C12 cells were treated with five fractions (25 μg / ml) for one day, and then cell proliferation was analyzed by the MTT method.

[0161] Figure 4 shows the results of treatment with fractions of licorice extract to determine whether or not myoblast proliferation occurred. Referring to this, in addition to cells treated with licorice hot water extract (EX, 10%), cells treated with ethyl acetate fraction of licorice ( Depositphotos, 12%) or n-butanol fraction (BuOH, 12%) also showed increased cell proliferation compared to cells that were not treated with the extract or fraction. However, cells treated with dichloromethane (DCM) fraction showed decreased cell proliferation compared to untreated cells (27%).

[0162] Furthermore, when cells were scratched and treated with the aforementioned fraction, and cultured for one day, it was observed that cells treated with ethyl acetate fraction showed greater regenerative capacity at the scratched areas compared to untreated cells.

[0163] 2-4. Confirmation of myoblast differentiation by treatment with licorice extract fractions. To confirm the differentiation of C2C12 cells by treatment with a fraction of licorice extract, cells were treated with 2% FBS differentiation medium and the fraction (25 μg / ml) along with the cells, and cultured for 4 days.

[0164] Figure 5 shows the results of myoblast differentiation observations after treatment with a fraction of licorice extract. Referring to this, it can be confirmed that treatment with the ethyl acetate (siRNA) fraction of licorice increased the formation of myotubes.

[0165] In addition, we confirmed an increase in the expression of MYOD, MYOG, and MYL2 genes and proteins related to muscle differentiation, and a decrease in the expression of Atrogin 1, MuRF1, and MSTN genes related to protein degradation, muscle atrophy, and differentiation inhibition. Furthermore, we confirmed a decrease in the expression of MuRF1 and MSTN proteins.

[0166] Treatment with the n-butanol (BuOH) fraction of licorice also increased myotube formation. In addition, the expression of the MYOG and MYL2 genes and proteins increased, while the expression of the Atrogin 1 and MuRF1 genes decreased. Treatment with licorice hot water extract (EX) also increased the expression of the MYL2 gene and decreased the expression of the Atrogin 1 and MuRF1 genes. Treatment with the water (H2O) fraction slightly increased the expression of the MYOG gene, but there were no changes in the expression of other related genes.

[0167] 2-5. Confirmation of cell proliferation by final single-substance treatment. Figure 6 shows the chemical structure of a single substance separated from an ethyl acetate (SiO) fraction according to one embodiment of the present invention. Ten final substances were separated from the fraction through several fractionation steps, and then structural analysis was performed.

[0168] Table 2 below shows the 10 final substances separated and their analysis results.

[0169] [Table 2]

[0170] To observe the proliferation of C2C12 cells after the aforementioned final substance treatment, the cells were treated with 0.5 ng / ml of the final substance for one day, and cell proliferation was observed using the MTT method.

[0171] Figure 7 shows the observation results of myoblast proliferation and differentiation after treatment with a single final substance. Referring to (a), cells treated with 0.5 ng / ml of liquiritigenin (10%), tetrahydroxymethoxychalcone (8%), or licochalcone B (11%) showed increased cell proliferation compared to cells not treated with the substance.

[0172] Referring to Figure 7b, it was observed that myotubation and lysis indices increased with treatment with liquiritigenin (13%), tetrahydroxymethoxychalcone (28%), or licochalcone B (19%). Referring to Figure 7c, it was observed that cells treated with purchased pure liquiritigenin (0.25 ng / ml, 5%), tetrahydroxymethoxychalcone (0.25 ng / ml, 5%), or licochalcone B (1 ng / ml, 11%) showed increased cell proliferation compared to untreated cells. Furthermore, referring to Figure 7d, it was observed that myotubation and lysis indices increased with treatment with liquiritigenin (20%), tetrahydroxymethoxychalcone (23%), or licochalcone B (20%).

[0173] 2-6. Confirmation of muscle regeneration effects by liquiritigenin treatment. To confirm the muscle regeneration effect of liquiritigenin treatment, mice were given liquiritigenin, and then cardiotoxin was injected into the mouse muscles. The diameter of the regenerated muscles was measured, and the degree of regeneration of damaged muscles was observed.

[0174] Figure 8 shows the results of an analysis of muscle changes after mice were given purchased liquiritigenin. Referring to this, in (a), there was almost no change in body weight and muscle mass between mice that ingested liquiritigenin and those that did not. However, it was observed that the rate of muscle loss was less in the mice that ingested the substance compared to the mice that did not.

[0175] Referring to Figures 8b and 8c, it was confirmed that the muscle diameter of mice that ingested liquilitigenin was larger than that of mice that did not ingest it (muscle diameter of mice that did not ingest it: 78±4 μm, muscle diameter of mice that ingested it: 91±3 μm). In the muscles of mice that were not injected with cardiotoxin, it was also confirmed that the muscle diameter of mice that ingested it was larger than that of mice that did not ingest it (muscle diameter of mice that did not ingest it: 124±4 μm, muscle diameter of mice that ingested it: 139±4 μm).

[0176] Although specific aspects of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. In other words, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of muscle diseases, containing a fraction of licorice extract as an active ingredient, The licorice extract is a hot water extract obtained by placing the licorice in distilled water and heating it at 110-120°C for 2-4 hours. The aforementioned fraction is obtained by fractionation of the licorice extract with ethyl acetate. A pharmaceutical composition for the prevention or treatment of muscle diseases, wherein the muscle disease is one or more selected from the group consisting of muscle atrophy, muscle dystrophy, sarcopenia, myopathy, myasthenia, and muscle injury.

2. The licorice extract fraction is characterized by promoting the proliferation of myoblasts, the formation of myotubes, and the differentiation into muscle cells, as described in claim 1, for the prevention or treatment of muscle diseases.

3. The licorice extract fraction is characterized by regenerating damaged muscles, as described in claim 1, for the pharmaceutical composition for the prevention or treatment of muscle diseases.

4. The licorice extract fraction is characterized by inhibiting muscle protein breakdown or muscle atrophy, as described in claim 1, for the prevention or treatment of muscle diseases.

5. The fraction of the licorice extract is A pharmaceutical composition for the prevention or treatment of muscle disease according to claim 1, characterized by increasing the expression of one or more muscle differentiation-related factors selected from the group consisting of MYOG, MYOD, MYL2, and Pax7, and decreasing the expression of one or more muscle protein degradation-related factors selected from the group consisting of MSTN, MuRF1, Atrogin 1, and nitrotyrosine.

6. A health functional food composition for preventing or improving muscle diseases, containing a fraction of licorice extract as an active ingredient, The licorice extract is a hot water extract obtained by placing the licorice in distilled water and heating it at 110-120°C for 2-4 hours. The aforementioned fraction is obtained by fractionation of the licorice extract with ethyl acetate. A functional health food composition for preventing or improving muscle diseases, wherein the muscle disease is one or more selected from the group consisting of muscle atrophy, muscle dystrophy, sarcopenia, myopathy, myasthenia, and muscle injury.

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