Composition for preventing or treating muscle diseases containing Citrus aurantium extract

Abstract

The present invention relates to a composition for preventing or treating muscle diseases, which contains an extract of Citrus aurantium dulcis (Citrus aurantium dulcis) and has the effects of increasing myotube differentiation and the expression of genes related to athletic performance, increasing muscle mass, and improving athletic performance.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2023-0018844 filed on February 13, 2023, and Korean Patent Application No. 10-2023-0085747 filed on July 3, 2023, the entire specifications of which are incorporated herein by reference.

[0002] The present invention relates to a composition for preventing or treating muscle diseases, which comprises an extract of Citrus Fruit. [Background technology]

[0003] Muscles are mainly divided into skeletal, cardiac, and smooth muscles, of which skeletal muscle is the most abundant tissue in the human body, accounting for 40-45% of body weight. Skeletal muscle is attached to bones via tendons and plays a role in generating bone movement or force. A muscle is composed of numerous muscle fibers, which in turn are made up of numerous myofibrils composed of actin and myosin. When actin and myosin overlap and move, the muscle length shortens and lengthens, inducing overall muscle contraction and relaxation. An increase in myofibril size means an increase in muscle fiber thickness, resulting in muscle mass growth.

[0004] Muscle diseases weaken skeletal muscles, gradually impairing walking and mobility, making activities of daily living (ADL) difficult, and ultimately leading to the inability to live independently. Furthermore, they can cause cardiopulmonary dysfunction and other complications, so it is important to accurately understand the characteristics of each muscle disease and take appropriate approaches.

[0005] In 2000, South Korea entered an aging society, with the elderly population accounting for 7.2% of the total population. By 2050, the country is predicted to become a super-aging society (over 20%) (Statistics Korea, 2013 Elderly Statistics). Human muscle mass decreases with age (by about 10-15% between the ages of 50 and 70, and by over 30% between the ages of 70 and 80). This leads to a decline in muscle strength and function, a condition known as sarcopenia. Sarcopenia is a major cause of activity and walking disorders, limiting the ability of elderly people to live independently. Furthermore, sarcopenia reduces primary mortality, increases insulin resistance, promotes the development of type 2 diabetes, and increases the risk of hypertension and cardiovascular disease by three to five times. Currently, there are no medications approved for the treatment of sarcopenia, and drug repositioning technology is under development to apply myostatin inhibitors or other existing FDA-approved treatments to sarcopenia.

[0006] Ponciri Fructus (Ponciri Fructus) is the dried unripe fruit of the Rutaceae plant, Poncirus trifoliata Rafinesque, and is distinct from Aurantii Fructus (Aurantii Fructus). Ponciri Fructus was used to treat symptoms such as flatulence, constipation, allergies, and inflammation. Recent research into its biological activity has revealed its anticancer, antibacterial, antiviral, melanin synthesis inhibitory effects, uterine contraction, and allergy and hypersensitivity suppression effects. It has also been reported that the coumarin component in Ponciri Fructus inhibits platelet aggregation. The Donguibogam Materia Medica (Compendium of Materia Medica) describes Ponciri Fructus as a medicinal herb with downward-acting properties, used to treat chest and stomach disorders, while Aurantii Fructus, unlike Ponciri, has upward-acting properties, used to treat skin and thoracic disorders.

[0007] The present inventors have made efforts to develop natural products that have preventive or therapeutic effects on muscle diseases, and have experimentally confirmed that an extract of Citrus Fruit can increase the expression of genes related to myotube differentiation and athletic performance, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases, which comprises an extract of Citrus Fruit, a fraction thereof, or auraptene as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing and / or improving muscle diseases, increasing muscle mass, and / or improving athletic performance, which contains an extract of Citrus aurantium ginseng, a fraction thereof, or auraptene as an active ingredient.

[0010] The object of the present invention is to provide a quasi-drug composition for preventing and / or improving muscle diseases, increasing muscle mass, and / or improving athletic ability, which contains an extract of Citrus aurantium ginseng, a fraction thereof, or auraptene as an active ingredient.

[0011] However, the technical problems that the present invention aims to achieve are not limited to the above problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0012] The present invention provides a food composition for preventing and / or improving muscle diseases, increasing muscle mass, and / or improving athletic performance, which comprises an extract of Citrus Fruit, a fraction thereof, or auraptene as an active ingredient.

[0013] The present invention also provides a quasi-drug composition for preventing and / or improving muscle diseases, increasing muscle mass, and / or improving athletic ability, which contains an extract of Citrus Fruit, a fraction thereof, or auraptene as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating muscle diseases, which comprises an extract of Citrus Fruit, a fraction thereof, or auraptene as an active ingredient.

[0014] The muscle disease may be one or more diseases selected from the group consisting of sarcopenia, muscle atrophy, myasthenia, muscle dystrophy, myotonia, muscle hypotonia, muscle weakness, muscle degenerative atrophy, atony, amyotrophic lateral sclerosis, and inflammatory muscle diseases.

[0015] The composition can increase the expression of one or more genes selected from the group of myotube differentiation markers consisting of Myf5, Myf6, Myod, and Myog genes. The composition can increase the expression of the Ppard and / or Pgc1a genes, which are markers of athletic performance. The extract may be extracted with a solvent selected from the group consisting of water, organic solvents and mixtures thereof.

[0016] The organic solvent may be one or more solvents selected from the group consisting of lower alcohols having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, chloroform, and diethyl ether.

[0017] The present invention also provides a method for preventing, treating, or ameliorating muscle diseases, and / or a method for increasing muscle mass, and / or a method for improving athletic ability, which includes a step of administering or having a subject ingest a composition containing a Citrus Fruit extract, a fraction thereof, or auraptene as an active ingredient.

[0018] Furthermore, the present invention provides a use of a composition containing an extract of Citrus Fruit, a fraction thereof, or auraptene as an active ingredient for preventing, treating, or improving muscle diseases, and / or for increasing muscle mass, and / or for improving athletic performance. [Effects of the Invention]

[0019] The Citrus aurantium extract, a fraction thereof, or auraptene of the present invention has the effect of increasing myotube differentiation and the expression of genes related to athletic performance, thereby increasing muscle weight and improving athletic performance, etc. Furthermore, the Citrus aurantium extract, a fraction thereof, or auraptene of the present invention as an active ingredient can be applied as a natural medicine to various products such as pharmaceutical compositions, food compositions, and quasi-drug compositions for the prevention, treatment, or improvement of muscle diseases. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows the effect of Ponciri Fructus extract on root canal cell viability (Con: control group; Dex: dexamethasone; PT 20: Ponciri (Ponciri Fructus) extract 20 μg / ml; PT 10: Ponciri extract 10 μg / ml; PT 5: Ponciri extract 5 μg / ml; *, p <0.05; ***, p <0.001). [Figure 2] Figure 2 shows the results of confirming the effect of Citrus aurantium extract in promoting the expression of genes related to myotube differentiation (Con: control group, Dex: dexamethasone, PT 20: Citrus aurantium extract 20 μg / ml, PT 10: Citrus aurantium extract 10 μg / ml, PT 5: Citrus aurantium extract 5 μg / ml). [Figure 3] Figure 3 shows the results of confirming the effect of Citrus aurantium extract on promoting the expression of genes related to athletic performance (Con: control group; Dex: dexamethasone; PT 20: Citrus aurantium extract 20 μg / ml; PT 10: Citrus aurantium extract 10 μg / ml; PT 5: Citrus aurantium extract 5 μg / ml). [Figure 4] Figure 4 shows the results of confirming the grip strength-increasing effect of Citrus aurantium extract (Con: control group; Dex: dexamethasone; Oxy: oxymetholone; PT 100: Citrus aurantium extract 100 mg / kg; PT 50: Citrus aurantium extract 50 mg / kg; PT 10: Citrus aurantium extract 10 mg / kg; ** p <0.01; ***, p <0.001). [Figure 5] Figure 5 shows the results of confirming the grip strength-increasing effect of Citrus aurantium extract (Con: control group; Dex: dexamethasone; Oxy: oxymetholone; PT 100: Citrus aurantium extract 100 mg / kg; PT 50: Citrus aurantium extract 50 mg / kg; PT 10: Citrus aurantium extract 10 mg / kg; ** p <0.01; ***, p <0.001). [Figure 6]Figure 6 shows the effect of Citrus aurantium extract on increasing quadriceps muscle weight (Con: control group; Dex: dexamethasone; Oxy: oxymetholone; PT 100: Citrus aurantium extract 100 mg / kg; PT 50: Citrus aurantium extract 50 mg / kg; PT 10: Citrus aurantium extract 10 mg / kg; ** p <0.01; ***, p <0.001). [Figure 7] Figure 7 shows the effect of Citrus aurantium extract on increasing gastrocnemious muscle weight (Con: control group; Dex: dexamethasone; Oxy: oxymetholone; PT 100: Citrus aurantium extract 100 mg / kg; PT 50: Citrus aurantium extract 50 mg / kg; PT 10: Citrus aurantium extract 10 mg / kg; *p<0.05). [Figure 8] Figure 8 shows the effect of Citrus aurantium extract on increasing tibialis muscle weight (Con: control group; Dex: dexamethasone; Oxy: oxymetholone; PT 100: Citrus aurantium extract 100 mg / kg; PT 50: Citrus aurantium extract 50 mg / kg; PT 10: Citrus aurantium extract 10 mg / kg; *, p <0.05; ** p <0.01). [Figure 9] Figure 9 shows the results of confirming the efficacy of Citrus Fruit Extract in improving motor performance (Con: control group, Dex: dexamethasone, Oxy: oxymetholone, PT 100: Citrus Fruit Extract 100 mg / kg, PT 50: Citrus Fruit Extract 50 mg / kg, PT 10: Citrus Fruit Extract 10 mg / kg; *, p<0.05). [Figure 10] FIG. 10 shows the results of confirming the efficacy of Citrus aurantium extract in improving motor performance (Con: control group, Dex: dexamethasone, Oxy: oxymetholone, PT 100: Citrus aurantium extract 100 mg / kg, PT 50: Citrus aurantium extract 50 mg / kg, PT 10: Citrus aurantium extract 10 mg / kg; *, p<0.05). [Figure 11]FIG. 11 shows the results of confirming the efficacy of Citrus aurantium extract in improving motor performance (Con: control group, Dex: dexamethasone, Oxy: oxymetholone, PT 100: Citrus aurantium extract 100 mg / kg, PT 50: Citrus aurantium extract 50 mg / kg, PT 10: Citrus aurantium extract 10 mg / kg; *, p<0.05). [Figure 12] Figure 12 shows the results of examining the effects of fractions of the Citrus Fruit extract on root canal cell viability (*, p <0.05; ** p <0.01; ***, p <0.001). [Figure 13] Figure 13 shows the results of confirming the effect of compounds derived from Citrus aurantium on root canal cell viability (*, p <0.05; ** p <0.01; ***, p <0.001). [Figure 14] Figure 14 shows the results of examining the effect of auraptene on root canal cell viability (Con: control group, Dex: dexamethasone, AUR 20: auraptene 20 μg / ml, AUR 10: auraptene 10 μg / ml, AUR 5: auraptene 5 μg / ml; *, p <0.05; ** p <0.01; ***, p <0.001). [Figure 15] Figure 15 shows the results of confirming the muscle fiber strengthening efficacy of auraptene (Con: control group; Dex: dexamethasone; AUR 20: auraptene 20 μg / ml; AUR 10: auraptene 10 μg / ml; AUR 5: auraptene 5 μg / ml; *, p <0.05; ** p <0.01). [Figure 16] Figure 16 shows the results of confirming the efficacy of auraptene in promoting muscle differentiation-related gene expression (Con: control group; Dex: dexamethasone; AUR 20: auraptene 20 μg / ml; AUR 10: auraptene 10 μg / ml; AUR 5: auraptene 5 μg / ml; *, p <0.05; ** p <0.01). [Figure 17]Figure 17 shows the results of confirming the efficacy of auraptene in promoting the expression of genes related to motor performance (Con: control group; Dex: dexamethasone; AUR 20: auraptene 20 μg / ml; AUR 10: auraptene 10 μg / ml; AUR 5: auraptene 5 μg / ml *, p<0.05; ** p<0.01). [Figure 18] Figure 18 shows the results of confirming the effects of auraptene on improving oxidative stress and mitochondrial function (Con: control group, Dex: dexamethasone, AUR 20: auraptene 20 μg / ml, AUR 10: auraptene 10 μg / ml, AUR 5: auraptene 5 μg / ml *, p <0.05; ** p <0.01; p <0.001). [Figure 19] Figure 19 shows the results of confirming the grip strength-improving effect of auraptene (Con: control group, Dex: dexamethasone, AUR 50: auraptene 50 mg / kg, AUR 10: auraptene 10 mg / kg; *, p <0.05; ** p <0.01; p <0.001). [Figure 20] Figure 20 shows the results of confirming the effect of auraptene on increasing muscle weight (quadriceps, gastrocnemius, tibialis, and soleus muscle weights) (Con: control group, Dex: dexamethasone, AUR 50: auraptene 50 mg / kg, AUR 10: auraptene 10 mg / kg; *, p <0.05; ** p <0.01; p <0.001). DETAILED DESCRIPTION OF THE INVENTION

[0021] The inventors have experimentally confirmed that the extract of Citrus Fruit, a natural product with little or no side effects, has the effect of increasing duct cell differentiation and the expression of genes related to athletic performance, increasing muscle weight, and improving athletic performance, and have thus completed the present invention. The present invention will be described in detail below.

[0022] The present invention provides a composition for preventing, improving or treating muscle diseases, and / or increasing muscle mass, and / or improving athletic performance, which comprises an extract of Ponciri Fructus or a fraction thereof as an active ingredient.

[0023] The muscle disease may be one or more diseases selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, and inflammatory myopathy.

[0024] The composition can increase the expression of one or more genes selected from the group of myotube differentiation markers consisting of Myf5, Myf6, Myod, and Myog genes.

[0025] The composition can increase the expression of the Ppard and / or Pgc1a genes, which are markers of athletic performance.

[0026] The composition may be a food composition, a functional health food composition, a pharmaceutical composition, or a quasi-drug composition.

[0027] As used herein, "prevention" refers to any action that delays the onset of a muscle disease by administering the composition of the present invention, and "treatment" and "improvement" refer to any action that improves or beneficially changes the symptoms of a muscle disease by administering the composition of the present invention.

[0028] The above extract can be extracted using polar and / or non-polar solvents.

[0029] The polar solvent can include one or more selected from the group consisting of (i) water, (ii) alcohol (preferably methanol, ethanol, propanol, butanol, normal propanol, isopropanol, normal butanol, 1-pentanol, 2-butoxyethanol, or ethylene glycol), (iii) acetic acid, (iv) DMFO (dimethylformamide), and (v) DMSO (dimethyl sulfoxide). The non-polar solvent may include one or more selected from the group consisting of acetone, acetonatryl, ethyl acetate, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

[0030] More specifically, the extract may be extracted using water, an organic solvent, or a mixture thereof as the solvent, and according to a specific embodiment of the present invention, the extract of the present invention may be obtained by treating the extract with ethanol, more preferably with 70 to 100% (v / v) ethanol.

[0031] The organic solvent can include one or more solvents selected from the group consisting of lower alcohols having 1 to 6 carbon atoms (such as methanol, ethanol, propanol, and butanol), hexane, acetone, ethyl acetate, chloroform, and diethyl ether.

[0032] The Citrus Fruit Extract can be prepared as a fraction according to a method commonly used in the art, and the solvent used to fractionate the Citrus Fruit Extract can be at least one selected from the group consisting of water, C1-C4 lower alcohols, n-hexane, ethyl acetate, acetone, acetonitrile, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixtures thereof, but is not limited thereto. However, methylene chloride is preferably used.

[0033] As used herein, the term "extract" has the same meaning as a crude extract in the art, as described above, but in a broader sense also includes fractions obtained by further fractionation of the extract. That is, the above-mentioned Citrus Fruit extract includes not only those obtained using the above-mentioned extraction solvents, but also those obtained by additionally applying a purification step thereto. For example, fractions obtained by passing the above-mentioned extract through an ultrafiltration membrane with a certain molecular weight cutoff value, and fractions obtained by various additional purification methods, such as separation by various chromatographies (designed for separation by size, charge, hydrophobicity, or affinity), are also included in the natural product extract of the present invention.

[0034] The extracts utilized in the present invention can be produced in powder form by vacuum distillation and additional steps such as freeze drying or spray drying.

[0035] Specifically, the extract can be prepared by adding water, an organic solvent, or a mixture thereof to the fruit in an amount 5 to 50 times the weight of the fruit, preferably 10 to 30 times, but not limited to this. The extraction temperature can be 10 to 150°C, more preferably 15 to 120°C, but not limited to this. The extraction time is preferably 1 to 20 hours, more preferably 2 to 8 hours, but not limited to this. The extraction method can include, but is not limited to, cold maceration, ultrasonic extraction, or reflux cooling extraction. The number of extractions is preferably 1 to 5 times, more preferably 2 to 3 times, but not limited to this. Furthermore, the extract can be diluted or concentrated, or purified and dried after dilution or concentration for use.

[0036] The Citrus Fruit extract and / or fraction according to the present invention may contain one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 13 (see Table 1 below). Preferably, it may contain one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 2, 3, 9, and 12. More preferably, it may contain the compound represented by Chemical Formula 2.

[0037] As used herein, "containing as an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of the Citrus Fruit extract. The present invention is a composition extracted from Citrus Fruit, a natural plant material, and the upper limit of the amount of the Citrus Fruit extract contained in the composition of the present invention can be selected within an appropriate range by those skilled in the art.

[0038] A pharmaceutical composition for preventing or treating muscle diseases, comprising an extract of Citrus aurantium as an active ingredient. The compositions of the present invention can be prepared as pharmaceutical compositions.

[0039] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

[0040] According to a preferred embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the above-mentioned Citrus Fruit Extract of the present invention; and (b) a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to achieve the efficacy or activity of the above-mentioned Citrus Fruit Extract.

[0041] Pharmaceutically acceptable carriers are those commonly used in pharmaceutical formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl methylhydroxybenzoate, methyl propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0042] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0043] The appropriate dosage of the pharmaceutical composition of the present invention can be formulated in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, condition, diet, administration time, administration route, excretion rate, and reaction sensitivity. The typical dosage of the active ingredient contained in the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day, for adults. Administration can be once a day or in several divided doses. However, the scope of the present invention is not limited to the above dosage.

[0044] The pharmaceutical compositions of the present invention can be prepared in unit dose form or in multi-dose containers by formulating them using pharmaceutically acceptable carriers and / or excipients according to a method easily performed by a person skilled in the art to which the invention pertains. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer.

[0045] Quasi-drug composition for preventing or improving muscle disorders containing Citrus aurantium extract as an active ingredient The composition of the present invention can be provided as a quasi-drug composition.

[0046] The above-mentioned Citrus Fruit Extract may be added as is or may be used in combination with other quasi-drug ingredients, etc., and may be used appropriately according to conventional methods. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). The above-mentioned quasi-drug composition can be used to manufacture external preparations, patches, ointments, etc., but is not limited thereto.

[0047] Food composition for preventing or improving muscle diseases containing citrus fruit extract as an active ingredient

[0048] The composition of the present invention can be provided as a food composition or a functional health food composition. When the composition for preventing, improving, or treating muscle disorders containing the Citrus Fruit extract of the present invention as an active ingredient is prepared as a food composition, the active ingredients include not only the Citrus Fruit extract but also ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavors. Examples of carbohydrates include monosaccharides, such as monosaccharides like glucose and fructose; disaccharides, such as disaccharides like maltose, sucrose, and oligosaccharides; and polysaccharides, such as common sugars like dextrin and cyclodextrin, and sugar alcohols like xylitol, sorbitol, and erythritol. Natural flavors [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavors [saccharin, aspartame, etc.] can be used as flavors. For example, when the food composition of the present invention is manufactured as a drink, in addition to the natural product extract of the present invention, it may further contain citric acid, liquid sugar, sugar, glucose, acetic acid, malic acid, fruit juice, Houttuynia cordata extract, jujube extract, licorice extract, etc.

[0049] The food composition or health functional food composition may be formulated in the form of powder, granules, pills, tablets, or capsules, as well as in the form of a general food or beverage.

[0050] There are no particular limitations on the type of food, and examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and can include all foods in the usual sense.

[0051] Generally, when producing a food or beverage, the above-mentioned Citrus Fruit Extract can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, for health or hygiene purposes, or for long-term intake for health regulation purposes, the amount may be less than the above range. Furthermore, since the present invention utilizes natural products, there are no safety issues, and an amount greater than the above range can also be used.

[0052] The present invention also provides a composition for preventing, improving, or treating a muscle disease, comprising auraptene as an active ingredient, wherein the composition may be a food composition, a quasi-drug composition, or a pharmaceutical composition, the details of which are as described above. The auraptene may be a compound derived from a citrus fruit.

[0053] The muscle disease is one or more diseases selected from the group consisting of sarcopenia, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, muscular weakness, muscular dystrophy, atony, amyotrophic lateral sclerosis, and inflammatory myopathy.

[0054] The present invention also provides a composition for building muscle, comprising auraptene as an active ingredient, wherein the composition may be a food composition, a quasi-drug composition, or a pharmaceutical composition, the details of which are as described above.

[0055] The composition can increase the expression of one or more genes selected from the group of myotube differentiation markers consisting of Myod1, Myog, and Myf5 genes.

[0056] The composition is capable of increasing muscle mass.

[0057] The present invention will be described in more detail with reference to the following examples. Objects, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the examples described herein and may be embodied in other forms. The examples introduced herein are provided to fully convey the concept of the present invention to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples. [Example]

[0058] [Production Example 1] Preparation of Citrus Fruit Extract Ponciri Fructus was purchased from Gyeongdong Market in Seoul, cut into appropriate sizes, and then 6 kg of Ponciri Fructus was added to an extraction vessel with 10 L of 70% (v / v) ethanol for cold immersion extraction. The extract was then filtered through Whatman extraction solvent filter paper. The extraction process was repeated three times, and the solvent was then concentrated under reduced pressure and dried to obtain 600 g of extract.

[0059] [Production Example 2] Production of fractionated products The extract obtained in Preparation Example 1 was subjected to solvent fractionation to obtain methylene chloride (MC), butanol (BuOH), and ethyl acetate (EA) fractions. Each fraction was prepared by suspending the 70% ethanol extract in distilled water (10 L x 3 times), adding an equal volume of methylene chloride (MC), separating the methylene chloride layer from the aqueous layer, filtering, and concentrating under reduced pressure to obtain a methylene chloride fraction (150 g). The methylene chloride fraction was then removed, and the same volume of butanol (BuOH) was added to the remaining aqueous layer to obtain a butanol fraction (300 g) using the same method as above. The butanol fraction was then removed, and the same volume of ethyl acetate was added to the remaining aqueous layer to obtain an ethyl acetate fraction (30 g) using the same method as above.

[0060] [Production Example 3] Separation of compounds Compounds were separated from the methylene chloride fraction obtained in Production Example 2. Specifically, the methylene chloride fraction of Production Example 2 was separated using a silica gel open column under conditions of MC:MeOH = 15:1 to 0:15, and then separated according to molecular weight using a Sephadex open column under conditions of 50% methanol, and the following 13 compounds were obtained using prep-MPLC.

[0061] [Table 1]

[0062] [ka] TIFF2026505115000004.tif234161 TIFF2026505115000005.tif30161

[0063] [Example 1] The effect of Citrus aurantium extract on improving myotube cell viability in dexamethasone-induced muscle atrophy C2C12 myotube cells Cell culture and myoblast differentiation: Myoblasts derived from C2C12 mice (CRL1772; American Type Cell Collection, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 (v / v)% FBS. After reaching 90% confluence, they were transferred to DMEM supplemented with 2 (v / v)% horse serum and cultured for 7 days to differentiate into myotubes. C2C12 myoblasts were differentiated into myotubes, and then treated with 100 μM dexamethasone for 24 hours to induce myoatrophy. The muscle atrophy was then alleviated by treatment with 5, 10, and 20 μg / ml of PT. As a result, as shown in Figure 1, the viability of root canal cells, which had been reduced by dexamethasone treatment, was restored in the PT treatment group.

[0064] [Example 2] The effect of Citrus aurantia japonica extract on promoting myotube differentiation and motor performance-related gene expression in dexamethasone-induced muscle atrophy C2C12 myotubes After treating C2C12 mouse root canal cells with 10 μM dexamethasone and washing them twice with PBS, total RNA was extracted according to the user's recommended protocol for the RNA isolation kit (GeneAll RNA isolation kit, Seoul, South Korea). cDNA was synthesized from the extracted RNA and subjected to real-time quantitative polymerase chain reaction (RT-qPCR). PCR analysis was performed according to the user's recommended cycling conditions for the analytical instrument (Applied Biosystems 7900 HT thermal cycler, Applied Biosystems). To confirm the expression level of each mRNA, fold change values ​​were calculated compared to the control group using beta-actin as an endogeneous control, and relative expression levels were compared. The primer sequences are as follows: Myf5 forward primer: TCC AGG TAT TAT TCC CAC CTG CT (SEQ ID NO: 1), Myf5 reverse primer: CAC CTG GAG CGG ATG AAG AAG AAC (SEQ ID NO: 2), Myf6 forward primer: ACA GAT CGT CGG AAA GCA GC (SEQ ID NO: 3), Myf6 reverse primer: CAC TCC GCA GAA TCT CCA CC (SEQ ID NO: 4), Myod forward primer: CAT AGA CTT GAC AGG CCC CG (SEQ ID NO: 5), Myod reverse primer: CGG GTC CAG CAG GTC CTC AAA AA (SEQ ID NO: 6), Myog forward primer: AGC TAT CCG GTT CCA AAG CC (SEQ ID NO: 7), Myog reverse primer: GCA CAG GAG ACC TTG GTC AG (SEQ ID NO: 8), Ppard forward primer: GTA TGC GCA TGG GAC TCA CT (SEQ ID NO: 9), Ppard reverse primer: ACT GCC TTT ACC GTG GGT TT (SEQ ID NO: 10), Ppargc1a forward primer: GTT GCC TGC ATG AGT GTG TG (SEQ ID NO: 11), Ppargc1a reverse primer: CAC ATG TCC CAA GCC ATC CA (SEQ ID NO: 12). As a result, as shown in Figures 2 and 3, it was confirmed that the gene expression of myotube differentiation markers Myf5, Myf6, Myod, and Myog, which was decreased by dexamethasone treatment, was increased by treatment with the Citrus aurantium extract, and the gene expression of motor performance markers Ppard and Pgc1a was increased by treatment with the Citrus aurantium extract.

[0065] [Example 3] Confirmation of changes in grip strength in mice with sarcopenia induced by dexamethasone administration using Citrus aurantium extract C57BL / 6J mice (male, 10 weeks old) were obtained from Doyeol Biotech Co., Ltd. and, after a two-week adaptation period, were divided into six test groups (n = 10 per group) based on body weight. The diets, dosages, and administration methods used in the experimental groups are shown in the table below. The test substances were prepared as liquid suspensions using 0.5% CMC and administered for four weeks. To compensate for the placebo effect of 0.5% CMC and the weight loss effects caused by administration stress, the vehicle control group received daily oral administration of 0.5% CMC. Two weeks after test substance administration, the experimental groups were administered dexamethasone intraperitoneally for two weeks to induce sarcopenia, and the normal control group received saline intraperitoneally. All mice were fed the same AIN76 diet (Research Diets) and were administered the test substances for an additional two weeks, for a total of four weeks. The dark:light cycle was maintained at 12 hours, and water was available ad libitum. As a result, as shown in Figures 4 and 5, the grip strength of the dexamethasone (Dex) group decreased over time, while the grip strength of the positive control group of mice given oxymetholone and cinnamon extract increased compared to the group treated with dexamethasone alone.

[0066] [Table 2]

[0067] [Example 4] Confirmation of muscle type weight increase by Citrus aurantium extract in mice with sarcopenia induced by dexamethasone administration In the above [Example 3], after the administration of the Citrus Fruit extract for a total of 4 weeks, the weight of each type of muscle tissue in all administration groups was confirmed. As a result, as shown in Figures 6 to 8, it was confirmed that the weights of the quadriceps, gastrocnemious, and tibialis muscles in the dexamethasone-administered group were reduced compared to the control group administered with saline, and that the weights of these muscles were significantly increased by the administration of oxymetholone and Citrus Fruit Extract, which were the positive control groups.

[0068] [Example 5] Confirmation of the efficacy of Citrus Fruit Extract in improving the exercise performance of mice with sarcopenia induced by dexamethasone administration In Example 3, after a total of 4 weeks of administration of the Citrus Fruit extract, the maximum running distance (Distance to exhaustion), maximum running speed (Maximal speed capacity), and time to exhaustion, which correspond to the exercise performance capacity, were measured for all treatment groups. As a result, as shown in Figures 9 to 11, it was confirmed that the administration of the Citrus Fruit extract improved exercise capacity.

[0069] [Example 6] Analysis of cell viability using fractions The effect of each fraction (concentration: 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml) prepared according to Preparation Example 2 on the viability of myotubes in which muscle atrophy was induced with dexamethasone was analyzed. The analysis was performed in the same manner as in Example 1. As shown in Figure 12, it was confirmed that the methylene chloride fraction best restored the viability of root canal cells that had been reduced by dexamethasone treatment.

[0070] [Example 7] Cell viability analysis using compounds derived from Citrus aurantium

[0071] The effects of the compounds isolated in Preparation Example 3 (each at a concentration of 20 μM) on the viability of myotubes in which muscle atrophy was induced with dexamethasone were analyzed. The analysis was performed in the same manner as in Example 1. As shown in Figure 13, most of the compounds were confirmed to restore the viability of root canal cells reduced by dexamethasone treatment, and compounds 2, 3, 9, and 12 in particular were confirmed to restore the viability of root canal cells reduced by dexamethasone treatment.

[0072] [Example 8] Efficacy analysis of auraptene Example 8-1. Cell viability analysis Oraptene (Compound 2), the compound that showed the most excellent effect in Example 7, was analyzed for cell viability at various concentrations. The analysis was performed in the same manner as in Example 1. As shown in Figure 14, oraptene was confirmed to effectively restore the viability of root canal cells that had been reduced by dexamethasone treatment in all experimental groups at 5 μM, 10 μM, and 20 μM.

[0073] Example 8-2. Analysis of root canal cell diameter The effect of oraptene (5 μM, 10 μM, 20 μM) on the diameter of root canal cells after muscle atrophy was induced with 100 μM dexamethasone was analyzed. The analysis results confirmed that the diameter of root canal cells reduced by dexamethasone treatment was restored by oraptene treatment (Figure 15).

[0074] Example 8-3. Effect of promoting myotube differentiation and exercise performance-related gene expression The effect of oraptene on promoting myotube differentiation and the expression of genes related to athletic performance was analyzed in dexamethasone-treated C2C12 myotubes. The analysis was performed using the same method as in Example 2. The primer sequences are as follows: Myod1 forward primer: CAT AGA CTT GAC AGG CCC CG (SEQ ID NO: 5), Myod1 reverse primer: CGG GTC CAG GTC CTC AAA AA (SEQ ID NO: 6), Myog forward primer: AGC TAT CCG GTT CCA AAG CC (SEQ ID NO: 7), Myog reverse primer: GCA CAG GAG ACC TTG GTC AG (SEQ ID NO: 8); Myf5 forward primer: TCC AGG TAT TCC CAC CTG CT (SEQ ID NO: 1), Myf5 reverse primer: CAC CTG GAG CGG ATG AAG AAG AAC (SEQ ID NO: 2), Pparg1a forward primer: GTT GCC TGC ATG AGT GTG TG (SEQ ID NO: 11), Pparg1a reverse primer: CAC ATG TCC CAA GCC ATC CA (SEQ ID NO: 12), Ucp3 forward primer: GTT TTG CGG ACC TCC TCA CT (SEQ ID NO: 13), Ucp3 reverse primer: CTC TGT GCG CAC CAT AGT CA (SEQ ID NO: 14), Nrf1 forward primer: CCC GTG TTC CTT TGT GGT GA (SEQ ID NO: 15), Nrf1 reverse primer: ATT CCA TGC TCT GCT GCT GG (SEQ ID NO: 16), Tomm20 forward primer: TGT GCG GTG TGT TGT TGT CTG TT (SEQ ID NO: 17), Tomm20 reverse primer: TAA GTG CCC AGA GCA CAG GA (SEQ ID NO: 18). As shown in Figures 16 and 17, gene expression of myotube differentiation markers Myod1, Myog, and Myf5, which were decreased by dexamethasone treatment, and motor performance differentiation markers Pparg1a, UCP3, Nrf1, and Tomm20, were confirmed to be increased by oraptene treatment (Figures 16 and 17).

[0075] Example 8-4. Improvement of oxidative stress and mitochondrial function C2C12 myoblasts were differentiated into myotubes as in Example 1, and then treated with 10 μM dexamethasone for 24 hours to induce oxidative stress or mitochondrial dysfunction. Oraptene treatment was then used to confirm its efficacy in improving function. Dexamethasone treatment reduced the amount of glutathione (GSH), which has antioxidant properties, but oraptene treatment increased the amount of glutathione (GSH), confirming that oraptene treatment improved the mitochondrial dysfunction induced by dexamethasone treatment (FIG. 18).

[0076] Example 8-5. Confirmation of changes in grip strength in mice with sarcopenia induced by dexamethasone administration Changes in the grip strength of the mice were confirmed using the same method as in Example 3. The diets administered to the experimental groups, their dosages, and administration methods are shown in the table below.

[0077] [Table 3]

[0078] As a result, as shown in Figure 19, it was confirmed that the grip strength of the dexamethasone (Dex) group decreased over time, while the grip strength of the mice given auraptene increased compared to the group treated with dexamethasone alone.

[0079] Example 8-6. Confirmation of weight gain by muscle type in mice with sarcopenia induced by dexamethasone administration In the above [Example 8-5], after a total of 4 weeks of orapten administration, the weights of each type of muscle tissue in all administration groups were confirmed. As a result, as shown in Figure 20, it was confirmed that the weights of the quadriceps, gastrocnemius, tibialis, and soleus muscles in the dexamethasone-administered group were reduced compared to the control group administered with saline, but that the weights of these muscles were significantly increased through the administration of auraptene.

Claims

1. A food composition for preventing or improving muscle diseases, comprising an extract of Citrus aurantium or a fraction thereof as an active ingredient.

2. The food composition of claim 1, wherein the muscle disease is one or more diseases selected from the group consisting of sarcopenia, muscle atrophy, myasthenia, muscle dystrophy, myotonia, hypotonia, muscle weakness, muscle degeneration, atony, amyotrophic lateral sclerosis, and inflammatory muscle diseases.

3. The food composition according to claim 1, characterized in that the composition increases the expression of one or more genes selected from the group of myotube cell differentiation markers consisting of Myf5, Myf6, Myod and Myog genes.

4. The food composition according to claim 1, characterized in that the composition increases the expression of Ppard or Pgc1a genes, which are markers of athletic performance.

5. 2. The food composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, organic solvents and mixtures thereof.

6. The food composition according to claim 5, wherein the organic solvent is one or more solvents selected from the group consisting of lower alcohols having 1 to 6 carbon atoms, hexane, acetone, ethyl acetate, chloroform, and diethyl ether.

7. A food composition for preventing or improving muscle diseases, comprising auraptene as an active ingredient.

8. A quasi-drug composition for preventing or improving muscle diseases, comprising an extract of Citrus aurantium or a fraction thereof as an active ingredient.

9. A quasi-drug composition for preventing or improving muscle diseases, comprising auraptene as an active ingredient.

10. A pharmaceutical composition for preventing or treating muscle diseases, comprising an extract of Citrus aurantium or a fraction thereof as an active ingredient.

11. The pharmaceutical composition for preventing or treating muscle diseases according to claim 10, characterized in that the muscle disease is one or more diseases selected from the group consisting of sarcopenia, muscle atrophy, myasthenia, muscle dystrophy, myotonia, hypotonia, muscle weakness, muscle degenerative atrophy, atony, amyotrophic lateral sclerosis, and inflammatory muscle diseases.

12. A pharmaceutical composition for preventing or treating muscle diseases, comprising auraptene as an active ingredient.

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