Pharmaceutical composition for preventing or treating of muscle diseases

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases. Specifically, the elderberry extract or the monosaccharide and amino acid compound according to the present invention can be usefully used for the treatment of muscle diseases by inhibiting the death of muscle cells, inhibiting the expression of inflammatory cytokines, promoting the secretion of testosterone, inhibiting the expression of muscle atrophy marker proteins and myostatin, and exhibiting the effect of restoring muscle in an animal model of sarcopenia.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of muscle diseases. Background Technology

[0003] Muscle is the most abundant tissue in the human body, and maintaining an appropriate muscle mass is necessary to sustain functional capacity and prevent metabolic diseases. Muscle size is regulated by intracellular signaling processes that induce anabolism or catabolism within the muscle. If signaling responses inducing synthesis of muscle protein occur more frequently than those inducing breakdown, muscle protein synthesis increases, leading to hypertrophy or hyperplasia.

[0004] Meanwhile, muscles promote the influx of calcium, thereby increasing bone density. However, as the body ages, its composition changes, resulting in a redistribution of body fat and body protein. Furthermore, after the age of 50, the rate of protein synthesis within muscle cells slows down compared to the rate of breakdown, causing muscles to begin to degenerate rapidly; consequently, one may become susceptible to diseases caused by muscle loss.

[0005] Sarcopenia, one of the diseases caused by muscle loss, is a condition in which the body's muscle mass decreases by approximately 13–24% of body mass. This indicates a decline not only in muscle mass but also in protein content, fiber diameter, muscle strength production, and fatigue resistance. Sarcopenia is caused by various factors, including sepsis, cancer, renal failure, excess glucocorticoids, neuropathy, muscle disuse, and aging. The primary causes are the gradual decrease in the quantity and quality of skeletal muscle due to aging or weight loss involving fat and body fat components resulting from an inappropriate diet.

[0006] Sarcopenia results from an imbalance between protein synthesis and breakdown. The presence of sarcopenia leads to a significant decrease in physical activity, resulting in a decline in quality of life and increased susceptibility to injury during daily activities. Furthermore, excessive exercise can induce muscle fatigue and damage, which in turn manifests as pain and temporary loss of mobility.

[0007] In this regard, Korean Patent Publication No. 10-2022-0113912 relates to a composition for the prevention, improvement, or treatment of muscle diseases comprising castor oil extract as an active ingredient, and discloses that castor oil extract can be used to prevent, improve, or treat muscle diseases without side effects by increasing the size of muscle cells and improving exercise performance. Prior art literature

[0009] Republic of Korea Patent Publication No. 10-2022-0113912 The problem to be solved

[0010] The object of the present invention is to provide elderberry extract for the prevention, improvement, or treatment of muscle diseases.

[0011] Another objective of the present invention is to provide a muscle-strengthening use of elderberry extract.

[0012] Another object of the present invention is to provide a use of a monosaccharide and amino acid combined compound, or a pharmaceutically acceptable salt thereof, for the prevention, improvement, or treatment of muscle diseases.

[0013] Another object of the present invention is to provide a muscle-enhancing use of a monosaccharide and amino acid combined compound, or a pharmaceutically acceptable salt thereof. means of solving the problem

[0015] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising elderberry extract as an active ingredient.

[0016] In addition, the present invention provides a health functional food for the prevention or improvement of muscle diseases comprising elderberry extract as an active ingredient.

[0017] In addition, the present invention provides a health functional food for muscle strengthening comprising elderberry extract as an active ingredient.

[0018] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0019] In addition, the present invention provides a health functional food for the prevention or improvement of muscle diseases comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0020] Furthermore, the present invention provides a muscle-strengthening health functional food comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient. Effects of the invention

[0022] The elderberry extract or the monosaccharide and amino acid compound according to the present invention can be usefully used in the treatment of muscle diseases by inhibiting the death of muscle cells, inhibiting the expression of inflammatory cytokines, promoting the secretion of testosterone, inhibiting the expression of muscle atrophy marker proteins and myostatin, and exhibiting the effect of restoring muscle in an animal model of sarcopenia. Brief explanation of the drawing

[0024] Figure 1 is a diagram showing the results of observing the morphology of a myoblast before (A) and after (B) differentiation of a myoblast into a myoblast in one embodiment of the present invention using a microscope. Figure 2 is a diagram showing the results of observing the morphology of myotube cells before (A) and after (B) treatment with dexamethasone in one embodiment of the present invention using a microscope. Figure 3 is a graph showing the results of confirming the inhibitory effect on muscle cell death by elderberry hot water extract (A), elderberry 50% ethanol extract (B), or elderberry 100% ethanol extract (C) in one embodiment of the present invention. Figure 4 is a graph showing the results of confirming the inhibitory effect of FL (A), FV (B), or FI (C) on muscle cell death in one embodiment of the present invention. Figure 5 is a graph showing the results of confirming the inhibitory effect of various types of amino acids (leucine, valine, arginine, tyrosine, methionine, or phenylalanine) on muscle cell death in one embodiment of the present invention. Figure 6 is a graph showing the results of confirming the inhibitory effect of various types of amino acids (isoleucine, tryptophan, glycine, lysine, or threonine) on muscle cell death in one embodiment of the present invention. Figure 7 is a graph showing the results of confirming the inhibitory effect of elderberry hot water extract (A), elderberry 50% ethanol extract (B), or elderberry 100% ethanol extract (C) in one embodiment of the present invention on the expression of TNFα. Figure 8 is a graph showing the results of confirming the inhibitory effect of FL (A), FV (B), or FI (C) on the expression of TNFα in one embodiment of the present invention. Figure 9 is a graph showing the results of confirming the testosterone secretion-promoting effect by elderberry hot water extract (A) or FL (B) in one embodiment of the present invention. Figure 10 is a graph showing the results of confirming the effect of elderberry hot water extract (A), elderberry 50% ethanol extract (B), or elderberry 100% ethanol extract (C) on the expression of the MuRF-1 gene in one embodiment of the present invention. Figure 11 is a graph showing the results of confirming the effect of elderberry hot water extract (A), elderberry 50% ethanol extract (B), or elderberry 100% ethanol extract (C) on the expression of the atrogen-1 gene in one embodiment of the present invention. Figure 12 is a graph showing the results of confirming the inhibitory effect on the expression of the myostatin gene by elderberry hot water extract (A), elderberry 50% ethanol extract (B), or elderberry 100% ethanol extract (C) in one embodiment of the present invention. Figure 13 is a graph showing the result of confirming the effect of FL (A), FV (B), or FI (C) on the expression inhibition of the MuRF-1 gene in one embodiment of the present invention. Figure 14 is a graph showing the result of confirming the effect of FL (A), FV (B), or FI (C) on the expression inhibition of the atrogen-1 gene in one embodiment of the present invention. Figure 15 is a graph showing the result of confirming the inhibitory effect of FL (A), FV (B), or FI (C) on the expression of the myostatin gene in one embodiment of the present invention. FIG. 16 is a schematic diagram showing an animal experiment plan using a sarcopenia animal model in one embodiment of the present invention. Figure 17 is a graph showing the results of confirming the change in body weight of an animal model of sarcopenia caused by elderberry hot water extract (A) or FL (B) in one embodiment of the present invention. Specific details for implementing the invention

[0025] The present invention will be described in detail below.

[0026] The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising elderberry extract as an active ingredient.

[0027] As used in this specification, the term "elderberry" refers to a berry fruit that is purple-black in color and is also called black elder because it typically bears fruit in the autumn. In addition, in North America, elderberry extract is also called sambucol. Elderberries are known to be rich in vitamins A, B, and C and anthocyanins, and have been reported to be effective in preventing colds and strengthening immunity.

[0028] The above elderberry extract can be prepared by a manufacturing method comprising the following steps:

[0029] 1) A step of preparing an extract by adding an extraction solvent to elderberries;

[0030] 2) A step of filtering the extract of step 1); and

[0031] 3) A step of drying the filtered filtrate from step 2) after concentrating it under reduced pressure.

[0032] Additionally, the extraction solvent may be water, alcohol, or a mixture thereof. The alcohol may be a C1 to C2 lower alcohol, and specifically, the C1 to C2 lower alcohol may be ethanol, methanol, or spirit. The extraction solvent may be added in an amount of 1 to 100 times, 1 to 70 times, 1 to 50 times, 1 to 30 times, or 1 to 15 times the weight of the elderberry. When the extraction solvent is used as an alcohol, the alcohol may be 10 to 100%, 20 to 100%, 30 to 100%, or 40 to 100% alcohol.

[0033] The above extraction method may be shaking extraction, cold maceration extraction, reflux extraction, or ultrasonic extraction. In this case, the extraction time may be 1 to 20 hours, 2 to 20 hours, 1 to 10 hours, 2 to 10 hours, 1 to 5 hours, or 2 to 5 hours. The above extraction may be repeated one or more times.

[0034] Meanwhile, the vacuum concentration in step 3) above may utilize a vacuum vacuum concentrator or a vacuum rotary evaporator. Additionally, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze-drying, and specifically, freeze-drying.

[0035] The aforementioned muscle disease may be a disease caused by muscle loss. In another aspect, the aforementioned muscle disease may be a progressive disease including loss of walking ability, weakening of respiratory muscle strength, and weakening of cardiac function resulting from a gradual decrease in muscle strength. Additionally, the aforementioned muscle disease may be a congenital or acquired disease. For example, the aforementioned disease caused by muscle loss may be stony, muscular atrophy, muscular dystrophy, muscle degeneration, rigidity, muscular dystrophy, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, or sarcopenia.

[0036] The pharmaceutical composition according to the present invention may contain 10 to 95 weight percent of an elderberry extract, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may additionally include one or more active ingredients that exhibit the same or similar functions in addition to the above active ingredient.

[0037] The pharmaceutical composition of the present invention may comprise a carrier, diluent, excipient, or mixture thereof that is commonly used in biological preparations. Any pharmaceutically acceptable carrier suitable for delivering the composition in vivo may be used. Specifically, said carrier may be a compound listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. Additionally, conventional additives such as antioxidants, buffers, bacteriostatic agents, etc., may be added as needed.

[0038] When formulating the above composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

[0039] The composition of the present invention may be formulated into an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. Additionally, the solid preparation may include a lubricant, examples of which include magnesium styrate, talc, etc. Meanwhile, the liquid preparation may be a suspension, an oral liquid, an emulsion, or a syrup. In this case, the liquid preparation may include excipients such as humectants, sweeteners, flavorings, preservatives, etc.

[0040] The above parenteral preparations may include injectables, suppositories, powders for respiratory inhalation, aerosols for spray, powders, and creams. The above injectables may include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, etc. In this case, as non-aqueous solvents or suspension solvents, vegetable oils such as propylene glycol, polyethylene glycol, or olive oil, or injectable esters such as ethyl oleate may be used.

[0041] The composition of the present invention may be administered orally or parenterally according to the intended method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, or intrathoracic injection methods.

[0042] The above composition may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, drugs used concurrently, etc. However, for a desirable effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg / kg, specifically 0.001 to 500 mg / kg. The administration may be once or several times a day.

[0043] The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, the administration may be sequential or simultaneous.

[0045] In addition, the present invention provides a health functional food for the prevention or improvement of muscle diseases comprising elderberry extract as an active ingredient.

[0046] The elderberry extract included in the health functional food according to the present invention may have the characteristics described above. The muscle disease may also have the characteristics described above.

[0047] The elderberry extract of the present invention may be added to food as is or used together with other food or food ingredients. At this time, the content of the added active ingredient may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total weight of the health functional food.

[0048] The form and type of the health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional food may include various flavoring agents, sweeteners, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of natural sweeteners include taumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrate may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, and sugar alcohols.

[0049] In addition to the additional ingredients described above, the health functional food of the present invention may further include nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, Pextan and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, etc. These ingredients may be used independently or in combination. The proportion of the additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0051] In addition, the present invention provides a health functional food for muscle strengthening comprising elderberry extract as an active ingredient.

[0052] The elderberry extract included in the health functional food according to the present invention may have the characteristics described above. The health functional food may also have the characteristics described above.

[0054] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0055] As used herein, the term "monosaccharide" refers to the most basic unit of carbohydrates, also known as simple sugar. The monosaccharide is generally a colorless, water-soluble crystalline solid and includes glucose, fructose, galactose, etc. In one embodiment of the present invention, the monosaccharide may be fructose. The term "fructose" refers to a monosaccharide containing six carbon atoms, which is a ketose having a ketone group, C6H 12 It is represented by the chemical formula O6. The above fructose is an intermediate product in the breakdown of glucose or the synthesis of glycogen in the sugar metabolism of living organisms.

[0056] In addition, the term "amino acid" as used in this specification refers to a compound that contains both a chemically basic amino group (-NH2) and an acidic carboxyl group (-COOH), serving as the basic building blocks of proteins that constitute living organisms. Generally, when proteins are hydrolyzed by acids or enzymes such as pepsin or trypsin, they are broken down into various types of amino acids. The amino acids thus produced are absorbed into the body, rearranged, and synthesized into proteins that constitute the body. These amino acids are classified into non-essential amino acids and essential amino acids.

[0057] The above amino acid may include all types of amino acids known in the ordinary art, and specifically, the above amino acid may be a branched-chain amino acid (BCAA). The term "BCAA (branched-chain amino acid)" means an amino acid having a branched chain, which is a central carbon atom bonded to three or more carbon atoms, and an aliphatic side chain. Specifically, the above BCAA may be leucine, valine, or isoleucine.

[0058] The monosaccharide and amino acid bonding compound according to the present invention refers to a compound obtained by bonding a monosaccharide and an amino acid having the characteristics described above by a method well known in the ordinary art. For example, the monosaccharide and amino acid bonding compound may be a compound in which BCAA is bonded to fructose. Specifically, the monosaccharide and amino acid bonding compound may be a compound in which leucine, valine, or isoleucine is bonded to fructose, and more specifically, the monosaccharide and amino acid bonding compound may be a compound represented by the following chemical formulas 1 to 3.

[0059] [Chemical Formula 1]

[0060] ,

[0061] [Chemical Formula 2]

[0062] , and

[0063] [Chemical Formula 3]

[0064] .

[0066] In addition, the present invention may include not only the monosaccharide and amino acid combined compounds, but also pharmaceutically acceptable salts thereof.

[0067] Here, "pharmaceuticalally acceptable salt" refers to a salt suitable for use in contact with human and lower animal tissues within the scope of purely medical judgment, without causing excessive toxicity, irritation, or allergic reactions. The above-mentioned pharmaceutically acceptable salt is well known in the field, and, for example, in the literature (SM Berge et al. , J. Pharmaceutical SciencesThis is described in detail in , 66, 1, 1977). The salt can be prepared in the same reaction system during the final separation and purification of the compound of the present invention, or separately by reacting with an inorganic base or an organic base. Specific examples of base addition salts include alkali salts and alkaline earth metal salts such as ammonium salts, lithium, sodium, potassium, magnesium, calcium, etc., salts with organic bases, for example, primary, secondary, and tertiary aliphatic and aromatic amines, for example, methylamine, ethylamine, propylamine, isopropylamine, four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidein, pyridine, quinoline and isoquinoline, benzathine, N-methyl-D-glucarmine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydravamin salts, and salts with amino acids such as arginine and lysine.

[0068] In addition, the present invention may include hydrates or solvates of the monosaccharide and amino acid bonding compounds, and derivative compounds thereof. Among the solvates, the solvent is not particularly limited and may include all conventional solvents known in the art.

[0069] The aforementioned muscle disease may be a disease caused by muscle loss. In another aspect, the aforementioned muscle disease may be a progressive disease including loss of walking ability, weakening of respiratory muscle strength, and weakening of cardiac function resulting from a gradual decrease in muscle strength. Furthermore, the aforementioned muscle disease may be a congenital or acquired disease. For example, the aforementioned disease caused by muscle loss may be hypotonia, muscular atrophy, muscular dystrophy, muscle degeneration, rigidity, muscular dystrophy, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, or sarcopenia.

[0070] The pharmaceutical composition according to the present invention may comprise 10 to 95 weight percent of a monosaccharide and amino acid compound, which is an active ingredient, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further comprise one or more active ingredients exhibiting the same or similar functions in addition to the above active ingredients.

[0071] The pharmaceutical composition of the present invention may comprise a carrier, diluent, excipient, or mixture thereof that is commonly used in biological preparations. Any pharmaceutically acceptable carrier suitable for delivering the composition in vivo may be used. Specifically, said carrier may be a compound listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. Additionally, conventional additives such as antioxidants, buffers, bacteriostatic agents, etc., may be added as needed.

[0072] When formulating the above composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

[0073] The composition of the present invention may be formulated into an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. Additionally, the solid preparation may include a lubricant, examples of which include magnesium styrate, talc, etc. Meanwhile, the liquid preparation may be a suspension, an oral liquid, an emulsion, or a syrup. In this case, the liquid preparation may include excipients such as humectants, sweeteners, flavorings, preservatives, etc.

[0074] The above parenteral preparations may include injectables, suppositories, powders for respiratory inhalation, aerosols for spray, powders, and creams. The above injectables may include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, etc. In this case, as non-aqueous solvents or suspension solvents, vegetable oils such as propylene glycol, polyethylene glycol, or olive oil, or injectable esters such as ethyl oleate may be used.

[0075] The composition of the present invention may be administered orally or parenterally according to the intended method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, or intrathoracic injection methods.

[0076] The above composition may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, drugs used concurrently, etc. However, for a desirable effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg / kg, specifically 0.001 to 500 mg / kg. The administration may be once or several times a day.

[0077] The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, the administration may be sequential or simultaneous.

[0079] In addition, the present invention provides a health functional food for the prevention or improvement of muscle diseases comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0080] The monosaccharide and amino acid combined compound included in the health functional food according to the present invention, or a pharmaceutically acceptable salt thereof, may have the characteristics described above. The muscle disease may also have the characteristics described above.

[0081] The monosaccharide and amino acid compound of the present invention, or a pharmaceutically acceptable salt thereof, may be added to food as is or used together with other food or food ingredients. In this case, the content of the added active ingredient may be determined according to the purpose and, generally, may be 0.01 to 90 parts by weight of the total weight of the health functional food.

[0082] The form and type of the health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills. The health functional food may include various flavoring agents, sweeteners, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of natural sweeteners include taumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrate may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, and sugar alcohols.

[0083] In addition to the additional ingredients described above, the health functional food of the present invention may further include nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, Pextan and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, etc. These ingredients may be used independently or in combination. The proportion of the additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0085] Furthermore, the present invention provides a muscle-strengthening health functional food comprising a monosaccharide and amino acid compound, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0086] The monosaccharide and amino acid combined compound included in the health functional food according to the present invention, or a pharmaceutically acceptable salt thereof, may have the characteristics described above. The health functional food may also have the characteristics described above.

[0088] The present invention is described in detail below by way of the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention. Any configuration having substantially the same structure as the technical concept described in the claims of the present invention and achieving the same functional effect is included within the technical scope of the present invention.

[0090] Example 1. Preparation of Elderberry Hot Water Extract

[0091] A hot water extract was prepared using dried elderberries in the following manner.

[0092] First, 10 times the weight of the dried elderberries was added to the purified water, and the mixture was extracted at 80°C for 3 hours to obtain an extract. The obtained extract was filtered using a metal detector and concentrated to obtain an elderberry concentrate. 30% maltodextrin was mixed with the elderberry concentrate and freeze-dried to prepare a powder of the elderberry hot water extract.

[0094] Example 2. Preparation of Elderberry 50% Ethanol Extract

[0095] A powder of elderberry 50% ethanol extract was prepared using the same method and conditions as in Example 1, except that 50% ethanol was added instead of purified water and extracted for 6 hours.

[0097] Exempl 3. Preparation of Elderberry 100% Ethanol Extract

[0098] A powder of elderberry 100% ethanol extract was prepared using the same method and conditions as in Example 1 above, except that 100% ethanol was added instead of purified water and extracted for 6 hours.

[0100] Example 4. Component analysis of elderberry extract

[0101] Liquid chromatography / mass spectrometry (LC / MS) was performed using the elderberry hot water extract prepared above to confirm the presence of FL (fructose-leucine) in the extract.

[0102] Specifically, 200 mg of elderberry hot water extract powder was dissolved in water, methanol was added, and the mixture was sonicated for at least 60 minutes to prepare a sample. Meanwhile, a calibration curve was constructed using FL of 0.2 mg / L to establish the quantitative range. Analysis was performed using the sample prepared under the above conditions, and this process was repeated three times. As a result, the FL content present in the elderberry hot water extract was calculated using the obtained peak area and test solution RT by a conventional method, and the results are shown in Table 1.

[0103] Sample Peak area Test solution RT FL content (%) Average FL content (%) #1 7844 2.899 1.02 1.02 #2 7713 2.892 1.01 #3 7869 2.904 1.03

[0105] As shown in Table 1, it was found that the average FL content in the samples was 1.02%.

[0107] Experimental Example 1. Inhibition of muscle cell death

[0108] Whether the elderberry extract prepared above and the compound combining monosaccharides and amino acids inhibit muscle cell death was confirmed by the following method.

[0110] 1-1. Preparation of Myotubes

[0111] First, the C2C12 cell line (ATCC, USA), a myoblast cell, was cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotic-antifungal agent at 37°C under 5% CO2 conditions. The culture medium was replaced with a culture medium containing 2% HS (horse serum), and the cells were cultured for an additional 72 hours. The cultured cells were observed under a microscope to confirm whether the myoblasts had differentiated into myotubes, and 10 μM of dexamethasone was added and the cells were incubated for 24 hours. The cells were observed under a microscope again after the reaction was complete. As a result, the results confirming differentiation into myotubes are shown in Figure 1, and the results of microscopic observation after treatment with dexamethasone are shown in Figure 2.

[0112] As shown in Fig. 1, the myoblasts differentiated into myotubes. In addition, as shown in Fig. 2, it was confirmed that the muscle was reduced as the thickness of the myotubes decreased due to treatment with dexamethasone.

[0114] 1-2. Inhibition of muscle cell apoptosis by elderberry extract

[0115] Cell lines treated with dexamethasone as described above were treated with 12.5, 25, 50, or 100 μg / ml of elderberry hot water extract, or 100, 200, 300, or 400 μg / ml of elderberry 50% ethanol extract, or 100, 200, 300, or 400 μg / ml of elderberry 100% ethanol extract. After 24 hours of treatment, the cell culture medium was removed, 5 mg / ml of MTT reagent was added per well, and the cells were cultured for 4 more hours. Subsequently, DMSO was added to dissolve the precipitate, and the absorbance was measured at a wavelength of 570 nm. Cell viability was calculated from the measured values ​​using a conventional method, and the results are shown in Figure 3.

[0116] As shown in Figure 3, cell viability reduced by dexamethasone was significantly restored by treatment with elderberry extract.

[0118] 1-3. Inhibition of Muscle Cell Apoptosis by Monosaccharide and Amino Acid-Bound Compounds

[0119] Inhibition of muscle cell death by FL, which was confirmed to be present in elderberry extract, as well as by FV (fructose-valine) and FI (fructose-isoleucine) compounds, in which the branched-chain amino acid (BCAA) valine or isoleucine is bound to the monosaccharide fructose, was confirmed. The experiment was conducted under the same method and conditions as Experimental Examples 1-2 above, except that FL, FV, or FI were treated at concentrations of 2.5, 5, 10, or 20 μg / ml instead of elderberry extract. At this time, leucine, valine, isoleucine, tryptophan, arginine, tyrosine, glycine, lysine, methionine, phenylalanine, or threonine were used as controls. As a result, the cell viability calculated from treatment with FL, FV, or FI is shown in Fig. 4, and the cell viability calculated from treatment with amino acids is shown in Figs. 5 and 6.

[0120] As shown in Figure 4, cell viability reduced by dexamethasone was significantly restored by FL, FV, or FI. On the other hand, as shown in Figures 5 and 6, cell viability was not restored by treatment with amino acids.

[0122] Experimental Example 2. Inhibition of inflammatory cytokine expression

[0123] It was confirmed by the following method whether the elderberry extract prepared above and the compound combining monosaccharides and BCAAs inhibit the expression of inflammatory cytokines.

[0125] 2-1. Inhibition of TNFα Expression by Elderberry Extract

[0126] In the same manner as in Experimental Examples 1-2 above, cells treated with elderberry extract were treated with trypsin to recover only the cells. The recovered cells were centrifuged at 3,000 rpm for 5 minutes to remove the supernatant, and total RNA was extracted by sequentially treating with RiboEx™ (JINOL, Korea) and Hybrid-R™ (JINOL, Korea). The concentration of the extracted RNA was measured using the nanodrop method, and cDNA was synthesized using this as a template. cDNA was synthesized using a mixture of RNA, oligo-(dT) primers, and 2×HyperScript™ RT Master Mix (JINOL, Korea). Subsequently, real-time PCR (qPCR) was performed under standard conditions using Power SYBR™ Green PCR Master Mix with the synthesized cDNA as a template to confirm the expression of TNFα. As a result, the graph confirming the expression level of TNFα mRNA is shown in Figure 7.

[0127] As shown in Figure 7, the expression level of TNFα, which was increased by dexamethasone, was significantly inhibited by treatment with elderberry extract.

[0129] 2-2. Inhibition of TNFα Expression by Monosaccharide and Amino Acid-Bound Compounds

[0130] In the same manner as in Experimental Examples 1-3 above, cells treated with FL, FV, or FI were treated with trypsin to recover only the cells, and the expression level of TNFα was confirmed under the same conditions and methods as described in Experimental Example 2-1. The resulting graph is shown in FIG. 8.

[0131] As shown in Figure 8, the expression level of TNFα increased by dexamethasone was significantly inhibited by treatment with FL, FV, or FI.

[0132] Therefore, from the above results, it was found that the expression of inflammatory cytokines that directly induce muscle wasting was significantly inhibited by elderberry extract and monosaccharide and amino acid-binding compounds, indicating that these components inhibit muscle cell death induced by inflammatory cytokines.

[0134] Experimental Example 3. Promotion of male hormone secretion

[0135] The elderberry extract prepared above and the compound combining monosaccharides and BCAAs were confirmed as follows using the ELISA analysis method to inhibit muscle loss caused by the male hormone testosterone.

[0136] First, mouse Leydig cell line TM3 (ATCC, USA) was cultured using conventional methods. The cultured cell line was treated with 500 μM hydrogen peroxide (H2O2) for 6 hours, and 12.5, 25, 50, or 100 μg / ml of elderberry hot water extract, or 1.25, 2.5, 5, or 10 μg / ml of FL was added. Subsequently, ELISA was performed using the cell culture medium using conventional methods to determine the levels of testosterone, and the results are shown in Figure 9.

[0137] As shown in Figure 9, the concentration of testosterone inhibited by hydrogen peroxide was significantly restored by elderberry hot water extract or FL.

[0139] Experimental Example 4. Inhibition of muscle protein expression

[0140] We confirmed by the qPCR method whether the elderberry extract and the compound combined with monosaccharides and BCAAs prepared above inhibit the expression of muscle proteins. Specifically, as muscle proteins, we used the muscle atrophy markers MuRF-1 (muscle RING-finger protein-1) and atrogin-1, and the myostatin protein, a negative regulator of skeletal muscle mass.

[0142] 4-1. Inhibition of Muscle Protein Expression by Elderberry Extract

[0143] As described in Experimental Examples 1-2, qPCR was performed using cells treated with elderberry hot water extract, 50% ethanol extract, or 100% ethanol extract. As a result, graphs showing the expression levels of MuRF-1, atrogen-1, and myostatin mRNA were confirmed are shown in Figures 10 to 12.

[0144] As shown in Figures 10 to 12, the mRNA expression levels of MuRF-1, atrozine-1, and myostatin, which were increased by dexamethasone, were significantly reduced by elderberry extract.

[0146] 4-2. Inhibition of Muscle Protein Expression by Monosaccharide and Amino Acid-Bound Compounds

[0147] As described in Experimental Examples 1-3, qPCR was performed using cells treated with FL, FV, or FI as described above. As a result, graphs showing the expression levels of MuRF-1, atrogen-1, and myostatin mRNA were confirmed are shown in Figures 10 to 12.

[0148] As shown in Figures 10 to 12, the mRNA expression levels of MuRF-1, atrozine-1, and myostatin, which were increased by dexamethasone, were significantly reduced by FL, FV, or FI.

[0149] Therefore, from the above results, it was found that elderberry extract, or monosaccharide and amino acid-binding compounds, exhibit a therapeutic effect on sarcopenia by inhibiting the expression of muscle atrophy markers and myostatin.

[0151] Experimental Example 5. Confirmation of effects in an animal model of sarcopenia

[0152] The therapeutic effect of the elderberry extract prepared above and the compound combining monosaccharides and BCAAs on sarcopenia was confirmed in an animal model of sarcopenia.

[0153] Specifically, 7-week-old male C57BL / 6 mice (Samtaco Bio Korea, Korea) were acclimatized, and body weight (g), lean body weight (g), fat weight (g), and body fat percentage (%) were measured via DXA (dual-energy-X-ray absorptiometry). Based on these measurements, the mice were grouped into sets of seven, and sarcopenia was induced in the grouped mice by administering 20 mg / kg of dexamethasone via intraperitoneal injection once daily for 14 days (Fig. 13). In addition, while administering dexamethasone, 300 mg / kg of elderberry hot water extract or 0.5 mg / kg of FL was administered orally once daily. At this time, mice on day 0 of administration (normal mice) were used as the control group. The results of measuring body weight, lean body weight, fat weight, and body fat percentage by DXA imaging of mice on days 7 and 14 of administration are shown in Fig. 14, Tables 2 and 3. Meanwhile, the results of sacrificing mice by conventional method on day 14 of administration, extracting the thymus, spleen, liver, and muscle, and measuring their weights are shown in Table 4.

[0154] Normal control group Dexamethasone administration group Elderberry hot water extract administration group Day 0 lean body weight(g) 18.84±1.03 18.93±1.42 18.65±0.71 fat(g) 1.28±0.16 1.44±0.16 1.4±0.19 fat(%) 6.37±0.79 7.1±1.07 6.98±1.00 tissue area (㎠) 13.71±0.62 13.32±2.03 13.98±0.84 BMD(g / ㎠) 0.053±0.003 0.057±0.012 0.055±0.004 BMC(g) 0.18±0.02 0.23±0.13 0.18±0.02 Day 7 lean body weight(g) 21.14±1.32 17.53±1.36 17.79±1.71 fat(g) 1.60±0.43 1.13±0.34 1.68±0.57 fat(%) 6.98±1.61 5.99±1.53 8.5±2.3 tissue area (㎠) 13.69±1.27 12.41±1.31 13.88±1.17 BMD(g / ㎠) 0.057±0.007 0.052±0.004 0.054±0.007 BMC(g) 0.26±0.08 0.16±0.02 0.19±0.04 Day 14 lean body weight(g) 22.47±0.59 17.47±0.29 19.22±0.5 fat(g) 1.65±0.39 1.56±0.34 1.79±0.45 fat(%) 6.79±1.37 8.17±1.58 8.49±1.94 tissue area (㎠) 15.12±1.01 13.97±0.51 15.29±0.50 BMD(g / ㎠) 0.064±0.004 0.057±0.004 0.058±0.002 BMC(g) 0.29±0.09 0.16±0.01 0.18±0.01

[0155] Normal control group Dexamethasone administration group FL administration group Day 0 lean body weight(g) 18.84±1.03 18.93±1.42 18.31±1.43 fat(g) 1.28±0.16 1.44±0.16 1.27±0.25 fat(%) 6.37±0.79 7.1±1.07 6.49±1.03 tissue area (㎠) 13.71±0.62 13.32±2.03 13.13±1.08 BMD(g / ㎠) 0.053±0.003 0.057±0.012 0.053±0.004 BMC(g) 0.18±0.02 0.23±0.13 0.19±0.04 Day 7 lean body weight(g) 21.14±1.32 17.53±1.36 18.05±0.44 fat(g) 1.60±0.43 1.13±0.34 1.43±0.53 fat(%) 6.98±1.61 5.99±1.53 7.31±2.47 tissue area (㎠) 13.69±1.27 12.41±1.31 13.83±0.53 BMD(g / ㎠) 0.057±0.007 0.052±0.004 0.059±0.009 BMC(g) 0.26±0.08 0.16±0.02 0.20±0.03 Day 14 lean body weight(g) 22.47±0.59 17.47±0.29 18.34±0.56 fat(g) 1.65±0.39 1.56±0.34 1.29±0.19 fat(%) 6.79±1.37 8.17±1.58 6.57±0.76 tissue area (㎠) 15.12±1.01 13.97±0.51 13.66±0.92 BMD(g / ㎠) 0.064±0.004 0.057±0.004 0.056±0.006 BMC(g) 0.29±0.09 0.16±0.01 0.19±0.04

[0156] group Normal control group Dexamethasone administration group Elderberry hot water extract administration group FL administration group Thymus (g) 0.036±0.006 0.009±0.004 0.011±0.003 0.009±0.002 Spleen (g) 0.075±0.007 0.026±0.01 0.026±0.009 0.028±0.009 Liver (g) 1.338±0.106 1.514±0.134 1.661±0.302 1.375±0.117 Muscle (g) 0.608±0.045 0.501±0.064 0.598±0.142 0.683±0.18

[0158] As shown in Figure 14, Tables 2 and 3, the body weight, lean body weight, and tissue area of ​​mice were significantly reduced by dexamethasone, but were restored by the administration of elderberry hot water extract or FL. In addition, the weight of each tissue was also reduced by dexamethasone, but was restored to a level similar to that of the normal control group by the administration of elderberry hot water extract or FL.

[0159] Therefore, from the above results, it was found that elderberry extract, or monosaccharide and amino acid combined compounds, exhibited a significant effect in the treatment of sarcopenia.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of a muscle disease comprising an elderberry hot water extract as an active ingredient, wherein the muscle disease is a disease caused by muscle loss, and the disease caused by muscle loss is sarcopenia. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A health functional food for the prevention or improvement of muscle diseases comprising an elderberry hot water extract as an active ingredient, wherein the muscle disease is a disease caused by muscle loss, and the disease caused by muscle loss is sarcopenia. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete

Images