Composition for preventing and alleviating or treating skeletal muscle atrophy or sarcopenia, comprising extract of stellaria dichotoma l. var. lanceolata bunge or compound isolated therefrom as active ingredient

Abstract

Provided is a pharmaceutical composition for preventing and treating or alleviating skeletal muscle atrophy or sarcopenia, comprising an extract of Stellaria dichotoma L. var. lanceolata Bunge as an active ingredient. A composition for preventing, alleviating, or treating muscle atrophy or sarcopenia of the present invention comprising, as an active ingredient, an extract of Stellaria dichotoma L. var. lanceolata Bunge, a fraction thereof, or dichotomine B, glucodichotomine B, shaftoside, or isoshaftoside isolated therefrom increases the thickness of muscle fibers, has few side effects due to being derived from natural products, and has high safety, thereby being able to be useful for preventing, alleviating, or treating muscle atrophy or sarcopenia.

Description

A composition for preventing, improving or treating skeletal muscle atrophy or sarcopenia containing an extract of Eunshiho or a compound isolated therefrom as an active ingredient

[0001] The present invention relates to a composition for preventing and treating skeletal muscle atrophy or sarcopenia, and more specifically, to a pharmaceutical composition, food composition or feed composition for preventing, improving or treating skeletal muscle atrophy or sarcopenia, containing an extract of Stellaria dichotoma L. var. lanceolataBunge or a compound isolated therefrom as an active ingredient.

[0002] Skeletal muscle is the largest tissue in the human body, comprising approximately 600 muscles and accounting for 40-50% of body weight. They play a crucial role in bodily functions such as movement, breathing, posture maintenance, body temperature maintenance, and energy metabolism. Skeletal muscle mass is regulated by the balance between protein synthesis and breakdown within the muscle. If breakdown exceeds synthesis, skeletal muscle mass declines, with a rate of approximately 8% per decade beginning in the 40s. By the age of 70, the rate of decline has increased to 15% per decade.

[0003] Muscle atrophy is caused by various factors, including aging, long-term muscle disuse, lack of exercise, hormonal deficiencies, and various diseases (cachexia, sepsis, starvation, cancer, diabetes, chronic inflammation, degenerative diseases, etc.). When muscle atrophy occurs, the muscle strength required for physical activity is weakened, and it further leads to musculoskeletal degeneration, which leads to decreased exercise capacity, decreased physical function, increased risk of falls and fractures, decreased ability to perform daily activities, and increased risk of death, all of which drastically reduce the quality of life. Furthermore, the loss of skeletal muscle mass and musculoskeletal degeneration increases the risk of various diseases, including fractures, joint damage, metabolic syndrome, obesity, dyslipidemia, and cardiovascular disease. Therefore, reducing the occurrence of muscle atrophy and skeletal muscle loss caused by various factors and increasing regeneration are crucial for leading a healthy and happy life.

[0004] Muscle atrophy is primarily caused by inhibition of muscle fiber synthesis through the inhibition of the PI3K / Akt / mTOR pathway and its downstream factors, such as 4E-BP1 and p70S6K, and increased protein degradation through activation of the ubiquitin-proteasome pathway. Muscle atrophy F-box (MAFbx) / atrogin-1 and Muscle RING finger-1 (MuRF1) are representative protein degradation factors, and increased expression of these factors directly affects muscle protein degradation.

[0005] For the regeneration of damaged skeletal muscle, proliferation and differentiation of myoblasts, as well as hypertrophic growth of myotubes, are essential. Myoblasts secrete various growth factors for proliferation and induce differentiation into myotubes through the expression of myogenic proteins such as MyoD, Myogenin, and MHC (Myosin Heavy Chain). Skeletal muscle anabolism is regulated by mTOR (Mammalian target of rapamycin) signaling. Phosphorylation (activation) of mTOR induces phosphorylation of p70S6K (Ribosomal p70 S6 kinase), which ultimately increases the synthesis of myogenic proteins.

[0006] Research has reported the efficacy of various natural products (Coptis chinensis, ginseng, black soybean, and black ginseng) and their constituents in suppressing muscle atrophy and inducing skeletal muscle cell differentiation and hypertrophy. These results demonstrate that natural products can effectively suppress muscle atrophy and induce skeletal muscle cell differentiation and hypertrophy, and hold great potential for development as health functional foods.

[0007] Stellaria dichotoma L. var.lanceolataBunge is the dried root of Stellaria dichotoma L. var.lanceolataBunge, a member of the Caryophyllaceae family, cultivated in China, Europe, Russia, and Mongolia. It is nearly cylindrical and branched, 15-40 cm long, and 10-25 mm in diameter. The outer surface is light yellow or yellowish white, and has twisted longitudinal wrinkles and traces of lateral roots. It is concave and hollow like a hole. When cut lengthwise near this area, a powdery substance comes out and a brown gap can be seen. At the upper end, you can see traces of a stem that was attached in a dense, warty shape. The texture is hard but brittle and easy to cut. The cut surface has crevices, the skin is very thin, and the xylem has a radial pattern of yellow and white alternating. Other names include Baekgeunja, Sasam-a, Udungun, Eunhasiho, Eunho, and Tosam. The roots are dug up in spring or fall, washed in water, trimmed, and dried in the sun for medicinal use.

[0008] The main compounds of Eunshiho are β-carboline alkaloids, cyclic peptide alkaloids, and various compounds such as flavonoids, neolignans, sterols, and terpenoids have been reported. Eunshiho has the effects of reducing fever and clearing heat, so it is mainly used as an antipyretic in oriental medicine, and it is also used as a herbal medicine to treat wasting diseases and infant malnutrition syndrome. In addition, recent studies have reported that it has the effects of anti-allergy, vasorelaxation, anti-inflammation, and improving atopic dermatitis.

[0009] However, the preventive and improving effects of Eunshiho extract on skeletal muscle atrophy have not yet been reported. Accordingly, the inventors of the present invention conducted extensive research on natural ingredients capable of preventing muscle atrophy and, as a result, confirmed that a composition containing Eunshiho extract has an excellent effect in preventing muscle atrophy, thereby completing the present invention.

[0010] Accordingly, the problem to be solved by the present invention is to provide a composition for preventing, improving or treating skeletal muscle atrophy or sarcopenia using an extract, fraction or compound isolated therefrom of Eunshiho.

[0011] In order to solve the above problem, the present invention provides a pharmaceutical composition for preventing and treating or improving skeletal muscle atrophy or sarcopenia, which contains an extract of Eunshiho as an active ingredient.

[0012] In one embodiment of the present invention, the silver ginseng extract is extracted with one or more solvents selected from the group consisting of water (H2O), lower alcohols of C1 to C4, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

[0013] In one embodiment of the present invention, the pharmaceutical composition is a fraction fractionated from the extract of the silver ginseng.

[0014] The present invention also provides a pharmaceutical composition for preventing or treating skeletal muscle atrophy, comprising as an active ingredient at least one selected from the group consisting of Dichotomine B, Glucodichotomyne B, Shaftoside, Isoshaftoside and pharmaceutically acceptable salts thereof.

[0015] In one embodiment of the present invention, the Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

[0016] In one embodiment of the present invention, the muscle atrophy is at least one selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, cachexia, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

[0017] The present invention also provides a food composition for preventing or improving skeletal muscle atrophy or sarcopenia, comprising an extract of Eunshiho as an active ingredient.

[0018] The present invention also provides a feed composition for preventing or improving skeletal muscle atrophy or sarcopenia, comprising an extract of Eunshiho as an active ingredient.

[0019] The present invention also provides a food composition for preventing or improving skeletal muscle atrophy, comprising as an active ingredient at least one selected from the group consisting of Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside, wherein Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

[0020] The present invention also provides a feed composition for preventing or improving skeletal muscle atrophy, comprising as an active ingredient at least one selected from the group consisting of Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside, wherein Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

[0021] The composition for preventing, improving or treating muscular dystrophy or sarcopenia, which contains the extract or fraction of the present invention or Dichotomine B, Glucodichotomycin B, Shaftoside or Isoshaftoside isolated therefrom as an active ingredient, increases the thickness of muscle fibers and, since it is derived from a natural product and has almost no side effects and high safety, can be usefully utilized for preventing, improving or treating muscular dystrophy or sarcopenia.

[0022] Figure 1 shows the method for preparing the extract and fraction of Eunshiho, Figure 2 shows the HPLC chromatography results of the extract and fraction, Figure 3 shows the photograph of the extract and fraction treated on C2C12 myotube cells, and Figure 4 shows the thickness of the extract and fraction treated on C2C12 myotube cells.

[0023] Figure 5 is a schematic diagram illustrating the process of separating and purifying compounds 1 to 5 from the n-butanol fraction of Eunshiho.

[0024] Figure 6 is an HPLC chromatogram of (A) butanol fraction and purified compounds (1-5) in the extract of Eunshilho, Figure 7 is the structure of purified compounds 1-5, Figure 8 is a photograph of myotubes treated with n-BuOH fraction and compounds 1-5, Figure 9 shows the degree of cell death and cell thickness after treating C2C12 myotubes with 30 μg / mL of n-BuOH fraction and 30 μM of compounds 1-5.

[0025] Figure 10 shows cell viability in 30 μM treatment with dichotomine B (2), Figure 11 shows MHC staining photographs after 10 μM and 30 μM treatment with dichotomine B following dexamethasone treatment, Figure 12 shows cell thickness, and Figure 13 shows nuclei infusion index.

[0026] Figure 14 shows western blot images of MHC (Myosin heavy chain), Atrogin-1, and MuRF-1 after treatment with 10 μM and 30 μM dichotomine B following dexamethasone treatment, and Figure 15 shows the degree of their expression.

[0027] Figure 16 shows the evaluation of the efficacy of dichotomine B treatment at 1 μM and 10 μM in the Starvation model of C2C12 myotube cells, including cell viability by treatment with 1 nM - 10 μM of dichotomine B, Figure 17 shows the effect of dichotomine B on cell viability in the Starvation model, Figure 18 shows an MHC staining photograph, Figure 19 shows the thickness of myotube cells, and Figure 20 shows the relative MHC staining degree.

[0028] Figure 21 shows the results of western blot of MHC, FoxO3a, Atrogin-1 and MuRF-1 by treatment with 1 μM and 10 μM of dichotomine B in the Starvation model of C2C12 myotube cells. Western blot by treatment with 1 μM and 10 μM of dichotomine B, Figure 22 shows the level of MHC expression, Figure 23 shows the level of FoxO3a expression, Figure 24 shows the level of Atrogin-1 expression, and Figure 25 shows the level of MuRF-1 expression.

[0029] Figure 26 shows the change in body weight due to administration of 10 mg / kg of dichotomine B to mice starved for 48 hours, and Figure 27 shows the change in grip strength.

[0030] Figure 28 shows the change in skeletal muscle weight due to administration of 10 mg / kg of dichotomine B to mice starved for 48 hours. The pictures are of TA, EDL, GA, and SOL, and Figure 29 shows the weight.

[0031] Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0032] Before describing the present invention in detail, it should be noted that the terms or words used in this specification should not be interpreted as being unconditionally limited to their usual or dictionary meanings, and the inventor of the present invention may appropriately define and use the concepts of various terms in order to describe his or her invention in the best possible manner.

[0033] Furthermore, it should be noted that these terms and words should be interpreted with meanings and concepts that are consistent with the technical idea of ​​the present invention.

[0034] That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention.

[0035] It should be noted that these terms are defined taking into account the various possibilities of the present invention.

[0036] Additionally, in this specification, a singular expression may include a plural expression unless the context clearly indicates a different meaning.

[0037] Throughout this specification, whenever a component is described as "including" another component, it may mean that the component may further include any other component, rather than excluding any other component, unless specifically stated otherwise.

[0038] The present invention is intended to solve the above-described problem, and as a result of extensive research to search for a natural ingredient that can alleviate skeletal muscle loss, it was confirmed that the diameter reduction of C2C12 cells caused by dexamethasone can be effectively prevented by treatment with an extract of Eunshiho.

[0039] In addition, a fraction was prepared from the extract of Eunssiho by solvent-solvent fractionation, and this was also confirmed to have the effect of improving skeletal muscle loss.

[0040] In one embodiment of the present invention, five compounds were separated and purified through various chromatography and HPLC fractionation from the n-butanol fraction, which is an active fraction, and the structures of the five compounds were identified as Glucodichotomyne B, Dichotomine B, Shaftoside, Dichotomine A, and Isoshaftoside using NMR and MASS, respectively. Among them, Glucodichotomyne B, Dichotomine B, Shaftoside, and Isoshaftoside effectively prevented the reduction in diameter of C2C12 myotube cells caused by dexamethasone.

[0041] In one embodiment of the present invention, the Glucodichotomyne B, Dichotomine B, Shaftoside, and Isoshaftosid are as shown in FIG. 7.

[0042] In particular, the present invention confirmed that Dichotomine B, which was isolated and purified as an active ingredient of the extract of Eunssiho and had a structure identified, effectively prevented a decrease in cell diameter using a starvation model in C2C12 myotube cells, and also improved the area of ​​MHC staining sites. In addition, it was confirmed that intraperitoneal administration of 10 mg / kg of Dichotomine B improved the decrease in calf muscle weight and grip strength caused by 48 hours of starvation in 7-week-old C57BL / 6 mice, thereby completing the present invention.

[0043] Accordingly, the present invention provides a method for preventing, improving, and / or treating muscle atrophy, comprising an extract or fraction of Stellaria dichotoma Linne var. lanceolata Bunge, or Dichotomine B, Glucodichotoma B, Shaftoside, or Isoshaftoside isolated therefrom as an active ingredient.

[0044] The extract of the silver ginseng of the present invention can be extracted using a conventional solvent under conditions of conventional temperature and pressure according to a conventional method known in the art for extracting extracts from natural materials. For example, the extract of the silver ginseng of the present invention can be extracted using at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof. Preferably, the extract can be extracted using a mixed solvent of water and alcohols having 1 to 4 carbon atoms. In this case, the water and alcohol may be mixed in a volume ratio of 1:5 to 1:1, but more preferably, the extract can be extracted using 75% ethanol or 100% ethanol (ethanol). The extraction efficiency of the effective substance of the silver ginseng that improves muscle atrophy may vary depending on the mixing ratio.

[0045] In addition, the method of extracting the extract from the silver lining can be extracted through various methods such as hot water extraction, cold extraction, reflux extraction, and ultrasonic extraction, but is not limited thereto, and preferably, the reflux extraction method can be used.

[0046] The above-mentioned prepared extract can then be filtered, concentrated, or dried to remove the solvent. Filtration, concentration, and drying can all be performed, and the filtration, concentration, or drying processes can be performed multiple times. For example, filtration can be performed using filter paper or a vacuum filter, concentration can be performed using a vacuum concentrator, and drying can be performed using spray drying, freeze drying, etc., but are not limited thereto.

[0047] In addition, the extract extracted with the above solvent may be further subjected to a fractionation process with a solvent selected from the group consisting of butanol, n-hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof, but is not limited thereto.

[0048] In addition, in the present invention, when extracting the silver ginseng extract, an acid for edible purposes may be added to the solvent for extraction, and preferably, at least one acid selected from the group consisting of acetic acid, citric acid, malic acid, succinic acid, fumaric acid, tartaric acid, amino acid, and lactic acid may be added to the solvent, but preferably, acetic acid may be added, and in this case, the acetic acid added to the solvent may be added in an amount of 0.1 to 1 wt%, preferably 0.5 wt%.

[0049] Accordingly, the present invention provides a method for preparing a silver ginseng extract for preventing, improving or treating muscular atrophy, comprising the steps of (a) preparing a powder by crushing dried silver ginseng; and mixing the crushed silver ginseng powder with at least one solvent selected from the group consisting of water (H2O), lower alcohols having 1 to 4 carbon atoms, and mixtures thereof to prepare an extract.

[0050] In the above manufacturing method, the extraction method for manufacturing the extract may be a reflux extraction method, and may additionally include a step of concentrating the extracted extract under reduced pressure and then freeze-drying it.

[0051] In the present invention, the term 'muscle atrophy' refers to a state in which muscle mass is gradually reduced due to a decrease in the number of muscle cells and fibers and a decrease in the cross-sectional area of ​​the muscle due to a bias toward muscle breakdown rather than muscle synthesis, and refers to muscle weakness and degeneration. Muscle atrophy is promoted by inactivity, disease, oxidative stress, and chronic inflammation, and refers to a decrease in muscle function and exercise capacity. In general, muscle atrophy is accompanied by physiological, histochemical, and biochemical changes due to a decrease in muscle protein, and may cause intrinsic functional impairment of skeletal muscle. The extract of the present invention can be used for the purpose of maintaining and preserving the intrinsic function of skeletal muscle caused by said muscle atrophy through the activity enhancement and protective function of muscle fibers.

[0052] In the present invention, the sarcopenia or muscular atrophy may be characterized by at least one selected from the group consisting of sarcopenia, muscular dystrophy, dystonia, disuse atrophy, muscular atrophy due to absence of mechanical stimulation, denervational atrophy, cachexia, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy, but is not necessarily limited thereto.

[0053] The above-mentioned disuse muscle atrophy includes a decrease in muscle thickness due to restrictions on activity due to aging or disease, prolonged bed rest, cast or plaster fixation, etc. In addition, it is accompanied by various clinical diseases such as peripheral nerve damage, cancer, sepsis, as well as long-term bed rest and the aging process. The above-mentioned composition can be used for the purpose of helping prevent and improve symptoms of skeletal muscle atrophy that may be caused by these causes. The above-mentioned muscle atrophy due to the absence of mechanical stimulation and denervation-induced muscle atrophy cause muscle atrophy by increasing the quantitative decrease of mitochondria, dysfunction, increased production of reactive oxygen species (ROS) in mitochondria, and apoptosis of muscle cells due to oxidative damage. The above-mentioned composition can be used for the purpose of helping prevent and improve symptoms of skeletal muscle atrophy that may be caused by these causes.

[0054] Rapid emaciation is one of the symptoms of cachexia, a severe systemic weakness that can be seen in the terminal stages of cancer, tuberculosis, hemophilia, etc., and the composition can be used to help prevent and improve symptoms of skeletal muscle atrophy that may be caused by this cause. The muscular dystrophy is a disease caused by muscle atrophy and muscle weakness as the main symptoms due to the absence of the dystrophin-glycoprotein complex, a component of the striated muscle plasma membrane, caused by a genetic mutation. The composition can be used to help prevent and improve symptoms of skeletal muscle atrophy that may be caused by this cause.

[0055] Cortisol is a hormone secreted from the adrenal cortex in response to acute stress. It provides energy to combat stress, but depending on its mechanism, it can also cause muscle atrophy in the body. The composition described above can be used to help prevent and improve symptoms of skeletal muscle atrophy caused by such factors. Synthetic adrenocortical hormones can cause adverse reactions such as steroid myopathy and muscle damage. The composition described above can be used to help prevent and improve symptoms of skeletal muscle atrophy caused by such factors.

[0056] The term “prevention” used in the present invention means any act of inhibiting or delaying the onset of muscle atrophy or muscle loss by administering a pharmaceutical composition according to the present invention.

[0057] The term "treatment" as used in the present invention means any action by which symptoms of muscular atrophy are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

[0058] The term "alleviation" or "improvement" as used in the present invention means any action that reduces a parameter related to muscle atrophy or muscle loss, for example, the degree of symptoms, by administration of a composition according to the present invention.

[0059] The term "pharmaceutical composition" in the present invention refers to a composition manufactured for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0060] In the present invention, "including as an active ingredient" means including the ingredient in an amount necessary or sufficient to realize the desired biological effect. In actual application, the amount included as an active ingredient may be determined by considering the amount for treating the target disease and not causing other toxicities, and may vary depending on various factors such as the disease or condition being treated, the form of the composition being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art to which the present invention pertains can empirically determine the effective amount of an individual composition without undue experimentation.

[0061] In addition, the composition of the present invention may, depending on each formulation, additionally contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients. The pharmaceutically acceptable carriers may be saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and may further contain other conventional additives such as antioxidants, buffers, and bacteriostatic agents, if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).

[0062] The composition of the present invention can be administered orally or parenterally in a pharmaceutically effective amount depending on the intended method, and the term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined based on factors including the patient's health condition, severity, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0063] In the present invention, the term "subject" may be a mammal such as a rat, livestock, mouse, or human, and preferably a human. The pharmaceutical composition of the present invention may be formulated in various forms for administration to a subject, and a representative form for parenteral administration is an injectable formulation, preferably an isotonic aqueous solution or suspension. The injectable formulation may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or a buffer solution to be formulated for injection. In addition, oral administration dosage forms include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers, which may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine) and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and may optionally further contain disintegrating agents such as starch, agar, alginic acid or its sodium salt, absorbents, coloring agents, flavoring agents, and / or sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0064] In addition, the pharmaceutical composition of the present invention may further include auxiliary agents such as binders, lubricants, disintegrants, colorants, preservatives, sweeteners, flavoring agents, stabilizers, diluents, antiseptics, wetting agents, emulsifying agents, salts for osmotic pressure control, or buffers, and other therapeutically useful substances, and may be formulated according to conventional methods.

[0065] The pharmaceutical composition according to the present invention can be administered via various routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular. The dosage of the active ingredient can be appropriately selected based on various factors, such as the route of administration, the patient's age, sex, weight, and severity of the condition. Furthermore, the composition of the present invention can be administered in combination with known compounds capable of enhancing the desired effect.

[0066] The pharmaceutical composition according to the present invention may be administered to humans or animals orally or parenterally, such as intravenously, subcutaneously, intranasally, or intraperitoneally. Oral administration includes sublingual administration. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection, as well as drip methods. In addition, the present invention can be used as a health functional food for preventing or improving sarcopenia or muscular dystrophy, containing the extract of Eunssiho as an active ingredient.

[0067] In the present invention, the term "health functional food composition" refers to a food prepared by adding the extract of the silver ginseng to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and means that when consumed, it brings about a specific health effect, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material. The health functional food composition may be any one selected from meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, vitamin complexes, and other health supplements.

[0068] The present invention is susceptible to various modifications and embodiments, and will be described in more detail below through specific examples. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0069] Example 1. Preparation of Eunshiho extract

[0070] Dried silver moss was purchased and ground to an average particle size of 0.30 to 0.50 mm using a grinder. 3 kg of the ground silver moss was extracted three times with 5 L of EtOH at 75°C for 3 hours. The extract was concentrated using a rotary vacuum evaporator and then freeze-dried using Heto Power Dry LL3000 to obtain 366.3 g of the extract.

[0071] HPLC analysis of the extract of Eunshiho was performed using a Capcell pak C18 UG120 (5 μm, 4.6 x 250 mm, Shiseido, Japan) column. The mobile phases used were A: acetonitrile and B: water, and each solvent contained 0.1% formic acid. The chromatogram was obtained at a flow rate of 1 mL / min with a gradient elution condition of 0-5 min, 5-5% A; 5-45 min, 5-30% A; 45-85 min, 30-95% A; 85-90 min, 95-100% A, at a detection wavelength of 265 nm, and the results are shown in Figures 1 to 4.

[0072]

[0073] Example 2. Preparation of the silver ash fraction

[0074] To separate the extract of Eunshiho according to polarity, sequential polar solvent fractionation was performed using organic solvents n-hexane (n-HX), ethyl acetate (EA), n-butanol (n-BuOH), and water. 366.3 g of the ethanol extract of Eunshiho was made into a suspension in 1 L of water, and fractionation was repeated three times in the order of n-HX-EA-n-BuOH, and then concentrated to obtain fractions. As a result of solvent fractionation of 366.3 g of the ethanol extract of Eunshiho, fractions of 2.6181 g (n-HX), 3.9553 g (EA), 6.4428 g (n-BuOH), and 103.52 g (water) were obtained, and the results are shown in Fig. 5.

[0075]

[0076] Example 3. In vitro confirmation of the effect of silver ginseng extract or fraction on prevention, improvement, or treatment of skeletal muscle atrophy.

[0077] 3-1. Culturing and differentiation of C2C12 cells

[0078] C2C12 mouse myoblasts used in this experiment were purchased from ATCC (American Type Culture Collection) and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin as a growth medium under 5% CO2, 37℃ conditions. C2C12 myoblasts were seeded in 12-well plates and cultured to divide until the cell density reached 85-90%. Afterwards, differentiation was induced using differentiation medium (DMEM containing 2% horse serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), and the medium was replaced every two days, and differentiation was performed for a total of 6 days.

[0079]

[0080] 3-2. Induction of muscle atrophy with dexamethasone and treatment with extracts and fractions of Eunssiho

[0081] Dexamethasone induces muscle atrophy by inhibiting muscle synthesis and causing protein breakdown. Dexamethasone treatment induced muscle atrophy in C2C12 cells differentiated in Example 3-1, and then treatment with extracts and / or fractions of Eunssiho to the C2C12 cells in which muscle atrophy had been induced. The specific experimental conditions are as follows.

[0082] (1) The control group was treated with differentiation medium for 24 hours to differentiated C2C12 myotube cells.

[0083] (2) The negative control was prepared by adding dexamethasone to the differentiation medium to C2C12 myotube cells that had completed differentiation to a final concentration of 10 μM, and then treating the cells for 24 hours.

[0084] (3) The dexamethasone and silver ginseng extract treatment group was prepared under the same conditions as the negative control group of (2) above, with an additional composition of 30 μg / mL of silver ginseng extract, and then treated for 24 hours.

[0085] (4) The group treated with dexamethasone and the hexane (n-Hexane) fraction of the extract of Eunshiho was prepared under the same conditions as the negative control group of (2) above, with an additional composition of 30 μg / mL of the hexane fraction of the extract of Eunshiho, and then treated for 24 hours.

[0086] (5) The group treated with dexamethasone and the ethyl acetate fraction of the extract of Eunshiho was prepared under the same conditions as the negative control group of (2) above, with an additional composition of 30 μg / mL of the ethyl acetate fraction of the extract of Eunshiho, and then treated for 24 hours.

[0087] (6) The group treated with dexamethasone and the butanol (n-BuOH) fraction of the extract of Eunshiho was prepared under the same conditions as the negative control group of (2) above, and was additionally prepared to have a composition of 30 μg / mL of the butanol fraction of the extract of Eunshiho, and then treated for 24 hours.

[0088] (7) The water fraction treatment group of dexamethasone and silver ginseng extract was prepared under the same conditions as the negative control group of (2) above, with an additional composition of 30 μg / mL of the water fraction of silver ginseng extract, and then treated for 24 hours.

[0089]

[0090] 3-3. Measurement of C2C12 myotube diameter

[0091] In the above Example 3-2, each control group and experimental group that had been treated with samples for 24 hours were photographed using the automated cell imaging system, JuLi Stage, and the thickness of each cell was measured for 50 cells per cell group using the cell processing program, Image J, and the data were organized by calculating the average and standard error (see Table 1 below). Each experiment was repeated three times.

[0092] Control groupDex treatment groupDEX + Silver ginseng extractC2C12 myotube diameter(% of control)10079.9 a 96.8 b

[0093] (Dex: dexamethasone (10 μM), silver ginseng extract (30 μg / mL), a , significant compared to the control group (p< 0.01); b , significant (p<0.01) compared to the Dex treatment group (negative control group)

[0094] From the above experimental results, it was confirmed that the thickness of C2C12 myotube cells was reduced by dexamethasone treatment and that the treatment with the Eunssiho extract had an improving effect (see Table 1 and Fig. 4). More specifically, (1) compared to the control group, which is the basic differentiation medium administration group, the thickness of C2C12 myotube cells was reduced by 21.1% in the 10 μM dexamethasone administration group (p<0.01). (2) compared to the negative control group administered dexamethasone to the basic differentiation medium, the thickness of C2C12 myotube cells was additionally increased by 21.2% in the Eunssiho extract administration group 30 μg / mL (p<0.01). Therefore, it was confirmed that the Eunssiho extract of the present invention significantly restored the thickness damage of C2C12 myotube cells in the dexamethasone-induced muscle atrophy model.

[0095]

[0096] Control group Dex (10 μM) treatment group n-HX fraction EA fraction n-BuOH fraction water fraction C2C12 myotube diameter (% of control) 100 79.9 a 73.766.794.1 c 104.6 c

[0097] (Dex: dexamethasone (10 μM), n-HX fraction: hexane fraction of the extract of Eunshiho (30 μg / mL), EA fraction: ethyl acetate fraction of the extract of Eunshiho (30 μg / mL), n-BuOH fraction: butanol fraction of the extract of Eunshiho (30 μg / mL), water fraction: water fraction of the extract of Eunshiho (30 μg / mL)) a , significant compared to the control group (p< 0.01); c , compared to the Dex treatment group (negative control group) (significantly (p<0.001)) As a result of the above experiment, it was confirmed that the thickness of C2C12 myotube cells decreased by dexamethasone treatment, and that a significant improvement effect was observed when treated with not only the extract of the silver ginseng of the present invention but also its fractions (see Table 2 and Fig. 4).

[0098] More specifically, (1) compared to the control group administered with basic differentiation medium, the thickness of C2C12 myotubes decreased by 21.1% in the group administered with 10 μM dexamethasone (p<0.01). (2) compared to the negative control group administered with dexamethasone in the basic differentiation medium, the thickness of C2C12 myotubes increased by 17.8% in the group administered with 30 μg / mL of the butanol fraction of the extract of Euphorbia militaris (p<0.001). (3) compared to the negative control group administered with dexamethasone in the basic differentiation medium, the thickness of C2C12 myotubes increased by 30.9% in the group administered with 30 μg / mL of the water fraction of the extract of Euphorbia militaris (p<0.001). Therefore, it was confirmed that the butanol and water fractions of the extract of the present invention significantly restored the thickness damage of C2C12 myotube cells in a dexamethasone-induced muscle atrophy model.

[0099]

[0100] Example 4. Separation, purification, and structural identification of compounds 1-5 from the n-butanol fraction of silver ginseng

[0101] Among the fractions of Eunssiho, the n-BuOH layer, which showed strong physiological activity, was subjected to open column chromatography using Diaion HP-20 macroporous resin. MeOH was used as the mobile phase, and the MeOH ratio was increased from 0% to 100% in 5% increments, with 1 L flowing three times. Afterwards, the column was washed by flowing 1 L of acetone three times. The solvent was removed using a vacuum rotary evaporator and a freeze dryer. Among the 14 fractions initially separated through open column chromatography, fractions 8 and 13 were separated into single components using preparative HPLC. Separation was performed at 265 nm, the wavelength at which β-carboline alkaloids are detected, and ACN and water containing 0.1% TFA were used as the mobile phase. Fraction 8 eluted compounds (1-5) by flowing 5-20% ACN for 50 minutes using a Cosmosil C18 column (20x150 mm) under gradient elution conditions, and fraction 14 separated compounds (2 and 4) by flowing 18% ACN under isocratic elution conditions using an INNO C18 column (20x250 mm) (see Figure 5).

[0102]

[0103] Example 5. Structural analysis of compounds isolated and purified from silver moss in vitro

[0104] The isolated compounds were structurally identified as Glucodichotomyne B (compound 1), Dichotomine B (compound 2), Shaftoside (compound 3), Dichotomine A (compound 4), and Isoshaftoside (compound 5) by spectroscopic methods such as NMR and MASS (see Figures 6 and 7).

[0105]

[0106] 글루코디코토민 B(Glucodichotomine B)

[0107] 1 H NMR (400 MHz, DMSO-d6): δ ppm 4.05 (2H, d, J= 7.9 Hz, H-15), 4.52 (1H, d, J=7.6 Hz, G-1'), 5.42 (1H, m, H-14), 7.42 (1H, t, H-6), 7.62 (1H, t, H-7), 7.71 (1H, d, J=7.4 Hz, H-8), 8.40 (1H, d, J=7.4 Hz, H-5), 8.87 (1H, s, H-4), 11.79 (1H, s, NH-9).

[0108] 13 C NMR (100 MHz, DMSO-d6): δ ppm 61.71 (G-6'), 64.95 (C-15), 70.63 (G-4'), 74.32 (G-2'), 76.88 (G-5'), 77.24 (C-14), 77.59 (G-3'), 102.00 (G-1'), 112.95 (C-8), 116.96 (C-4), 120.59 (C-6), 121.10 (C-12), 122.43 (C-5), 129.06 (C-11), 129.36 (C-7), 135.74 (C-10), 136.74 (C-3), 141.45 (C-13), 143.21 (C-1), 167.79 (C-16)

[0109] MS (ESI, positive) m / z = 435.2 [M+H] + , (ESI, negative) m / z = 433.2 [M-H] -

[0110] UV (MeOH) emax = 236 nm, 269 nm

[0111]

[0112] 디코토민 B(Dichotomine B)

[0113] 1 H NMR (400 MHz, DMSO-d6): δ ppm 3.85 (2H, d, J=7.9 Hz, H-15), 5.19 (1H, m, H-14), 7.31 (1H, t, H-6), 7.60 (1H, t, H-7), 7.75 (1H, d, J=7.5 Hz, H-8), 8.39 (1H, d, J=7.5 Hz, H-5), 8.85 (1H, s, H-4), 11.83 (1H, s, NH-9).

[0114] 13 C NMR (100 MHz, DMSO-d6): δ ppm 66.85 (C-15), 73.90 (C-14), 113.19 (C-8), 116.63 (C-4), 120.62 (C-6), 121.15 (C-12), 122.46 (C-5), 129.13 (C-11), 129.13 (C-7) 135.31 (C-10), 135.51 (C-3), 141.66 (C-13), 145.69 (C-1), 166.82 (C-16).

[0115] MS (ESI, positive) m / z = 273.2 [M+H] + , MS (ESI, negative) m / z = 271.2 [M-H] -

[0116] UV (MeOH) emax = 236 nm, 269 nm

[0117]

[0118] 샤프토사이드(Schaftoside)

[0119] 1 H NMR (400 MHz, DMSO-d6): δ ppm 4.53 (1H, d, J=8.7 Hz, G-1'), 4.96 (1H, d, J=9.3 Hz, A-1'), 6.81 (1H, s, H-3), 6.95 (2H, d, J=8.0 Hz, H-3', 5'), 7.98 (2H, d, J=8.0 Hz, H-2', 6'), 13.80 (1H, s, OH-5)

[0120] 13 C NMR (100 MHz, DMSO-d6): δ ppm 61.32 (G-2), 66.92 (G-6), 70.31 (A-2), 70.49 (A-4), 70.79 (A-5, G-4), 74.22 (A-3), 74.77 (G-1), 75.42 (A-1), 79.88 (G-3, G-5), 108.08 (C-3), 108.54 (C-10), 104.20 (C-8), 109.83 (C-6), 116.47 (C-3', 5'), 121.80 (C-1'), 129.24 (C-2',6'), 153.89 (C-9), 161.70 (C-4'), 164.11 (C-5, C-7), 182.88 (C-4).

[0121] MS (ESI, positive) m / z = 565.3 [M+H]+, MS (ESI, negative) m / z = 563.3 [M-H] -

[0122] UV (MeOH) emax = 270 nm, 330 nm

[0123]

[0124] 디코토민 A(Dichotomine A)

[0125] 1 H NMR (400 MHz, DMSO-d6): δ ppm 1.58 (3H, d, J=7.2 Hz, H-15), 5.23 (1H, m, H-14), 7.26 (1H, t, H-6), 7.60 (1H, t, H-7), 7.74 (1H, d, J=7.0 Hz, H-8), 8.37 (1H, d, J=7.0 Hz, H-5), 8.81 (1H, s, H-4), 11.83 (1H, s, NH-9)

[0126] 13C NMR (100 MHz, DMSO-d6): δ ppm 25.04 (C-15), 64.80 (C-14), 113.14 (C-8), 116.27 (C-4), 120.52 (C-6), 121.29 (C-12), 122.34 (C-5), 128.63 (C-7), 128.70 (C-11), 129.37 (C-10), 129.61 (C-3), 137.91 (C-13), 141.56 (C-1), 169.20 (C-16).

[0127] MS (ESI, positive) m / z = 257.2 [M+H]+, MS (ESI, negative) m / z = 255.2 [M-H] -

[0128] UV (MeOH) emax = 237 nm, 269 nm

[0129]

[0130] 이소샤프토사이드(Isoschaftoside)

[0131] 1 H NMR (400 MHz, DMSO-d6): δ ppm 4.72 (1H, d, J=8.5 Hz, G-1'), 4.78 (1H, d, J=9.1 Hz, A-1'), 6.83 (1H, s, H-3), 6.95 (2H, d, J=7.9 Hz, H-3', 5'), 7.95 (2H, d, J=7.9 Hz, H-2', 6'), 13.69 (1H, s, OH-5)

[0132] 13C NMR (100 MHz, DMSO-d6): δ ppm 61.25 (G-6), 68.88 (G-2), 70.96 (A-2), 71.07 (A-4), 74.77 (A-3, G-1), 74.99 (A-1), 79.17 (G-3), 79.48 (G-5), 108.23 (C-3), 104.13 (C-10), 104.95 (C-8), 109.67 (C-6), 116.40 (C-3', 5'), 121.93 (C-1'), 129.11 (C-2', 6'), 156.28 (C-9), 160.84 (C-4), 161.71 (C-5, C-7), 163.51 (C-2), 182.76 (C-4).

[0133] MS (ESI, positive)m / z=565.3 [M+H] + , MS (ESI, negative)m / z= 563.3 [MH] -

[0134] UV (MeOH) emax = 270 nm, 330 nm

[0135]

[0136] Example 6. Confirmation of the effect of compounds 1 to 5 isolated and purified from Eunshiho on preventing, improving, or treating skeletal muscle atrophy.

[0137] 6-1. Culturing and differentiation of C2C12 cells

[0138] The same experimental method as in Example 3-1 above was used.

[0139]

[0140] 6-2. Induction of muscle atrophy with dexamethasone and treatment with compounds 1 to 5 isolated and purified from silver ginseng

[0141] The experiment was conducted similarly to Example 3-2 above. However, similarly to the extract and fraction treatment groups, compounds 1 to 5 were each treated at 30 μM.

[0142]

[0143] 6-3. Measurement of C2C12 myotube diameter

[0144] The cell thickness was measured using the same method as in Example 3-3, and the data were organized by calculating the mean and standard error (see Table 3, Figures 8 and 9). Each experiment was repeated three times.

[0145]

[0146] Control group Dex (10 μM) Treatment group - n-BuOH fraction (30 μg / mL) Glucodichotomine B (1) 30 μM Ichotomine B (2) 30 μM Shaftoside (3) 30 μM Ichotomine A (4) 30 μM Soshaftoside (5) 30 μM MC2C12 myotube diameter (% of control) 100.081.3 a 100.7 b 102.0 b 98.2 b 92.7 c 87.995.7 c

[0147] (Dex: dexamethasone (10 μM), a , significant compared to the control group (p< 0.01); b , significant (p<0.01) compared to the Dex treatment group (negative control group), c , compared to the Dex treatment group (negative control group) (significantly (p<0.05)) As a result of the above experiment, it was confirmed that the thickness of C2C12 myotube cells decreased by dexamethasone treatment, and that the improvement effect was achieved by compounds 1-5, whose structure was identified by separating and purifying them from Eunshiho (Table 3 and Figures 8 and 9).

[0148] Example 7. Confirmation of the improvement or therapeutic effect of compound 2 (dichotomycin B) isolated and purified from Eunshiho on skeletal muscle loss.

[0149]

[0150] 7-1. Culturing and differentiation of C2C12 cells

[0151] The same experimental method as in Example 3-1 above was used.

[0152]

[0153] 7-2. Induction of muscle atrophy with dexamethasone and treatment with dicotomin B isolated and purified from silver ginseng

[0154] The experiment was conducted similarly to Example 3-2 above. However, dicotomin B was treated at 10 μM and 30 μM.

[0155]

[0156] 7-3. MHC immunofluorescence staining of C2C12 myotubes

[0157] After the reaction, the cell medium of each experimental group was removed, washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 minutes, and washed three times with PBS. After treating with 0.1% TritonX-100 for 10 minutes, the cells were washed twice with PBS, and blocked with 3% horse serum solution for 2 hours. Mouse anti-MHC was diluted 1:100 in 3% BSA, incubated overnight at 4°C, and washed three times with 0.1% PBST. Afterwards, goat-anti-mouse IgG-FITC secondary antibody (Thermo Fisher Scientific) was diluted 1:500 in 3% BSA, reacted at 37°C for 1 hour, and washed three times with 0.1% PBST. After treating with a 1 μg / ml Hoechst33342 solution and incubating at room temperature for 10 minutes, cells were photographed and images were acquired using JuLiTM. MyHC immunofluorescence results were visualized in green, and Hoechst33342-labeled cell nuclei were visualized in blue. Numerical analysis was performed using the Image J program, and the results are shown in Figure 11.

[0158]

[0159] 7-4. Analysis of the multinucleated myotube formation index (Fusion index)

[0160] From the results of 7-3 above, multinucleated myotube cells were randomly selected and the formation index of multinucleated myotube cells was calculated. After analyzing the number of multinucleated myotube cells, the relative number of multinucleated-MHC-positive myotube cells (MHC-positive myotubes) in each cell was calculated by applying the total number of cells (see Table 4 and Fig. 13).

[0161] Control group Dex (10 μM) treatment group - Dichotomine B (2) 10 μMDichotomine B (2) 30 μMC2C12 myotube diameter (% of control) 100.0 80.0 a 90.3 b 92.3 b

[0162] (Dex: dexamethasone (10 μM), a , significant compared to the control group (p< 0.01); b , compared to the Dex treatment group (negative control group) (significantly (p<0.01)) As a result of the above experiment, it was confirmed that the multinucleated myotube formation index of C2C12 myotube cells was reduced by dexamethasone treatment, and that the improvement effect was shown by Dichotomine B (compound 2), which was isolated and purified from Eunshiho and the structure was identified (see Table 4 and Figures 12 and 13).

[0163] 7-4. Measurement of C2C12 myotube diameter

[0164] The thickness of the cells was measured using the same method as in Example 3-3 above, and the average and standard error were calculated, and the data were organized in Table 4. Each experiment was performed three times.

[0165]

[0166] Control group Dex (10 μM) treatment group - Dichotomine B (2) 10 μMDichotomine B (2) 30 μMC2C12 myotube diameter (% of control) 100.0 80.0a 97.9 b 100.6 b

[0167] (Dex: dexamethasone (10 μM), a , significant compared to the control group (p< 0.01); b , compared to the Dex treatment group (negative control group) (significantly (p<0.01)) As a result of the above experiment, it was confirmed that the thickness of C2C12 myotube cells was reduced by dexamethasone treatment, and that the improvement effect was achieved by Dichotomine B (compound 2), which was isolated and purified from Eunshiho and its structure was identified (see Table 5).

[0168]

[0169] 7-5. Western Blotting

[0170] C2C12 cells were seeded in a 6-well plate at 1 x 105 / well, and differentiated C2C12 myotube cells were treated with dexamethasone and samples according to each experimental group. The prepared cells were lysed using a cell lysis solution containing LIPA lysis buffer, protease inhibitors, and phosphatase inhibitors. 200 μl of the cell lysis solution was applied to each well, and the cells were lysed using a cell scraper. The cells were centrifuged at 13,000 rpm for 20 minutes to obtain the supernatant. The concentration was measured using the BCA protein quantitation method, and then equal amounts of protein were separated using 8% SDS-PAGE. The proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, blocked with 3% BSA buffer, and then treated with various primary antibodies (MyHC, FoxO3a, MuRF1 GAPDH, etc.), washed, treated with secondary antibodies, and washed sequentially. Finally, after expression by ECL (enhanced chemiluminescence), images were acquired using ChemiDoc, and the expression levels of each protein were quantified using the Image J program.

[0171] As a result of the above experiment, it was confirmed that Atrogin-1 and MuRF-1 were significantly increased and MHC was decreased in C2C12 myotube cells by dexamethasone treatment, and that MHC, which was decreased by Dichotomine B (compound 2) treatment, was increased in a concentration-dependent manner and that increased Atrogin-1 and MuRF-1 were decreased (see Figures 14 to 15).

[0172]

[0173] Example 8. Confirmation of the prevention, improvement, or treatment effect of skeletal muscle atrophy in a starvation model of Dichotomine B isolated and purified from Eunshiho

[0174] 8-1. Culturing and differentiation of C2C12 cells

[0175] The same experimental method as in Example 3-1 above was used.

[0176]

[0177] *8-2. Dichotomine B treatment in an in vitro starvation model

[0178] Culturing differentiated C2C12 myotubes in serum-free / low glucose medium induces muscle atrophy by inhibiting muscle synthesis and causing protein degradation. C2C12 cells, which had been differentiated in Example 8-1, were cultured in serum-free / low glucose medium to induce muscle atrophy, and the C2C12 cells in which muscle atrophy was induced were treated with Dichotomine B. The specific experimental conditions are as follows.

[0179] (1) The control group was treated with differentiation medium for 48 hours to differentiated C2C12 myotube cells.

[0180] (2) For the negative control, differentiated C2C12 myotube cells were cultured in serum-free / low glucose medium for 48 hours.

[0181] (3) The Dichotomine B treatment group was additionally treated with Dichotomine B at a concentration of 1 μM or 10 μM for 48 hours under the same conditions as the negative control group in (2).

[0182]

[0183] 8-3. MyHC immunofluorescence staining of C2C12 myotubes

[0184] It was performed in the same manner as Example 7-3 above.

[0185]

[0186] 8-4. Measurement of C2C12 myotube diameter and comparison of MHC staining area

[0187] The thickness and staining area of ​​the cells were measured using the same method as in Example 3-3 above, and the average and standard error were calculated, and the data were organized in Table 6. Each experiment was performed three times.

[0188]

[0189] Control Starvation (Serum-free / Low glucose medium)-Dichotomine B (2) 1 μMDichotomine B (2) 10 μMC2C12 myotube diameter (% of control) 100.0 ± 9.2 80.9 ± 8.2 a 98.6 + 9.3 b 100.4 ± 10.8 b MHC positive area (% of control)100 ± 8.187.1 ± 5.4 a 93.6 + 7.8 b 97.1 ± 8.4 b

[0190] (Dex: dexamethasone (10 μM), a , significant compared to the control group (p< 0.01); b , compared to the Dex treatment group (negative control group) (significantly (p<0.01)) In the above experiment, it was confirmed that dichotomine B significantly increased the thickness of myotubes and the area through immunofluorescence with MHC (see Table 6 and Figures 16 to 20). 8-5. Confirmation of the expression level of proteins related to muscle atrophy

[0191] The amount of protein expression was confirmed by performing western blotting in the same manner as in Example 7-5 above.

[0192] As a result of the above experiment, in the starvation model of C2C12 myotube cells, MHC decreased, and FOXO3a, Atrogin-1, and MuRF-1 significantly increased, and it was confirmed that this was effectively improved by Dichotome B treatment (see Figures 21 to 25).

[0193]

[0194] Example 9. In vivo confirmation of the effects of Dicotomine B on preventing and improving muscle atrophy.

[0195] 9-1. Experimental animals

[0196] Experimental animals were 6-week-old male C57BL / 6 mice obtained from Coretech (Pyeongtaek, Gyeonggi-do, Korea). After one week of quarantine, acclimatization, and breeding at the Hanyang University ERICA Laboratory Animal Center (Animal Facility Registration No. 445), the animals were tested. The lighting period was set to 12 h (07:00–19:00), and food and water were provided ad libitum. The following experiments were conducted in accordance with the policies and regulations of the Hanyang University Institutional Animal Care and Use Committee.

[0197] 9-2. Muscle loss model derivation and sample administration

[0198] Experiments were conducted after obtaining laboratory animals and acclimation for 7 days. Solid feed for laboratory animals (Woojung Bio, Hwaseong, Gyeonggi-do, Korea) was provided, and the animals were treated within a specific time period (10:00 AM-12:00 PM). Body weight changes were measured three times in total. Animals in the fasting group were housed in individual cages during the fasting period. Samples were administered twice: once at the start of the fasting period and once 24 hours later. The experimental results are expressed as the mean and standard deviation, and the significance of each group was confirmed using the Anova t-test in the Statview program.

[0199]

[0200] 9-3. Checking weight changes

[0201] The non-fasting group showed normal body weight changes, but the 48-hour fasting resulted in a significant decrease in body weight in group F (Table 7).

[0202] GroupBody weight (g)Start of sample administrationEnd of sample administrationFeed21.167 ± 0.33921.950 ± 0.404Feed+ Dichotomine B21.080 ± 0.37021.400 ± 0.339Starvation21.800 ± 0.66618.028 ± 1.254 *** Starvation+Dichotomine B22.214 ± 0.70117.743 ± 0.885 ***

[0203] (***p<0.001 vs. Feed) The results of the above experiment confirmed that the body weight of the mice used in the experiment decreased due to fasting for 48 hours, and administration of dicotomin B did not affect the body weight (see Table 7 and Fig. 26)).

[0204] 9-4. Checking changes in grip strength

[0205] After fasting, grip strength was assessed using a grip strength meter.

[0206] GroupGrip Strength (kgf)Feed192.100 ± 6.973Feed+ Dichotomine B190.400 ± 11.747Starvation154.393 ± 12.630 *** Starvation+Dichotomine B187.114 ± 10.724 ###

[0207] (***p<0.001 vs. Feed, ###p<0.001 vs. Starvation) The above experimental results showed that grip strength was significantly reduced by fasting for 48 hours, and significantly increased by administration of dicotomin B compared to the fasting group (see Table 8 and Figure 27).

[0208] 9-5. Checking the weight of muscle tissue

[0209] To determine muscle loss, the weights of the tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GA), and soleus (SOL) muscles were measured after autopsy, and the results are summarized in Table 9.

[0210] Could be.

[0211] GroupTibialis anterior (g)Extensor digitorum longus (g)Gastrocnemius (g )Soleus (g)Feed0.061 ± 0.0050.015 ± 0.0030.180 ± 0.0150.013 ± 0.003Feed+ Dichotomine B0.058 ± 0.0060.012 ± 0.0020.169 ± 0.0140.010 ± 0.001Starvation0.054 ± 0.002 ** 0.012 ± 0.003 * 0.162 ± 0.017 ** 0.009 ± 0.003 * Starvation+Dichotomine B.058 ± 0.005 # 0.014 ± 0.0040.165 ± 00150.013 ± 0.005 #

[0212] (*p<0.05 vs. Feed, **p<0.01 vs. Feed, #p<0.05 vs. Starvation) Referring to the above results, the weight of the tibialis anterior muscle, which was reduced due to fasting, increased with the administration of 10 mg / kg of dicotomin B (p<0.05), and the same result (p<0.05) was confirmed in the soleus muscle. The above results confirmed that dicotomin B improved muscle loss due to fasting (see Table 9, Figures 28 and 29).

[0213]

[0214] The present invention was achieved by synthesizing dicotomin B, a new component capable of alleviating skeletal muscle loss among the above-described compounds, having the following chemical formula.

[0215]

[0216] Example 10. Preparation of dicotomin B

[0217] I purchased Dicotomin B through the announced website (https: / www.medchemexpress.com)

[0218]

[0219] Example 2 Structural identification of the compound

[0220] 1 H NMR (400 MHz, DMSO-d6): δ ppm 3.85 (2H, d, J=7.9 Hz, H-15), 5.19 (1H, m, H-14), 7.31 (1H, t, H-6), 7.60 (1H, t, H-7), 7.75 (1H, d, J=7.5 Hz, H-8), 8.39 (1H, d, J=7.5 Hz, H-5), 8.85 (1H, s, H-4), 11.83 (1H, s, NH-9).

[0221] 13C NMR (100 MHz, DMSO-d6): δ ppm 66.85 (C-15), 73.90 (C-14), 113.19 (C-8), 116.63 (C-4), 120.62 (C-6), 121.15 (C-12), 122.46 (C-5), 129.13 (C-11), 129.13 (C-7) 135.31 (C-10), 135.51 (C-3), 141.66 (C-13), 145.69 (C-1), 166.82 (C-16).

[0222] MS (ESI, positive) m / z = 273.2 [M+H] + , MS (ESI, negative) m / z = 271.2 [MH] -

[0223] UV (MeOH) emax = 236 nm, 269 nm

[0224]

[0225] Treatment and prevention of skeletal muscle atrophy or sarcopenia with dicotomin B are as described above.

[0226] The pharmaceutical composition according to the present invention can be used for the treatment and prevention of skeletal muscle atrophy or sarcopenia, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for preventing, treating or improving skeletal muscle atrophy or sarcopenia, containing an extract of Eunshiho as an active ingredient.

2. In paragraph 1, A pharmaceutical composition for preventing and treating or improving skeletal muscle atrophy or sarcopenia, characterized in that the above-mentioned silver moss extract is extracted with one or more solvents selected from the group consisting of water (H2O), lower alcohols of C1 to C4, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixed solvents thereof.

3. In paragraph 2, A pharmaceutical composition for preventing and treating or improving skeletal muscle atrophy or sarcopenia, characterized in that the pharmaceutical composition is a fraction fractionated from the extract of the silver ginseng.

4. A pharmaceutical composition for preventing or treating skeletal muscle atrophy, comprising as an active ingredient at least one selected from the group consisting of Dichotomine B, Glucodichotomyne B, Shaftoside, Isoshaftoside and pharmaceutically acceptable salts thereof.

5. In paragraph 4, A pharmaceutical composition for preventing or treating skeletal muscle atrophy, characterized in that the above-mentioned Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

6. In paragraph 1, A pharmaceutical composition characterized in that the above muscle atrophy is at least one muscle atrophy selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, cachexia, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

7. A food composition for preventing or improving skeletal muscle atrophy or sarcopenia, containing an extract of Eunshiho as an active ingredient.

8. A feed composition for preventing or improving skeletal muscle atrophy or sarcopenia, containing an extract of Eunshiho as an active ingredient.

9. A food composition for preventing or improving skeletal muscle atrophy, comprising at least one active ingredient selected from the group consisting of Dichotomine B, Glucodichotomycin B, Shaftoside, and Isoshaftoside.

10. In paragraph 9, A food composition for preventing or improving skeletal muscle atrophy, characterized in that the above-mentioned Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

11. A feed composition for preventing or improving skeletal muscle atrophy, comprising at least one active ingredient selected from the group consisting of Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside.

12. In paragraph 10, A food composition for preventing or improving skeletal muscle atrophy, characterized in that the above-mentioned Dichotomine B, Glucodichotomyne B, Shaftoside, and Isoshaftoside are extracted from Eunshiho.

13. In paragraph 11, The above Dichotomine B is a food composition for preventing or improving skeletal muscle atrophy, which is chemically synthesized.

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