Combination for preventing, ameliorating, and / or treating muscle diseases or muscle disorders

Abstract

Use of a compound represented by formula (I) in the prevention, amelioration, and / or treatment of muscle diseases or muscle disorders.

Description

Combination for preventing, improving and / or treating muscle diseases or muscle disorders Technical Field

[0001] The present invention relates to the field of muscle diseases or muscle disorders, and more particularly to the use of compounds of formula (I) in preventing, ameliorating, and / or treating muscle diseases or muscle disorders, particularly in increasing muscle cell survival, promoting muscle cell proliferation, increasing myotube size, slowing down and / or preventing myotube size decline, improving muscle mass, improving muscle endurance, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial count, regulating energy metabolism, and / or reducing protein degradation.

[0002] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is Background Art

[0003] Skeletal muscle is attached to bones through myotomes and moves bones and joints to enable movement throughout the body. Skeletal muscle is not only essential for various bodily functions but is also closely linked to energy storage and metabolism. When the body undergoes major physiological changes, skeletal muscle is lost before other organs. Severe loss can lead to muscle disorders or diseases such as muscle atrophy and sarcopenia.

[0004] Sarcopenia has many causes, including degeneration or alterations in the neuromuscular machinery, a decrease in anabolic hormones (i.e., signaling molecules that promote muscle development), an increase in catabolic hormones (i.e., signaling molecules that promote muscle degradation), muscle disuse, and inflammatory responses. Examples of degeneration or alterations in the neuromuscular machinery include a decrease in the number and efficiency of motor units, a weakening of excitation-contraction coupling, and changes in muscle structure, orientation, and fiber type.

[0005] Muscle atrophy can be broadly divided into pathological and disuse-related muscle atrophy. The causes of the former include genetics, aging, malnutrition, decreased anabolic hormones, increased catabolic hormones, inflammatory responses, and cachexia, while the latter is caused by long-term lack of muscle activity (such as prolonged bed rest and postoperative immobilization). For example, see: Development of Pharmacotherapies for the Treatment of Sarcopenia. J Frailty Aging. 2019; 8(3): 120-130. and Atenolol ameliorates skeletal muscle atrophy and oxidative stress induced by cast immobilization in rats. Biomedicines. 2023 Apr 25; 11(5): 1269. The full texts of these documents are incorporated herein by reference.

[0006] Currently, the treatment for muscle atrophy is mainly through exercise, nutritional supplements, acupuncture therapy, hormone therapy, etc., and less through drug intervention. Taking spinal muscular atrophy (SMA) as an example, the US Food and Drug Administration (FDA) has approved only three drugs for the treatment of SMA in recent years: Nusinersen onasemnogene abeparvovec and risdiplam

[0007] As developed nations generally face the problem of aging populations, the impact of muscle atrophy on human health is receiving increasing attention. In particular, the World Health Organization (WHO) officially recognized sarcopenia as a disease (International Classification of Diseases code M62.84) in 2016. Consequently, the industry is actively researching effective treatments and therapies for muscle atrophy and related diseases and disorders, such as sarcopenia.

[0008] Summary of the Invention

[0009] The inventors of this case have discovered that the compound of formula (I) can increase muscle cell survival rate, promote muscle cell proliferation, increase myotube size, slow down and / or prevent myotube size decline, improve muscle mass, enhance muscle endurance, increase muscle fiber volume, promote protein synthesis, increase mitochondrial number, regulate energy metabolism, and / or reduce protein degradation. Therefore, it can be used to prevent, improve and / or treat muscle diseases or muscle disorders:

[0010] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is

[0011] The inventors of this case have further discovered that both the compound of formula (II) and the compound of formula (III) can significantly enhance the efficacy of the compound of formula (I), and that the use of the compound of formula (II) or the compound of formula (III) alone can also provide benefits such as increasing muscle cell survival rate, promoting muscle cell proliferation, increasing myotube size, slowing down and / or preventing the decrease in myotube size, improving muscle mass, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial number, and / or regulating energy metabolism:

[0012] wherein R5, R6 and R7 are each independently H or OH, and R8 is

[0013] Therefore, an object of the present invention is to provide a use of a composition containing a compound of formula (I) in preparing a preparation, wherein the preparation is used to prevent, improve and / or treat muscle diseases or muscle disorders:

[0014] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is Preferably, the preparation is a medicine or a food. When the preparation is a medicine, it is provided in the form of oral administration or injection; or when the preparation is a food, it is a health food, dietary supplement, functional food, nutritional supplement food or special nutritional food.

[0015] Another object of the present invention is to provide a combination comprising a compound of formula (I) for preventing, improving and / or treating muscle diseases or muscle disorders:

[0016] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is Preferably, the combination is a pharmaceutical composition, a food composition, or a set. When the combination is a pharmaceutical composition, it is in the form of oral administration or injection; or when the combination is a food composition, it is a health food, dietary supplement, functional food, nutritional supplement, or special nutritional food.

[0017] Another object of the present invention is to provide a method for preventing, improving and / or treating muscle diseases or muscle disorders, comprising administering to a subject in need thereof an effective amount of a composition containing a compound of formula (I):

[0018] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is Preferably, the composition is a pharmaceutical composition or a food composition. When the composition is a pharmaceutical composition, it is administered to the subject orally or by injection. Furthermore, when the composition is a food composition, it is a health food, dietary supplement, functional food, nutritional supplement, or special nutritional food.

[0019] In the above-mentioned use, combination or method according to the present invention, the preparation, combination or composition may further contain the following compound of formula (II) and / or compound of formula (III):

[0020] wherein R5, R6 and R7 are each independently H or OH, and R8 is

[0021] In the above-mentioned uses, combinations or methods according to the present invention, the compound of formula (I) is preferably one in which R1 and R2 are both OH, and R3 is a C1-4 alkyl group (preferably CH3, i.e., swertiajaponin is the compound of formula (I)); the compound of formula (II) is preferably one in which R5, R6 and R7 are all OH, i.e., isoorientin 2"-O-xyloside is the compound of formula (II).

[0022] Furthermore, in the use, combination or method according to the present invention, only the compound of formula (II), or only the compound of formula (III), or both the compound of formula (II) and the compound of formula (III) may be used in the preparation, combination or composition without using the compound of formula (I).

[0023] The preparations, combinations or methods provided herein are preferably used to prevent, improve and / or treat one or more of the following: decreased muscle cell number, muscle atrophy, decreased muscle mass, decreased muscle endurance, and decreased muscle fiber size; more preferably, they are used to prevent, improve and / or treat sarcopenia.

[0024] The preparations, combinations, or methods provided herein are preferably used for one or more of the following: increasing myocyte survival, promoting myocyte proliferation, increasing myotube size, slowing and / or preventing myotube size reduction, improving muscle mass, improving muscle endurance, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial count, regulating energy metabolism, and reducing protein degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1A to 1C are bar graphs showing the relative cell proliferation rate (%) of C2C12 cells treated with various compounds at different concentrations, compared to a control C2C12 cell line (labeled "Control"). Figure 1A shows C2C12 cells treated with different concentrations of SW, Figure 1B shows C2C12 cells treated with different concentrations of I2, and Figure 1C shows C2C12 cells treated with different concentrations of 5H.

[0026] Figures 2A to 2E are bar graphs showing the relative cell viability (%) of the "Dexa-induced C2C12 cell line (hereinafter referred to as the "Dexa-C2C12 cell line," labeled Dexa in the figure)" and the Dexa-C2C12 cell line treated with different compounds at different concentrations, compared to the control C2C12 cell line (labeled "Control" in the figure). Among them, Figure 2A shows that the Dexa-C2C12 cell line was treated with different concentrations of SW; Figure 2B shows that the Dexa-C2C12 cell line was treated with different concentrations of I2; Figure 2C shows that the Dexa-C2C12 cell line was treated with different concentrations of 5H; Figure 2D shows that the Dexa-C2C12 cell line was treated with a combination of SW and different concentrations of I2; Figure 2E shows that the Dexa-C2C12 cell line was treated with a combination of SW and different concentrations of 5H, among which, "***" indicates that the p value compared with the control group is <0.005, "#" indicates that the p value compared with the "Dexa" group is <0.05, and "##" indicates that the p value compared with the "Dexa" group is <0.01.

[0027] 3A to 3E show the myotube sizes of the control C2C12 cell line (labeled as Control), the Dexa-C2C12 cell line (labeled as Dexa), and the Dexa-C2C12 cell line treated with different compounds at different concentrations. Figures 3A and 3C are photographs showing the staining results of each cell line; Figures 3B and 3D are bar graphs showing the myotube counts (%) of different diameters for each cell line, with the distribution results for diameters ">30 μm (micrometers)", "25-30 μm", "20-25 μm", "15-20 μm", "10-15 μm", and "<10 μm" from top to bottom, respectively; Figure 3E is a bar graph showing the weighted mean number of myotube sizes (micrometers) for each cell line, where "**" indicates a p-value <0.01 compared to the control group; "***" indicates a p-value <0.005 compared to the control group; "#" indicates a p-value <0.05 compared to the "Dexa" group; "##" indicates a p-value <0.01 compared to the "Dexa" group; and "###" indicates a p-value <0.005 compared to the "Dexa" group.

[0028] 4A to 4C show the myotube sizes of the control C2C12 cell line (labeled as Control in the figure), the Dexa-C2C12 cell line (labeled as Dexa in the figure), and the Dexa-C2C12 cell line treated with different compounds. Figure 4A is a photograph showing the staining results for each cell line; Figure 4B is a bar graph showing the myotube count (%) of different diameters for each cell line, with the distribution results for diameters ">30 μm," "25-30 μm," "20-25 μm," "15-20 μm," "10-15 μm," and "<10 μm" from top to bottom; Figure 4C is a bar graph showing the weighted mean number of myotube sizes (micrometers) for each cell line, where "**" indicates a p-value <0.01 compared to the control group; "***" indicates a p-value <0.005 compared to the control group; "#" indicates a p-value <0.05 compared to the "Dexa" group; and "##" indicates a p-value <0.01 compared to the "Dexa" group.

[0029] Figures 5A and 5B show the protein levels of the Dexa-C2C12 cell line (labeled "Dexa"), the Dexa-C2C12 cell line (labeled "Dexa"), and the Dexa-C2C12 cell line treated with different compounds, compared to the control C2C12 cell line (labeled "Control" in the figure). Figure 5A is a bar graph showing the relative ratio of p-Akt protein to total Akt protein, and Figure 5B is a bar graph showing the relative ratio of p-AMPK protein to total AMPK protein. "*" indicates a p-value < 0.05 compared to the control group, "**" indicates a p-value < 0.01 compared to the control group, and "#" indicates a p-value < 0.05 compared to the "Dexa" group.

[0030] Figure 6 shows the relative expression of PGC-1α protein in the Dexa-C2C12 cell line (labeled "Dexa"), as well as in the Dexa-C2C12 cell line treated with various compounds, compared to the control C2C12 cell line (labeled "Control" in the figure). "*" indicates a p-value < 0.05 compared to the control group, and "#" indicates a p-value < 0.05 compared to the "Dexa" group.

[0031] Figures 7A to 7C show the changes in different physical quantities in mice without any treatment (control group), mice treated with immobilization ("IM" group), and IM mice injected with 20 mg / kg or 50 mg / kg SW ("IM+SW(20)" group, "IM+SW(50)" group), wherein Figure 7A shows the changes in body weight of mice in each group, Figure 7B shows the changes in food intake of mice in each group, and Figure 7C shows the changes in water intake of mice in each group.

[0032] Figure 8 shows the changes in muscle endurance of mice in each group in Figure 7, wherein a higher number of electrical stimulations indicates lower muscle endurance, and wherein, "*" indicates a p-value <0.05 compared with the control group; "***" indicates a p-value <0.001 compared with the control group; "#" indicates a p-value <0.05 compared with the "IM" group; and "###" indicates a p-value <0.001 compared with the "IM" group.

[0033] Figures 9A to 9C show the myotube sizes of the gastrocnemius muscles of the mice in each group in Figure 7, wherein Figure 9A is a photograph showing the results of gastrocnemius muscle section staining, and Figure 9B is a bar graph showing the myotube counts (%) of gastrocnemius muscle at different cross-sectional areas, from top to bottom, for cross-sectional areas ">2800 μm 2 (μm 2 ))、2300-2800μm 2 ”, “1800-2300μm 2 ”, “1300-1800μm 2 ”, “<1300μm 2 9C shows the weighted average size of gastrocnemius muscle tubes of each group of mice (μm 2 ), where “*” indicates p-value < 0.05 compared with the control group; “#” indicates p-value < 0.05 compared with the “IM” group.

[0034] Figures 10A to 10C are bar graphs showing the protein content of gastrocnemius muscle of the mice in each group shown in Figure 7. Figure 10A shows the relative ratio of p-Akt protein to total Akt protein, Figure 10B shows the relative ratio of p-mTOR protein to total mTOR protein, and Figure 10C shows the relative expression of MuRF-1 protein. "*" indicates a p-value < 0.05 compared to the control group; "#" indicates a p-value < 0.05 compared to the "IM" group. DETAILED DESCRIPTION

[0035] The following describes the detailed technical content and some specific implementation plans of the present invention so that those skilled in the art can understand the characteristics of the present invention. However, without departing from the spirit of the present invention, the present invention can be practiced in different forms, and the scope of protection of the present invention should not be interpreted as limited to what is specifically described in the specification.

[0036] Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the claims) should be understood to include both singular and plural forms; the so-called "individual" refers to mammals, including humans and non-human mammals (e.g., dogs and cats).

[0037] The inventors of this application have discovered that compounds of formula (I) can enhance muscle cell survival, promote muscle cell proliferation, increase myotube size, slow down and / or prevent myotube size decline, improve muscle mass, enhance muscle endurance, increase muscle fiber volume, promote protein synthesis, increase mitochondrial count, regulate energy metabolism, and / or reduce protein degradation. Therefore, they can be used to prevent, improve, and / or treat muscle diseases or disorders. Furthermore, compounds of formula (II) and formula (III) can significantly enhance the efficacy of compounds of formula (I).

[0038] Therefore, the present invention relates to the use of a compound of formula (I) in preventing, ameliorating and / or treating muscle diseases or muscle disorders:

[0039] wherein R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is

[0040] The above applications include: (i) use of a composition containing a compound of formula (I) in the preparation of a preparation for preventing, improving and / or treating a muscle disease or muscle disorder, (ii) a combination containing a compound of formula (I) for preventing, improving and / or treating a muscle disease or muscle disorder, and (iii) a method for preventing, improving and / or treating a muscle disease or muscle disorder, comprising administering an effective amount of a composition containing a compound of formula (I) to a subject in need thereof.

[0041] In some specific embodiments of the present invention, the preparation, combination or composition containing the compound of formula (I) may further contain the following compound of formula (II) and / or compound of formula (III):

[0042] wherein R5, R6 and R7 are each independently H or OH, and R8 is

[0043] The inventors of this application have also discovered that the compounds of formula (II) and formula (III) themselves can also increase muscle cell survival rate, promote muscle cell proliferation, increase myotube size, slow down and / or prevent myotube size decline, improve muscle mass, increase muscle fiber volume, promote protein synthesis, increase mitochondrial number, and / or regulate energy metabolism. Therefore, the compounds of formula (II) and / or formula (III) can also be used as active ingredients for the prevention, improvement, and / or treatment of muscle diseases or muscle disorders, including: (i) using a composition containing the compound of formula (II) and / or formula (III) in the preparation of a preparation for the prevention, improvement, and / or treatment of muscle diseases or muscle disorders; (ii) a combination containing the compound of formula (II) and / or formula (III) for the prevention, improvement, and / or treatment of muscle diseases or muscle disorders; and (iii) a method for the prevention, improvement, and / or treatment of muscle diseases or muscle disorders, comprising administering an effective amount of the composition containing the compound of formula (II) and / or formula (III) to a subject in need thereof.

[0044] In some embodiments of the present invention, the formulations, combinations, and methods are used to prevent, improve, and / or treat at least one of the following: decreased muscle cell number, muscle atrophy, decreased muscle mass, decreased muscle endurance, and decreased muscle fiber size. In some embodiments of the present invention, the formulations, combinations, and methods are used to prevent, improve, and / or treat sarcopenia.

[0045] In the applications according to the present invention, the compound of formula (I) is preferably one in which R1 and R2 are both OH, and R3 is a C1-4 alkyl group (preferably CH3). For example, in some embodiments of the present invention, the compound of formula (I) is swertiajaponin.

[0046] In the application according to the present invention, the compound of formula (II) is preferably one in which R5, R6 and R7 are all OH, that is, isoorientin 2"-O-xyloside is the compound of formula (II).

[0047] In the use according to the present invention, the compound of formula (III) is 5-hydroxymethylfurfural.

[0048] In the application according to the present invention, the compounds of formula (I), formula (II) and formula (III) can be commercially available or prepared by synthetic methods known in the art.

[0049] In the use according to the present invention, the preparation may be a medicament or a food. In the method according to the present invention, the composition may be a pharmaceutical composition or a food composition. The combination provided according to the present invention may be a pharmaceutical composition, a food composition, or a set. When the combination provided according to the present invention is a set, the compounds contained are typically packaged separately and stored in different storage spaces (e.g., plastic bottles, glass bottles, ampoules), and can be shipped or sold separately, or they can be combined and distributed and sold together as a set. In addition, the set may also include instructions for use to facilitate the user to mix the ingredients at the site of use according to the procedures and processes set out therein for the desired processing and application.

[0050] The compound components of the medicaments, pharmaceutical compositions, or kits provided herein can be administered systemically or locally and can be delivered via various drug delivery systems (DDS). For example, but not limited to, the compound components of the medicaments, pharmaceutical compositions, or kits provided herein can be delivered via an oral drug delivery system or an injectable drug delivery system.

[0051] The compound components of the medicaments, pharmaceutical compositions, or kits provided herein may be in any suitable form, without particular limitation, and may be in a suitable dosage form depending on the intended use. For example, but not limited to, the compound components of the medicaments, pharmaceutical compositions, or kits may be administered to a subject in need thereof orally, intravenously (including intravenous infusion and bolus injection), intramuscularly, subcutaneously, intraperitoneally, or intraperitoneally. Preferably, the compound components of the medicaments, pharmaceutical compositions, or kits are administered to a subject in need thereof by intramuscular injection. Depending on the form of use and intended use, the compound components of the medicaments, pharmaceutical compositions, or kits may be provided in a pharmaceutically acceptable carrier. Such carriers are known in the art and include excipients, diluents, adjuvants, stabilizers, absorption enhancers, disintegrants, solubilizers, emulsifiers, antioxidants, adhesives, binders, viscosity-increasing agents, dispersants, suspending agents, lubricants, hygroscopic agents, and the like.

[0052] Taking oral dosage forms as an example, the compound components of the medicament, pharmaceutical composition, or kit can be provided in a dosage form suitable for oral administration by any convenient method. Liquid dosage forms suitable for oral administration include syrups, oral solutions, suspensions, elixirs, etc., and solid dosage forms suitable for oral administration include powders, granules, buccal tablets, sugar-coated tablets, enteric-coated tablets, chewable tablets, blister tablets, film-coated tablets, capsules, and long-acting sustained-release tablets. The compound components of the medicament, pharmaceutical composition, or kit provided by the present invention may contain any pharmaceutically acceptable carrier that does not adversely affect the desired efficacy of the compound of Formula (I), Formula (II), and / or Formula (III). For example, but not limited to, pharmaceutically acceptable carriers for the aforementioned liquid dosage forms include: water, saline, dextrose, glycerol, ethanol or its analogs, oils (such as olive oil, castor oil, cottonseed oil, peanut oil, corn oil, and germ oil), polyethylene glycol, and combinations thereof; and pharmaceutically acceptable carriers for the aforementioned solid dosage forms include: cellulose, starch, kaolinite, bentonite, sodium citrate, gelatin, agar, carboxymethyl cellulose, gum arabic, algin, glyceryl monostearate, calcium stearate, and combinations thereof.

[0053] For injections or drips suitable for injection, the compound components of the medicament, pharmaceutical composition, or kit provided by the present invention may contain one or more ingredients such as an isotonic solution, a salt buffer (such as a phosphate buffer or a citrate buffer), a solubilizer, an emulsifier, a 5% sugar solution, and other carriers, and the compound components of the medicament, pharmaceutical composition, or kit may be provided in dosage forms such as an intravenous infusion solution, an emulsion intravenous infusion solution, a dry powder injection, a suspension injection, or a dry powder suspension injection. Alternatively, the compound components of the medicament, pharmaceutical composition, or kit may be prepared as a pre-injection solid and, prior to administration to a subject in need thereof, the pre-injection solid may be dissolved in another solution or suspension or emulsified to provide the desired injection. Preferably, the compound components of the medicament, pharmaceutical composition, or kit are in the form of a suspension injection.

[0054] Optionally, the compound components of the medicament, pharmaceutical composition, or kit provided by the present invention may further contain appropriate amounts of additives, such as toners and colorants that can enhance the feel of the medicament, pharmaceutical composition, or kit during use, as well as buffers, preservatives, preservatives, antibacterial agents, antifungal agents, etc. that can improve the stability and storage properties of the medicament, pharmaceutical composition, or kit.

[0055] The food or food composition provided by the present invention can be a health food, dietary supplement, functional food, nutritional supplement, or specialty food. For example, but not limited to, the food can include dairy products, processed meat products, bread, noodles, biscuits, ice cream, lozenges, capsules, juices, teas, sparkling water, alcoholic beverages, sports drinks, nutritional beverages, and the like.

[0056] Furthermore, depending on the intended use and needs, the food or food composition provided by the present invention may contain any suitable food additives. For example, such food additives include, but are not limited to, preservatives, antiseptics, bactericides, antioxidants, bleaching agents, colorants, bulking agents, nutritional additives, colorants, flavorings (e.g., sweeteners), thickeners, binders, food industry chemicals, emulsifiers, and agents for quality improvement, brewing, and food manufacturing.

[0057] The outer packaging of the health food, dietary supplement, functional food, nutritional supplement, or special nutritional food provided by the present invention may be labeled with recommended dosage, usage standards and conditions, or recommendations for co-administration with other foods or medications, to facilitate self-administration without the guidance of a physician, pharmacist, or related personnel without safety concerns.

[0058] The compound components of the formulations, compositions, or kits provided by the present invention may optionally contain one or more other active ingredients to further enhance the efficacy of the formulation, composition, or kit or to increase the flexibility and adjustability of the formulation, as long as the other active ingredients do not adversely affect the benefits of the compounds of Formula (I), Formula (II), and / or Formula (III) contained in the formulation, composition, or kit of the present invention.

[0059] In the compound components of the preparations, compositions or kits provided by the present invention, the content of the compound of formula (I), formula (II) and / or formula (III) is at least about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 0.11, 0.12, 0.13, 0.14, 0.15 %, about 0.01 wt % to about 10 wt %, about 0.01 wt % to about 5 wt %, about 0.05 wt % to about 1 wt %, and about 0.05 wt % to about 0.5 wt %.

[0060] The formulations, compositions, or kits provided herein can be administered at various frequencies, such as once daily, multiple times daily, or once every few days, depending on the needs, age, weight, and health status of the individual being administered, as well as the intended purpose of administration. The content of the compound of Formula (I), Formula (II), and / or Formula (III) in the formulations, compositions, or kits provided herein can also be adjusted based on actual application needs, for example, to a daily dosage for oral or topical use. For example, when used in humans by injection to prevent, improve and / or treat muscle diseases or muscle disorders, the dosage of the preparation, composition or kit of the present invention, based on the weight of the compound of formula (I), formula (II) and / or formula (III), is from about 0.1 mg / kg body weight to about 50 mg / kg body weight per day, preferably from about 1 mg / kg body weight to about 20 mg / kg body weight per day, more preferably from about 2 mg / kg body weight to about 8 mg / kg body weight per day; or, from about 0.2 mg / kg body weight to about 100 mg / kg body weight every two days, preferably from about 2 mg / kg body weight to about 40 mg / kg body weight every two days, more preferably from about 4 mg / kg body weight to about 16 mg / kg body weight every two days. Furthermore, when administered orally to humans for the prevention, improvement, and / or treatment of muscle diseases or disorders, the dosage of the preparations, compositions, or kits of the present invention, based on the weight of the compound of Formula (I), Formula (II), and / or Formula (III), is from about 0.5 mg / kg body weight to about 1000 mg / kg body weight per day, preferably from about 2 mg / kg body weight to about 500 mg / kg body weight per day, and more preferably from about 4 mg / kg body weight to about 300 mg / kg body weight per day; or, from about 1 mg / kg body weight to about 2000 mg / kg body weight every two days, preferably from about 4 mg / kg body weight to about 1000 mg / kg body weight every two days, and more preferably from about 8 mg / kg body weight to about 600 mg / kg body weight every two days. The unit "mg / kg body weight" refers to the dosage per kilogram of body weight of the individual. However, for acute patients, the dosage may be increased as needed, for example, by several or even dozens of times. In one embodiment of the present invention, a preparation, composition or kit for preventing, ameliorating and / or treating muscle diseases or muscle disorders contains a compound of formula (I) as an active ingredient. The preparation, composition or kit is in the form of an injection, and the dosage thereof is about 2.2 mg / kg body weight or about 5.5 mg / kg body weight per day, based on the weight of the compound of formula (I).

[0061] In the method according to the present invention, the ingredients, dosage form, administration method, administration frequency, and dosage range of the composition are as described above.

[0062] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is as set forth in the claims.

[0063] Example

[0064] In the following examples, the materials and equipment used are as follows:

[0065] 1. C2C12 cell line (a muscle fibroblast): obtained from Bioresource Collection and Research Center (BCRC; website: www.bcrc.firdi.org.tw / ); accession number: BCRC 60083.

[0066] 2. Dulbecco's Modified Eagle Medium (DMEM) culture medium: purchased from ThermoFisher (USA); product number: 11995-040.

[0067] C2C12 cell line growth medium: DMEM supplemented with 3.7 g / L sodium bicarbonate, 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0068] 4. C2C12 cell line differentiation culture medium: DMEM culture medium supplemented with 2% horse serum.

[0069] 5. MTS cell survival assay kit: purchased from Promega.

[0070] 6. Microplate spectrophotometer: purchased from Molecular Devices (USA); Model: M3.

[0071] 7. Swertiajaponin (hereinafter referred to as “SW”): purchased from KM3 SCIENTIFIC CORP. (website: https: / / www.km3.com.tw / ); product number: CAS No. 6980-25-2.

[0072] 8. Isoorientin 2"-O-xyloside (hereinafter referred to as "I2"): Cecropia pachystachya leaves were dried at room temperature and ground. 35 g of the leaves were extracted with 300 mL of methanol at 65°C in a Soxhlet extractor for 12 hours. The extract was cooled to room temperature, filtered, and concentrated under reduced pressure on a rotary evaporator until some of the methanol was removed. After evaporation of the residual methanol, the extract was freeze-dried and I2 was isolated and purified using a preparative HPLC column. Differentiation of the phenolic chemical profiles of Cecropia pachystachya and Cecropia hololeuca. Phytochemical analysis. 2019July; 30(1): 73-82. The full text of this document is incorporated herein by reference.

[0073] 9. 5-Hydroxymethylfurfural (5-hydroxymethylfurfural; referred to herein as “5H”): purchased from Sigma-Aldrich (USA); product number: CAS NO. 67-47-0.

[0074] 10. Dexamethasone (hereinafter referred to as "Dexa"): purchased from Sigma-Aldrich (USA); product number: CAS NO. 50022. Dexa is a long-acting synthetic glucocorticoid that causes skeletal muscle atrophy by stimulating myoprotein hydrolysis (degradation) and inhibiting protein synthesis. For example, see: Glucocorticoid-induced skeletal muscle atrophy. Int J Biochem Cell Biol. 2013 Oct; 45(10): 2163-2172. The full text of this article is incorporated herein by reference.

[0075] 11. Phase-contrast microscope: purchased from ZEISS Microscopy (USA); model: Axio Observer 3.

[0076] 12. Antibodies used for Western blotting: anti-PGC-1α antibody (purchased from Abcam (USA); product number: ab191838); anti-β-actin antibody (purchased from Abcam (USA); product number: ab8226); anti-Akt (pan) (c67E7) antibody (purchased from Cell Signaling Technologies (USA); product number: #4691); anti-p-Akt (Ser473) (193H12) antibody (purchased from Cell Signaling Technologies (USA); product number: #4058); anti-mTOR (7C10) antibody (purchased from Cell Signaling Technologies (USA); product number: #2983); anti-p-mTOR (Ser2448) antibody (purchased from Cell Signaling Technologies (USA); product number: #5536); anti-AMPK antibody (purchased from Cell Signaling Technologies (USA); product number: #5537); Technologies (USA); Product No.: #2793); anti-p-AMPK antibody (purchased from Cell Signaling Technologies (USA); Product No.: #2535); anti-MuRF-1 antibody (purchased from Proteintech (USA); Product No.: 55456-1-AP).

[0077] 13. Other materials used in Western blotting: Protein quantification kit (Bio-Rad Protein Assay; purchased from Bio-Rad (USA); product number: #5000006); Clarity Western ECL substrate kit (purchased from Bio-Rad (USA); product number: #1705061); Multiplexed fluorescent luminescence imaging system (ChemiDoc imaging system; purchased from Bio-Rad (USA)).

[0078] 14. Rodent Treadmill: purchased from Ugo Basile (Italy); model: 47300.

[0079] Example 1: Effects of the compounds of the present invention on promoting myocyte proliferation and enhancing myocyte survival

[0080] This example uses the MTS "a tetrazolium compound, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]" cell survival assay (MTS assay) to study the effectiveness of the compounds of the present invention in promoting muscle cell proliferation and improving muscle cell survival.

[0081] The cell cultures in this example were all carried out in an environment of 95% relative humidity, 5% CO 2 , and 37° C.

[0082] 1-1. Cell Culture

[0083] C2C12 cells were first cultured in C2C12 cell culture medium to a confluence of 70-80%, and then plated in a 10 cm culture dish at 4 × 10 5 Continue to culture C2C12 cells at a starting cell density of cells / well until they reach 80-90% confluence.

[0084] 1-2. Cell proliferation analysis

[0085] The cells provided in Example 1-1 were taken, the C2C12 cell line growth medium was removed, and the cells were rinsed with DPBS (Dulbecco's phosphate-buffered saline). The cells were then divided into groups and cultured for 24 hours in the following culture media:

[0086] 1. Control group: C2C12 cell line differentiation culture medium.

[0087] 2. SW-2.5, SW-5, SW-10, SW-20, SW-50, SW-100, or SW-200 groups: C2C12 cell differentiation medium supplemented with 2.5, 5, 10, 20, 50, 100, or 200 μg / mL SW.

[0088] 3. "I2-2.5" group, "I2-5" group, "I2-10" group, "I2-20" group, "I2-50" group, "I2-100" group, or "I2-200" group: C2C12 cell line differentiation culture medium supplemented with 2.5, 5, 10, 20, 50, 100, or 200 μg / mL of I2.

[0089] 4. 5H-2.5, 5H-5, 5H-10, 5H-20, 5H-50, 5H-100, or 5H-200 groups: C2C12 cell differentiation medium supplemented with 2.5, 5, 10, 20, 50, 100, or 200 μg / mL of 5H.

[0090] Then, each group took 1×10 4 Cells were plated in different wells of a 96-well microtiter plate (each well containing 100 μl of C2C12 cell line differentiation medium). 20 μl of MTS reagent was added to each well. After incubation for 1 hour, the 96-well microtiter plate was placed in a microtiter plate spectrometer and the absorbance at 490 nanometers (nm) was measured. The relative cell proliferation rate (percentage) was calculated based on the absorbance. The results are shown in Figures 1A to 1C.

[0091] As shown in Figures 1A to 1C, compared to the control group, the cell proliferation rates of the groups supplemented with SW, I2, or 5H were higher. These results indicate that SW, I2, and 5H can promote myocyte proliferation, and that this effect is evident even at a low dose (2.5 μg / mL).

[0092] 1-3. Cell Survival Analysis

[0093] The cells provided in Example 1-1 were taken, the C2C12 cell line growth medium was removed, and the cells were rinsed with DPBS. The cells were then divided into groups and cultured for 24 hours in the following culture medium:

[0094] 1. Control group: C2C12 cell line differentiation culture medium.

[0095] 2. "Dexa" group: C2C12 cell line differentiation culture medium containing 10 micromolar (μM) Dexa.

[0096] 3. "SW-2.5" group, "SW-5" group, "SW-10" group, "SW-20" group, "SW-50" group, "SW-100" group, or "SW-200" group: SW (2.5, 5, 10, 20, 50, 100, or 200 μg / ml) was further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0097] 4. "I2-2.5" group, "I2-5" group, "I2-10" group, "I2-20" group, "I2-50" group, "I2-100" group or "I2-200" group: I2 (2.5, 5, 10, 20, 50, 100 or 200 μg / ml) was further added to the C2C12 cell line differentiation culture medium containing 10 μM concentration of Dexa.

[0098] 5. 5H-2.5, 5H-5, 5H-10, 5H-20, 5H-50, 5H-100, or 5H-200 groups: 5H (2.5, 5, 10, 20, 50, 100, or 200 μg / ml) was further added to the C2C12 cell line differentiation medium containing 10 μM Dexa.

[0099] 6. "SW-2.5+I2-2.5" group, "SW-2.5+I2-5" group, and "SW-2.5+I2-25" group: SW (2.5 μg / ml) and I2 (2.5, 5, or 25 μg / ml) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0100] 7. "SW-2.5+5H-2.5" group, "SW-2.5+5H-5" group, and "SW-2.5+5H-25" group: SW (2.5 μg / ml) and 5H (2.5, 5, or 25 μg / ml) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0101] Then, each group took 1×10 4 Cells were plated in separate wells of a 96-well microtiter plate (each well containing 100 μl of C2C12 cell line differentiation medium). 20 μl of MTS reagent was added to each well. After incubation for 1 hour, the 96-well microtiter plate was placed in a microtiter plate spectrometer and absorbance at 490 nm was measured to determine cell survival. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. Relative cell survival rates of the remaining groups were calculated using the control group as the baseline (i.e., the control group survival rate was set at 100%). The results are shown in Figures 2A to 2E.

[0102] As shown in Figures 2A to 2C, cell viability in the "Dexa" group was significantly decreased compared to the control group. However, cell viability in groups supplemented with SW, I2, or 5H was higher than that in the "Dexa" group. These results demonstrate that SW, I2, and 5H can enhance myocyte survival, with an effect observed even at a low dose (2.5 μg / mL).

[0103] As shown in Figures 2D and 2E , in culture medium containing Dexa, the cell survival rates in the groups supplemented with both SW and I2, or with SW and 5H, were higher than in the group supplemented with SW alone. These results demonstrate that the combined use of SW with I2 and / or 5H offers superior results compared to SW alone.

[0104] The results of this example show that the compounds of formula (I), formula (II) and / or formula (III) of the present invention can be used to prevent, improve and / or treat decreased muscle cell count.

[0105] Example 2: Effectiveness of the compounds of the present invention in restoring / increasing myotube size, slowing down / preventing myotube size decline, and increasing muscle fiber volume

[0106] The cell cultures in this example were all carried out in an environment of 95% relative humidity, 5% CO 2 , and 37° C.

[0107] 2-1. Restore / increase myotube size

[0108] The cells provided in Example 1-1 were taken, the C2C12 cell line growth medium was removed, and the cells were washed with DPBS. The cells were then divided into groups and cultured in the following culture medium for 24 hours:

[0109] 1. Control group: C2C12 cell line differentiation culture medium.

[0110] 2. “Dexa” group: C2C12 cell line differentiation culture medium containing 10 micromolar concentration of Dexa.

[0111] 3. "Dexa+SW" group, "Dexa+I2" group, or "Dexa+5H" group: 50 μg / mL SW, I2, or 5H was further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0112] 4. "SW-2.5" group: SW (2.5 μg / ml) was further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0113] 5. "SW-2.5+I2-2.5" group, "SW-2.5+I2-5" group, or "SW-2.5+I2-25" group: SW (2.5 μg / mL) and I2 (2.5, 5, or 25 μg / mL) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0114] 6. "SW-2.5+5H-2.5" group, "SW-2.5+5H-5" group, or "SW-2.5+5H-25" group: SW (2.5 μg / mL) and 5H (2.5, 5, or 25 μg / mL) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0115] The cells were then fixed with 10% neutral buffered formalin and stained with hematoxylin-eosin (H&E). Afterwards, each group of cells was imaged using phase contrast microscopy (20x magnification; five images per group). The diameters (μm) of the thickest portion of 100 randomly selected myotubes in each image were measured using ImageJ software (website: rsb.info.nih.gov / ij / ). The measured diameters were weighted and statistically analyzed using one-way analysis of variance and Dunnett's multiple comparison test. The images are shown in Figures 3A and 3C, the myotube diameter counts are shown in Figures 3B and 3D, and the weighted analysis results are shown in Figure 3E.

[0116] Figures 3A and 3B show that compared to the control group, the myotube diameter in the "Dexa" group was significantly reduced. However, the myotube diameters in the "Dexa + SW" group, the "Dexa + I2" group, and the "Dexa + 5H" group were all significantly higher than those in the "Dexa" group. These results demonstrate that SW, I2, and 5H can restore or increase myotube size, thereby increasing muscle fiber volume.

[0117] As shown in Figures 3C and 3E , myotube sizes in the SW-2.5 + I2-2.5, SW-2.5 + I2-5, SW-2.5 + I2-25, SW-2.5 + 5H-2.5, SW-2.5 + 5H-5, and SW-2.5 + 5H-25 groups were significantly larger than those in the SW-2.5 group. These results further demonstrate that the combined use of SW with I2 and / or 5H provides superior results compared to SW alone.

[0118] 2-2. Slowing / Preventing the Decrease in Myotube Size

[0119] Take the cells provided in Example 1-1, remove the C2C12 cell line growth medium, rinse the cells with DPBS, and then divide the cells into groups and perform the following culture operations:

[0120] 1. Control group: After culturing C2C12 cells with differentiation medium for 6 hours, the cells were replaced with new differentiation medium and cultured for another 24 hours.

[0121] 2. "Dexa" group: After culturing the cells in the C2C12 cell line differentiation medium for 6 hours, the differentiation medium was replaced with fresh one, and Dexa (10 μM concentration) was added to the culture medium, and the cells were cultured for another 24 hours.

[0122] 3. "Dexa + SW" group: After culturing C2C12 cells in differentiation medium containing 50 μg / mL SW for 6 hours, the differentiation medium was replaced with fresh one, and Dexa (10 μM concentration) and SW (50 μg / mL) were added to the culture medium, and the culture was continued for another 24 hours.

[0123] 4. "Dexa + I2" group: After culturing C2C12 cells in differentiation medium containing 50 μg / mL I2 for 6 hours, the differentiation medium was replaced with fresh one, and Dexa (10 μM concentration) and I2 (50 μg / mL) were added to the culture medium, and the culture was continued for 24 hours.

[0124] 5. "Dexa + 5H" group: After culturing C2C12 cells in differentiation medium containing 50 μg / mL 5H for 6 hours, the differentiation medium was replaced with fresh one, and Dexa (10 μM concentration) and 5H (50 μg / mL) were added to the culture medium, and the culture was continued for 24 hours.

[0125] The cells were then fixed with 10% neutral buffered formalin and stained with hematoxylin and eosin (H&E). Afterwards, each group of cells was imaged using phase contrast microscopy (20x magnification; five images per group). The diameters (μm) of the thickest portion of 100 randomly selected myotubes in each image were measured using ImageJ software (website: rsb.info.nih.gov / ij / ). The measured diameters were weighted and statistically analyzed using one-way analysis of variance and Dunnett's multiple comparison test. The images are shown in Figure 4A, the myotube diameter counts are shown in Figure 4B, and the weighted analysis results are shown in Figure 4C.

[0126] As shown in Figures 4A to 4C, myotube diameter in the Dexa group was significantly reduced compared to the control group. However, myotube diameters in the Dexa + SW, Dexa + I2, and Dexa + 5H groups were significantly higher than those in the Dexa group. These results suggest that pretreatment with SW, I2, or 5H can slow or prevent the decrease in myotube size.

[0127] The results of this example show that the compounds of formula (I), formula (II) and / or formula (III) of the present invention can be used to prevent, improve and / or treat muscle atrophy and muscle fiber volume reduction.

[0128] Example 3: The effectiveness of the compounds of the present invention in promoting protein synthesis, increasing muscle mass, increasing mitochondrial count, and regulating energy metabolism

[0129] Studies have shown that muscle hypertrophy is related to protein synthesis, myotube formation, and mitochondrial formation; while the causes of muscle atrophy include apoptosis, protein degradation, and mitochondrial degeneration.

[0130] 3-1. p-Akt protein and p-AMPK protein

[0131] It is known that an increase in the expression level of p-Akt protein (i.e., increased phosphorylation of Akt protein) indicates that the signal transduction pathway involved in protein synthesis is activated. For example, see: PI3K / AKT / mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer. 2023 Aug 18; 22(1): 138., and Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech. 2013 Jan; 6(1): 25-39., the entire texts of which are incorporated herein by reference; an increase in the expression level of p-AMPK protein (i.e., increased phosphorylation of AMPK protein) indicates that the signal transduction pathway involved in regulating energy metabolism and mitochondrial formation is activated. For example, see: AMP-activated protein kinase and its downstream transcriptional pathways. Cellular and Molecular Life Sciences. 2010; 67: 3407-3423., the entire text of which is incorporated herein by reference.

[0132] To understand the benefits of the compounds of the present invention in promoting protein synthesis and mitochondrial formation in muscle cells, the cells provided in Example 1-1 were removed from the C2C12 cell line culture medium, washed with DPBS, and then divided into groups and cultured in the following culture medium for 24 hours:

[0133] 1. Control group: C2C12 cell line differentiation culture medium.

[0134] 2. “Dexa” group: C2C12 cell line differentiation culture medium containing 10 micromolar concentration of Dexa.

[0135] 3. "Dexa+SW" group, "Dexa+I2" group, or "Dexa+5H" group: 50 μg / mL SW, I2, or 5H was further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0136] Next, protein extraction was performed, and Western blotting was used to measure the expression of p-Akt, total Akt, p-AMPK, and total AMPK proteins in each protein sample. The ratios of p-Akt / total Akt and p-AMPK / total AMPK were calculated for each group. The relative protein expression ratios of the control group were then calculated using the control group as a baseline (i.e., the protein expression ratio of the control group was set to 1). Statistical analysis was performed using one-way analysis of variance and Dunnett's multiple comparison test. The results are shown in Figures 5A and 5B.

[0137] As shown in Figures 5A and 5B, the ratios of p-Akt protein to total Akt protein and p-AMPK protein to total AMPK protein were significantly decreased in the "Dexa" group compared to the control group. However, the ratios of p-Akt protein to total Akt protein and p-AMPK protein to total AMPK protein were higher in the "Dexa + SW" and "Dexa + I2" groups compared to the "Dexa" group. These results indicate that both SW and I2 can activate the Akt and AMPK signaling pathways, thus promoting protein synthesis and mitochondrial formation, thereby increasing muscle mass, increasing mitochondrial number, and regulating energy metabolism.

[0138] 3-2. PGC-1α protein

[0139] PGC-1α is known to play a crucial role in mitochondrial formation and is closely related to skeletal muscle mass and energy metabolism. Increased PGC-1α protein expression indicates an increase in mitochondrial number and improved muscle mass. For more information, see, for example, AMP-activated protein kinase and its downstream transcriptional pathways. Cellular and Molecular Life Sciences. 2010;67:3407-3423, which is incorporated herein by reference in its entirety. To further explore the potential benefits of the compounds of the present invention in promoting mitochondrial formation, increasing mitochondrial number, improving muscle mass, and regulating energy metabolism, the cells provided in Example 1-1 were removed from the C2C12 cell line culture medium, washed with DPBS, and then divided into groups and cultured for 24 hours in the following culture medium:

[0140] 1. Control group: C2C12 cell line differentiation culture medium.

[0141] 2. “Dexa” group: C2C12 cell line differentiation culture medium containing 10 micromolar concentration of Dexa.

[0142] 3. “Dexa+SW” group: 2.5 μg / mL SW was further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0143] 4. "Dexa+SW+I2" group: SW (2.5 μg / mL) and I2 (2.5 μg / mL) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0144] 5. "Dexa+SW+5H" group: SW (2.5 μg / mL) and 5H (2.5 μg / mL) were further added to the C2C12 cell line differentiation culture medium containing 10 μM Dexa.

[0145] Next, protein extraction was performed, and the expression of PGC-1α protein in each protein sample was measured by Western blotting. Statistical analysis was then performed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. The relative protein expression levels of the remaining groups were calculated using the control group as a baseline (i.e., the protein expression in the control group was set to 1-fold higher). Statistical analysis was also performed using one-way ANOVA and Dunnett's multiple comparison test. The results are shown in Figure 6.

[0146] As shown in Figure 6, PGC-1α protein expression levels were significantly higher in the "Dexa" group, the "Dexa+SW" group, the "Dexa+SW+I2" group, and the "Dexa+SW+5H" group compared to the "Dexa" group. These results demonstrate that SW alone, or combined with I2 and / or 5H, can increase PGC-1α protein expression, potentially promoting mitochondrial formation, increasing mitochondrial number, improving muscle mass, and regulating energy metabolism.

[0147] The results of this example show that the compounds of formula (I), formula (II) and / or formula (III) of the present invention can be used to prevent, improve and / or treat muscle atrophy and decreased muscle mass.

[0148] Example 4: The benefits of the compounds of the present invention in improving muscle endurance, increasing myotube size, increasing muscle fiber volume, promoting protein synthesis, reducing protein degradation, and improving muscle mass

[0149] This example was conducted in accordance with the relevant regulations of the Institutional Animal Care and Use Committee (IACUC) of the Pharmaceutical Industry Technology Development Center (website: www.pitdc.org.tw / ) (IACUC approval number: PITDC1120006).

[0150] 4-1. Establishment of animal model

[0151] C57BL / 6 male mice (24±1 g) were caged for two weeks to acclimate to the laboratory environment: the temperature was maintained at 23±2°C and a 12-hour day-night cycle, and food and water were provided ad libitum.

[0152] Each mouse then underwent treadmill training for two days. Afterward, the mice were randomly divided into four groups (10 mice per group) and housed under the following conditions (maintaining a temperature of 23±2°C and a 12-hour day / night cycle, with food and water provided ad libitum throughout the experiment) for 14 days:

[0153] 1. Control group: no immobilization (IM) was performed.

[0154] 2. “IM” group: The right hind limbs of mice (under anesthesia) were fixed with 3M tape on day 1, and the tape was removed on day 7.

[0155] 3. “IM+SW(20)” group: The treatment was similar to that of the “IM” group, but 20 mg / kg SW was administered to the mice via intraperitoneal injection daily.

[0156] 4. “IM+SW(50)” group: The procedure was similar to that of the “IM” group, but 50 mg / kg SW was administered to the mice via intraperitoneal injection daily.

[0157] 4-2. Observation of body weight, food intake, and water intake

[0158] During the group-rearing process of Example 4-1, the body weight and food / water intake of the mice were measured every two to three days. The observation results of body weight, food intake, and water intake are shown in Figures 7A to 7C, respectively.

[0159] As shown in Figures 7A to 7C, compared with the control group, there were no significant changes in body weight, food intake, and water intake in the "IM" group, the "IM+SW(20)" group, and the "IM+SW(50)" group.

[0160] 4-3. Muscle endurance test

[0161] It is known that immobilizing an animal's foot can reduce its muscle mass and endurance. For example, see: Atenolol ameliorates skeletal muscle atrophy and oxidative stress induced by cast immobilization in rats. Biomedicines. 2023 Apr 25; 11(5): 1269. The full text of this document is incorporated herein by reference.

[0162] Therefore, on days 7 and 14 of group-rearing as described in Example 4-1, mice in each group underwent a treadmill test (a mild electrical stimulation device at 1.1 milliamperes (mA) was installed at the end of the treadmill; the first phase was a speed of 15 m / min for 5 minutes; the second phase was a speed of 18 m / min for 25 minutes). The number of electrical stimulations during the second phase (i.e., placing the hind leg on the electrical stimulation device for 3 seconds) was recorded. The results are shown in Figure 8. A greater number of electrical stimulations indicates lower muscle endurance and a higher degree of muscle fatigue.

[0163] As shown in Figure 8, the number of electrical stimulations in the "IM" group increased significantly compared to the control group; however, the number of electrical stimulations in the "IM+SW(20)" and "IM+SW(50)" groups was significantly lower than that in the "IM" group. The above results show that SW can reduce muscle fatigue and improve muscle endurance in animal models of muscle damage / atrophy, and therefore can be used to prevent, improve, and / or treat decreased muscle endurance.

[0164] 4-4. Sample Collection

[0165] After the treadmill test in Example 4-3, the mice were sacrificed and the gastrocnemius muscles of their right hind legs were removed for subsequent experimental analysis.

[0166] 4-5. Section staining analysis

[0167] The gastrocnemius muscle of Example 4-4 was fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned. The sections were stained with hematoxylin-eosin (H&E) and photographed using a phase contrast microscope (20x magnification; 5 images per group). The cross-sectional area (μm) of the thickest part of 100 random myotubes in each group of images was measured using ImageJ software (website: rsb.info.nih.gov / ij / ). 2 ; μm 2The measured cross-sectional areas were subjected to weighted analysis, and statistical analysis was performed using one-way analysis of variance and Dunnett's multiple comparison test. The aforementioned images are shown in Figure 9A , the myotube cross-sectional area counts are shown in Figure 9B , and the weighted analysis results are shown in Figure 9C .

[0168] As shown in Figures 9A to 9C , the myotube cross-sectional area of ​​the "IM" group was significantly reduced compared to the control group; however, the myotube cross-sectional area of ​​the "IM+SW(20)" and "IM+SW(50)" groups was significantly increased compared to the "IM" group. These results again demonstrate that SW has the effect of increasing myotube size and muscle fiber volume, and therefore can be used to prevent, improve, and / or treat muscle atrophy and muscle fiber volume reduction.

[0169] 4-6. Protein expression analysis

[0170] It is known that the activation of the PI3K / Akt / mTOR signaling pathway is closely related to cell survival, cell growth, and cell cycle progression. When the PI3K / Akt / mTOR signaling pathway is phosphorylated, protein synthesis will increase. In addition, the degeneration of skeletal muscle is related to the action of the ubiquitin-proteasome system, among which MAFbx and MuRF-1 are negative regulators of muscle mass (that is, increased expression of MAFbx and / or MuRF-1 will promote protein degradation, leading to decreased muscle mass). For the above, see, for example: PI3K / AKT / mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer. 2023 Aug 18; 22(1): 138., and Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech. 2013 Jan; 6(1): 25-39., the full text of these documents is hereby incorporated by reference.

[0171] After protein extraction from the gastrocnemius muscle of Example 4-4, the protein samples were analyzed by Western blotting for p-Akt, total Akt, p-mTOR, total mTOR, and MuRF-1 protein expression. The ratios of p-Akt / total Akt and p-mTOR / total mTOR were calculated for each group. Subsequently, relative protein expression ratios or protein expression levels were calculated for each group using the control group as a baseline (i.e., the protein expression ratio or protein expression level of the control group was set to 1). Statistical analysis was performed using one-way analysis of variance and Dunnett's multiple comparison test. The results are shown in Figures 10A to 10C.

[0172] As shown in Figures 10A and 10B, the ratios of p-Akt protein / total Akt protein and p-mTOR protein / total mTOR protein in the "IM+SW(20)" and "IM+SW(50)" groups were higher than those in the "IM" group. These results indicate that SW can activate the Akt / mTOR signaling pathway and thus can be used to promote protein synthesis and increase muscle mass, thereby preventing, improving, and / or treating muscle atrophy and decreased muscle mass.

[0173] As shown in Figure 10C, the relative expression of MuRF-1 protein in the "IM" group was significantly higher than that in the control group; however, the relative expression of MuRF-1 protein in the "IM+SW(20)" and "IM+SW(50)" groups was significantly lower than that in the "IM" group. These results indicate that SW can inhibit the ubiquitin-proteasome system and can therefore be used to reduce protein degradation, improve muscle mass, and thereby prevent, improve, and / or treat muscle atrophy and decreased muscle mass.

[0174] The results of the above examples indicate that the compounds of Formula (I), Formula (II), and / or Formula (III) of the present invention can effectively increase muscle cell survival rate, promote muscle cell proliferation, increase myotube size, slow down and / or prevent myotube size decline, improve muscle mass, enhance muscle endurance, increase muscle fiber volume, promote protein synthesis, increase mitochondrial number, regulate energy metabolism, and / or reduce protein degradation, thereby achieving the effect of preventing, ameliorating, and / or treating muscle diseases or disorders.

Claims

1. Use of a composition containing a compound of formula (I) in the preparation of a preparation, characterized in that: This preparation is used to prevent, improve and / or treat muscle diseases or muscle disorders: in, R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is 2. The use according to claim 1, characterized in that The preparation is used to prevent, improve and / or treat one or more of the following: decreased number of muscle cells, muscle atrophy, decreased muscle mass, decreased muscle endurance, and decreased muscle fiber size.

3. The use according to claim 1, characterized in that The preparation is used for preventing, improving and / or treating sarcopenia.

4. The use according to claim 1, characterized in that The preparation is used for one or more of the following: improving muscle cell survival rate, promoting muscle cell proliferation, increasing myotube size, slowing down and / or preventing myotube size decline, improving muscle quality, improving muscle endurance, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial number, regulating energy metabolism, and reducing protein degradation.

5. The method according to any one of claims 1 to 4, characterized in that R1 and R2 are both OH.

6. The use according to claim 5, characterized in that R3 is CH3.

7. The method according to any one of claims 1 to 4, characterized in that The composition further contains a compound of formula (II) and / or a compound of formula (III): wherein R5, R6 and R7 are each independently H or OH, and R8 is 8. The use according to claim 7, characterized in that R5, R6 and R7 are all OH.

9. The method according to any one of claims 1 to 4, characterized in that The preparation is a medicine or a food.

10. The use according to claim 9, characterized in that The preparation is the medicine and is provided in the form of oral administration or injection.

11. The use according to claim 9, characterized in that The preparation is the food, which is a health food, a dietary supplement, a functional food, a nutritional supplement food or a special nutritional food.

12. A combination comprising a compound of formula (I), characterized in that It is used to prevent, improve and / or treat muscle diseases or muscle disorders: in, R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is 13. The combination according to claim 12, characterized in that It is used to prevent, improve and / or treat one or more of the following: decreased number of muscle cells, muscle atrophy, decreased muscle mass, decreased muscle endurance, and decreased muscle fiber size.

14. The combination according to claim 12, characterized in that It is used for preventing, improving and / or treating sarcopenia.

15. The combination of claim 12, wherein: It is used for one or more of the following: improving muscle cell survival rate, promoting muscle cell proliferation, increasing myotube size, slowing down and / or preventing myotube size decline, improving muscle mass, improving muscle endurance, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial number, regulating energy metabolism, and reducing protein degradation.

16. A combination according to any one of claims 12 to 15, characterised in that R1 and R2 are both OH.

17. The combination according to claim 16, characterized in that R3 is CH3.

18. A combination according to any one of claims 12 to 15, characterised in that Containing a compound of formula (II) and / or a compound of formula (III): wherein R5, R6 and R7 are each independently H or OH, and R8 is 19. The combination according to claim 18, characterized in that R5, R6 and R7 are all OH.

20. A combination according to any one of claims 12 to 15, characterised in that It is a pharmaceutical composition, a food composition, or a set.

21. The combination of claim 20, wherein: It is the pharmaceutical composition and is in the form of oral administration or injection.

22. The combination of claim 20, wherein: The food composition is a health food, a dietary supplement, a functional food, a nutritional supplement or a special nutritional food.

23. A method for preventing, improving and / or treating muscle diseases or muscle disorders, characterized in that: The method comprises administering an effective amount of a composition containing a compound of formula (I) to a subject in need thereof: in, R1 and R2 are each independently H or OH; R3 is C1-4 alkyl; and R4 is 24. The method of claim 23, wherein: It is used to prevent, improve and / or treat one or more of the following: decreased number of muscle cells, muscle atrophy, decreased muscle mass, decreased muscle endurance, and decreased muscle fiber size.

25. The method of claim 23, wherein: It is used for preventing, improving and / or treating sarcopenia.

26. The method of claim 23, wherein: It is used for one or more of the following: improving muscle cell survival rate, promoting muscle cell proliferation, increasing myotube size, slowing down and / or preventing myotube size decline, improving muscle mass, improving muscle endurance, increasing muscle fiber volume, promoting protein synthesis, increasing mitochondrial number, regulating energy metabolism, and reducing protein degradation.

27. The method according to any one of claims 23 to 26, characterized in that R1 and R2 are both OH.

28. The method of claim 27, wherein: R3 is CH3.

29. The method according to any one of claims 23 to 26, characterized in that The composition further contains a compound of formula (II) and / or a compound of formula (III): wherein R5, R6 and R7 are each independently H or OH, and R8 is 30. The method of claim 29, wherein: R5, R6 and R7 are all OH.

31. The method according to any one of claims 23 to 26, characterized in that The composition is a pharmaceutical composition or a food composition.

32. The method of claim 31, wherein: The composition is the pharmaceutical composition and is administered to the subject orally or by injection.

33. The method of claim 31, wherein: The composition is the food composition, which is a health food, a dietary supplement, a functional food, a nutritional supplement food or a special nutritional food.

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