Use of calycosin in the preparation of a drug for preventing and treating muscle atrophy and / or promoting muscle growth

Abstract

The application discloses a new application of calycosin in preparation of medicines for preventing and treating muscle atrophy and / or promoting muscle growth, and belongs to the technical field of biological medicines. The application finds for the first time that calycosin can significantly promote degradation of myostatin protein in skeletal muscle, activate a protein synthesis pathway, and simultaneously down-regulate expression of atrophy-related genes, thereby playing a double role of reducing muscle atrophy and promoting muscle growth. In vivo experiments verify that calycosin can effectively restore muscle strength and weight, increase gastrocnemius muscle weight, and increase muscle fiber cross-sectional area through various animal models such as dexamethasone induction, high-fat diet-induced obesity, and pregnancy induction. The medicine can be prepared into oral or non-gastrointestinal administration preparations, is safe and rich in source, is suitable for prevention and treatment of various types of muscle atrophy, and can be used for maternal supplementation to promote muscle growth of offspring, and has a wide clinical conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of Calycosin in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth. Background Technology

[0002] Skeletal muscle is one of the largest organs in the human body, accounting for about 40% of total body weight, and plays a crucial role in maintaining bodily movement, metabolic balance, and energy homeostasis. Skeletal muscle atrophy refers to a pathological state in which the rate of muscle protein degradation exceeds the rate of synthesis, resulting in the progressive loss of muscle mass, strength, and function.

[0003] Muscle atrophy can be induced by a variety of factors, including aging (sarcopenia), prolonged bed rest or limb immobilization (disuse atrophy), malignant tumors (cachexia), chronic heart failure, diabetes, obesity, and long-term use of glucocorticoids. Muscle atrophy not only seriously affects patients' quality of life and independence, but also significantly increases the rates of falls, fractures, hospitalizations, and even all-cause mortality, making it a major public health problem that urgently needs to be addressed.

[0004] Currently, clinical interventions for muscle atrophy are very limited and significantly inadequate. Non-pharmacological treatments, such as resistance training, have some effect, but are difficult to implement for many patients. Regarding pharmacological treatment, while direct supplementation with anabolic hormones (such as testosterone and growth hormone) can increase muscle mass to some extent, long-term use can lead to serious side effects such as cardiovascular disease and prostate cancer. Therefore, developing safe, effective, and versatile novel anti-muscle atrophy drugs has significant clinical importance and broad market prospects.

[0005] Myostatin (MSTN, also known as GDF-8) is a member of the transforming growth factor-β (TGF-β) superfamily and a key negative regulator of skeletal muscle mass. MSTN knockout mice exhibit a significant increase in overall skeletal muscle mass, with individual muscle volumes approximately twice that of normal mice. This finding reveals the immense potential of MSTN as a target for muscle growth inhibition. MSTN primarily binds to its receptor ActRIIB, activating the downstream Smad2 / 3 signaling pathway, thereby inhibiting Akt / mTOR-mediated protein synthesis and upregulating the expression of atrophy-related E3 ubiquitin ligases Atrogin-1 and MuRF-1 in the ubiquitin-proteasome system, leading to skeletal muscle atrophy. Therefore, developing drugs that can inhibit MSTN activity and / or promote MSTN degradation is an important strategy for treating muscle atrophy.

[0006] Calycosin is a natural isoflavone compound mainly derived from the rhizomes of the legumes *Astragalus membranaceus* and *Hedysarum polybotrys*. Modern pharmacological studies have shown that calycosin possesses various biological activities, including antioxidant, anti-inflammatory, antitumor, estrogen-like effects, and cardiovascular protection. In recent years, some studies have reported that calycosin can improve osteoblast differentiation and reduce neuronal damage through the PI3K / Akt pathway. However, there are currently no literature reports or patent publications regarding the role of calycosin in skeletal muscle atrophy and muscle growth, or its relationship with MSTN protein. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide the use of verbascoside in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth. This aims to address the shortcomings of existing clinical interventions for muscle atrophy, which are very limited and have significant deficiencies.

[0008] To achieve the above objectives, the present invention provides the following technical solutions:

[0009] Through long-term research, the inventors have discovered that verbascoside can significantly promote the degradation of MSTN protein in skeletal muscle, activate protein synthesis, and downregulate the expression of key muscle atrophy markers Atrogin-1 and MuRF-1, ultimately achieving the dual effects of reducing muscle atrophy and promoting muscle growth. Based on this, the present invention proposes the application of verbascoside in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth.

[0010] Furthermore, pharmaceutically acceptable salts of verbascoside are used in the preparation of drugs for preventing and / or promoting muscle growth.

[0011] In the technical solution of the present invention, the verrucous isoflavone has the chemical structural formula as shown in Formula I:

[0012] ;

[0013] Formula I.

[0014] Preferably, the muscle atrophy is one or more of the following: glucocorticoid-induced muscle atrophy, obesity-related muscle atrophy, pregnancy-induced muscle atrophy, age-related muscle atrophy, disuse muscle atrophy, cachexia-related muscle atrophy, and denervation-related muscle atrophy.

[0015] Preferably, the muscle atrophy is manifested as a decrease in skeletal muscle mass, a decrease in the cross-sectional area of ​​muscle fibers, a decrease in muscle strength, and / or a decrease in exercise endurance.

[0016] Preferably, the promotion of muscle growth manifests as increased skeletal muscle mass, increased cross-sectional area of ​​muscle fibers, conversion of muscle fiber type to oxidized muscle fibers, and / or enhanced muscle strength.

[0017] In the application of this invention, verbascoside exerts its effect by promoting the degradation of myostatin (MSTN), and isoflavonoids are used in the preparation of agents that promote the degradation of MSTN. Specifically, verbascosides promote the degradation of MSTN protein in skeletal muscle, reduce MSTN protein expression levels, and activate muscle protein synthesis pathways; simultaneously, it inhibits the expression of Atrogin-1 and MuRF-1 in the ubiquitin-proteasome system, reducing skeletal muscle protein degradation.

[0018] This invention provides the first discovery of the effect of verbascoside on promoting offspring muscle growth through maternal supplementation; more preferably, the verbascoside is used in the preparation of a drug that promotes offspring muscle growth through maternal supplementation.

[0019] In another embodiment, this application discloses the use of verbascoflavonoids or pharmaceutically acceptable salts thereof in the preparation of medicaments for preventing and / or promoting muscle growth, primarily manifested in the ability of said medicaments to restore muscle strength and reduce weight loss, increase gastrocnemius muscle weight, and increase muscle fiber area.

[0020] When the vernixone or its pharmaceutically acceptable salt is used as a medicine, it is applied either directly or as a pharmaceutical composition; the pharmaceutical composition further comprises a pharmaceutically acceptable, non-toxic, and inert pharmaceutically acceptable carrier and / or excipient, or is used in combination with other therapeutic agents. More preferably, the vernixone or its pharmaceutically acceptable salt constitutes 0.1%-99% of the pharmaceutical composition.

[0021] The pharmaceutical composition of this invention is in the form of an oral or non-gastrointestinal dosage form; the oral dosage form is one of capsules, tablets, powders, granules, or syrups; the non-gastrointestinal dosage form is one of injections, ointments, or suppositories. More preferably, the pharmaceutical composition further comprises an excipient, which is at least one of a binder, excipient, stabilizer, disintegrant, flavoring agent, or lubricant. The pharmaceutical composition is in the form of a controlled-release dosage form, a sustained-release dosage form, or various particulate delivery systems.

[0022] This invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of vernix isoflavone or a pharmaceutically acceptable salt thereof to a subject requiring treatment. The subject includes humans and non-human mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, with human patients being preferred.

[0023] Compared with the prior art, the present invention has the following outstanding advantages:

[0024] 1. This invention reveals for the first time a novel mechanism by which isoflavones inhibit muscle atrophy and promote muscle growth by promoting the degradation of MSTN protein, providing a new intervention strategy for the treatment of various types of muscle atrophy and filling a technological gap in this field.

[0025] 2. This invention systematically verifies the positive regulatory effect of isoflavones on skeletal muscle mass from two dimensions: “reversing pathological atrophy” and “promoting physiological growth”, through a dexamethasone-induced muscle atrophy model and a model in which pregnant and lactating female mice were supplemented with isoflavones to promote muscle growth in pups. It has a wide range of indications.

[0026] 3. The verbascosides used in this invention are natural isoflavone compounds extracted from the traditional Chinese medicines Astragalus membranaceus and Astragalus rubrum. They are characterized by high safety, no obvious toxic side effects, and good patient compliance with long-term use. Furthermore, verbascosides are abundant (Astragalus membranaceus is a commonly used Qi-tonifying Chinese medicine with sufficient resources), the extraction and purification process is mature, the production cost is controllable, and they have good potential for clinical translation. Attached Figure Description

[0027] Figure 1 This is an example of the effect of isoflavones on the proliferation and differentiation of C2C12 cells in Example 1 of the present invention; wherein, Figure A is a line graph of cell viability; Figures BC are cell scratch and scratch area statistics; Figure D is a methylene blue staining image; Figures EF are photographs of cells at different stages of differentiation and myotube length statistics; and Figure G is a Giemsa staining image.

[0028] Figure 2 This invention illustrates the effect of isoflavones from *Variegata* on a dexamethasone-induced muscle atrophy model in C2C12 cells, as described in Example 2. Figure A shows immunofluorescence images of Myh4 and Myh7 cells; Figure B shows mitochondrial DNA quantification; Figure C shows mitochondrial fluorescence quantification; Figure D shows fluorescence images of reactive oxygen species (ROS) and mitochondrial ROS; Figure E shows mitochondrial ROS fluorescence quantification; Figure F shows MDA content; Figure G shows GSH content; Figure H shows cellular glycogen content; Figure I shows cellular glucose uptake; Figure J shows cellular lactate content; and Figure K shows cellular lactate dehydrogenase content.

[0029] Figure 3 The figures shown in Example 3 of this invention illustrate the therapeutic effects of isoflavones on dexamethasone-induced muscle atrophy in mice, including changes in body weight, fat mass, and grip strength. Figure A is a flowchart of the mouse rearing process; Figure B is a typical mouse image; Figure C shows the changes in mouse body weight during the experiment; Figures D and E show the mouse fat percentage and lean meat percentage, respectively; Figure F is a MicroCT image; and Figures G and H show the grip strength of the mouse's forelimbs and limbs, respectively.

[0030] Figure 4 This invention illustrates the effects of isoflavones from *Verbena* on dexamethasone-induced muscle atrophy in mice, as described in Example 3. The effects include muscle weight, gastrocnemius muscle sections, oxidative stress, lactate levels, and lactate dehydrogenase levels. Figure A shows photographs of various mouse muscles; Figure B shows the weight and relative weight of the soleus, gastrocnemius, tibialis anterior, and extensor digitorum longus muscles, respectively; Figure J shows HE staining and fast and slow-twitch fluorescence staining of the gastrocnemius muscle; Figure K shows the ROS content of the gastrocnemius muscle; Figure L shows the MDA content of the gastrocnemius muscle; and Figures M and N show the lactate and lactate dehydrogenase levels in the gastrocnemius muscle.

[0031] Figure 5 The following figures illustrate the effects of supplementing lactating mother mice with isoflavones on the weight gain and skeletal muscle development of pups in Example 4 of this invention, including pup weight change curves, fat mass, lean meat mass, and gastrocnemius muscle weight. Figures A and B are weekly weight bar charts for mice; Figures C and D show the fat percentage and lean meat percentage of mice; Figures E and F show the grip strength of the forelimbs and limbs of mice; Figure G shows photographs of various mouse muscles; Figures HK show the weights of the soleus, gastrocnemius, tibialis anterior, and extensor digitorum longus muscles of mice, respectively; and Figure LO shows the levels of MDA, GSH, lactate, and lactate dehydrogenase in the gastrocnemius muscle of mice.

[0032] Figure 6 This document presents the interaction analysis of verbascoside and MSTN protein in Example 5 of the present invention. Figures AD show the molecular docking and interaction analysis between verbascoside and MSTN protein; Figure E shows the thermal displacement of cells after treatment with verbascoside; Figure F shows the BLI analysis of verbascoside and MSTN; and Figure G shows the Western blot and grayscale quantification of MSTN in cells. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The described embodiments are only some, not all, of the embodiments of this invention. The following pharmacodynamic verification is based solely on dexamethasone-induced and maternal supplementation models, but is not limited thereto. Unless otherwise specified, conventional methods are used in the following embodiments. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0034] Example 1: Effects of verbascoside on the proliferation and differentiation of C2C12 cells.

[0035] 1.1 Cell Culture and Differentiation

[0036] C2C12 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. When the cells reached 80%–90% confluence, they were digested with trypsin and passaged for experiments. When the cells reached 80%–90% confluence, the medium was replaced with DMEM containing 2% horse serum for differentiation, and differentiation was induced for 6 days to form multinucleated myotubes. After differentiation, the cells were divided into a blank group (containing the same amount of DMSO as the treatment group, Con), a model group (containing 25 μM dexamethasone, Dex), and a verbascoside treatment group (containing 25 μM dexamethasone and 25 μM verbascoside, Cal). After 12 h of treatment, the cells were harvested.

[0037] 1.2. Cell viability assay

[0038] Cells were cultured in 96-well plates for 24 h, and then different concentrations of verbenafil (0, 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 200 μM, 250 μM, 300 μM) were added and cultured for another 24 h. The viability of C2C12 cells at different concentrations of verbenafil was then assessed using a CCK-8 assay kit. Absorbance values ​​were recorded at 490 nm using a microplate reader and calculated. Results are as follows: Figure 1 As shown in Figure A, treatment with isoflavone at concentrations below 50 μM did not significantly affect cell viability, and the IC50 value of isoflavone for C2C12 cells was 145.6 μM.

[0039] 1.3 Cell Scratch Assay

[0040] Cells were cultured in 24-well plates for 24 hours until they reached confluence. Then, the cells were streaked using a pipette tip. The culture medium was then replaced with standard medium containing 50 μM verbascoside, with an equal volume of DMSO as a control. Cells were observed and photographed under a microscope at 4, 8, 12, and 24 hours, and the streak area was analyzed using ImageJ. Results are as follows: Figure 1 As shown in Figure BC, the scratch healing rate of the 50 μmol / L verbascoside treatment group was not significantly different from that of the control group at any time point. This result indicates that 50 μmol / L verbascoside has no significant effect on the migration and proliferation of C2C12 myoblasts.

[0041] 1.4 Methylene blue staining

[0042] Cells were cultured in 96-well plates for 24 hours, then the medium was replaced with regular medium containing 50 μM verbascoside, with an equal volume of DMSO as a control. After 24 hours of culture, cells were stained with methylene blue and photographed. Results are as follows: Figure 1As shown in D, this result is consistent with the CCK-8 and scratch assays, further confirming that 50 μmol / L verbascoside has no significant effect on the proliferation of C2C12 cells.

[0043] 1.5 Analysis of Myotube Length

[0044] When cells reached 80%–90% confluence, the medium was replaced with differentiation medium containing different concentrations of verbascoside, with an equal volume of DMSO as a control. Images were taken every two days, and myotube diameter was measured using ImageJ software. Furthermore, after differentiation, cells were fixed with paraformaldehyde and then stained with Giemsa stain at room temperature for 30 minutes. The cells were gently washed with deionized water until the background was clear. Observation and photography were performed under a microscope. The results are shown in Figure EG; treatment with 25 μM and 50 μM verbascoside significantly promoted cell differentiation and increased myotube length.

[0045] Example 2: Therapeutic effect of verbascoside on a dexamethasone-induced C2C12 cell muscle atrophy model

[0046] 2.1 Immunofluorescence,

[0047] Cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. Primary antibodies against Myh4 (a marker for fast-twitch muscle fibers) and Myh7 (a marker for slow-twitch muscle fibers) were added, and the cells were incubated overnight at 4°C. The next day, secondary antibodies labeled with Alexa Fluor 488 / 594 (1:500) were added, and the cells were incubated at room temperature in the dark for 1 hour. Cells were then observed and photographed under a fluorescence microscope.

[0048] The results are as follows Figure 2 As shown in Figure A, compared with the control group, the positive staining areas of slow-twitch markers Myh7 and fast-twitch markers Myh4 in the C2C12 myotubes of the dexamethasone (DEX) treatment group were significantly reduced, and the number of myotube fusions was decreased, indicating that dexamethasone induced overall myotube atrophy and a decrease in myosin heavy chain expression. However, after treatment with verbascoside (50 μmol / L), the positive areas of Myh7 and Myh4 were significantly restored, indicating that verbascoside reversed the dexamethasone-induced myotube atrophy.

[0049] 2.2 Mitochondrial DNA Quantification and Staining

[0050] After culturing the cells as described in 1.1, they were washed with PBS and then an appropriate amount of mitochondrial DNA extraction solution was added. The cells were then incubated at 37°C for lysis. Subsequently, the lysis solution was inactivated at 80°C, and the supernatant was collected by centrifugation. The mitochondrial DNA content was then determined by fluorescence quantitative PCR using specific primers.

[0051] After culturing cells as described in section 1.1, they were stained with serum-free DMEM containing 100 nM MitoTracker Green at 37°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS, leaving an equal amount of PBS in each well. The fluorescence intensity was measured using a microplate reader. Subsequently, the cells were lysed, and the supernatant was used to determine the total protein content using the BCA method. Finally, the mitochondrial fluorescence intensity was expressed as "fluorescence intensity / total protein".

[0052] The results are as follows Figure 2 As shown in BC, dexamethasone-induced mitochondrial DNA and mitochondrial fluorescence intensity decreased in cells, while treatment with verbascoside restored mitochondrial DNA content and fluorescence intensity, indicating that verbascoside can inhibit the reduction in mitochondrial numbers caused by dexamethasone.

[0053] 2.3 Detection of Oxidative Stress Indicators

[0054] After culturing cells as described in section 1.1, intracellular and mitochondrial reactive oxygen species (ROS) were detected using DCFH-DA and MitoSOX staining. Cells were observed and photographed under a fluorescence microscope, and fluorescence intensity was measured using a microplate reader. Cells were then lysed, and the supernatant was used to determine total protein content using the BCA method. Results are expressed as "fluorescence intensity / total protein".

[0055] After collecting cells from each group, the cells were disrupted by ultrasound, and the intracellular MDA and GSH contents were measured according to the lipid peroxidation detection kit and the glutathione detection kit, and the results were calibrated using protein content.

[0056] The results are as follows Figure 2 As shown in the DG, the levels of reactive oxygen species (ROS), MitoSOX fluorescence intensity, and MDA content significantly increased in the dexamethasone-treated group, while GSH content significantly decreased. However, these indicators returned to control levels after treatment with verbascoside. These results indicate that verbascoside can enhance intracellular antioxidant capacity and effectively scavenge dexamethasone-induced cellular oxidative stress.

[0057] 2.4 Glycogen Detection

[0058] After culturing the cells as described in section 1.1, the cells were lysed using glycogen extraction buffer and then sonicated. After thorough lysis, the supernatant was collected by centrifugation and the glycogen content in the cells was determined using a glycogen assay kit. The glycogen content was calculated based on the absorbance values ​​and calibrated using protein concentration.

[0059] The results are as follows Figure 2 As shown in H, the glycogen content in the dexamethasone group decreased, while verbascoside was able to restore intracellular glycogen content, indicating that verbascoside can improve the reduction in cellular glycogen storage caused by dexamethasone and restore energy reserves.

[0060] 2.5 Sugar intake detection

[0061] After culturing the cells as described in section 1.1, the cells were washed with PBS and then stained with a glucose-free culture medium containing 10 μM 2-NBDG at 37°C in the dark for 30 minutes. After incubation, the cells were washed with PBS, leaving an equal amount of PBS in each well. The fluorescence intensity was measured using a microplate reader. Subsequently, the cells were lysed, and the supernatant was used to determine the total protein content using the BCA method for calibration.

[0062] The results are as follows Figure 2 As shown in Figure I, dexamethasone treatment led to a decrease in cellular glucose uptake, while verbascoside was able to restore it to control levels, revealing that verbascoside can improve dexamethasone-induced insulin resistance and glucose uptake impairment.

[0063] 2.6 Lactate and lactate dehydrogenase assay

[0064] After the cells were cultured as described in 1.1, the intracellular lactate and lactate dehydrogenase levels were measured according to the instructions of the Nanjing Jiancheng lactate test kit and lactate dehydrogenase assay kit.

[0065] The results are as follows Figure 2 As shown in JK, dexamethasone treatment led to an increase in lactate and lactate dehydrogenase levels, while verbascoside treatment reduced these levels to control levels. This indicates that dexamethasone induces increased glycolysis and severely damages muscle cells, while verbascoside can protect muscle cells, reduce cytotoxicity, and restore dexamethasone-induced abnormal glycolysis.

[0066] The above results indicate that verbascoside can reverse dexamethasone-induced muscle atrophy, reduce oxidative stress levels, restore antioxidant capacity, and improve cellular energy metabolism. This provides evidence for the efficacy of verbascoside in combating dexamethasone-induced muscle atrophy.

[0067] Example 3: Therapeutic effect of verbascoside on a mouse model of dexamethasone-induced muscular atrophy

[0068] 3.1 Establishment and grouping of animal models

[0069] Six-week-old male C57BL / 6J mice (weighing 20-22g) were acclimatized for two weeks in an environment with a temperature of 24±2℃, relative humidity of 70%-80%, and a light / dark cycle of 12h / 12h. They were then randomly divided into three groups of eight mice each. The normal control group (Con) and the dexamethasone model group (Dex) were fed a standard diet, while the verbascoside treatment group (Cal) was fed a standard diet supplemented with 0.3g / kg verbascoside. After six weeks of feeding, the Dex and Cal groups were injected with dexamethasone solution (dexamethasone dissolved in DMSO, prepared according to the ratio of 10% DMSO + 40% PEG 300 + 5% Tween-80 + 45% Saline, administered intraperitoneally at a dose of 25mg / kg / day for the first week and 5mg / kg / day for the following two weeks). The control group received an equal volume of the DMSO-containing solution. Two weeks before the experiment, body weight, fat percentage, and lean meat percentage were measured every two weeks. After the injection of dexamethasone solution, body weight, fat percentage, and lean meat percentage were measured weekly.

[0070] The results are as follows Figure 3 As shown in the AE, during the first 6 weeks of feeding, mice treated with verbascoside showed a slight increase in body weight, with no significant difference in fat percentage and lean meat percentage. This indicates that verbascoside supplementation alone has no significant adverse effect on normal growth. After the start of dexamethasone injection, the body weight of mice in the Dex and Cal groups decreased slightly, and the fat percentage of mice in the Dex group increased significantly while the lean meat percentage decreased significantly. Although the body weight of mice in the Cal group decreased slightly, the fat percentage and lean meat percentage did not change significantly compared to the Con group. This indicates that verbascoside can significantly alleviate the increase in fat percentage and decrease in lean meat percentage caused by dexamethasone injection, antagonizing the dexamethasone-induced changes in body composition.

[0071] 3.2 Mouse Micro CT

[0072] Mice were anesthetized by intraperitoneal injection of tribromoethanol (2.4%) at a dose of 10 μL / g body weight after fasting for 6 hours. The mice were then placed supine in a detection chamber for scanning. 3D reconstruction of the scan results was performed using NRECON software, and the results were analyzed using DATAVIEWER, CTVOX, and CTAN software.

[0073] The results are as follows Figure 3 As shown in Figure F, the hind limb muscles of mice in the Con group were full and had a large cross-sectional area. The cross-sectional area of ​​the leg muscles of mice in the Dex group was smaller than that in the Con group, suggesting that dexamethasone induced significant muscle atrophy. The cross-sectional area of ​​the muscles in mice in the Cal group recovered significantly compared with the Dex group, approaching the level of the Con group.

[0074] 3.3 Grasp Force Experiment

[0075] The grip strength test was conducted using a mouse grip strength meter. The mouse's forelimbs or limbs were placed on the metal grid of the grip strength meter, and the mouse's tail was gently pulled back. The reading was recorded when the mouse's grip strength reached its peak. Each mouse was measured three times, and the average value was taken as the final grip strength value (unit: N).

[0076] The results are as follows Figure 3 As shown in GH, there were no significant differences in grip strength among the three groups of mice in the initial stage. After the onset of dexamethasone injection, the grip strength in the Dex group was significantly lower than that in the Con group, but after supplementation with verbascoside isoflavone, the grip strength returned to the control level. This indicates that verbascoside isoflavone can effectively alleviate dexamethasone-induced muscle weakness.

[0077] 3.4 Skeletal muscle weight measurement and histochemical analysis

[0078] Gastrocnemius muscle samples were harvested from mice, and surrounding connective tissue and fat were removed. The wet weight was accurately measured using an electronic analytical balance, and the ratio of wet weight of the gastrocnemius muscle to the mouse's body weight was calculated. After collection, the gastrocnemius muscle samples were fixed in 4% paraformaldehyde for 24 hours and then sent to the company for hematoxylin and eosin (HE) section staining. Additionally, a portion of the gastrocnemius muscle was taken for immunofluorescence staining. The muscle fiber morphology was observed and photographed under a microscope.

[0079] The results are as follows Figure 4 As shown in AJ, the gastrocnemius muscle weight of mice in the Dex group was significantly lower than that in the Con group, with a decrease in muscle fiber area, a decrease in the proportion of fast-twitch fibers, and an increase in the proportion of slow-twitch fibers. After treatment with vernix isoflavones, the gastrocnemius muscle weight of mice significantly recovered, the proportion of fast-twitch fibers increased, and the proportion of slow-twitch fibers decreased. This indicates that vernix isoflavones can effectively antagonize dexamethasone-induced muscle atrophy and restore normal muscle fiber composition.

[0080] 3.5 Determination of skeletal muscle oxidative stress markers, lactate, and lactate dehydrogenase

[0081] A certain amount of gastrocnemius muscle tissue was taken, homogenized in pre-cooled PBS, sonicated, and centrifuged to collect the supernatant for analysis. The levels of reactive oxygen species, MDA, lactate, and lactate dehydrogenase in the skeletal muscle were measured according to the manufacturer's instructions. Results are as follows: Figure 4 As shown in the KN diagram, the levels of reactive oxygen species, MDA, lactate, and lactate dehydrogenase were significantly increased in the Dex group compared to the Con group. Treatment with verbascoside significantly reduced these increases, restoring them to control levels. These results indicate that verbascoside can inhibit dexamethasone-induced muscle oxidative stress and metabolic disorders.

[0082] Example 4: Effects of maternal supplementation with verbascoside on fetal rats

[0083] 4.1 Establishment and grouping of animal models

[0084] Eight-week-old female C57BL / 6J mice were mated with males at a 2:1 ratio. The day the vaginal plug was found was day 0.5 of gestation. Pregnant mice were randomly divided into two groups of six mice each: control group: fed a normal diet with verbascoside during gestation and lactation; treatment group: fed a normal diet supplemented with 0.3 g / kg verbascoside during gestation and lactation. After gestation, the pups were raised with their mothers and allowed to nurse freely. The pups were weighed on days 7, 14, 21, and 28 postpartum (six pups per litter, half male and half female). Starting from week 5, the fat percentage and lean meat percentage of the pups were measured weekly by CT scan. The grip strength of the mice was measured in weeks 6 and 7. At week 9, the pups were sacrificed, and the gastrocnemius muscle was harvested, weighed, and MDA, GSH, lactate, and lactate dehydrogenase were measured according to the method described in section 3.5.

[0085] The results are as follows Figure 5 As shown, compared with the control group, pups in the cucurbitacin group showed a significant increase in body weight from the first week to the end of the eighth week postpartum, but the fat percentage and lean meat percentage did not change significantly. These results indicate that cucurbitacin supplementation during maternal pregnancy and lactation significantly promotes postnatal weight gain in pups, and the effect persists until weaning. The proportions of body components remained unchanged, suggesting that the weight gain mainly comes from the increase in lean tissue (muscle) rather than fat accumulation. Furthermore, the forelimb and quadripleg grip strength of female pups in the cucurbitacin group was significantly improved. Male pups showed a significant improvement in forelimb grip strength, and a slight but not significant improvement in quadripleg grip strength. These results indicate that maternal supplementation with cucurbitacin has a beneficial effect on offspring muscle strength, with a more significant effect in female offspring, which may be related to sex-related hormonal differences. After sampling, the gastrocnemius, tibialis anterior, and extensor digitorum longus muscles of female pups significantly increased, while the extensor digitorum longus muscle of male pups significantly increased, and the gastrocnemius and tibialis anterior muscles showed slight but not significant increases. These results indicate that verbascoside can promote muscle growth in mice. Furthermore, there were no significant changes in MDA, GSH, lactate, and lactate dehydrogenase levels in the pups, suggesting that maternal supplementation with verbascoside did not adversely affect the redox balance and glycolytic metabolism of skeletal muscle in the pups, further supporting its safety.

[0086] Example 5: Verification of the interaction between verbascoside and MSTN

[0087] 5.1 Molecular docking of verbascoside and MSTN

[0088] The crystal structure of the MSTN protein (PDB ID: 3HH2) was downloaded from the PDB database. The protein underwent pretreatment using Discovery Studio software, including dehydration, hydrogenation, and repair of missing amino acid side chains. Semi-flexible molecular docking was performed using CDOCKER. Based on CDOCKER energy and interacting residue analysis, the optimal binding mode was screened, and the interacting residues were analyzed.

[0089] The results are as follows Figure 6 As shown in AD, verbascoside has a high affinity for MSTN. Verascoside forms hydrogen bonds with Gln53 and Trp31 of the MSTN protein, and hydrophobic interactions and van der Waals forces with Trp29, Lys54, and Val50.

[0090] 5.2 Cell thermal displacement analysis (CETSA) to verify the binding of verbascoside isoflavones to endogenous MSTN in cells.

[0091] C2C12 cells were collected by adding RIPA lysis buffer containing the protease inhibitor. The cells were divided into three equal groups: one group was treated with 50 μM isoflavone, and the other two groups were treated with equal amounts of DMSO. After incubation at 37°C for 2 hours, the mixture was aliquoted into PCR tubes and heated at 37°C, 42°C, 47°C, 52°C, 57°C, 62°C, 67°C, and 72°C for 5 minutes each, followed by cooling at 4°C for 3 minutes. The cells were then centrifuged again to remove the denatured protein precipitate. The supernatant was analyzed by SDS-PAGE and Western blot using anti-MSTN antibody (1:1000) and anti-GAPDH antibody (1:5000).

[0092] The results are as follows Figure 6 As shown in Figure E, the thermal stability of MSTN protein was significantly improved in cells treated with verbascoside, and its denaturation temperature curve shifted to the right. This result indicates that verbascoside directly binds to endogenous MSTN protein, stabilizing its three-dimensional conformation and making it more resistant to thermal denaturation.

[0093] 5.3 Biomembrane interferometry (BLI) was used to determine the binding energy between verbascoside and MSTN.

[0094] MSTN protein was biotinylated and labeled. Using an SA-XT sensor with PBST as a baseline, the biotinylated MSTN protein was then immobilized on the sensor. The sensor was subsequently immersed in solutions containing different concentrations of verbascoside to detect binding energies, followed by dissociation in PBST. A protein-free sensor was used as a reference to calculate the KD.

[0095] The results are as follows Figure 6 As shown in Figure F, the BLI experiment confirmed the concentration-dependent binding and dissociation behavior between verbascoside and MSTN. The KD value calculated based on global fitting was 3.01 μM.

[0096] 5.4 Effects of verbascoside on MSTN protein expression

[0097] After culturing cells as described in section 1.1, total protein was extracted using RIPA lysis buffer, and protein concentration was determined by the BCA method. An equal volume of protein (20 μg) was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk at room temperature for 1 hour. Anti-MSTN antibody (1:1000) and anti-β-actin antibody (1:5000) were added, and the membrane was incubated overnight at 4°C. The next day, after washing three times with TBST, secondary antibody was added and the membrane was incubated at room temperature for 1 hour. Subsequently, after washing three times with TBST, the membrane was developed using ECL chemiluminescence immunoassay, and the grayscale values ​​of the protein bands were analyzed using ImageJ software.

[0098] The results are as follows Figure 6 As shown in G, compared with the Con group, the MSTN protein expression level in the Dex group was significantly increased, while the MSTN protein expression level in C2C12 cells treated with verbenafil was significantly decreased. These results indicate that verbenafil can effectively reduce the MSTN protein expression level in C2C12 cells.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of verbascoside in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth.

2. Pharmaceutically acceptable salts of verbascoside are used in the preparation of drugs for the prevention and / or promotion of muscle atrophy.

3. The use of the verbascoflavonoids according to claim 1 in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The verbascoflavonoid has the chemical structural formula as shown in Formula I: ； Formula I.

4. The use of the verbascoflavonoids according to claim 1 in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The muscle atrophy is one or more of the following: glucocorticoid-induced muscle atrophy, obesity-related muscle atrophy, pregnancy-induced muscle atrophy, age-related muscle atrophy, disuse muscle atrophy, cachexia-related muscle atrophy, and denervation-related muscle atrophy.

5. The use of the verbascoflavonoids according to claim 1 in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The muscle atrophy is characterized by a decrease in skeletal muscle mass, a decrease in the cross-sectional area of ​​muscle fibers, a decrease in muscle strength, and / or a decrease in exercise endurance.

6. The use of the verbascoflavonoids according to claim 1 in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The promotion of muscle growth is manifested in increased skeletal muscle mass, increased cross-sectional area of ​​muscle fibers, conversion of muscle fiber type to oxidative muscle fibers, and / or enhanced muscle strength.

7. Application of verbascoside in the preparation of degrading agents that promote myostatin.

8. The application of verbascoside in the preparation of drugs that promote muscle growth in offspring through maternal supplementation.

9. The use of the verbascoflavonoids according to claim 1 in the preparation of drugs for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The isoflavone is used either directly or in the form of a pharmaceutical composition; the pharmaceutical composition further comprises a pharmaceutically acceptable, non-toxic and inert pharmaceutically acceptable carrier and / or excipient, or used in combination with other therapeutic agents.

10. The use of the verbascoflavonoids according to claim 9 in the preparation of a drug for preventing and treating muscle atrophy and / or promoting muscle growth, characterized in that: The pharmaceutical composition is administered in an oral or non-gastrointestinal dosage form; the oral dosage form is one of capsules, tablets, powders, granules, or syrups; the non-gastrointestinal dosage form is one of injections, ointments, or suppositories. The pharmaceutical composition further comprises an excipient, which is at least one of a binder, excipient, stabilizer, disintegrant, flavoring agent, or lubricant.

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