Pharmaceutical composition for preventing or alleviating sarcopenia comprising soybean leaf extract

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing, treating, or alleviating sarcopenia, which, by comprising a soybean leaf extract, prevent the loss of muscle fibers and help the recovery and regeneration of muscles, thus exhibiting excellent medicinal effects.

Description

Pharmaceutical composition for preventing or improving sarcopenia containing soybean leaf extract

[0001] The present invention relates to a pharmaceutical composition for preventing or improving sarcopenia comprising a soybean leaf extract.

[0002]

[0003] Muscle atrophy is a complex physiological process that occurs under various conditions, including aging, disease, a sedentary lifestyle, and long-term use of glucocorticoids. Sarcopenia, a natural process associated with aging, is characterized by a decrease in muscle mass and function and is associated with decreased physical function, loss of independence, increased mortality, and increased healthcare costs in the elderly. Despite its numerous clinical benefits, the steroid hormone dexamethasone (Dexa) has the side effect of inducing muscle atrophy with long-term use, which increases muscle protein breakdown and leads to muscle damage. Maintaining the structural integrity and functional integrity of muscles is essential for maintaining an individual's independence and quality of life.

[0004] Loss of muscle mass is a major cause of symptoms such as sarcopenia and frailty, and is closely related to the aging process. While the mechanisms underlying this loss of muscle mass remain unclear, various physiological changes, including mitochondrial dysfunction, insulin resistance, chronic inflammation, increased oxidative stress, neuromuscular damage, and adipose tissue infiltration, have been reported to be major contributors to sarcopenia. Furthermore, chronic diseases associated with aging accelerate this loss of muscle mass. During this process, the E3 ubiquitin ligases MuRF1 and MAFbx play a pivotal role in muscle atrophy by activating the ubiquitin-proteasome pathway, which promotes protein degradation in muscle fibers and leads to muscle wasting. Therefore, inhibiting their activity may help maintain muscle mass and slow the progression of sarcopenia.

[0005] The development of treatments for muscle atrophy has been a focus of numerous studies, with recent research particularly focusing on the therapeutic potential of natural substances. Soybean leaf extract (SL) is known to possess various biological activities, including antioxidant, anti-inflammatory, and anticancer properties. These biological activities of SL suggest a role in protecting muscle fibers and regulating muscle atrophy. However, research on the effects of SL on the molecular pathways associated with muscle atrophy remains limited.

[0006]

[0007] The purpose of the present invention is to provide a pharmaceutical composition for preventing or improving sarcopenia containing a soybean leaf extract.

[0008]

[0009] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia comprising a soybean leaf extract.

[0010] In the composition of the present invention, the soybean leaf extract may be a hot water extract.

[0011] In the composition of the present invention, the sarcopenia may be at least one selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

[0012] The present invention relates to a health functional food for preventing or improving sarcopenia containing a soybean leaf extract.

[0013] In the health functional food of the present invention, the soybean leaf extract may be a hot water extract.

[0014] In the health functional food of the present invention, the sarcopenia may be at least one selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

[0015]

[0016] The pharmaceutical composition of the present invention prevents muscle fiber loss and promotes muscle recovery and regeneration. Therefore, the pharmaceutical composition of the present invention may exhibit excellent efficacy against various muscle diseases.

[0017]

[0018] Figure 1. The mitigating effect of soybean leaf extract (SL) on dexamethasone (Dexa)-induced atrophy in C2C12 myotubes. (A) Phase-contrast microscopy at 100x magnification captured the morphology of undifferentiated C2C12 myoblasts treated with various concentrations of SL (0, 50, 100, 200, 500, and 1000 μg / ml) for 24 h. (B) Cell viability was quantitatively assessed using the CCK-8 assay. (C) Differentiated C2C12 myotubes were treated with SL in the presence or absence of 1 μM Dexa, and then Giemsa and May-Grunwald staining was used to evaluate myotube morphology and density. (D) Myotube diameters were measured, and the results were graphically presented to highlight the effect of SL on myotube size and differentiation. Statistical significance was indicated as **p<0.01 and ****p<0.0001 versus the control group. ##p<0.01 and ####p<0.0001 versus the Dexa alone group.

[0019] Figure 2. SL-mediated attenuation of muscle atrophy regulatory proteins. (A-B) Expression levels of MuRF1 and MAFbx, key ubiquitin ligases associated with muscle atrophy, were quantified by qPCR in C2C12 myotubes treated with SL in the presence of Dexa. Results were normalized to the reference gene and expressed relative to the untreated control. (C) Western blot analysis shows protein expression of MuRF1 and MAFbx after treatment with SL and Dexa. (D) Densitometric quantification of Western blot signals is displayed in bar graph format to indicate changes in protein levels after treatment. Statistical annotations are as follows: ****p<0.0001 compared to the control group (CTL); #p<0.05, ####p<0.0001 compared to Dexa alone.

[0020] Figure 3. SL attenuates Dexa-induced weight loss and muscle wasting in a mouse model. (A) Graphical representation of body weight changes in mice that experienced Dexa-induced weight loss treated with SL at concentrations of 250 and 500 μg / ml. The results demonstrate the ability of SL to attenuate the effects of Dexa on weight loss throughout the study. (B) Gastrocnemius muscle (GA) weight measured at the end of the study suggests that SL preserved muscle mass, in contrast to the Dexa monotherapy group. (C) Similarly, assessment of tibialis anterior (TA) mass demonstrates a protective effect of SL on muscle mass similar to that observed with GA. Statistical significance is indicated as **p<0.01, ****p<0.0001 versus control (CTL). #p<0.05, ##p<0.01, ####p<0.0001 versus Dexa monotherapy group.

[0021] Figure 4. SL prevents Dexa-induced muscle atrophy in mice. (A) Immunofluorescence staining of GA muscle fiber cross-sections using a fluorescence microscope. Images were taken at 100x magnification. The graph on the right shows the distribution of cross-sectional area (CSA) of GA muscle fibers. (B) Similar immunofluorescence staining of TA muscle fiber cross-sections. The graph on the right shows CSA measurements of TA muscles, demonstrating changes in muscle fiber size under various treatment conditions.

[0022] Figure 5. Enhancement of muscle strength and endurance by SL in a mouse model of atrophy. (A) Results of a grip strength test performed on mice to assess muscle function after SL treatment. The bars represent the average maximal force generated by the mice in each group. (B) Results of a treadmill endurance test to measure physical fitness in SL-treated mice. The bars represent the total time (in minutes) that the mice ran before reaching fatigue. Statistical significance is indicated as follows: **p<0.01, ****p<0.0001 vs. CTL; #p<0.05, ##p<0.01 vs. Dexa alone.

[0023] Figure 6. Inhibition of MAFbx expression by SL in skeletal muscle atrophy. (A) Representative Western blot bands for MuRF1 and MAFbx proteins from gastrocnemius muscle (with densitometric analysis on the right) are shown. Treatment with SL demonstrates a decrease in MAFbx expression levels. (B) Similar Western blot analysis for tibialis anterior muscle with densitometric quantification on the right. In both muscle types, SL treatment was associated with a decrease in the expression of atrophy-related proteins, indicating a potential protective effect against Dexa-induced muscle atrophy. Data are normalized to the loading control. Statistical significance is indicated as follows: **p<0.01 vs. CTL; #p<0.05 vs. Dexa alone group.

[0024]

[0025] The present invention is described in detail below.

[0026]

[0027] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia comprising a soybean leaf extract.

[0028] Soybean leaf extract can be obtained using various solvents and methods.

[0029] Any known extraction solvent may be used without limitation. For example, water, C1 to C4 alcohol, hexane, or a mixture thereof may be used, preferably water, but is not limited thereto.

[0030] Any known extraction method may be used without limitation. Examples of such extraction methods include hot water extraction, ultrasonic extraction, distillation extraction, or reflux heating extraction. Preferably, hot water extraction is used, but is not limited thereto.

[0031] Sarcopenia is a disease that requires management and treatment as it causes abnormal decrease in muscle mass, resulting in decreased muscle strength, which in turn leads to decreased physical function, such as difficulty walking, falling, and inability to move on one's own.

[0032] Sarcopenia can be, for example, sarcopenia, disuse atrophy, muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

[0033] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the above compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0034] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, but may be administered once or several times daily at a dosage of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. Furthermore, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0035]

[0036] In addition, the present invention relates to a health functional food for preventing or improving sarcopenia, comprising a soybean leaf extract.

[0037] Soybean leaf extract may be an example of the above.

[0038] Sarcopenia can be, for example, sarcopenia, disuse atrophy, muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

[0039] The health functional food of the present invention can be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, by further including one or more of a carrier, a diluent, an excipient, and an additive.

[0040] As additives that may be further included in the present invention, one or more ingredients selected from the group consisting of natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonating agents, and fruit pulp may be used.

[0041] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavoring agent, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0042] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0043] Specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, and it is preferable to use at least one selected from the group consisting of:

[0044] When formulating the health functional food of the present invention, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0045] The content of the extract according to the present invention as an effective ingredient in the above-described formulation can be appropriately adjusted depending on the form and purpose of use, the patient's condition, the type and severity of symptoms, etc., and may be 0.001 to 99.9 wt%, preferably 0.01 to 50 wt%, based on the solid weight, but is not limited thereto.

[0046] The dosage of the health functional food of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the daily dosage may be 0.05 to 500 mg / kg, preferably 0.1 to 300 mg / kg, based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dosage of the health functional food of the present invention is less than the above dosage, a significant effect may not be obtained, and if it exceeds it, it is not only uneconomical but also goes beyond the range of commercial dosage, so undesirable side effects may occur.

[0047]

[0048] The present invention will be described in more detail with reference to the following examples.

[0049]

[0050] Materials and Methods

[0051] Manufacturing of soybean leaf extract (SL)

[0052] Soybean leaf extract (SL) was prepared according to a standardized method. 200 g of dried soybean leaves were added to 5 L of purified water, and the mixture was heated at 95-100°C for 60 minutes for extraction. After cooling to room temperature, the extract was first filtered using a 200-mesh filter. The filtrate was centrifuged at 5,000 kHz for 10 minutes to separate the supernatant, which was then concentrated at 60°C using a vacuum rotary evaporator (EYELA, JAPAN). The concentrated extract was sterilized at 90°C for 20 minutes and then freeze-dried. Finally, the dried soybean leaf extract was ground to a 60-mesh filter and prepared for use in the experiment.

[0053]

[0054] Cell culture, myotube differentiation, and atrophy induction

[0055] C2C12 cell line, a mouse myoblast, was purchased from the American Type Culture Collection (ATCC, CRL-1772; Manassas, VA, USA). C2C12 cells were cultured in growth medium (GM) supplemented with 10% (v / v) Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.) in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were maintained at 37°C and 5% CO2 in a humidified incubator. Cells were replaced with fresh medium every two days, and passaged when 80–90% confluent for use in experiments.

[0056] When C2C12 myoblasts reached 80–90% confluency, differentiation into myotubes was initiated by replacing GM with differentiation medium (DM) containing 2% horse serum (HS; Thermo Fisher Scientific, Inc.). Cells were induced to differentiate into myotubes for 6 days, with DM replaced every 2 days.

[0057] To induce muscle atrophy, mature muscle fibers differentiated for 6 days were treated with 1 μM dexamethasone for 48 h to activate the proteolytic pathway and mimic atrophy. After treatment, cells were analyzed for morphological changes, and the expression of MuRF1 and MAFbx, markers of muscle atrophy, was assessed by Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), confirming the induction of a muscle atrophy state.

[0058]

[0059] Cell viability measurement

[0060] C2C12 cells were seeded at 5 Х 10 in 24-well plates 4 Cells were seeded at a density of 10 cells / well. After cell attachment, SL was treated at concentrations of 0, 50, 100, 200, 500, and 1000 μg / ml. After a certain period of time, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). After adding 10 μl of CCK-8 solution to each well, the cells were further incubated at 37°C for 1 h, and the absorbance of the produced formazan was measured at 450 nm using a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA, USA). The viability was expressed as a percentage by dividing the absorbance of the treatment group by the absorbance of the control group and multiplying the result by 100.

[0061]

[0062] May-Grunwald and Giemsa staining

[0063] Differentiated myotubes were washed with phosphate-buffered saline (PBS) and fixed with 100% methanol. The cells were stained with May-Grünwald's solution diluted 1:3 in phosphate buffer (1 mM NaH2PO4·H2O and 1 mM Na2HPO4, pH 6.0) for 5 minutes. After a brief rinse with distilled water, the cells were stained with Giemsa's solution diluted 1:10 in distilled water for 10 minutes. The stained cells were randomly divided into four sections and images were obtained using a camera-equipped microscope (Eclipse 80i; Nikon, Tokyo, Japan). The width of the myotubes was measured using NIS Elements software (NIS-Elements Advanced Research, Melville, NY, USA) by randomly selecting cells from each section.

[0064]

[0065] RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0066] C2C12 myotube cells were washed twice with cold PBS, and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). mRNA expression was assessed using a ViiATM7 Real-Time PCR system (Applied Biosystems Inc., Waltham, MA, USA) using a TaqMan assay. The amplification settings were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Primers and probes for MAFbx (Mm00499523_m1; Applied Biosystems Inc.), MuRF1 (Mm01185221_m1; Applied Biosystem Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Mm99999915_g1; Applied Biosystem Inc.) were used.

[0067]

[0068] Western blot

[0069] Prepared cells and mouse muscle tissues were washed with phosphate-buffered saline (PBS) and lysed using RIPA buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). After homogenization using an ultrasonicator, the samples were centrifuged at 13,000 x g for 15 min at 4°C, and the protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein were mixed with 4x laemmli sample buffer (Bio-Rad Laboratories), boiled, and subjected to electrophoresis on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked for 1 h at room temperature with Tris-buffered saline with 0.05% Tween 20 (TBST) buffer containing 5% skim milk (Difco, Detroit, MI, USA) and incubated with primary antibodies, including MuRF1 (Santa Cruz Biotechnology Inc.), MAFbx (Santa Cruz Biotechnology Inc.), and β-actin (Sigma-Adrich, St Louis, MO, USA). After overnight incubation, the membranes were washed with TBST and incubated with peroxidase-conjugated secondary antibodies. Protein bands were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories), and images were captured using a ChemiDocTM Touch Imaging System (Bio-Rad Laboratories).

[0070]

[0071] Dexamethasone-induced muscular dystrophy mouse model

[0072] This study was conducted under the approval of the Animal Experiment Ethics Committee of Gyeongsang National University (GNU-230526-M0104). Six-week-old male C57BL / 6 mice used in the experiment were purchased from Coretech Co., Ltd. (Seoul, Korea). The mice were acclimated for 1 week in an environment with a temperature of 24±2℃, a relative humidity of 40-60%, and an illumination of 150-300 lux, with a 12-h light / dark cycle. The mice were divided into four groups: a normal group (Control), a dexamethasone-treated control group (Dexa), and an experimental group treated with a combination of dexamethasone and SL (250, 500 mg / kg) (D + SL 250, D + SL 500). To induce muscle atrophy, 10 mg / kg of dexamethasone was administered intraperitoneally daily between 10 and 11 am for 3 weeks. The SL group received oral dexamethasone once daily at doses of 250 mg / kg and 500 mg / kg, starting one week before dexamethasone administration and continuing until the end of the experiment. During the same period, the normal and control groups received oral saline. Body weights were measured before, and on days 7, 14, and 21 after dexamethasone administration.

[0073]

[0074] Histological analysis

[0075] At the end of the experiment, the tibialis anterior muscle and gastrocnemius muscle were collected from the control and experimental mice after euthanasia, embedded in OCT compound (Lab-Tek; Miles Laboratories, Inc., Naperville, IL, USA), and flash-frozen. Five-μm-thick tissue slides were prepared using a cryostat (Leica CM 1950; Heidelberg, Germany). After staining overnight at 4°C with Alexa Fluor488 fluorescently labeled protein conjugated to wheat germ agglutinin (W11261; ThermoFisher Scientific Inc.), the specimens were observed and images were acquired using a fluorescence microscope (Nikon Eclipse NI DSRi2; Nikon, Tokyo, Japan). The cross-sectional area (CSA) of muscle fibers was measured using MyoVision v1.0 software.

[0076]

[0077] Endurance measurement

[0078] Endurance of the mice was assessed by a running test using a treadmill (Panlab, Barcelona, ​​Spain). All mice ran at an acclimation speed of 10 cm / sec for 3 minutes, and then increased the speed by 4 cm / sec every 4 minutes until exhaustion. The treadmill speed was controlled using software (SeDaCom v2.0.02; Panlab, Barcelona, ​​Spain). This acclimation walking speed and acceleration conditions were applied equally to all experimental groups, and a 1.1 mA electrical stimulus was applied behind each treadmill rail to encourage continuous running. The time to exhaustion (time to exhaustion) was recorded, and this was used to assess individual motor ability. The time of exhaustion was defined as the time during which the forelimbs were placed on the rails and the hindlimbs were placed on the electrical device for 3 seconds.

[0079]

[0080] Grip strength measurement

[0081] The grip strength of experimental animals was measured in grams using a Bioseb Grip Strength Test (BIO-GS3; BIOScience and Experimental Biology, Florida, USA). To measure this, a stainless steel T-bar was attached to a gauge, and the animal was asked to grasp the T-bar with both forearms. The animal's tail was then pulled at a constant speed (2 cm / s) to measure the force required to release the grip. Five trials were performed for each animal, and the average value was used to determine grip strength.

[0082]

[0083] Statistical analysis

[0084] Statistical analysis was performed using GraphPad Prism software (version 5.01; GraphPad Software, San Diego, CA, USA). Cell experiments were performed in triplicate, and results are expressed as the mean ± standard deviation. Animal model results are expressed as the mean ± standard error for each group. To analyze between-group differences, statistical significance was analyzed at the 5% level using one-way ANOVA, and the Mann-Whitney test was performed as a post-hoc test.

[0085]

[0086] Results and Discussion

[0087] Inhibitory effect of SL on muscle atrophy in C2C12 myotube cells

[0088] To investigate the inhibitory effect of SL on dexamethasone-induced muscle atrophy, C2C12 myoblasts were treated with SL at various concentrations (50-1000 μg / ml) for 24 h to determine the optimal concentration range of SL, and cell viability was measured. Significant morphological changes were observed at concentrations of 500 μg / ml and 1000 μg / ml of SL (Fig. 1A), and cell viability also decreased in a concentration-dependent manner, indicating cytotoxicity consistent with the morphological observation (Fig. 1B). Therefore, in subsequent experiments, SL was used at a maximum concentration of 500 μg / ml.

[0089] Next, to evaluate the anti-myofibrillar effect of SL in myotubes with dexamethasone-induced muscle atrophy, differentiated and mature myotubes were treated with SL together with dexamethasone, and the myotube diameters of the myotubes were measured using May-Grunwald and Giemsa staining. SL treatment was found to attenuate the muscle atrophy effect, as clearly observed in morphological observations (Fig. 1C). Furthermore, measurements of myofibrillar diameters showed that SL had a positive effect on myofibrillar size and differentiation (Fig. 1D). These results demonstrate that SL treatment inhibits dexamethasone-induced muscle atrophy, suggesting its potential as a promising natural substance for the prevention of muscle atrophy.

[0090]

[0091] Regulation of MuRF1 and MAFbx expression by SL

[0092] The ubiquitin-proteasome pathway is responsible for protein degradation and plays a crucial role in muscle mass regulation. MuRF1 and MAFbx are key components in this regulatory process. Therefore, we investigated the effect of SL on the expression of MuRF1 and MAFbx, which are factors that regulate muscle atrophy. qPCR analysis revealed that when SL was treated with various concentrations in dexamethasone-treated C2C12 myotubes, the mRNA levels of MuRF1 and MAFbx decreased in a dose-dependent manner (Fig. 2A-B). This decrease suggests that SL is effective in regulating genes related to muscle atrophy. Western blot analysis confirmed that SL treatment significantly reduced the protein levels of MuRF1 and MAFbx (Fig. 2C). Furthermore, a bar graph of the relative abundance of proteins obtained by densitometry analysis confirmed that protein levels were significantly reduced in the group treated with dexamethasone and SL together (Fig. 2D). These results suggest that SL may have a potential therapeutic role in preventing muscle atrophy by suppressing the expression of MuRF1 and MAFbx by modulating the ubiquitin-proteasome pathway.

[0093]

[0094] Muscle mass protective effect of SL in a mouse model of muscular dystrophy

[0095] A variety of medical conditions can lead to muscle atrophy, including sepsis, cachexia, starvation, metabolic acidosis, and severe insulin deficiency. These conditions are often associated with increased levels of circulating glucocorticoids, hormones that promote muscle atrophy. Unlike fixed, denervated, or disused atrophy models, the dexamethasone-induced muscle atrophy mouse model has been reported to induce changes in muscle fiber types similar to the loss of heterotypic fibers seen in human sarcopenia. This study evaluated the anti-atrophy effect of SL using a dexamethasone-induced muscle atrophy mouse model. Results showed that SL-treated groups attenuated dexamethasone-induced body weight loss, and this effect was observed at both administered concentrations (250 and 500 μg / ml) (Figure 3A). At the end of the experiment, the weight of the gastrocnemius muscle measured showed a significant increase in the SL-treated group compared to the dexamethasone-treated group (Fig. 3B). The weight of the tibialis anterior muscle also showed a muscle mass preservation effect by SL treatment, similar to the protective effect observed in the gastrocnemius muscle (Fig. 3C). These results demonstrate that SL has a significant protective effect against dexamethasone-induced weight loss and muscle atrophy.

[0096]

[0097] Muscle fiber protective effect of SL in a mouse model of muscular dystrophy

[0098] Sarcopenia typically results in a loss of muscle mass, with a decrease in the number and size of muscle fibers. To assess these morphological changes, cross-sections of muscle fibers from the gastrocnemius and tibialis anterior muscles were observed under a fluorescence microscope after immunofluorescence staining. Images taken at 100x magnification showed a significant increase in the cross-sectional area (CSA) of muscle fibers treated with SL compared to the dexamethasone-treated group (Fig. 4A). Furthermore, the size and distribution of muscle fibers in the SL-treated group were observed over a wider range than in the dexamethasone-treated group (Fig. 4B). These results suggest that SL may be effective in preventing muscle atrophy and maintaining muscle fiber size. Furthermore, these results suggest that SL may play a key role in preserving the structural integrity of muscle fibers against muscle atrophy in vivo.

[0099]

[0100] SL's protective effect on muscle function in a mouse model of muscular dystrophy

[0101] To investigate the effects of SL on muscle function, mice with dexamethasone-induced muscle atrophy were treated with SL, and muscle function was assessed. First, the grip strength test results showed that the SL-treated group significantly increased the average maximal force compared to the control group that did not receive dexamethasone, suggesting that SL can improve muscle strength (Figure 5A). In an experiment comparing the time to exhaustion (TTE) during treadmill exercise in a mouse model of dexamethasone-induced muscle atrophy, SL-treated mice showed improved endurance compared to mice treated only with dexamethasone, and the TTE was longer (Figure 5B). These results indicate that SL can positively affect the improvement of muscle function, including strength and endurance, and suggest that SL may be a promising therapeutic option for patients with muscle-related diseases, including sarcopenia.

[0102]

[0103] Regulation of MAFbx expression by SL in a mouse model of muscular dystrophy

[0104] The effect of SL on the expression of muscle atrophy-related proteins in mouse skeletal muscle was evaluated. MuRF1 and MAFbx protein levels in the gastrocnemius and tibialis anterior muscles were measured by Western blot, and the expression of MAFbx was significantly reduced in the SL-treated group compared to the dexamethasone-only group (Fig. 6A-B).

[0105] In conclusion, we demonstrated that SL protects against dexamethasone-induced muscle atrophy by suppressing MAFbx expression in both in vitro and in vivo muscle atrophy models. This suggests that SL may have a mechanism to prevent muscle fiber loss by modulating protein degradation pathways within muscle cells. Furthermore, SL was shown to have a positive effect on muscle recovery and regeneration, suggesting its potential for use not only in the treatment but also in the prevention of muscle wasting diseases.

Claims

1. A pharmaceutical composition for preventing or treating sarcopenia containing a soybean leaf extract.

2. A pharmaceutical composition for preventing or treating sarcopenia according to claim 1, wherein the soybean leaf extract is a hot water extract.

3. A pharmaceutical composition for preventing or treating sarcopenia according to claim 1, wherein the sarcopenia is at least one selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

4. Health functional food containing soybean leaf extract for preventing or improving sarcopenia.

5. In claim 4, the soybean leaf extract is a health functional food for preventing or improving sarcopenia, which is a hot water extract.

6. A health functional food for preventing or improving sarcopenia according to claim 4, wherein the sarcopenia is at least one selected from the group consisting of sarcopenia, disuse atrophy, muscle atrophy due to the absence of mechanical stimulation, denervational atrophy, drug-induced atrophy, malnutritional atrophy, and muscular dystrophy.

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