Pharmaceutical composition for preventing or alleviating sarcopenia, comprising acetylgenistin

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia, the composition comprising acetylgenistin or a pharmaceutically acceptable salt thereof, and thus exhibiting excellent medicinal effects.

Description

Pharmaceutical composition for preventing or improving sarcopenia containing acetylgenistin

[0001] The present invention relates to a pharmaceutical composition for preventing or improving sarcopenia, comprising acetylgenistin.

[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]

[0006] The purpose of the present invention is to provide a pharmaceutical composition for preventing or improving sarcopenia containing acetylgenistin.

[0007]

[0008] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia, comprising acetylgenistin or a pharmaceutically acceptable salt thereof.

[0009] 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.

[0010] The present invention relates to a health functional food for preventing or improving sarcopenia, comprising acetylgenistin or a pharmaceutically acceptable salt thereof.

[0011] 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.

[0012]

[0013] 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.

[0014]

[0015] Figure 1. Acetylgenistin promotes C2C12 myotube differentiation. (a) Chemical structure of acetylgenistin. (b) C2C12 cells were cultured in medium containing various concentrations (0, 1, 5, 10, 20, and 40 μM) of acetylgenistin for 24 h. Cell viability was measured using the CCK-8 (Cell Counting Kit-8) assay. Data are expressed as a percentage of viability relative to the control group, and bars in the graph represent the mean ± standard deviation (SD) (n = 4 per group). (c) Cells were stained using the May-Grunwald and Giemsa staining methods on days 2, 4, and 6. At each time point, cells were cultured in the presence or absence of 40 μM acetylgenistin and 1 μM dexamethasone. (d) Myotube width measurements based on images stained with May-Grunwald and Giemsa. Data presented represent the mean ± SD (n = 100). *** p < 0.001 vs. CTL. (e) Fusion index. The total number of nuclei incorporated into myotubes and the total number of nuclei were calculated. The fusion index was then determined as the percentage of total nuclei incorporated into myotubes. * p < 0.05 vs. CTL. CTL, control; AG, acetylgenitin.

[0016] Figure 2. Acetylgenistin enhances the expression of MHC, MyoD, and MyoG. (a) Quantitative evaluation of myogenic marker genes (MyoD and MyoG) at three time points (days 2, 4, and 6) in cells exposed to control or acetylgenistin treatment. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. Data are expressed as the mean ± standard deviation (SD) (n = 4 per group). ** p < 0.01 and *** p < 0.001 versus CTL. (b) Expression of MHC, MyoD, and MyoG proteins in C2C12 myotubes was estimated by Western blot analysis. β-Actin was used as a control for protein loading. Data are expressed as the mean ± SD (n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTL. CTL, control; AG, acetylgenistin.

[0017] Figure 3. AG attenuates dexamethasone-induced muscle atrophy in C2C12 myotubes by suppressing MAFbx and MuRF1 expression. (a) May-Grunwald and Giemsa staining. Cells were cultured with 40 μM acetylgenistin in the presence or absence of 1 μM dexamethasone for 48 h. (b) MAFbx and MuRF1 mRNA levels were analyzed by quantitative polymerase chain reaction. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. Data are expressed as the mean ± standard deviation (SD) (n = 4 per group). ** p < 0.01 and *** p < 0.001 vs. CTL; ## p < 0.01 vs. Dexa. (c) Expression of MAFbx and MuRF1 proteins in C2C12 myotubes was estimated by Western blot analysis. β-Actin was used as a control for protein loading. Data are expressed as mean ± SD (n = 3 per group). *** p < 0.001 vs. CTL, ## p < 0.01 vs. Dexa. CTL, control; Dexa, dexamethasone; AG, acetylgenistin; DAG, dexamethasone + acetylgenistin.

[0018] Figure 4. Acetylgenistin inhibits dexamethasone-induced atrophy of C2C12 myotubes through AMPK / FoxO-dependent signaling. (a) Representative images of Western blot analysis for AMPK, FoxO1, and FoxO3 in C2C12 myotubes. The lower panels show quantification of the indicated proteins. β-Actin was used as a control for protein loading. Data are expressed as the mean ± standard deviation (SD) (n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL; ## p < 0.01, and ### p < 0.001 vs. Dexa. CTL, control; Dexa, dexamethasone; AG, acetylgenistin; DAG, dexamethasone + acetylgenistin.

[0019] Figure 5. Comprehensive acetylgenistin mechanism of action in dexamethasone-induced muscle atrophy. Acetylgenistin influences muscle physiology. A potent compound, acetylgenistin, plays a pivotal role in promoting myoblast differentiation into myotubes by increasing the expression of key markers such as MHC, MyoD, and MyoG. Acetylgenistin promotes myoblast differentiation into myotubes and serves as a natural defense against dexamethasone-induced muscle atrophy. This defense involves acetylgenistin's unique ability to modulate the central upstream regulator AMPK, which reduces FoxO1 / 3 activity and consequently suppresses MAFbx expression. This dual function highlights the potential of acetylgenistin to promote muscle differentiation and prevent muscle atrophy, contributing to overall muscle health and function.

[0020]

[0021] The present invention is described in detail below.

[0022]

[0023] The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia, comprising acetylgenistin or a pharmaceutically acceptable salt thereof.

[0024] Acetylgenistine may be monoacetylgenistine or diacetylgenistine.

[0025] The position of the acetyl group is not particularly restricted and may be bonded to various positions.

[0026] Specifically, acetyl genistin may be, but is not limited to, 6''-O-acetyl-genistin.

[0027] Acetylgenistin is available in the form of pharmaceutically acceptable salts.

[0028] The term pharmaceutically acceptable salt means any organic or inorganic addition salt of acetylgentistin at a concentration that is relatively non-toxic and harmless to the patient and that does not diminish the beneficial effects of acetylgentistin due to side effects attributable to this salt.

[0029] The salt may be an acid addition salt or a metal salt obtained using a base. Any acid or base known in the art may be used without limitation.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034]

[0035] In addition, the present invention relates to a health functional food for preventing or improving sarcopenia, comprising acetylgenistin or a pharmaceutically acceptable salt thereof.

[0036] Acetylgenistin or a pharmaceutically acceptable salt thereof may be any of those exemplified above.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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:

[0043] 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.

[0044] The content of acetyl genistein or its salt as an active 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.

[0045] 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.

[0046]

[0047] The present invention will be described in more detail with reference to the following examples.

[0048]

[0049] Materials and Methods

[0050] Cell culture and maintenance

[0051] 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.

[0052]

[0053] Cell viability measurement

[0054] C2C12 cells were seeded at 5 Х 10 in 24-well plates 4Cells were seeded at a density of 10 cells / well. After cell attachment, acetylgenistin was treated at concentrations of 0, 1, 5, 10, 20, and 40 μM and cultured in a humidified incubator at 37°C in 5% CO2 for 24 h. After incubation, 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 value by 100.

[0055]

[0056] Induction of myotube differentiation and atrophy

[0057] When C2C12 myoblasts reached 80–90% confluency, differentiation into myotubes was initiated by replacing the GM with DMEM differentiation medium (DM) supplemented with 2% horse serum (HS; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). Cells were induced to differentiate into myotubes for 6 days, with the DM replaced every 2 days.

[0058] 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.

[0059]

[0060] May-Grunwald and Giemsa staining

[0061] 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 washed, visualized, and photographed at 100x magnification. The width of the myotubes was measured in randomly selected sections using ImageJ software.

[0062]

[0063] Calculating the fusion index

[0064] To quantify myogenic differentiation efficiency, random fields of view were captured from May-Grünwald and Giemsa-stained images. The number of nuclei within the myotubes and the total number of nuclei were counted. The fusion index was calculated as follows:

[0065] Fusion index (%) = (number of nuclei in the root canal / total number of nuclei) Х 100

[0066]

[0067] Western blot

[0068] 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). The proteins were electrophoresed on a 10% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking, the membranes were incubated with primary antibodies, including MuRF1 (Santa Cruz Biotechnology Inc.), MAFbx (Santa Cruz Biotechnology Inc.), FoxO1 (Cell Signaling Technology, Beverly, MA, USA), FoxO3 (Cell Signaling Technology), AMPK (Cell Signaling Technology), and β-actin (Sigma-Adrich, St Louis, MO, USA). After overnight incubation, the membranes were washed 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).

[0069]

[0070] quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0071] 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 analyzed using the ViiA TaqMan assay. TM 7 Real-Time PCR system (Applied Biosystems Inc., Waltham, MA, USA). 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 for MuRF1 (Mm01185221 m1), MAFbx (Mm00499523 m1), MyoD (Mm00440387 m1), MyoG (Mm00446194 m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915 g1) were used.

[0072]

[0073] Statistical analysis

[0074] Statistical analysis was performed using GraphPad Prism software (version 5.01; GraphPad Software, San Diego, CA, USA). Cell experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation. The results of the animal model were expressed as the mean ± standard error of the mean for each group. Significance between two groups was assessed using the Student's t-test, and statistical significance was set at *p<0.05.

[0075]

[0076] result

[0077] Acetyl genistin promotes C2C12 myotube differentiation

[0078] To elucidate the potential effects of AG on myogenic differentiation and molecular properties, a comprehensive experimental approach was designed and implemented, including molecular characterization, viability analysis, morphological assessment, and fusion efficiency evaluation. The molecular structure of AG was elucidated, highlighting its unique chemical composition (Fig. 1a). Viability analysis of C2C12 cells along an AG concentration gradient demonstrated robust cell survival, with an upward trend in viability observed with increasing AG concentrations (Fig. 1b). Morphological evaluation over a time gradient after AG exposure revealed enhanced myogenic differentiation, particularly at day 6 (Fig. 1c), an effect that persisted even in the presence of dexa, a known myogenic antagonist. Quantification of myotube width demonstrated enhanced myogenic differentiation by AG, with a clear increase in width compared to the control group (Fig. 1d). Concurrently, the Fusion Index, a quantitative measure of myoblast fusion efficiency, demonstrated an enhanced fusion profile in the AG-treated cohort, suggesting superior nuclear integration within the emergent myotubes compared to the control group (Fig. 1e). Collectively, these findings demonstrate a robust role for AG in enhancing C2C12 myotube differentiation, a phenomenon robust against dextran-mediated side effects.

[0079]

[0080] Regulatory effect of acetyl genistin on the expression of major myogenic markers

[0081] MyoD represents an early marker for the myogenic lineage, MyoG regulates terminal differentiation of myoblasts, and MHC confirms mature myotube formation. Examining the regulatory effects of AG on these key myogenic markers revealed distinct transcriptional and translational alterations. Sequential evaluations on days 2, 4, and 6 post-treatment revealed a robust upregulation of MyoD and MyoG gene expression in AG-treated cells compared to the control group, with GAPDH serving as a normalization control (Fig. 2a). Consistent with this, Western blot analysis confirmed these findings at the protein level, demonstrating enhanced expression of MHC, MyoD, and MyoG in AG-treated C2C12 myotubes, with equal loading of β-actin (Fig. 2b). These results demonstrate a robust role for AG in the regulation of myogenic differentiation markers.

[0082]

[0083] Alleviation of dexa-induced muscle atrophy through downregulation of MAFbx and MuRF1 in AG

[0084] MAFbx and MuRF1 are key E3 ubiquitin ligases involved in skeletal muscle protein degradation. Their elevation is particularly associated with muscle atrophy induced by glucocorticoids such as dexamethasone. As shown in Figure 3, careful analysis highlighted the protective effect of AG against the detrimental effects of dexamethasone on skeletal muscle cells. Histological examination using May-Grunwald and Giemsa staining revealed the potent muscle-sparing effect of AG. Notably, dexamethasone exposure promoted discernible muscle atrophy, whereas co-treatment with AG significantly suppressed this atrophic response, as evidenced by dense myotube structures (Figure 3a). Molecular investigations confirmed these morphological observations, providing mechanistic insights. Quantitative PCR revealed a marked upregulation of MAFbx and MuRF1 mRNA levels upon dexamethasone exposure, both essential markers of muscle degradation. Interestingly, AG treatment significantly attenuated the expression of these atrophy-related genes, suggesting a direct involvement in the inhibition of muscle protein degradation (Fig. 3b). Western blot analysis confirmed this observation. A marked increase in the protein levels of MAFbx and MuRF1 was observed in dexamethasone-treated cells. However, co-administration of AG significantly suppressed the expression of these catabolic markers, indicating its potential to counteract dexamethasone-induced protein degradation (Fig. 3c). AG has emerged as a promising therapeutic agent with potent muscle-sparing effects, particularly against glucocorticoid-induced muscle atrophy, by modulating the expression of central muscle degradation markers.

[0085]

[0086] AG's response to dexa-mediated atrophy through modulation of AMPK / FoxO signaling

[0087] The AMP-activated protein kinase (AMPK) and forkhead box O (FoxO) signaling pathways are central to muscle metabolism and atrophy. AMPK, known as the "master regulator" of cellular energy, is crucial for muscle energy balance, whereas FoxO transcription factors, particularly FoxO1 and FoxO3, regulate genes associated with muscle atrophy under stress conditions. Figure 4 illustrates the complex molecular basis of AG's action on dexa-induced skeletal muscle atrophy. Western blotting revealed that dexa exposure suppressed AMPK activation while simultaneously upregulating the expression of FoxO1 and FoxO3 transcription factors, key regulators of muscle catabolism. Remarkably, AG administration not only restored AMPK activity but also reduced the increased levels of FoxO1 and FoxO3 (Figure 4a). Accompanying quantification further supported these observations, highlighting the significant inhibitory effect of AG on the atrophic effects of dexa via the AMPK / FoxO axis. Collectively, these results suggest that AG is a potent regulator of glucocorticoid-induced muscle wasting, acting primarily through an AMPK / FoxO-dependent pathway.

[0088] These examples clearly demonstrate the therapeutic efficacy of AG on muscle differentiation and its protective effect against muscle atrophy. When exposed to AG, enhanced myoblast differentiation was observed, along with upregulation of key markers, MHC, MyoD, and MyoG. Furthermore, in the presence of dexa, a known muscle atrophy inducer, AG exerted its protective effect primarily through modulation of the AMPK / FoxO signaling pathway and inhibition of atrogenes MAFbx and MuRF1. The presented diagram provides comprehensive insights into the molecular mechanisms of these effects (Fig. 5).

Claims

1. A pharmaceutical composition for preventing or treating sarcopenia comprising acetylgenistin or a pharmaceutically acceptable salt thereof.

2. 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.

3. A health functional food for preventing or improving sarcopenia containing acetylgenistin or a pharmaceutically acceptable salt thereof.

4. A health functional food for preventing or improving sarcopenia according to claim 3, 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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