Isolation and Characterization of Compounds from Glycyrrhiza uralensis as Therapeutic Agents for the Muscle Disorders

Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds (4-hydroxybenzoic acid, liquiritigenin, (R)-(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.


Introduction
Skeletal muscle is composed of multinucleated myofibers and accounts for about half of the total body weight [1]. Myogenesis refers to myofiber producing processes such as those that occur during embryogenesis, postnatal growth, and muscle tissue regeneration [2]. Myogenesis is an extremely coordinated process and is associated with various transcriptional networks, the important components of which are Pax7, Myf5, MYOD, myogenin (MYOG), and Myf6 [3]. Post-injury repair of skeletal muscle involves a series of complex events, which can be broadly categorized into three steps. The first involves the activation of quiescent muscle satellite cells (MSCs). In response to damage,

The CWE of G. uralensis Promoted Myoblast Proliferation and Differentiation
The CWE of G. uralensis was added at different concentrations (0, 50, 100, or 200 µg/mL) to C2C12 cells cultured in growth media for one day. Cell proliferation was significantly increased by 50 and 100 µg/mL treatments versus non-treated controls ( Figure 1A). In addition, expression of Ki67 (cellular marker for proliferation), CyclinA2 (regulator of cell cycle), and MSTN (inhibitor of myoblast cell proliferation) mRNA was analyzed in G. uralensis CWE treated cells (0 or 100 µg/mL for 1 day). Ki67 expression was significantly increased and MSTN expression was decreased with G. uralensis CWE treatment (Supplementary Figure S1). Next, scratch testing was performed on 100% confluent cells, which were incubated with 0 or 100 µg/mL of the CWE of G. uralensis for one day. Cell recoveries of treated cells (100 µg/mL) were better than those of non-treated controls ( Figure 1B). Treatment with the CWE of G. uralensis induced myotube formation and elevated the mRNA and protein expression of myogenic factors. However, the expressions of atrophy and protein degradation related marker genes (Atrogin1 and MurF1) were lower in cells treated with G. uralensis CWE than in non-treated controls ( Figure 1C). These results show that G. uralensis CWE induced myoblast proliferation and differentiation by enhancing the expressions of myogenic genes and inhibiting atrophy-related genes expression. Scratch analysis was performed on ~100% confluent cells, incubated in growth medium containing 0 or 100 µg/mL of G. uralensis CWE for one day. Cell recovery was measured in treated and non-treated cells. (C) Cells were incubated with differentiation media supplemented with 100 µg/mL of G. uralensis CWE for four days. Myotube formation and fusion indices were determined by Giemsa staining, mRNA levels by real-time RT-PCR, and protein expressions by Western blotting. Non-treated cells were used as controls. Means ± SD (n = 6). * p ≤ 0.05, ** p ≤ 0.001.

The CWE of G. uralensis Enhanced Muscle Regeneration
In order to investigate the effects of G. uralensis CWE on muscle regeneration, it was administered once a day for nine days from one day before CTX injection. Pre-treatment of G. uralensis CWE along with muscle injury can induce faster and stronger cellular signaling preferentially due to the effect of G. uralensis extract. Muscle tissues were collected and body weights (g), percentage muscle mass reduction (%), and myogenic and atrophy related protein expression were analyzed. G. uralensis CWE did not influence body weight but suppressed reductions in muscle mass as compared with non-treated muscles ( Figure  2A). In addition, G. uralensis CWE enhanced the expressions of Pax7, MYOD, MYOG, and Scratch analysis was performed on~100% confluent cells, incubated in growth medium containing 0 or 100 µg/mL of G. uralensis CWE for one day. Cell recovery was measured in treated and non-treated cells. (C) Cells were incubated with differentiation media supplemented with 100 µg/mL of G. uralensis CWE for four days. Myotube formation and fusion indices were determined by Giemsa staining, mRNA levels by real-time RT-PCR, and protein expressions by Western blotting. Non-treated cells were used as controls. Means ± SD (n = 6). * p ≤ 0.05, ** p ≤ 0.001.
In addition, cells were cultured in differentiation media supplemented with 100 µg/mL of G. uralensis for four days to determine its effects on myoblast differentiation. Treatment with the CWE of G. uralensis induced myotube formation and elevated the mRNA and protein expression of myogenic factors. However, the expressions of atrophy and protein degradation related marker genes (Atrogin1 and MurF1) were lower in cells treated with G. uralensis CWE than in non-treated controls ( Figure 1C). These results show that G. uralensis CWE induced myoblast proliferation and differentiation by enhancing the expressions of myogenic genes and inhibiting atrophy-related genes expression.

The CWE of G. uralensis Enhanced Muscle Regeneration
In order to investigate the effects of G. uralensis CWE on muscle regeneration, it was administered once a day for nine days from one day before CTX injection. Pre-treatment of G. uralensis CWE along with muscle injury can induce faster and stronger cellular signaling preferentially due to the effect of G. uralensis extract. Muscle tissues were collected and body weights (g), percentage muscle mass reduction (%), and myogenic and atrophy related protein expression were analyzed. G. uralensis CWE did not influence body weight but suppressed reductions in muscle mass as compared with non-treated muscles (Figure 2A). In addition, G. uralensis CWE enhanced the expressions of Pax7, MYOD, MYOG, and MYL2 and reduced those of MSTN, Atrogin1, and MuRF1 and nitrotyrosine levels in regenerated muscle tissues as compared with non-CTX injected muscle ( Figure 2B-D). These observations show that G. uralensis CWE effectively promoted muscle regeneration by enhancing the expressions of myogenic genes and inhibiting those of atrophy-related genes. MYL2 and reduced those of MSTN, Atrogin1, and MuRF1 and nitrotyrosine levels in regenerated muscle tissues as compared with non-CTX injected muscle ( Figure 2B-D). These observations show that G. uralensis CWE effectively promoted muscle regeneration by enhancing the expressions of myogenic genes and inhibiting those of atrophy-related genes.

Figure 2.
Muscle regeneration by the CWE of G. uralensis. Mice were orally administered G. uralensis CWE (100 mg/kg) daily for nine days from one day before CTX injection. Seven days after CTX injection, gastrocnemius muscle tissues were Muscle regeneration by the CWE of G. uralensis. Mice were orally administered G. uralensis CWE (100 mg/kg) daily for nine days from one day before CTX injection. Seven days after CTX injection, gastrocnemius muscle tissues were collected from control (intact or CTX injected animals) and G. uralensis CWE treated animals and from treatment naïve controls. (A) Body weights (g) and reductions in muscle mass (%). (B,C) Protein expression was determined by Western blotting and band intensities using Multi Gauge 3.0 software. (D) H&E staining and protein expression as determined by immunohistochemistry. Con indicates control. Means±SDs (n = 9). ** p ≤ 0.001, *** p ≤ 0.0001.

Fractions Derived from G. uralensis CWE Enhanced Myoblast Proliferation
Five different fractions (water extracts; EX, Dichloromethane; DCM, Ethyl acetate; EtOAc, n-butanol; BuOH, and H 2 O; aqueous layer) from G. uralensis water extract were separated with different solvents (Supplementary Figure S2) and C2C12 cells were treated with each fraction for one day. Cell proliferation was significantly enhanced by the EX, EtOAc, and BuOH fractions, but decreased by the DCM fraction versus non-treated controls ( Figure 3A). Scratch analysis was carried out on 100% confluent cells incubated with each of the five fractions for one day. Recovered area was larger in the sample treated with the Ex, EtOAc, and BuOH fractions respect to control ( Figure 3B). These results show that the EX, EtOAc, and BuOH fractions of G. uralensis CWE enhance myoblast proliferation.
collected from control (intact or CTX injected animals) and G. uralensis CWE treated animals and from treatment naïve controls. (A) Body weights (g) and reductions in muscle mass (%). (B,C) Protein expression was determined by Western blotting and band intensities using Multi Gauge 3.0 software. (D) H&E staining and protein expression as determined by immunohistochemistry. Con indicates control. Means±SDs (n = 9). ** p ≤ 0.001, *** p ≤ 0.0001.

Fractions Derived from G. uralensis CWE Enhanced Myoblast Proliferation
Five different fractions (water extracts; EX, Dichloromethane; DCM, Ethyl acetate; EtOAc, n-butanol; BuOH, and H2O; aqueous layer) from G. uralensis water extract were separated with different solvents (Supplementary Figure S2) and C2C12 cells were treated with each fraction for one day. Cell proliferation was significantly enhanced by the EX, EtOAc, and BuOH fractions, but decreased by the DCM fraction versus non-treated controls ( Figure 3A). Scratch analysis was carried out on 100% confluent cells incubated with each of the five fractions for one day. Recovered area was larger in the sample treated with the Ex, EtOAc, and BuOH fractions respect to control ( Figure 3B). These results show that the EX, EtOAc, and BuOH fractions of G. uralensis CWE enhance myoblast proliferation.

Effects of G. uralensis CWE Derived Fractions on Myoblast Differentiation
C2C12 cells were incubated in differentiation media supplemented with the EX, DCM, EtOAc, BuOH, or H2O fractions for four days to determine their effects on myoblast differentiation. Myotube formation was enhanced by the EtOAc and BuOH fractions versus non-treated cells. When cells were treated with DCM during differentiation, most cells detached from plates ( Figure 4A). In addition, the expressions of myogenic marker genes were increased by EX (MYL2), EtOAc (MYOD, MYOG, and MYL2), BuOH (MYOD,

Effects of G. uralensis CWE Derived Fractions on Myoblast Differentiation
C2C12 cells were incubated in differentiation media supplemented with the EX, DCM, EtOAc, BuOH, or H 2 O fractions for four days to determine their effects on myoblast differentiation. Myotube formation was enhanced by the EtOAc and BuOH fractions versus non-treated cells. When cells were treated with DCM during differentiation, most cells detached from plates ( Figure 4A). In addition, the expressions of myogenic marker genes were increased by EX (MYL2), EtOAc (MYOD, MYOG, and MYL2), BuOH (MYOD, MYOG, and MYL2), and H 2 O (MYOG) fractions. Furthermore, the expression of atrophyrelated genes were significantly decreased by EX (MuRF1), EtOAc (Atrogin1 and MuRF1), and BuOH (MuRF1). Interestingly, MSTN and nitrotyrosine protein expression were lower in EtOAc fraction treated cells and higher in BuOH fraction treated cells than in controls ( Figure 4B). In addition, protein expression in EtOAc or BuOH fraction treated cells were consistent with their mRNA expressions ( Figure 4B). Following metabolite analysis, NH 3 concentrations (a by-product of protein degradation) were lower in EtOAc fraction than in non-treated culture media (Supplementary Figure S3). These results suggest that the EtOAc fraction inhibited protein degeneration by inhibiting Atrogin1 expression. Taken together, these results suggest that G. uralensis derived EtOAc fraction enhanced myoblast differentiation by inducing the expression of myogenic marker genes and inhibiting those of MSTN and atrophy-related genes.
MYOG, and MYL2), and H2O (MYOG) fractions. Furthermore, the expression of atrophyrelated genes were significantly decreased by EX (MuRF1), EtOAc (Atrogin1 and MuRF1), and BuOH (MuRF1). Interestingly, MSTN and nitrotyrosine protein expression were lower in EtOAc fraction treated cells and higher in BuOH fraction treated cells than in controls ( Figure 4B). In addition, protein expression in EtOAc or BuOH fraction treated cells were consistent with their mRNA expressions ( Figure 4B). Following metabolite analysis, NH3 concentrations (a by-product of protein degradation) were lower in EtOAc fraction than in non-treated culture media (Supplementary Figure S3). These results suggest that the EtOAc fraction inhibited protein degeneration by inhibiting Atrogin1 expression. Taken together, these results suggest that G. uralensis derived EtOAc fraction enhanced myoblast differentiation by inducing the expression of myogenic marker genes and inhibiting those of MSTN and atrophy-related genes.
Next, C2C12 cells were cultured in differentiation media supplemented with liquiritigenin, tetrahydroxymethoxychalcone, or licochalcone B for four days to determine their effects on myoblast differentiation. Myotube formation was increased by tetrahydroxymethoxychalcone versus non-treated controls ( Figure 5C). Three compounds (liquiritigenin, tetrahydroxymethoxychalcone, or licochalcone B) were commercially available. After acquiring these compounds, cell proliferation and differentiation were analyzed in proliferation medium for one day or differentiation medium for four days. Cell proliferation was significantly increased by liquiritigenin (0.25 ng/mL; 5%) and by licochalcone B (1 ng/mL; 11%) ( Figure 5D), and, interestingly, myogenic differentiation was also increased by each of the three commercial standards ( Figure 5E). These results show that liquiritigenin, tetrahydroxymethoxychalcone, or licochalcone B enhance myoblast proliferation and differentiation.

Liquiritigenin Enhanced Muscle Regeneration
In order to investigate the effects of liquiritigenin on muscle regeneration, mice were orally administered liquiritigenin daily for nine days from one day before CTX injection. Muscle tissues were collected and body weights (g), gastrocnemius muscle weights (g), and muscle mass reductions (%) were analyzed. Body and gastrocnemius muscle weights were similar for liquiritigenin treated and non-treated controls. However, muscle mass reductions in liquiritigenin treated mice were less than in non-treated controls ( Figure 6A). In addition, muscle fiber size is increased after liquiritigenin treatment, both in non-injected and in CTX-injected regenerating muscles compared to non-treated ones ( Figure 6B,C). These observations showed that liquiritigenin enhanced muscle regeneration. ol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 20  Muscle tissues were collected and body weights (g), gastrocnemius muscle weights (g), and muscle mass reductions (%) were analyzed. Body and gastrocnemius muscle weights were similar for liquiritigenin treated and non-treated controls. However, muscle mass reductions in liquiritigenin treated mice were less than in non-treated controls ( Figure  6A). In addition, muscle fiber size is increased after liquiritigenin treatment, both in noninjected and in CTX-injected regenerating muscles compared to non-treated ones ( Figure  6B,C). These observations showed that liquiritigenin enhanced muscle regeneration.

FAF-Drugs 4 Based Analysis of Liquiritigenin, Tetrahydroxymethoxychalcone, and Licochalcone B
In silico predictions of the biological properties and toxicities of chemical compounds provide a rapid, dependable means of assessment before further bench-work is conducted, and physicochemical and ADMET properties are essential considerations for any candidate drug. Liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were

FAF-Drugs 4 Based Analysis of Liquiritigenin, Tetrahydroxymethoxychalcone, and Licochalcone B
In silico predictions of the biological properties and toxicities of chemical compounds provide a rapid, dependable means of assessment before further bench-work is conducted, and physicochemical and ADMET properties are essential considerations for any candidate drug. Liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to be suitable for FAF-Drug4 analysis in terms of molecular complexity, number of aromatic rings, sp3 hybridized (Fsp3) carbon fractions, and ADMET properties [44], which indicated these compounds are non-toxic and might be therapeutically useful ( Figure 7A-C).
found to be suitable for FAF-Drug4 analysis in terms of molecular complexity, number of aromatic rings, sp3 hybridized (Fsp3) carbon fractions, and ADMET properties [44], which indicated these compounds are non-toxic and might be therapeutically useful (Figure 7A-C).

Discussion
Population aging is a comparatively recent global phenomenon, hence greater awareness is needed to ensure healthy aging and quality of life. Physical exercise and a high protein diet are known to contribute to the maintenance of muscle function in the elderly, but these approaches are restricted to healthy subjects rather than those suffering from disease or immobility [45]. Hence, the present study was undertaken to determine whether G. uralensis has the potential to promote myogenesis and muscle functions. In this study, we found that extracts of G. uralensis enhanced the proliferation and differentiation of C2C12 myoblasts, and that pre-treatment with G. uralensis extracts enhanced muscle regenerative ability in a CTX-induced mouse model of muscle injury. In a previous study, Hachimijiogan (a Japanese herbal medicine) has found to enhance C2C12 myoblast proliferation but not to affect or induce their differentiation [46]. In contrast, we found that the G. uralensis extract promoted the proliferation and differentiation of C2C12 myoblasts.
Furthermore, G. uralensis extract was found to promote myogenesis by upregulating the expressions of myogenic marker genes (MYOD, MYOG, and MYL2), which is important, as MYOD and MYOG are recognized myogenic regulatory factors that perform essential functions during myogenesis [47]. In addition, following muscle injury, pretreatment with G. uralensis extract enhanced muscle regeneration by inducing the expression of myogenic mRNA and proteins, which pointed the muscle regenerative ability of G. uralensis.
Cachexia, unlike sarcopenia, is an intricate metabolic syndrome primarily characterized by extreme loss of muscle mass with or without fat loss. Abnormal metabolism is a major characteristic of cachexia, and several metabolic pathways in different tissues are known to be disturbed in this condition [48]. Cachexia is related to multiple chronic diseases, most commonly cancer. Effective therapeutic strategies for cancer-associated cachexia include a combination of multiple approaches aimed at stabilizing metabolic alterations [48,49]. MSTN is an important target because it is overexpressed in many cachectic disorders [50]. We previously found that curcumin and gingerol potently inhibit MSTN and help suppress the expressions of advanced glycation end products and that of their receptor RAGE [51], which is also viewed as a potential therapeutic target for cancer cachexia [52]. Here, we report that the EtOAc derived fractions of G. uralensis reduced the expressions of MSTN, Atrogin1, and MurF1, which suggests it has therapeutic potential against muscle wasting in muscular dystrophy.
We found that G. uralensis extract reduced the level of nitrotyrosine, which suggests that the extract has anti-inflammatory properties. Licorice extract has been reported to reduce proinflammatory cytokine levels in serum [53], and studies on bioactive compounds in G. uralensis have shown they also exhibit anti-inflammatory effects. For instance, Su et al. [54] concluded that the antidepressant and antianxiety effects of liquiritigenin were associated with its anti-inflammatory effect, as liquiritigenin pretreatment reduced pro-inflammatory cytokine (IL-6 and TNF-α) levels. In another study, isoliquiritigenin was observed to exert anti-inflammatory effects by reducing lipopolysaccharide-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 expressions by inhibiting the activation of nuclear factor-kappa B in RAW264.7 macrophages [55]. Furthermore, in mice, licochalcone A in diet decreased the expressions of iNOS and cyclooxygenase-2 and reduced proinflammatory cytokine levels in colon tissues [56]. All of these studies and the present study indicate G. uralensis has anti-inflammatory properties.
In the present study, G. uralensis CWE enhanced the proliferation and differentiation of myoblast cells and muscle regeneration, and the EtOAc-fraction of G. uralensis CWE increased proliferation and differentiation significantly more than the other fractions. Therefore, we subjected the EtOAc-soluble fraction to further study to identify its bioactive constituents and determine their effects on myoblast proliferation and differentiation. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. This analysis showed these compounds to be a phenolic and nine flavonoid compounds, and, among these, liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation.
On a cautionary note, the presence of glycyrrhizin, a major active constituent of G. uralensis, suggests that excessive consumption of G. uralensis might have a toxic effect [57]. However, it should be emphasized that the compounds isolated from the EtOAc-fraction of G. uralensis that did not contain glycyrrhizin were found to be non-toxic by in silico analysis. Furthermore, myoblast proliferation and differentiation were increased by commercially procured liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B, and muscle regeneration was enhanced by orally administered liquiritigenin as compared with nontreated controls, which suggested that it plays important roles in myogenesis and muscle regeneration.

General Experimental Procedures
A BRUKER AVANCE III HD 600 unit (Bruker Biospin GmbH, Karlsruhe, Germany) was used to record 1 H-and 13 C NMR spectra at 600 and 150 MHz using tetramethylsilane as the internal standard. Medium-pressure liquid chromatography (MPLC) was carried out using an Isolera One machine (Biotage, Uppsala, Sweden) equipped with SNAP KP-SIL and SNAP Ultra C 18 cartridges. Silica gel (Kieselgel 60, 70-230, and 230-400 mesh, Merck, Darmstadt, Germany). Column chromatography was performed using YMC C 18 resins and thin-layer chromatography (TLC) was carried out using pre-coated silica gel 60 F 254 and RP-18 F 254S plates (0.25 mm, Merck, Darmstadt, Germany). Plates were treated with 10% H 2 SO 4 and visualized under UV light (254 and 365 nm).

Plant Material and Preparation of G. uralensis Water Extract (CWE)
The dried roots of G. uralensis Fischer were procured from the Kwangmyeong Herbal Store (KM Herb Co., Ltd., Busan, Korea). All voucher specimens were deposited in an herbal bank at the KM Application Center, Korea Institute of Oriental Medicine. To prepare a water extract, dried roots of G. uralensis (50 g) were added to 1000 mL distilled water and extracted by heating at 115 • C for 3 h. The extract obtained was filtered using standard testing sieves (150 µm) and freeze-dried. The lyophilized extract powder was then dissolved in tertiary distilled water and left at 4 • C for 24 h, centrifuged at 5000× g for 5 min, transferred to new tubes, and stored at −20 • C.

Scratch Experiment
Cells were cultured in DMEM+10% FBS+1% P/S until 100% confluent. Cell layers on plates were then scratched, media was added, and cells were incubated for 1 day. Cell recovery was observed using a light microscope (Nikon, Melville, NY, USA) and cell mobility from scratched part to recovered part was measured and then calculated as cell recovery value (%).

Fusion Index
Differentiated cells were washed with PBS, fixed with a methanol/PBS mixture for 2 min, and stained with Giemsa G250 (Sigma Aldrich). Random images were taken at three different regions using a microscope. Nuclei numbers in myotubes and total numbers of nuclei in cells were calculated in each image. Fusion indices were calculated by expressing numbers of nuclei integrated into myotubes as percentages of total numbers of nuclei. All experiments were performed in triplicate [58,59].

Metabolite Analysis
Cells were cultured in differentiation media supplemented with DMSO (EtOAc was dissolved in DMSO, DMSO was used as a control) or EtOAc for 4 days and concentrations of NH 3 in cultured media were determined using commercial reagent kits (Glucose Bio HT; Roche Diagnostics, Indianapolis, IN, USA) and a Cedex Bio-analyzer (Roche Diagnostics, Indianapolis, IN, USA).

Real-Time RT-PCR
Trizol reagent (Thermo Fisher Scientific) was used to extract total RNA from cells according to the manufacturer's instructions. RNA (2 µg) in 20 µL of reaction mixture was used to synthesize 1st strand cDNA using random hexamer and reverse transcriptase at 25 • C for 10 min, 37 • C for 120 min, and 85 • C for 5 min. Two micro liters of cDNA and 10 pmole of gene-specific primers were used to analyze gene expression by real-time RT-PCR, which was performed using a 7500 real-time PCR system and a power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Primer information is provided in Supplementary Table S1.

Western Blot
PBS was used to wash cells, which were then lysed with RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). Total protein concentrations were measured using the Bradford assay. Proteins (40 µg) were electrophoresed in 10 or 12% SDS-polyacrylamide gel and then transferred to PVDF membranes (EMS-Millipore, Billerica, MA, USA). Blots were blocked using 3% skim milk or BSA in Tris-buffered saline (

Analysis of Muscle Mass Reduction and Muscle Fiber Diameters
The reduction ratio of muscle mass (%) was calculated using a given formula: Reduction ratio of muscle mass (%) = 100 − (CTX injected gastrocnemius muscle/non-injected gastrocnemius muscle) × 100. (1) Muscle tissue sections were H&E stained, and images were captured under a light microscope. Muscle fiber diameters were measured using ImageJ software [61].

FAF-Drugs4 Analysis
The ADMET (absorption, distribution, metabolism, elimination, and toxicity) pharmacokinetic properties of compounds were analyzed using FAFdrugs4 (https://fafdrugs4 .rpbs.univ-paris-diderot.fr/), an open-access tool for the prediction of ADMET properties that allows filtering according to Egan's rule, Lipinski's rule of five, and Veber's rule for the prediction of bioavailabilities, and according to GSK and Pfizer's rules for overall toxicity predictions [62,63].

Statistical Analysis
Tukey's Studentized Range test (honest significance difference) was employed to analyze normalized expression mean values to categorize significant gene expression changes. Multi Gauge 3.0 software (Fujifilm, Tokyo, Japan) was used to quantify band intensities in Western blots. Gene and protein expressions were normalized versus GAPDH (the internal control), and the analysis was conducted using one-way ANOVA and PROC GLM in SAS, ver.9.0 (SAS Institute, Cary, NC, USA).

Conclusions
In summary, our results demonstrate that G. uralensis promotes the proliferation and differentiation of myoblasts and muscle regeneration by upregulating the expressions of the myogenic marker genes (MYOD, MYOG, and MYL2) and by downregulating the expression of MSTN (a muscle growth inhibitor). Based on the observed inhibitory potency of G. uralensis against atrophy and protein degradation-related marker genes (Atrogin1 and MuRF1), we suggest that G. uralensis be considered a therapeutic agent for the prevention and treatment of muscular dystrophy, sarcopenia, and cachexia. In addition, the observed anti-inflammatory effect of G. uralensis suggests therapeutic applications in the context of inflammatory disease.