In Vitro Study on the Effects of Rhododendron mucronulatum Branch Extract, Taxifolin-3-O-Arabinopyranoside and Taxifolin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript entitled “In vitro study on the effects of Rhododendron mucronulatum 2 branch Extract, Taxifolin-3-O-arabinopyranoside and Taxifolin 3 on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal 4 Muscle Cells”, the authors investigate the protective effects of Rhododendron mucronulatum branch extract (RMB), comparing its effects with those of its major flavonoids, taxifolin-3-O-arabinopyranoside (Tax-G) and taxifolin (Tax-A), against oxidative stress–induced apoptosis (H₂O₂) and dexamethasone-induced muscle atrophy using in vitro C2C12 murine skeletal muscle models.

Moreover, the manuscript highlights the importance of using plant branches rather than roots as a sustainable and eco-friendly source of bioactive compounds.

The paper is well written and the topic is quite interesting but there are some points to deepen before accepting the manuscript. The paper could be accepted for publication with major revision.

General considerations:

- The presentation is characterized by an exceptionally large number of figures and quantitative comparisons, which makes it difficult to clearly identify the key message. Authors should add a simpler explanation of what they demonstrate in the figures, especially in the first party (figure 1-8) or moving selected data to supplementary materials.

- While the in vitro data is extensive, the conclusions occasionally suggest translational or therapeutic relevance that would require in vivo validation, which is currently lacking.

- There are numerous minor grammatical errors and typographical issues (e.g., spacing, repeated symbols, encoding problems). Careful language editing by a native English speaker is recommended.

- Authors should refresh the bibliography: they add 40% of manuscript published before 2017. Except for the manuscript relevant for the published work, they should add or substitute them with more recent publications. For example, in the introduction section they refer to statistic data of 2014 and 2016, does not exist any other more recent data?

- Vehicle controls should be clearly described for all treatments, especially if authors used different solvents for extracts and isolated compounds.

- Positive controls for apoptosis inhibition or muscle protection (e.g., known antioxidants or Akt/mTOR activators) are not included. Their inclusion would strengthen the interpretation of efficacy.

Figure 1: authors should better describe the figure. The meaning of the acronyms RMB RF1 and RMB RF2 is explained only in the subsequent paragraph, the have to anticipate something referring to figure 1.

Figure 9: The observed increase in cell viability following RMB treatment under basal conditions is interpreted as a beneficial effect. However, it remains unclear whether this reflects enhanced proliferation, metabolic stimulation, or assay interference. Additional clarification or supportive experiments (e.g., proliferation markers or alternative viability assays) are required.

Moreover, authors should better explain why we can observe an increase in cell viability between 100 ad 600 ug/ml with RMB treatment and then cell viability decreases drastically? Authors should analyze, for example, the cell cycle to better understand what happens to the cell with these higher concentrations.

Figure 10: authors should explain the choice of the concentration of 100uM of H2O2 and show us a curve demonstrating a dose dependent effect and the efficacy of H2O2 at the dose selected.

Why did authors decide to cotreat cells with H2O2 and compounds or DEX and compounds and they do not use for example a pretreatment with H202 or DEX? Please explain.

Figure 11: authors should explain the choice of the concentration of 5uM of DEX and show us a curve demonstrating a dose dependent effect and the efficacy of DEX at the dose selected.

Authors could add a higher dose of DEX to show if the compounds are able to revert the effects observed with this higher dose of DEX ad can restore the cell viability.

Figure 13A: the western blot shows that Bax increase with 5-10 ug/ml of Tax-A and decrease with 50ug/ml, authors should explain this data. Probably authors should change the picture or repeat the experiment.

Figure 15: authors should enrich the data reported in this figure adding the corresponding fluorescent cell images of each condition.

Table 1-3: Authors evaluate the effects of the compounds on muscle degradation and synthesis through protein and mRNA analysis of the major proteins involved (Atrogin-1 and MuRF1, MyoD1 and Myogenin). Authors should add the mRNA analysis also for apoptosis markers that are analyzed only at protein level.

Figure 18-20: authors write in the discussion that: “These results are consistent with the mechanism by which DEX inhibits the Akt/mTOR pathway, a muscle synthesis signal, while activating the FoxO pathway, a muscle atrophy signal, leading to activation of the ubiquitin–proteasome system and sup- pression of muscle synthesis regulators. The RMB, Tax-G, and Tax-A treatment groups inhibited the expression of muscle atrophy factors FoxO3α, Atrogin-1, and MuRF1, while restoring the expression of muscle synthesis factors Akt, mTOR, MyoD, and Myogenin”.

This conclusion is mainly due to the results and changes in phosphorylation rate of the proteins analyzed. However, no functional or pharmacological validation (e.g., pathway inhibitors, siRNA knockdown) is provided. As a result, the causal relationship between RMB/Tax-G/Tax-A treatment and Akt/mTOR or FoxO3α signaling remains associative rather than mechanistically demonstrated.

Auhtors should add one of the experiments suggested and better explained this correlation in the discussion section.

Author Response

Reviewer 1

We sincerely thank you for taking the time to review our manuscript. Detailed responses to your comments are provided below, and all revisions and corrections are highlighted in the revised manuscript.

Q1. The presentation is characterized by an exceptionally large number of figures and quantitative comparisons, which makes it difficult to clearly identify the key message. Authors should add a simpler explanation of what they demonstrate in the figures, especially in the first party (figure 1-8) or moving selected data to supplementary materials.

A1. We appreciate your valuable feedback. As you pointed out, this paper contains a variety of experimental results and quantitative comparisons, and we recognize that the core message may be somewhat complex to convey in the early sections (Figures 1–8).

Consequently, repetitive or supplementary figures—Figure 2 (validation data for standard calibration), Figure 4 (LC–MS/MS spectra), Figure 6 (purity validation via TLC), Figure 7 (purity validation via HPLC), and Figure 8 (LC–MS/MS spectra)—have been moved to the Supplementary Materials, and the Supplementary Materials file will be submitted separately. As a result, the figure numbering in the revised manuscript differs from that in the original manuscript.

In addition, Phytochemical Analysis part in the Results section of the revised manuscript has been thoroughly revised and described more concisely, and the legends of Figures 1 and 3 (Figure 2 in the revised manuscript) have been modified to clearly include the main conclusions of the corresponding experiments. We hope these revisions will contribute to improving the readability and logical flow of the paper.

Q2. While the in vitro data is extensive, the conclusions occasionally suggest translational or therapeutic relevance that would require in vivo validation, which is currently lacking.

A2. Thank you for highlighting this important point. From the study design stage, the present work was intended to focus on elucidating protective effects and related mechanisms at the level of skeletal muscle cells based on in vitro experiments. As correctly pointed out by the reviewer, in vivo validation is beyond the scope of this study.

Accordingly, in the revised manuscript, statements in the Discussion and Conclusion section that implied preclinical or therapeutic applicability were comprehensively re-evaluated, and expressions that could lead to overinterpretation were revised or removed.

In addition, based on the results obtained in this study, we plan to conduct further validation experiments using in vivo models of sarcopenia in a stepwise manner. Through these future studies, we aim to more comprehensively verify the effects and mechanisms proposed herein. In this context, the limitations of in vitro studies and the necessity for in vivo validation have been clearly addressed in the revised Discussion section (line 590–596).

Through these revisions, we have carefully avoided overinterpreting the study results and refined the conclusions by limiting their interpretation to the level supported by the current data.

Q3. There are numerous minor grammatical errors and typographical issues (e.g., spacing, repeated symbols, encoding problems). Careful language editing by a native English speaker is recommended.

A3. Thank you for your careful review and valuable comments. The authors acknowledge that minor grammatical errors and typographical issues (such as spacing inconsistencies, duplicated symbols, or encoding-related problems) may be present throughout the manuscript. We fully agree on the necessity of English language editing.

Accordingly, in the revised manuscript, we have carefully reviewed and corrected grammatical and stylistic issues to the best of our ability at this stage.

However, as the manuscript is currently undergoing multiple rounds of revision, further content and structural modifications are expected at this stage. In addition, professional English language editing will require additional time. Therefore, we kindly ask for the reviewer’s understanding that comprehensive language polishing could not be fully completed at the present revision stage.

We assure the reviewer that the manuscript will be edited using a native-level English editing service provided by MDPI prior to final publication, in order to enhance linguistic accuracy, clarity, and readability.

Q4. Authors should refresh the bibliography: they add 40% of manuscript published before 2017. Except for the manuscript relevant for the published work, they should add or substitute them with more recent publications. For example, in the introduction section they refer to statistic data of 2014 and 2016, does not exist any other more recent data?

A4. Thank you for your careful and insightful comment. In response to this suggestion, we have updated the statistical and epidemiological background in the Introduction using more recent literature.

In the revised manuscript, we have reviewed and updated several older statistical sources (e.g., data from 2014 and 2016) cited in the Introduction section by incorporating more recently published epidemiological data and review articles. In particular, we have strengthened the background by adding recent literature addressing the prevalence of sarcopenia, its socioeconomic burden, pathophysiology, and current research trends, thereby ensuring that the study context more accurately reflects the current state of the field (line 59-73).

Meanwhile, certain earlier references that are directly related to the conceptual definitions, mechanistic foundations, or well-established experimental models were retained, as they continue to hold high relevance and authoritative value in this research area. By applying these criteria, we have revised the reference list to enhance both the timeliness and overall coherence of the manuscript.

Q5. Vehicle controls should be clearly described for all treatments, especially if authors used different solvents for extracts and isolated compounds.

A5. Thank you for this valuable comment. The Rhododendron mucronulatum branch extract (RMB) and the isolated single compounds (Tax-G and Tax-A) used in this study were prepared as dried powders after the complete removal of organic solvents. The sample treatment protocol was based on experimental procedures established in our previous studies [1–2]. For the cell-based experiments, all samples were uniformly dissolved in DMSO, and the final DMSO concentration in the culture medium was kept consistent across all experimental groups.

Numerous previous in vitro studies using C2C12 cells have demonstrated that a final DMSO concentration of ≤0.1% is widely accepted as an appropriate vehicle condition that does not significantly affect cell viability or key muscle-related parameters [3–7].

Accordingly, DMSO was consistently used as the vehicle in this study, with the final DMSO concentration maintained uniformly across all treatment groups at a level deemed unlikely to affect data interpretation. Under these experimental conditions, potential vehicle-related effects were minimized, and all treatment groups were compared and analyzed relative to their respective controls (H₂O₂-treated control or dexamethasone-treated control).

In response to the reviewer’s comment, this information has been clarified more explicitly in the Methods section of the revised manuscript (4.5).

1. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
2. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339.
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6. Yakabe, M.; Hosoi, T.; Sasakawa, H.; Akishita, M.; Ogawa, S. Kampo formula hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang, TJ-41) ameliorates muscle atrophy by modulating atrogenes and AMPK in vivo and in vitro. BMC Complementary Medicine and Therapies 2022, 22(1), 341.

Q6. Positive controls for apoptosis inhibition or muscle protection (e.g., known antioxidants or Akt/mTOR activators) are not included. Their inclusion would strengthen the interpretation of efficacy.

A6. Thank you for this insightful comment. As the reviewer correctly pointed out, a limitation of the present study is the absence of positive control, such as well-established antioxidants or compounds known to activate the Akt/mTOR signaling pathway, to evaluate anti-apoptotic or muscle-protective effects.

However, there are currently few commercially established natural product–based positive controls that are widely accepted for muscle atrophy and muscle loss models. We recognized that defining a specific reference compound for relative efficacy comparison could introduce additional limitations. Therefore, rather than focusing on direct comparisons with a positive control, we adopted an experimental design in which normal and negative controls were clearly defined, and the effects of each treatment were statistically evaluated relative to these controls.

This approach was designed to objectively assess whether apoptosis- and muscle atrophy–related markers were significantly modulated by the test substances, thereby ensuring the scientific validity of data interpretation. Consistent with this rationale, numerous previous studies with similar research objectives have employed experimental designs comparing normal and negative controls to validate treatment effects [1–9].

Nevertheless, we agree with the reviewer’s suggestion, and the limitation regarding the absence of positive control, along with recommendations for future research, has been explicitly addressed in the Discussion section of the revised manuscript (line 591–593).

1. Chen, C.; Yang, J.S.; Lu, C.C.; Chiu, Y.J.; Chen, H.C.; Chung, M.I.; Wu, Y.T.; Chen, F.A. Effect of quercetin on dexamethasone-induced C2C12 skeletal muscle cell injury. Molecules 2020, 25(14), 3267.
2. Zhiyin, L.; Jinliang, C.; Qiunan, C.; Yunfei, Y.; Qian, X. Fucoxanthin rescues dexamethasone induced C2C12 myotubes atrophy. Biomedicine & Pharmacotherapy 2021, 139, 111590.
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4. Ceci, R.; Duranti, G.; Giuliani, S.; Rossi, M.N.; Dimauro, I.; Sabatini, S.; Mariottini, P.; Cervelli, M. The impact of spermidine on C2c12 myoblasts proliferation, redox status and polyamines metabolism under H2o2 exposure. International Journal of Molecular Sciences 2022, 23(19), 10986.
5. Mizugaki, A.; Kato, H.; Takeda, T.; Inoue, Y.; Hasumura, M.; Hasegawa, T.; Murakami, H. Cystine reduces mitochondrial dysfunction in C2C12 myotubes under moderate oxidative stress induced by H2O2. Amino Acids 2022, 54(8), 1203-1213.
6. Kim, Y.I.; Lee, H.; Nirmala, F.S.; Seo, H.D.; Ha, T.Y.; Jung, C.H.; Ahn, J. Antioxidant activity of Valeriana fauriei protects against dexamethasone-induced muscle atrophy.Oxidative Medicine and Cellular Longevity 2022, 2022.1: 3645431.
7. Kim, S.M.; Kim, J.Y.; Jun, E.M.; Jaiswal, V.; Park, E.J.; Lee, H.J. Mealworm hydrolysate ameliorates dexamethasone-induced muscle atrophy via sirtuin 1-mediated signaling and Akt pathway. npj Science of Food 2025, 9(1), 72
8. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
9. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339.

Q7. Figure 1: authors should better describe the figure. The meaning of the acronyms RMB RF1 and RMB RF2 is explained only in the subsequent paragraph, they have to anticipate something referring to figure 1.

A7. Thank you for this valuable comment. As pointed out by the reviewer, the abbreviations RMB RF1 and RMB RF2 used in Figure 1 were not sufficiently explained in the original figure legend, which may have hindered readers’ understanding.

Accordingly, in the revised manuscript, the definitions of RMB RF1 and RMB RF2 have been clearly described in the Results section when referring to Figure 1. In addition, the Figure 1 legend and the Abbreviations section have been revised to explicitly state that RF stands for Rich Fraction.

Q8. Figure 9: The observed increase in cell viability following RMB treatment under basal conditions is interpreted as a beneficial effect. However, it remains unclear whether this reflects enhanced proliferation, metabolic stimulation, or assay interference. Additional clarification or supportive experiments (e.g., proliferation markers or alternative viability assays) are required.

Moreover, authors should better explain why we can observe an increase in cell viability between 100 and 600 μg/mL with RMB treatment and then cell viability decreases drastically? Authors should analyze, for example, the cell cycle to better understand what happens to the cell with these higher concentrations.

A8. Thank you for this valuable comment. As the reviewer correctly noted, the increase in cell viability observed under normal conditions following RMB treatment may result from multiple factors, including enhanced cell proliferation, changes in metabolic activity, or assay-dependent signal variations. We acknowledge that the current data alone are insufficient to definitively attribute this effect to a specific mechanism.

However, the primary purpose of this experiment was not to evaluate the proliferative effects of RMB under normal conditions, but rather to determine a safe concentration range that does not induce cytotoxicity prior to oxidative stress– and muscle atrophy–inducing conditions and to confirm that cell viability is not adversely affected within this range. In this context, the increased cell viability observed under normal conditions was interpreted as indicating that RMB treatment maintains normal C2C12 cell viability or supports relatively higher survival levels.

In contrast, the marked decrease in cell viability observed at higher RMB concentrations aligns with the concentration-dependent responses commonly reported for many substances, including natural product extracts. This effect may be associated with non-specific cellular stress or an increased metabolic burden at elevated doses [1]. Similar reductions in cell viability at high concentrations have been documented in previous studies and are generally observed in conventional in vitro cell-based experiments [1–5].

The primary objective of this study was not to characterize proliferative responses under normal conditions but to evaluate whether RMB, Tax-G, and Tax-A exert protective effects on muscle cells under conditions inducing muscle loss and muscle atrophy. Therefore, detailed analyses of cell proliferation, cell cycle regulation, or metabolic activity under normal conditions were considered beyond the scope of this study.

Nevertheless, we agree with the reviewer’s suggestion, and these limitations have been more thoroughly addressed in the Discussion sections of the revised manuscript. We have also noted that additional analyses will be necessary in future studies to more clearly elucidate these aspects.

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Q9. Figure 10: authors should explain the choice of the concentration of 100uM of H₂O₂ and show us a curve demonstrating a dose dependent effect and the efficacy of H₂O₂ at the dose selected.

Why did authors decide to cotreat cells with H₂O₂ and compounds or DEX and compounds and they do not use for example a pretreatment with H₂O₂ or DEX? Please explain.

A9. Thank you for this insightful comment. We would like to clarify the rationale for selecting an H₂O₂ concentration of 100 μM. This concentration has been widely used in numerous previous studies employing similar experimental models [1–7]. In this study, treatment with 100 μM H₂O₂ resulted in approximately 50% cell viability relative to the control group, indicating that this condition induces substantial oxidative stress and apoptosis while preserving sufficient viability to evaluate protective effects. Accordingly, this level of damage was considered appropriate for evaluating cytoprotective effects.

The experimental conditions were established based on previously reported studies using the same cell line and comparable analytical endpoints. Although the reviewer's suggestion to show a dose–response analysis is reasonable, the aim of this study was not to quantitatively compare the extent of H₂O₂-induced damage but rather to evaluate the protective effects of RMB, Tax-G, and Tax-A under a well-established oxidative stress–induced apoptosis model. Therefore, dose–response analysis was beyond the scope of the present study and is more appropriately addressed in future investigations.

Furthermore, a co-treatment strategy was chosen instead of a pretreatment approach with H₂O₂ or dexamethasone (DEX) to evaluate the mitigating or protective effects of the test substances under conditions of ongoing cellular damage. This co-treatment design has been widely used in oxidative stress–induced apoptosis models and DEX-induced muscle atrophy models to assess cytoprotective effects. The present experimental design followed these established methodologies [1–5, 8–9].

Details regarding the experimental conditions and treatment strategies have been clarified more explicitly in the Methods sections (4.5 and 4.5.1) of the revised manuscript to enhance the clarity of the experimental design.

1. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339.
2. Lee, C.H.; Kwon, Y.e.; Park, S.m.; Kim, T.H.; Kim, M.S.; Kim, E.J.; Jung, J.I.; Min, S.g.; Park, K.-H.; Jeong, J.H.; Choi, S.E. The Impact of Ulmus macrocarpa extracts on a model of sarcopenia-induced C57BL/6 mice. International Journal of Molecular Sciences 2024, 25, 6197.
3. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
4. Jang, H.D.; Lee, C.H.; Kwon, Y.E.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.I.; Cheong, E.J.; Jang, T.Y.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Pilot Scale Hot Water Extracts on a Model of Dexamethasone-Induced Muscle Loss and Muscle Atrophy in C57BL/6 Mice. International Journal of Molecular Sciences 2025, 26, 3656.
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Q10. Figure 11: authors should explain the choice of the concentration of 5uM of DEX and show us a curve demonstrating a dose dependent effect and the efficacy of DEX at the dose selected.

Authors could add a higher dose of DEX to show if the compounds are able to revert the effects observed with this higher dose of DEX ad can restore the cell viability.

A10. Thank you for this valuable comment. The rationale for selecting a dexamethasone (DEX) concentration of 5 μM was to establish a condition that reliably induces muscle atrophy in C2C12 cells without causing cytotoxicity effects. This concentration has been widely used in previous studies as a standard condition for inducing muscle atrophy in C2C12 myotubes [1–7].

In the present study, treatment with 5 μM DEX did not significantly affect cell viability; however, myotube diameter was reduced by approximately 70% compared to the control group. These findings confirm that this treatment induces pronounced muscle atrophy without causing overt cytotoxic effects, supporting its suitability as an in vitro model for evaluating protective effects against muscle atrophy.

Although the reviewer’s suggestion to include a dose–response analysis and to examine higher concentrations of DEX is reasonable, the aim of this experiment was not to establish conditions that induce severe cellular damage or apoptosis. Instead, it was designed to assess the ability of RMB, Tax-G, and Tax-A to attenuate muscle atrophy using a well-established DEX-induced muscle atrophy model, employing multiple morphological and molecular indicators. Accordingly, dose–response analyses or the use of higher DEX concentrations were considered beyond the scope of the present study. Nevertheless, we agree that evaluating treatment effects under more severe atrophy conditions or conducting concentration-dependent analyses would be valuable.

In the revised manuscript, the rationale for the selection of the DEX concentration was clearly explained in the Methods section (4.5.2) by citing supporting references, thereby more effectively conveying the validity of the experimental design.

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2. Kim, J.Y.; Kim, H.M.; Kim, J.H.; Lee, J.H.; Zhang, K.; Guo, S.; Lee, D.H.; Gao,E.M.; Son, R.H.; Kim, S.M.; Kim, C.Y. Preventive effects of the butanol fraction of Justicia procumbens against dexamethasone-induced muscle atrophy in C2C12 myotubes. Heliyon 2022, 8.
3. Lee, M.K.; Ryu, H.; Jeong, H.H.; Lee, B. Meroterpenoid-rich fraction of the ethanol extract from Sargassum serratifolium suppressed muscle atrophy induced by dexamethasone in C2C12 myotubes and C57BL/6 mice. Diabetes 2023, 72 (Suppl. 1), 1639-P.
4. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339.
5. Lee, C.H.; Kwon, Y.e.; Park, S.m.; Kim, T.H.; Kim, M.S.; Kim, E.J.; Jung, J.I.; Min, S.g.; Park, K.-H.; Jeong, J.H.; Choi, S.E. The Impact of Ulmus macrocarpa extracts on a model of sarcopenia-induced C57BL/6 mice. International Journal of Molecular Sciences 2024, 25, 6197.
6. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
7. Jang, H.D.; Lee, C.H.; Kwon, Y.E.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.I.; Cheong, E.J.; Jang, T.Y.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Pilot Scale Hot Water Extracts on a Model of Dexamethasone-Induced Muscle Loss and Muscle Atrophy in C57BL/6 Mice. International Journal of Molecular Sciences 2025, 26, 3656.

Q11. Figure 13A: the western blot shows that Bax increase with 5-10 ug/ml of Tax-A and decrease with 50ug/ml, authors should explain this data. Probably authors should change the picture or repeat the experiment.

A11. Thank you for this valuable comment. As the reviewer correctly noted, in Figure 13A(Figure 8 in the revised manuscript), the Bax band intensity in the Tax-A–treated groups appears increased at 5–10 μg/mL, whereas a decrease is observed at 50 μg/mL, which may lead to ambiguity in interpretation.

However, the changes in Bax protein levels observed at the 5–10 μg/mL were not statistically significant, and no clear dose-dependent pattern was identified. Therefore, the variability in Bax protein levels was not considered a meaningful change based on statistical analysis, and we did not interpret Tax-A as significantly modulating Bax expression under in vitro conditions.

It is well established that, in Western blot analyses for apoptosis assessment, the Bax/Bcl-2 ratio is generally considered a more informative indicator than changes in Bax or Bcl-2 expression alone [1–3]. Consistent with this approach, the present study emphasized the overall Bax/Bcl-2 balance, along with other key apoptosis-related markers, including caspase-3 and PARP, rather than relying solely on Bax expression.

Notably, statistically significant and consistent changes were observed in multiple apoptosis-related biomarkers other than Bax, supporting the conclusion that the anti-apoptotic effects described in this study are based on an integrated assessment of several relevant indicators rather than a single marker. This finding aligns with our previously reported in vitro studies, in which the test substances did not significantly alter Bax expression but effectively suppressed apoptosis by modulating multiple apoptosis-related factors [4–5].

Although we fully agree with the reviewer’s concern, repeating this specific experiment immediately during the current revision was not feasible. Considering these limitations, additional validation will be prioritized in future studies. To avoid overinterpretation of the Bax results, the interpretation of the Bax data and the need for further investigation have been clearly described in the Discussion section of the revised manuscript.

1. Zhang, Y.E.; Huang, G.Q.; Wu, B.; Lin, X.D.; Yang, W.Z.; Ke, Z.Y.; Liu, J. Hydrogen sulfide protects H9c2 cardiomyoblasts against H₂O₂-induced apoptosis. J. Med. Biol. Res. 2019, 52, e7626.
2. Siu, P.M.; Wang, Y.; Alway, S.E. Apoptotic signaling induced by H₂O₂-mediated oxidative stress in differentiated C2C12 myotubes. Life Sci. 2009, 84, 468–481.
3. Kim, S.M.; Kim, J.Y.; Jun, E.M.; Jaiswal, V.; Park, E.J.; Lee, H.J. Mealworm hydrolysate ameliorates dexamethasone-induced muscle atrophy via sirtuin 1-mediated signaling and Akt pathway. npj Science of Food 2025, 9(1), 72.
4. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339
5. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.

Q12. Figure 15: authors should enrich the data reported in this figure adding the corresponding fluorescent cell images of each condition.

A12. Thank you for this valuable suggestion. As recommended by the reviewer, we agree that including representative fluorescence cell images for each treatment condition would enhance the visual clarity and interpretability of Figure 10 (in the revised manuscript).

Accordingly, in the revised manuscript, Figure 10 has been supplemented with representative fluorescence images corresponding to each treatment condition. We believe that this addition will facilitate clearer interpretation of the data presented in Figure 10.

Q13. Table 1-3: Authors evaluate the effects of the compounds on muscle degradation and synthesis through protein and mRNA analysis of the major proteins involved (Atrogin-1 and MuRF1, MyoD1 and Myogenin). Authors should add the mRNA analysis also for apoptosis markers that are analyzed only at protein level.

A13. Thank you for this valuable comment. As the reviewer correctly noted, in the present study, key regulators of muscle protein degradation and synthesis (Atrogin-1, MuRF1, MyoD1, and Myogenin) were analyzed at both the protein and mRNA levels, while apoptosis-related markers were primarily evaluated at the protein level.

This approach was adopted because the regulators of muscle protein degradation and synthesis, which are directly associated with changes in muscle mass, represent the core indicators of this study. Accordingly, these factors were analyzed at both transcriptional and protein levels to enhance the reliability of the findings.

In contrast, although apoptosis of muscle cells is a significant factor contributing to the progression of sarcopenia, numerous previous studies have primarily assessed apoptosis at the protein level rather than at the mRNA level [1–7]. Consistent with these precedents, the present study prioritized the evaluation of key apoptosis-related biomarkers (Bax, Bcl-2, caspase-3, and PARP) that are directly associated with apoptotic processes using Western blot analysis.

Nevertheless, we appreciate the reviewer’s suggestion and agree that incorporating mRNA-level analyses of apoptosis-related markers would provide a more comprehensive understanding of the regulatory mechanisms involved in this pathway. This limitation has been recognized in the present study and addressed in the Discussion section of the revised manuscript, with a recommendation for further investigation in future research.

1. Zhang, Y.E.; Huang, G.Q.; Wu, B.; Lin, X.D.; Yang, W.Z.; Ke, Z.Y.; Liu, J. Hydrogen sulfide protects H9c2 cardiomyoblasts against H₂O₂-induced apoptosis. J. Med. Biol. Res. 2019, 52, e7626.
2. Wei, Z.J.; Sun, L.; Li, Y.L.; Muhammad, J.S.; Wang, Y.; Feng, Q.W.; Zhang, Y.Z.; Inadera, H.; Cui, H.G.; Wu, H.A. Low-calorie sweetener D-psicose promotes hydrogen peroxide-mediated apoptosis in C2C12 myogenic cells favoring skeletal muscle cell injury. Med. Rep. 2021, 24, 536.
3. Antinozzi, C.; Duranti, G.; Ceci, R.; Lista, M.; Sabatini, S.; Caporossi, D.; Luigi, L.D.; Sgrò, P.; Dimauro, I. Hydrogen peroxide stimulates dihydrotestosterone release in C2C12 myotubes: A new perspective for exercise-related muscle steroidogenesis? J. Mol. Sci. 2022, 23, 6566.
4. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
5. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339
6. Lee, C.H.; Kwon, Y.e.; Park, S.m.; Kim, T.H.; Kim, M.S.; Kim, E.J.; Jung, J.I.; Min, S.g.; Park, K.-H.; Jeong, J.H.; Choi, S.E. The Impact of Ulmus macrocarpa extracts on a model of sarcopenia-induced C57BL/6 mice. International Journal of Molecular Sciences 2024, 25, 6197.
7. Jang, H.D.; Lee, C.H.; Kwon, Y.E.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.I.; Cheong, E.J.; Jang, T.Y.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Pilot Scale Hot Water Extracts on a Model of Dexamethasone-Induced Muscle Loss and Muscle Atrophy in C57BL/6 Mice. International Journal of Molecular Sciences 2025, 26, 3656.

Q14.  Figure 18-20: authors write in the discussion that: “These results are consistent with the mechanism by which DEX inhibits the Akt/mTOR pathway, a muscle synthesis signal, while activating the FoxO pathway, a muscle atrophy signal, leading to activation of the ubiquitin–proteasome system and sup- pression of muscle synthesis regulators. The RMB, Tax-G, and Tax-A treatment groups inhibited the expression of muscle atrophy factors FoxO3α, Atrogin-1, and MuRF1, while restoring the expression of muscle synthesis factors Akt, mTOR, MyoD, and Myogenin”.

This conclusion is mainly due to the results and changes in phosphorylation rate of the proteins analyzed. However, no functional or pharmacological validation (e.g., pathway inhibitors, siRNA knockdown) is provided. As a result, the causal relationship between RMB/Tax-G/Tax-A treatment and Akt/mTOR or FoxO3α signaling remains associative rather than mechanistically demonstrated.

Authors should add one of the experiments suggested and better explained this correlation in the discussion section.

A14. Thank you for your valuable feedback. As the reviewer pointed out, the interpretations of the Akt/mTOR and FoxO pathways presented in Figures 18–20 in the original manuscript are primarily based on changes in the phosphorylation of related proteins, and functional validation using pathway inhibitors or siRNA was not included in this study. Accordingly, the authors acknowledge that the results suggest an association between treatment with the three experimental substances and changes in the activity of these signaling pathways, rather than providing direct mechanistic causal evidence.

Importantly, the evaluation strategy employed in this study is consistent with the guidelines for the evaluation of muscle function improvement provided by the Korean Ministry of Food and Drug Safety (MFDS) (answer sheet Figure 1). The approach of interpreting muscle atrophy or protective effects based on phosphorylation changes in the Akt/mTOR and FoxO pathways has been widely used as an initial mechanistic exploration step in in vitro studies related to muscle atrophy and sarcopenia (1–8). Therefore, in this study, we interpreted the potential regulation of pathway activity based on phosphorylation changes, referencing existing literature.

However, we fully agree with the reviewer’s comment and clearly recognize that the findings of the present study have limitations in definitively elucidating the mechanistic causal relationships underlying the regulation of the Akt/mTOR and FoxO pathways and muscle atrophy. Accordingly, in the revised manuscript, we have avoided definitive interpretations of these results in both the Results and Discussion sections. Specifically, in the revised Discussion section (line 568–572), we have explicitly described the lack of functional and pharmacological validation as a limitation of this study. We have also noted that further functional validation, including the use of pathway inhibitors or gene-level approaches, is necessary in future research.

Figure 1. Biomarkers for the evaluation of muscle function improvement proposed by the Korean Ministry of Food and Drug Safety (MFDS). According to these guidelines, oxidative stress markers, apoptosis-related factors, and biomarkers associated with muscle protein synthesis and degradation (including Akt/mTOR, FoxO, MuRF1, and Atrogin-1) are recommended as validated indicators for assessing muscle health and functional improvement in preclinical in vitro and in vivo models.

1. Kim, J.Y.; Kim, H.M.; Kim, J.H.; Lee, J.H.; Zhang, K.; Guo, S.; Lee, D.H.; Gao,E.M.; Son, R.H.; Kim, S.M.; Kim, C.Y. Preventive effects of the butanol fraction of Justicia procumbens against dexamethasone-induced muscle atrophy in C2C12 myotubes. Heliyon 2022, 8.
2. Wang, P.; Kang, S.Y.; Kim, S.J.; Park, Y.K.; Jung, H.W. Monotropein improves dexamethasone-induced muscle atrophy via the AKT/mTOR/FOXO3a signaling pathways. Nutrients 2022, 14, 1859.
3. Kim, J.Y.; Kim, H.M.; Kim, J.H.; Guo, S.; Lee, D.H.; Lim, G.M.; Kim, W.; Kim, C.Y. Salvia plebeia Br. and rosmarinic acid attenuate dexamethasone-induced muscle atrophy in C2C12 myotubes. Int. J. Mol. Sci. 2023, 24, 1876.
4. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339
5. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
6. Lee, C.H.; Kwon, Y.e.; Park, S.m.; Kim, T.H.; Kim, M.S.; Kim, E.J.; Jung, J.I.; Min, S.g.; Park, K.-H.; Jeong, J.H.; Choi, S.E. The Impact of Ulmus macrocarpa extracts on a model of sarcopenia-induced C57BL/6 mice. International Journal of Molecular Sciences 2024, 25, 6197.
7. Jang, H.D.; Lee, C.H.; Kwon, Y.E.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.I.; Cheong, E.J.; Jang, T.Y.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Pilot Scale Hot Water Extracts on a Model of Dexamethasone-Induced Muscle Loss and Muscle Atrophy in C57BL/6 Mice. International Journal of Molecular Sciences 2025, 26, 3656.
8. Kim, S.M.; Kim, J.Y.; Jun, E.M.; Jaiswal, V.; Park, E.J.; Lee, H.J. Mealworm hydrolysate ameliorates dexamethasone-induced muscle atrophy via sirtuin 1-mediated signaling and Akt pathway. npj Science of Food 2025, 9(1), 72

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript describes the preparation of Rhododendrom mucronulatum branch extract and their active compounds, Taxiforlin-3-O-aravinopyranoside and its aglycone, Taxifolin, and their biological activities. Authors provided very details of isolation and purification of the materials and identification of the compounds is clearly established. In activity part, this manuscript investigated various apsects of muscle atrophy and many related markers. The study was quite comprehensive; the effects on cell viability, apoptosis and muscle atrophy were beneficial. The anti- muscle atrophy activity was studied at mRNA and protein level.  

I think this manuscript can be suitable for publication only after authors address the following points. Every point should be addressed fully and clearly.

(1) Figure 15.
A: Y-axis mentions as myotube diameter but the author used percentage in the result (line 358). I recommend to convert Myotube diameter at Y-axis to the percent as the author described in the result. The number in the result's paragraphs should match with the bar graph. 
B: In the method, the author mentioned that the measurement of myotubes diameter was based on the immunofluorescent assay. Could the author also include fluorescent cell images in this figures?  

(2) In Figure 16, 17, 18, 19 and 20. Y-axis of bar graph is mentioned as "percent of control" but the scale is not the percent. Please change all Y-axis to the percentage according to the explaination in the result's paragraphs. The number in the result's paragraphs should match with the bar graph.

(3) In the result 2.5.2. (line 368), the author mentioned "myotube" but the figure legend of both figure 16 and 17 is wroten "myoblast". Please clarify the information.    

(4) In every western blot's results, does the author normalized the inthensity of each target protein's band with b-actin, or only comparing with untreated control? 

(5) Overall of western blot's results, the beta-actin's band in many membranes looks like too high signal. The bands were over bright and became white color with black cornor. I do recommend to select another picture if it's applicable. 

(6) The mRNA expression via RT-PCR, what is the value that the author used and showed in the table 1-3? Is it Ct value (Cycle Threshold), PCR Efficiency or relative quantification? Please explain how the author quatitate and calculate these value in the method part and also mention at the table's legends. 

(7) In the result's paragraph (2.5.3.) The author explains and compares group-to-group using the relative percent of expression comparing to control. However, these values are not presented in any figure or table. I recommend the author to describe the result using the value from the table 1-3 instread of the relative percent. Otherwise, replace the value in the table 1-3 with the relative percent of expression. Please use the same format between the result's paragraphs and the table. 

(8) In method, line 806, "C2C12 Myoblasts" >>> should it be myotube, as it's described in the following paragraph? 

(9) When expressing the concentrations of Tax A and Tax G, authors should do it in molar concentration, not ug/mL, since they are isolated single compounds and their molecular weights are determined.

(10) Line 24 or related lines, authors should include a reference for the use of 100 uM of H2O2  to induce apoptosis.

Author Response

Reviewer 2

We express our sincere gratitude for your careful evaluation of our manuscript. Comprehensive responses to your remarks are presented below, and all modifications and corrections have been clearly indicated in the revised version of the manuscript.

Q1. Figure 15.

A: Y-axis mentions as myotube diameter but the author used percentage in the result (line 358). I recommend to convert Myotube diameter at Y-axis to the percent as the author described in the result. The number in the result's paragraphs should match with the bar graph.

B: In the method, the author mentioned that the measurement of myotubes diameter was based on the immunofluorescent assay. Could the author also include fluorescent cell images in this figures?

1. As pointed out by the reviewer, although the Y-axis in Figure 10 (in the revised manuscript) was labeled as myotube diameter, the corresponding Results section described the changes as percentages relative to the control, which could potentially cause confusion. Accordingly, in the revised manuscript, the Y-axis of Figure 10 has been modified to percentages (% of control). Moreover, the presentation of the results was revised to focus on relative changes (%), thereby improving overall readability (line 337–342 in the revised manuscript).
2. In addition, considering that the myotube diameter measurements were performed based on immunofluorescence analysis, as described in the Methods section, representative fluorescence images for each treatment condition have been added to Figure 10 in the revised manuscript. These images were included to visually support and complement the quantitative results.

Through these revisions, we believe that the presentation and interpretation of Figure 10 have been clarified.

Q2. In Figure 16, 17, 18, 19 and 20. Y-axis of bar graph is mentioned as "percent of control" but the scale is not the percent. Please change all Y-axis to the percentage according to the explanation in the result's paragraphs. The number in the result's paragraphs should match with the bar graph.

A2. Thank you for this valuable comment. As pointed out by the reviewer, although the Y-axes in Figure 13-14 and 16–20 in original manuscript were labeled as “percent of control,” the actual tick marks were not displayed in a percentage format, resulting in insufficient consistency between the graphical presentation and the descriptions in the Results section.

Accordingly, in the revised manuscript, all Y-axis tick marks in Figure 8–9 and 11–15 have been modified to display values as percentage (% of control), and the corresponding descriptions in the Results section have been adjusted to ensure consistency with the revised graphs. These changes allow the data presented in each figure to be more clearly interpreted as relative changes compared with the control group.

We believe that these revisions improve the readability of Figure 8–9 and 11–15 and enhance the clarity of data interpretation.

Q3.  In the result 2.5.2. (line 368), the author mentioned "myotube" but the figure legend of both figure 16 and 17 is wroten "myoblast". Please clarify the information.    

A3. Thank you for your careful review. The typographical errors identified in the figure legends of Figure 11 and 12 (in the revised manuscript) have been corrected by replacing “myoblast” with “myotube.”

Q4. In every western blot's results, does the author normalized the intensity of each target protein's band with b-actin, or only comparing with untreated control? 

A4. Thank you for this valuable feedback. In all western blot analyses conducted in this study, the expression levels of target proteins were quantified after normalization to β-actin.

The relative expression of each target protein was calculated as the ratio to β-actin detected in the same experiment and subsequently expressed relative to the control group. Although this information was already stated in the legends of Figures 13–15, we recognized that the explanation was not sufficiently clear in the original manuscript. Accordingly, we revised the Methods section (4.7.2 and 4.8.3) and the figure legends (Figures 8–9 and 11–12) to explicitly state that normalization was performed using β-actin.

We expect this revision to provide clearer criteria for quantifying and interpreting the Western blot results.

Q5. Overall, of western blot's results, the beta-actin's band in many membranes looks like too high signal. The bands were over bright and became white color with black cornor. I do recommend to select another picture if it's applicable. 

A5. We appreciate your valuable feedback. As you pointed out, we acknowledge that in some Western blot results, the β-actin band signal appears relatively strong and may seem overexposed, and we fully understand the importance of this concern.

In this study, the figures were composed based on data in which the β-actin signal was most consistently detected among images obtained under the same experimental conditions. Reflecting the reviewer’s comments, we have selected and replaced Figure 8-9 and Figure 11-15 images with original data from the same experiments that show a relatively reduced level of overexposure.

However, unfortunately, the limited remaining amount of natural product samples used in this study posed practical constraints on performing additional Western blot experiments or obtaining entirely new images. Nevertheless, we have made every effort to ensure the objectivity and reproducibility of the data within these limitations.

β-actin was used as an internal control, and the expression levels of target proteins were quantitatively analyzed after normalization to β-actin as the reference protein. Therefore, we clarify that the conclusions of this study are based on the normalized quantitative analysis results rather than the visual intensity of the β-actin bands.

Q6. The mRNA expression via RT-PCR, what is the value that the author used and showed in the table 1-3? Is it Ct value (Cycle Threshold), PCR Efficiency or relative quantification? Please explain how the author quatitate and calculate these value in the method part and also mention at the table's legends. 

A6. Thank you for your valuable comment. As you have indicated, the values presented in Tables 1–3 from the RT-PCR analysis were not clearly clarified in the original manuscript as to whether they represent Ct values, PCR efficiency, or relative quantification values.

In the original version, Tables 1–3 included a brief statement in the table legends indicating that “the target mRNA’s expression was normalized to that of GAPDH”; however, a detailed description of the calculation method was missing from the Methods section (4.8.2).

Accordingly, in the revised manuscript, we have clearly added a description of the RT-PCR data calculation procedure in the Methods section. Specifically, after obtaining the Ct values through real-time PCR, the Ct value of the target gene was normalized to the Ct value of the reference gene (GAPDH), and the relative expression levels were calculated using the 2^−ΔΔCt method. All results are presented as percentages relative to the control group.

In addition, the legends of Tables 1–3 have been revised to explicitly state that the reported values represent relative expression normalized to GAPDH. These revisions were made to ensure an unambiguous interpretation of the RT-PCR results.

Q7. In the result's paragraph (2.5.3.) The author explains and compares group-to-group using the relative percent of expression comparing to control. However, these values are not presented in any figure or table. I recommend the author to describe the result using the value from the table 1-3 instead of the relative percent. Otherwise, replace the value in the table 1-3 with the relative percent of expression. Please use the same format between the result's paragraphs and the table. 

A7. Thank you for this valuable comment. As pointed out by the reviewer, in Section 2.5.3, group comparisons were described using relative expression percentages (%), whereas the corresponding values in the original tables were presented in a different format, resulting in a formatting inconsistency.

Accordingly, in the revised manuscript, the values in Table 1–3 have been converted and presented as relative expression percentages (% of the control group), and the table format has been unified with the description in the Results section. These revisions improve the consistency and readability between the tabulated data and the interpretation of the results.

Q8. In method, line 806, "C2C12 Myoblasts" >>> should it be myotube, as it's described in the following paragraph? 

A8. Thank you for this valuable comment. As pointed out by the reviewer, we identified an inconsistency in terminology between “C2C12 myoblasts” used in line 806 of the original Methods section (4.8.3.) and the subsequent use of the term “myotube.” In the revised manuscript, the terminology has been consistently corrected to clearly distinguish between the pre-differentiation and post-differentiation stages, using terms appropriate to each stage.

Q9.  When expressing the concentrations of Tax A and Tax G, authors should do it in molar concentration, not ug/mL, since they are isolated single compounds and their molecular weights are determined.

A9. Thank you for this valuable comment. As noted by the reviewer, because Tax-A and Tax-G are isolated single compounds, expressing their concentrations in molar units is theoretically more appropriate.

We acknowledge that the use of mass-based units for these compounds in the original manuscript resulted from an oversight during the manuscript editing process. In the revised manuscript, all such notations have been carefully reviewed and corrected to ensure accurate and consistent expression of concentration.

However, concentrations used for HPLC quantitative analysis were expressed in μg/mL, since chromatographic quantification and calibration curves are conventionally established on a mass basis, and this unit is appropriate for determining compound content.

Q10. Line 24 or related lines, authors should include a reference for the use of 100 uM of H₂O₂ to induce apoptosis.

A10. Thank you for this valuable comment. As pointed out by the reviewer, we recognized that the rationale for using 100 μM H₂O₂ as an apoptosis-inducing condition was not sufficiently supported by explicit literature citations in the original version.

Accordingly, in the revised manuscript (4.5.2), we have added relevant references reporting the use of 100 μM H₂O₂ to induce oxidative stress and apoptosis in C2C12 muscle cell models [1-7]. These previous studies demonstrated that treatment with 100 μM H₂O₂ induces reproducible apoptosis without causing excessive cell death under comparable experimental conditions.

We believe that these additions more clearly substantiate the rationale for selecting 100 μM H₂O₂ in the present study.

1. Kim, M.S.; Park, S.; Kwon, Y.; Kim, T.; Lee, C.H.; Jang, H.; Kim, E.J.; Jung, J.I.; Min, S.; Park, K.-H.; Choi, S.E. Effects of Ulmus macrocarpa extract and catechin 7-O-β-D-apiofuranoside on muscle loss and muscle atrophy in C2C12 murine skeletal muscle cells. Current Issues in Molecular Biology 2024, 46, 8320-8339.
2. Lee, C.H.; Kwon, Y.e.; Park, S.m.; Kim, T.H.; Kim, M.S.; Kim, E.J.; Jung, J.I.; Min, S.g.; Park, K.-H.; Jeong, J.H.; Choi, S.E. The Impact of Ulmus macrocarpa extracts on a model of sarcopenia-induced C57BL/6 mice. International Journal of Molecular Sciences 2024, 25, 6197.
3. An, D.H.; Lee, C.H.; Kwon, Y.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.; Cheong, E.J.; Kim, S.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Hot Water Extract and Oregonin on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells. Pharmaceuticals 2024, 17, 1661.
4. Jang, H.D.; Lee, C.H.; Kwon, Y.E.; Kim, T.H.; Kim, E.J.; Jung, J.I.; Min, S.I.; Cheong, E.J.; Jang, T.Y.; Kim, H.K.; Choi, S.E. Effects of Alnus japonica Pilot Scale Hot Water Extracts on a Model of Dexamethasone-Induced Muscle Loss and Muscle Atrophy in C57BL/6 Mice. International Journal of Molecular Sciences 2025, 26, 3656.
5. Zhang, Y.E.; Huang, G.Q.; Wu, B.; Lin, X.D.; Yang, W.Z.; Ke, Z.Y.; Liu, J. Hydrogen sulfide protects H9c2 cardiomyoblasts against H₂O₂-induced apoptosis. J. Med. Biol. Res. 2019, 52, e7626.
6. Wei, Z.J.; Sun, L.; Li, Y.L.; Muhammad, J.S.; Wang, Y.; Feng, Q.W.; Zhang, Y.Z.; Inadera, H.; Cui, H.G.; Wu, H.A. Low-calorie sweetener D-psicose promotes hydrogen peroxide-mediated apoptosis in C2C12 myogenic cells favoring skeletal muscle cell injury. Med. Rep. 2021, 24, 536.
7. Kang, J.S.; Kim, D.J.; Kim, G.Y.; Cha, H.J.; Kim, S.; Kim, H.S.; Park, C.; Hwang, H.J.; Kim, B.W.; Kim, C.M.; Choi, Y.H. Ethanol extract of Prunus mume fruit attenuates hydrogen peroxide-induced oxidative stress and apoptosis involving Nrf2/HO-1 activation in C2C12 myoblasts. Bras. Farmacogn. 2016, 26, 184–190.