Effects of Ulmus macrocarpa Extract and Catechin 7-O-β-D-apiofuranoside on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

While I found it very interesting to study the protective functions of plant extracts as a potential treatment for muscle atrophy, I found this manuscript confused in several sections and the results were not clearly described. In general, I found the structure of the text not flowing and not very understandable.

The aim of this study is to evaluate whether UME and catechin 7-O-b-D-apiofuranoside have protective effects on muscle atrophy, this point is not highlighted in the abstract. The focus of the study is the treatment, the evaluation of the expression of biomarkers associated with muscle apoptosis is a method to verify and confirm the thesis. Extensive editing of the English language is required throughout the text.

Using the term “apoptosis” is not properly correct in this context, muscle atrophy in general could be more appropriate. Rewriting the abstract section focusing on the aim, results, and conclusions could be an important improvement for the study.

The introduction should be rephrased to improve the flow of the text. 

 Why do the authors describe “aging” as a first point in the introduction? The focus of the study is muscle atrophy in general. I believe that in the introduction “muscle atrophy” as loss of muscle tissue should be better described. Moreover, “muscle atrophy” and “sarcopenia” are two different processes, and these terms cannot be used as synonyms. Moreover, it is not clear to me the description of UME and catechin. Is the catechin the active compound in UME extract? What is demonstrated by the authors in the recent study that they cited in line 59? What is meant for “Generally” in line 58?

In line 63 the authors write “Through numerous previous studies…”, at least two references should be added. 

Lines 68-79 could be rephrased to highlight the rationale and the focus of the study. 

Regarding the section of Materials and methods, I found it very confusing, and the methods are not adequately described. First of all, there are a lot of repetitions, cell culture and treatments must be described one time and the first paragraph should include: cell culture and treatment conditions in which growth and differentiation conditions must be defined in addition to explaining the treatments used (H2O2, dexamethasone, UME and catechin), then a section for the pilot scale, UME extraction and catechin purification should be added. 

Subsequently, the methods used for the analyses should be described i.e. MTT assay, protein extraction, quantification and WB analyses, RNA extraction and Real Time analysis.

Next, Measurements and Statistical analysis should be included. 

In this way, the section will be clear and understandable.

About the Results section, I found a lot of missing information and I believe that the results are not properly presented and explained. For instance, the paragraph 3.1 (Quantitative chromatographic analysis…) should be moved to the material section. The paragraph 3.2.1 should be the first paragraph of the results and the title should be more informative by introducing the result obtained (For example, UME improves cell viability in H2O2 treated cells). 

The analysis of cytotoxicity for UME and CAG should be presented as the first result, is important to verify the cytotoxicity of a potential treatment before using it. 

The WB results are not well presented, it is right to show the membrane, but it is difficult to evaluate the differences among the groups in this way. Please, insert a graph for each protein evaluated with the average for each group, the error bars, and statistical analysis. Moreover, in figure 7 the actin membrane is the same for all the panels (A-D), is this right? Please, add the marker for the molecular weight in all the membranes. 

The quality of the representative images in fig 10 is worse, please change the pictures and add the scale bar. 

In this form it is very difficult to understand the results obtained by the authors and to follow the discussion, first, the results should be presented in the right way and then the discussion could be rephrased. 

 

Comments on the Quality of English Language

Extensive editing of English language required

Author Response

While I found it very interesting to study the protective functions of plant extracts as a potential treatment for muscle atrophy, I found this manuscript confused in several sections and the results were not clearly described. In general, I found the structure of the text not flowing and not very understandable.

 

The aim of this study is to evaluate whether UME and catechin 7-O-β-D-apiofuranoside have protective effects on muscle atrophy; this point is not highlighted in the abstract. The focus of the study is the treatment, the evaluation of the expression of biomarkers associated with muscle apoptosis is a method to verify and confirm the thesis. Extensive editing of the English language is required throughout the text. Using the term “apoptosis” is not properly correct in this context, muscle atrophy in general could be more appropriate. Rewriting the abstract section focusing on the aim, results, and conclusions could be an important improvement for the study.

→ Thank you for this observation. We have revised the abstract to clearly state that the aim of the study is to develop a therapeutic agent to prevent muscle loss and atrophy using UME and catechin 7-O-β-D-apiofuranoside. We have also replaced the term “apoptosis” with “muscle atrophy” where appropriate and ensured the abstract focuses on the aim, results, and conclusions.

 

The introduction should be rephrased to improve the flow of the text. Why do the authors describe “aging” as a first point in the introduction? The focus of the study is muscle atrophy in general. I believe that in the introduction “muscle atrophy” as loss of muscle tissue should be better described. Moreover, “muscle atrophy” and “sarcopenia” are two different processes, and these terms cannot be used as synonyms.

 

→ Thank you for your feedback. We have revised the introduction to better describe muscle atrophy as the loss of muscle tissue. We have clarified the distinction between “muscle atrophy” and “sarcopenia” and refocused the introduction on muscle atrophy.

 

Moreover, it is not clear to me the description of UME and catechin. Is the catechin the active compound in UME extract? What is demonstrated by the authors in the recent study that they cited in line 59?

→ Through previous studies, we conducted a phytochemical analysis of UME using HPLC and NMR, demonstrating that the active extract of UME is catechin 7-O-β-D-apiofuranoside. We have added an explanation regarding this.

 

What is meant for “Generally” in line 58?

→ We have corrected the text to indicate that (-)-catechin, triterpene, and neolignan glycoside are the main constituents of Ulmus macrocarpa, eliminating any ambiguity.

 

In line 63 the authors write “Through numerous previous studies…”, at least two references should be added.

→ Thank you for your feedback. We have added the following references to support this statement:

1. Lee, C. H., et al. (2024). Chemotaxonomic significance of catechin 7-O-beta-D-apiofuranoside in Ulmus species native to Asia. Forest Science and Technology, 1-9.
2. Lee, C. H., et al. (2024). The Impact of Ulmus macrocarpa Extracts on a Model of Sarcopenia-Induced C57BL/6 Mice. International Journal of Molecular Sciences, 25(11), 6197.

 

Lines 68-79 could be rephrased to highlight the rationale and the focus of the study.

→ Thank you for your feedback. We have revised these lines to better highlight the rationale and focus of the study. Thank you for giving me the opportunity to edit again.

 

Regarding the section of Materials and Methods, I found it very confusing, and the methods are not adequately described. First of all, there are a lot of repetitions; cell culture and treatments must be described one time and the first paragraph should include: cell culture and treatment conditions in which growth and differentiation conditions must be defined in addition to explaining the treatments used (H2O2, dexamethasone, UME, and catechin), then a section for the pilot scale, UME extraction and catechin purification should be added. Subsequently, the methods used for the analyses should be described i.e., MTT assay, protein extraction, quantification and WB analyses, RNA extraction and Real-Time analysis. Next, Measurements and Statistical analysis should be included. In this way, the section will be clear and understandable.

→ Thank you for this observation. We have reorganized the Materials and Methods section to eliminate repetitions and improve clarity. The cell culture and treatment conditions, extraction and purification methods, and analytical methods are now clearly described in a logical order. Measurements and statistical analysis sections have also been included.

 

About the Results section, I found a lot of missing information and I believe that the results are not properly presented and explained. For instance, the paragraph 3.1 (Quantitative chromatographic analysis…) should be moved to the material section. The paragraph 3.2.1 should be the first paragraph of the results and the title should be more informative by introducing the result obtained (For example, UME improves cell viability in H2O2 treated cells).

→ We have moved the quantitative chromatographic analysis to the Materials section. The Results section has been restructured for better clarity and flow, with more informative titles.

 

The analysis of cytotoxicity for UME and CAG should be presented as the first result, is important to verify the cytotoxicity of a potential treatment before using it.

→ Thank you for your feedback. We have moved the cytotoxicity analysis to be the first result presented in the Results section.

 

The WB results are not well presented, it is right to show the membrane, but it is difficult to evaluate the differences among the groups in this way. Please, insert a graph for each protein evaluated with the average for each group, the error bars, and statistical analysis. Moreover, in figure 7 the actin membrane is the same for all the panels (A-D), is this right? Please, add the marker for the molecular weight in all the membranes.

→ Thank you for your feedback. We have included graphs for each protein evaluated, showing the average for each group, error bars, and statistical analysis. The actin membrane issue has been corrected, and molecular weight markers have been added to all membranes.

 

The quality of the representative images in fig 10 is worse, please change the pictures and add the scale bar.

→ We have improved the quality of the images in Figure 10 and added scale bars.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the opportunity to review this interesting report. The authors have collected a unique dataset to link extractions (UME and CAG) from Ulmus macrocarpa and muscle atrophy. Researching the effects of this plant component on muscle growth, development, and regeneration will greatly contribute to the development of biomedical materials and functional medicinal foods. s I read the manuscript, and I found some areas in which I would have appreciated greater clarity.

1. The introduction section discussed that Ulmus macrocarpa contains many constituents. However, it is necessary to cite references to clarify whether it is the plant components themselves or their metabolites that exert effects after being ingested and absorbed by the human digestive system. Can this be further explained by citing references?

2. Understanding the wording in line 78 (“natural compounds derived from muscle atrophy and muscle loss”) is rather difficult. Do you mean “UME” vs “muscle atrophy and muscle loss”?

3. Line 109-123: Should it be put in supplementary?

4. Some reagents, particularly antibodies, in the Materials and Methods section have specified supplier information while others have not. Please ensure consistency according to the journal's requirements.

5. Line 202-203: The study used GAPDH as an internal reference for RT-PCR, but it needs to be verified or clarified whether UME and CAG can alter GAPDH transcription levels by affecting energy metabolism.

6. Line 228: Isn’t “Fig. 2.” traditionally enclosed by a parenthesis?

7. Line 238-239: Please keep the concentration conditions consistent between the graph and figure legend.

8. Line 245: I suggest “added” instead of “treated”, or “cells were treated”

9. Figure 5A and B: Please mark the significance between different groups.

10. Please specify in the legend or materials and methods section the technical and biological replicates. (replicate=?)

11. Line 262: “treated with”, a confusing description.

12. Line 266-267: sentence should be optimized.

13. Figure 6A: Please mark the significance between different groups.

14. Line 274-275: It would look cleaner to specify as “[H₂O₂(-)/UME(-)] group” instead of  “[(-)/(-)] group” .

15. Result 3.2.2: When detecting the expression of apoptosis-related proteins at the molecular level, apoptosis detection should also be conducted at the cellular level. (e.g. Annexin V-PI, TUNEL)

16. Line 299-301: should it be “Bax (A) did not show a significant difference between the group without H2O2 treatment and groups treated with varying concentrations of CAG along with H2O2”?

17. This study involved a large number of Western blot experiments, but many experiments shared the same Actin as a reference, which may affect the transparency of the results. Please submit SDS-PAGE gel images from different batches of replicates and provide explanations to ensure the reliability of the experimental results.

18. Figure 10A and B: The picture quality is too low and lacks scale bar, and cell differentiation ability is usually measured using differentiation or fusion index.

19. Line 420-422: “Atrogin-1 increased by dexamethasone decreased”, a confusing description.

20. Line 483: “FoxO Signaling Pathway-Related Muscle Protein Muscle Expression Investigation”, remove the second “Muslce”.

Comments on the Quality of English Language

The English expression quality of the manuscript is generally smooth and easy to understand, but there are also some confusing descriptions that may cause difficulties for readers (examples are provided in the review comments).

Author Response

I appreciate the opportunity to review this interesting report. The authors have collected a unique dataset to link extractions (UME and CAG) from Ulmus macrocarpa and muscle atrophy. Researching the effects of this plant component on muscle growth, development, and regeneration will greatly contribute to the development of biomedical materials and functional medicinal foods. s I read the manuscript, and I found some areas in which I would have appreciated greater clarity.

 

1. The introduction section discussed that Ulmus macrocarpa contains many constituents. However, it is necessary to cite references to clarify whether it is the plant components themselves or their metabolites that exert effects after being ingested and absorbed by the human digestive system. Can this be further explained by citing references?

→ Your concerns are completely understandable. We have added references to clarify that the metabolites of plant components exert effects after being ingested and absorbed by the human digestive system:

1. Nikawa, T., Ulla, A., & Sakakibara, I. (2021). Polyphenols and their effects on muscle atrophy and muscle health. Molecules, 26(16), 4887.
2. Yin, M. C., Lin, M. C., Mong, M. C., & Lin, C. Y. (2012). Bioavailability, distribution, and antioxidative effects of selected triterpenes in mice. Journal of agricultural and food chemistry, 60(31), 7697-7701.
3. Understanding the wording in line 78 (“natural compounds derived from muscle atrophy and muscle loss”) is rather difficult. Do you mean “UME” vs “muscle atrophy and muscle loss”?

→ Thank you for your feedback. We confirmed that the wording was awkward and consequently revised the overall content for clarity.

 

3. Line 109-123: Should it be put in supplementary?

→ The data demonstrating that the compound isolated and purified from Ulmus macrocarpa is CAG is essential and should be included in the main text.

 

4. Some reagents, particularly antibodies, in the Materials and Methods section have specified supplier information while others have not. Please ensure consistency according to the journal's requirements.

→ Thank you for your feedback. We have reviewed the Materials and Methods section and added supplier information for all reagents and antibodies to ensure consistency.

 

5. Line 202-203: The study used GAPDH as an internal reference for RT-PCR, but it needs to be verified or clarified whether UME and CAG can alter GAPDH transcription levels by affecting energy metabolism.

→ We have verified that UME and CAG do not significantly alter GAPDH transcription levels. Preliminary experiments confirmed that GAPDH is a stable internal control under our experimental conditions. We have also added this information to the table legend.

 

6. Line 228: Isn’t “Fig. 2.” traditionally enclosed by a parenthesis?

→ Thank you for making the correction. We have revised the text to enclose “Fig. 2.” in parentheses, as suggested.

 

7. Line 238-239: Please keep the concentration conditions consistent between the graph and figure legend.

→ Thank you for making the correction. We have revised the text to ensure consistency between the graph and figure legend.

 

8. Line 245: I suggest “added” instead of “treated”, or “cells were treated”

→ Thank you for making the correction. We have changed the wording to “added” for clarity.

 

9. Figure 5A and B: Please mark the significance between different groups.

→ Thank you for your feedback. We have updated the graphs to include significance markers between different groups.

 

10. Please specify in the legend or materials and methods section the technical and biological replicates. (replicate=?)

→ We have added the number of technical and biological replicates to all figure legends and the Materials and Methods section.

 

11. Line 262: “treated with”, a confusing description.

→ Thank you for making the correction. We have removed the confusing description.

 

12. Line 266-267: sentence should be optimized.

→ Thank you for your feedback. We have optimized the sentence for clarity.

 

13. Figure 6A: Please mark the significance between different groups.

→ We have added significance markers to Figure 6A.

 

14. Line 274-275: It would look cleaner to specify as “[H2O2(-)/UME(-)] group” instead of “[(-)/(-)] group” .

→ Thank you for your feedback. We have revised the text to “[H2O2(-)/UME(-)] group”.

 

15. Result 3.2.2: When detecting the expression of apoptosis-related proteins at the molecular level, apoptosis detection should also be conducted at the cellular level. (e.g. Annexin V-PI, TUNEL)

→ Thank you for your feedback. We have conducted MTT assay experiments at the cellular level and added this data to section 3.2.1. Unfortunately, since the experiment has already been completed, it is difficult to incorporate your suggestions at this time. However, we will certainly consider your feedback for future experiments. Thank you very much for your valuable input.

 

16. Line 299-301: should it be “Bax (A) did not show a significant difference between the group without H2O2 treatment and groups treated with varying concentrations of CAG along with H2O2”?

→ Thank you for making the correction. We have revised the text as suggested.

 

17. This study involved a large number of Western blot experiments, but many experiments shared the same Actin as a reference, which may affect the transparency of the results. Please submit SDS-PAGE gel images from different batches of replicates and provide explanations to ensure the reliability of the experimental results.

→ We will provide the original SDS-PAGE gel images as supplementary data.

 

18. Figure 10A and B: The picture quality is too low and lacks scale bar, and cell differentiation ability is usually measured using differentiation or fusion index.

→ Thank you for your feedback. We have added a scale bar to the images and updated them with higher quality versions.

 

19. Line 420-422: “Atrogin-1 increased by dexamethasone decreased”, a confusing description.

→ Thank you for your feedback. We have revised the text for clarity.

 

20. Line 483: “FoxO Signaling Pathway-Related Muscle Protein Muscle Expression Investigation”, remove the second “Muslce”.

→ Thank you for making the correction. I have made the necessary corrections

Author Response File: Author Response.pdf