Phenolic Acid Profile and In Vitro Antioxidant and Anticholinesterase Activities of Romanian Wild-Grown Acer spp. (Sapindaceae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although the Introduction describes the context of this work compared to previous related studies, the following reference seems similar and could also be cited. A comparison of the results in this manuscript to those in this paper or other papers is suggested. Were the extraction conditions, HPLC method, and HPTLC method based on prior litertaure or developed by the authors? If based on prior methods, appropriate references need to be cited. Both the extraction and HPTLC avoided the use of chlorinated solvents and seem to be "green"; are these new approaches? It is not clear what information the HPTLC study provides?  The same compounds were separated and quantified by HPLC. Does the PCA plot represent important relationships not reported before? Its significance is unclear. On line 481, caffeic acid does not appear to be positioned positively with PC1. 

 

Polyphenol contents and radical scavenging capacities of red maple (Acer rubrum L.) extracts

M Royer, PN Diouf, T Stevanovic - Food and Chemical Toxicology, 2011 - Elsevier … Our results indicate potential utilisation of extracts as natural antioxidants. … Our results
demonstrate the richness in phenolic compounds (>100 mg/g of extract) of different red maple tree 

Author Response

Dear Reviewer,

First of all, we would like to address you many thanks for your accurate observations and valuable comments. We used all these and improved the paper accordingly.

All changes in the revised manuscript were marked up using the “Track Changes” function.

The following changes have been made for the Manuscript (ID: applsci-3423460):

 

Reviewer #1 questions/comments

Comments 1:

Although the Introduction describes the context of this work compared to previous related studies, the following reference seems similar and could also be cited.

Polyphenol contents and radical scavenging capacities of red maple (Acer rubrum L.) extracts. M Royer, PN Diouf, T Stevanovic - Food and Chemical Toxicology, 2011 - Elsevier… Our results indicate potential utilisation of extracts as natural antioxidants… Our results demonstrate the richness in phenolic compounds (>100 mg/g of extract) of different red maple tree.

Response 1:

Thank you very much for your suggestion. Ref. [11] (Royer et al., 2011) has been cited in the manuscript (See pages 2 & 16, lines 164, 178 & 906–907).

 

Comments 2:

A comparison of the results in this manuscript to those in this paper or other papers is suggested.

Response 2:

We appreciate your suggestion to include the additional reference, and we have now incorporated it into the manuscript – Ref. [11] (Royer et al., 2011). A comparison of the results of our study to those in this paper and other relevant literature has also been added to the “Discussion” section to provide a clearer context for our findings. (See pages 12, 13 & 16, lines 747–754 & 906–907).

 

Comments 3:

Were the extraction conditions, HPLC method, and HPTLC method based on prior literature or developed by the authors? If based on prior methods, appropriate references need to be cited.

Response 3:

Thank you very much for bringing this to our attention. The HPLC and HPTLC methods utilized in this study were developed in-house. For this reason, no references were cited for their development. A discussion on the limitations of the method validation has been added to “4.3. Study Limitation” section, addressing potential concerns about method reproducibility and standardization. (See page 14, lines 836–842).

 

Comments 4:

Both the extraction and HPTLC avoided the use of chlorinated solvents and seem to be “green”; are these new approaches?

Response 4:

Thank you very much for your observation. Indeed, both the extraction process and the HPTLC method avoided the use of chlorinated solvents, aligning with principles of green chemistry. While these methods are not entirely novel, they represent an intentional shift toward more sustainable approaches in phytochemical analysis. This point has been emphasized in the manuscript. (See sections “2.3. Sample Preparation” & “2.10. HPTLC–DPPH Fingerprint”).

 

Comments 5:

It is not clear what information the HPTLC study provides? The same compounds were separated and quantified by HPLC.

Response 5:

Thank you for pointing this out. The HPTLC study served multiple purposes in this research. Beyond its role as a rapid screening tool for identifying targeted phenolic acids across various extracts, it provided valuable insights into the distribution and relative abundance of these compounds. Notably, the HPTLC analysis highlighted that not all phenolic acids contribute equally to antioxidant activity. For example, p-coumaric acid was detected but did not show a positive response with the antioxidant assay, underscoring the nuanced relationship between individual phenolic acids and their bioactivity. This aspect has been clarified in the manuscript to emphasize the added value of HPTLC in complementing HPLC analysis. (See section “3.5. HPTLC–DPPH Fingerprint”).

 

Comments 6:

Does the PCA plot represent important relationships not reported before? Its significance is unclear. On line 481, caffeic acid does not appear to be positioned positively with PC1.

Response 6:

Thank you for your insightful comment. The PCA plot was included to provide a multivariate perspective on the relationship between phenolic acids and their associated biological activities. Specifically, the PCA analysis demonstrated the correlation between individual phenolic acids and antioxidant activities, helping to visualize trends and key contributors among the compounds. This analysis underscores the complex interplay between chemical composition and bioactivity, offering insights not readily apparent from single-dimensional analysis. For instance, the positioning of certain phenolic acids, such as caffeic acid and its positive association (on the PC2 axis) with antioxidant activity, contrasted with compounds like p-coumaric acid, highlights the variability in bioactive potential. (See pages 13 & 14, lines 772–789 & 790–824).

 

Authors very much appreciated the encouraging, critical, and constructive comments on this manuscript by the Reviewer. The comments have been very thorough and useful in improving the manuscript.

We would like to thank the Reviewer again for taking the time to review our manuscript.

 

Note from Assistant Editor

Comments:

There are some sections that have significant overlap with previously published articles, and we would therefore request rephrasing during the revision. We have highlighted these sentences and attached the file to this email.

Response:

Thank you very much for your observation. The sentences have been rephrased accordingly to remove the significant overlap with previously published articles. However, the names of institutions, manufacturers and analytical techniques could not be modified. (See pages 3–6, lines 317–321, 330–333, 356, 402–404, 429–432, 473–482 & 507–510).

 

We have also introduced other additions/modifications that we hope will improve the quality of the manuscript:

▪ Tables 1–5 have been renumbered accordingly: Tables 1–3, S1 and S2.

▪ Figures 5–11 have been renumbered accordingly: Figures 5–9, S1 and S2.

▪ One new citation has been introduced: Ref. [11] (Royer et al., 2011).

▪ The Reference list has been entirely checked and renumbered accordingly.

▪ All abbreviations have been defined the first time they appear in the text.

▪ Some grammar, stylistic or spelling errors have been corrected.

 

Kind regards,

Ludovic Everard BEJENARU, Associate Professor, PhD

Corresponding Author

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Title & abstract. Authors state that a polyphenol profile was assessed; however in line 24 they specified only phenolic acids. 

Abstract. Auhtors must include the main results obtained in this study.

Introduction. OK.

Materials & Methods. How many biological replicates were obtained rom each  specie? Authors must reconsider the use of the phrase 'For HPLC analysis, a comprehensive set of ...' Since the profiling was very limited. Authors must provide full validation parameters of the HPLC analysis as a supplementary table. Why authors assumed all variables were parametric?

Results. Authors must not repeat the description of the results already included in tables/figures. Authors must provide only a table or a figure for each results. For example the TPC values are included twice, in Table 2 and in Figure 1. This must be corrected in the whole paper. Section 3.4 must be moved after the description of the phenolic acids profile (Section 3.5). It is recommended to exclude Table 4 and Figures 8 and 9 from the main manuscript; these can be included as supplementary material. How where the standards selected? Are these compounds previously described as majoritarian of these plant species? Otherwise, authors must clearly describe the limitations of this analysis. Why authors did not include the plant species in the PCA plot? Authors must describe the explained variance in each component. Moreover, authors must clearly describe the limitations of this analysis.

Discussion. Lines 401-403 must be moved to the methodology section. 

Conclusions. OK

 

 

Author Response

Dear Reviewer,

First of all, we would like to address you many thanks for your accurate observations and valuable comments. We used all these and improved the paper accordingly.

All changes in the revised manuscript were marked up using the “Track Changes” function.

The following changes have been made for the Manuscript (ID: applsci-3423460):

 

Reviewer #2 questions/comments

Comments 1:

Title & abstract. Authors state that a polyphenol profile was assessed; however, in line 24 they specified only phenolic acids.

Response 1:

Thank you for your observation. We have clarified that our focus was on the profile and analysis of phenolic acids, a subclass of polyphenols. The term “polyphenol profile” has been adjusted to “phenolic acids profile” throughout the text to accurately reflect this focus, ensuring consistency and precision in our terminology. (See page 1, lines 2, 20, 24 & 25).

 

Comments 2:

Abstract. Authors must include the main results obtained in this study.

Response 2:

Thank you for your valuable suggestion. “Abstract” section has been rephrased accordingly (See page 1, lines 20–34).

 

Comments 3:

Introduction. OK.

Response 3:

Thank you very much for pointing this out.

 

Comments 4:

Materials & Methods. How many biological replicates were obtained from each specie? Authors must reconsider the use of the phrase ‘For HPLC analysis, a comprehensive set of...” Since the profiling was very limited. Authors must provide full validation parameters of the HPLC analysis as a supplementary table. Why authors assumed all variables were parametric?

Response 4:

Thank you very much for your suggestion. Regarding the number of biological replicates, these varied for each species based on geographical availability and environmental conditions and we did not consider this to be relevant to our work. Additionally, we agree with the suggestion to revise the phrasing related to our HPLC analysis. The term “comprehensive” has been removed to better reflect the scope of the profiling conducted. (See page 4, line 367).

As for the validation parameters of the HPLC analysis, we acknowledge this as an important consideration. However, since the primary objective of this study was not method validation but rather the profiling of phenolic acids, we followed in-house established protocols. While full validation parameters were not determined, we have acknowledged this as a limitation and noted it in the discussion. (See section “2.11. HPLC/UV/MS Analysis”, lines 483–504 and section “4.3. Study Limitation”, lines 836–842).

Finally, concerning the assumption of parametric variables, we relied on normality tests, which confirmed that the data followed a normal distribution. This rationale has been explicitly stated in the manuscript to ensure clarity and accuracy in our methodology. (See section “2.12. Statistical Analysis”).

 

Comments 5:

Results. Authors must not repeat the description of the results already included in tables/figures. Authors must provide only a table or a figure for each results. For example, the TPC values are included twice, in Table 2 and in Figure 1. This must be corrected in the whole paper. Section 3.4 must be moved after the description of the phenolic acids profile (Section 3.5). It is recommended to exclude Table 4 and Figures 8 and 9 from the main manuscript; these can be included as supplementary material. How where the standards selected? Are these compounds previously described as majoritarian of these plant species? Otherwise, authors must clearly describe the limitations of this analysis. Why authors did not include the plant species in the PCA plot? Authors must describe the explained variance in each component. Moreover, authors must clearly describe the limitations of this analysis.

Response 5:

Thank you for your thoughtful comments.

“Supplementary Materials” section and its corresponding files (which include Tables 2 and 4 as Tables “S1” and “S2”, and Figures 8 and 9 as Figures “S1” and “S2”) have been added. (See page 15, lines 868–872).

Section 3.4 has been moved after the description of the phenolic acids profile (Section 3.5) and the two sections have been renumbered accordingly. (See pages 9, 10 & 11, lines 606–629, 630–631, 632–647 & 652–666).

Regarding the selection of standards, there are very few studies in the literature analyzing these specific Acer spp. As a starting point, we selected some of the most common and widely studied phenolic acids, which provided a basis for comparison with other species. This approach allowed us to explore the chemical profile while acknowledging that these standards may not best represent the phenolic profile of these species. We have clarified this rationale in the manuscript and also acknowledged the limitations of this analysis. (See page 14, lines 836–842).

In response to the suggestion to include plant species in the PCA plot, we agree that this could provide additional insights. However, the statistical software we used for the PCA analysis did not offer the option to include plant species directly in this type of visualization. Our analysis instead focused on the relationship between phenolic acids, antioxidant activity, and enzyme inhibition. (See pages 13 & 14, lines 772–789 & 790–824).

We have also included the explained variance for each principal component in the manuscript to provide a clearer interpretation of the PCA. (See page 13, lines 782–787).

 

Comments 6:

Discussion. Lines 401-403 must be moved to the methodology section.

Response 6:

Thank you very much for your observation. The paragraph has been removed to “2.12. Statistical Analysis” section and rephrased accordingly. (See page 6, lines 507–510 & 515–517).

 

Comments 7:

Conclusions. OK

Response 7:

Thank you very much for pointing this out.

 

Authors very much appreciated the encouraging, critical, and constructive comments on this manuscript by the Reviewer. The comments have been very thorough and useful in improving the manuscript.

We would like to thank the Reviewer again for taking the time to review our manuscript.

 

Note from Assistant Editor

Comments:

There are some sections that have significant overlap with previously published articles, and we would therefore request rephrasing during the revision. We have highlighted these sentences and attached the file to this email.

Response:

Thank you very much for your observation. The sentences have been rephrased accordingly to remove the significant overlap with previously published articles. However, the names of institutions, manufacturers and analytical techniques could not be modified. (See pages 3–6, lines 317–321, 330–333, 356, 402–404, 429–432, 473–482 & 507–510).

 

We have also introduced other additions/modifications that we hope will improve the quality of the manuscript:

▪ Tables 1–5 have been renumbered accordingly: Tables 1–3, S1 and S2.

▪ Figures 5–11 have been renumbered accordingly: Figures 5–9, S1 and S2.

▪ One new citation has been introduced: Ref. [11] (Royer et al., 2011).

▪ The Reference list has been entirely checked and renumbered accordingly.

▪ All abbreviations have been defined the first time they appear in the text.

▪ Some grammar, stylistic or spelling errors have been corrected.

 

Kind regards,

Ludovic Everard BEJENARU, Associate Professor, PhD

Corresponding Author

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the manuscript under review claim to report, for the first time, the polyphenolic profile and the in vitroantioxidant and anticholinesterase activities of the leaves and fruits of Romanian wild-grown Acer species. However, I believe the article would improve significantly if the title were adjusted, as the work does not primarily focus on a mere description of phenolic content and antioxidant and anticholinesterase activities. Instead, the authors present a correlation between phenolic acids, total phenolic content (TPC), and the in vitro studies.

The title and abstract do not reflect the study's focus and specificity. As readers progress through the document, they are left with questions that are only addressed later. The title and abstract should unequivocally indicate that the profile is limited to phenolic acids (and specify which ones), that a TPC evaluation is performed, and that a correlation is established. They should not merely describe isolated results.
Given this specificity, including chromatograms seems excessive and out of context, especially considering the limited identification provided. They should be at SI.

I felt the lack of a significance analysis for the results obtained (TPC, DPPH, ABTS, FRAP, AChE inhibition). There is no indication of how many replicates were used for each assay, and I cannot find information on the mode (positive/negative) used for MS identification.

The authors state that "These findings highlight the multidimensional nature of phenolic-driven activities and the importance of exploring their combined effects rather than focusing solely on individual compounds." However, I believe the number of compounds studied does not constitute a metabolomic analysis and is somewhat limited, which may render some of the conclusions drawn overly broad. The discussion should be adjusted in light of this potential overstatement of scope.

I also felt the lack of a conclusion addressing the question: which species or species exhibit the highest bioactive properties in light of the studied attributes?

Author Response

Dear Reviewer,

First of all, we would like to address you many thanks for your accurate observations and valuable comments. We used all these and improved the paper accordingly.

All changes in the revised manuscript were marked up using the “Track Changes” function.

The following changes have been made for the Manuscript (ID: applsci-3423460):

 

Reviewer #3 questions/comments

The authors of the manuscript under review claim to report, for the first time, the polyphenolic profile and the in vitro antioxidant and anticholinesterase activities of the leaves and fruits of Romanian wild-grown Acer species. However, I believe the article would improve significantly if the title were adjusted, as the work does not primarily focus on a mere description of phenolic content and antioxidant and anticholinesterase activities. Instead, the authors present a correlation between phenolic acids, total phenolic content (TPC), and the in vitro studies.

Comments 1:

The title and abstract do not reflect the study’s focus and specificity. As readers progress through the document, they are left with questions that are only addressed later. The title and abstract should unequivocally indicate that the profile is limited to phenolic acids (and specify which ones), that a TPC evaluation is performed, and that a correlation is established. They should not merely describe isolated results. Given this specificity, including chromatograms seems excessive and out of context, especially considering the limited identification provided. They should be at SI.

Response 1:

Thank you very much for your insightful feedback. We have revised the title and abstract to clearly specify that the profile is focused on phenolic acids. The abstract also now explicitly mentions the evaluation of total phenolic content (TPC) and the correlations established with antioxidant and acetylcholinesterase inhibitory activities. These adjustments aim to provide readers with a more cohesive and comprehensive overview from the outset. (See page 1, lines 2 & 20–34).

Regarding the inclusion of chromatograms, we acknowledge the reviewer’s concern. To streamline the manuscript and maintain focus, we have moved the chromatograms to the “Supplementary Materials” section and its corresponding files (which include Figures 8 and 9 as Figures “S1” and “S2” and Tables 2 and 4 as Tables “S1” and “S2”). This allows readers who are interested in additional technical details to access the information without detracting from the main narrative of the paper. (See page 15, lines 868–872).

 

Comments 2:

I felt the lack of a significance analysis for the results obtained (TPC, DPPH, ABTS, FRAP, AChE inhibition). There is no indication of how many replicates were used for each assay, and I cannot find information on the mode (positive/negative) used for MS identification.

Response 2:

Thank you for highlighting these important details. Regarding the number of replicates, we have already clarified this in the manuscript that all assays, including total phenolic content (TPC), antioxidant activity assays (DPPH, ABTS, FRAP), and the acetylcholinesterase inhibition assay, were performed in triplicate (n=3). (See sections “2.5. Total Phenolic Content Assay”, “2.6. DPPH Antioxidant Assay”, “2.7. ABTS Antioxidant Assay”, “2.8. FRAP Antioxidant Assay”, and “2.9. Acetylcholinesterase Inhibition Assay”).

For the MS identification, we have specified in the manuscript that the analysis was conducted in negative ion mode. This choice was made because it is particularly suitable for the detection and characterization of phenolic acids. (See page 6, line 501).

 

Comments 3:

The authors state that “These findings highlight the multidimensional nature of phenolic-driven activities and the importance of exploring their combined effects rather than focusing solely on individual compounds.” However, I believe the number of compounds studied does not constitute a metabolomic analysis and is somewhat limited, which may render some of the conclusions drawn overly broad. The discussion should be adjusted in light of this potential overstatement of scope.

Response 3:

Thank you very much for your observation. We agree that the number of compounds studied does not constitute a comprehensive metabolomic analysis. We have removed the statement, “These findings highlight the multidimensional nature of phenolic-driven activities and the importance of exploring their combined effects rather than focusing solely on individual compounds” to avoid overstating the scope of the study. (See page 14, the removed sentence before “4.3. Study Limitation” section).

Additionally, we have revised the limitations paragraph to reflect this adjustment, explicitly acknowledging the limited number of compounds analyzed and the need for a more extensive profiling in future research. These changes ensure that the discussion aligns more accurately with the study’s scope and findings. (See page 14, lines 836–842).

 

Comments 4:

I also felt the lack of a conclusion addressing the question: which species or species exhibit the highest bioactive properties in light of the studied attributes?

Response 4:

Thank you very much for bringing this to our attention. We have added a new paragraph to the “Conclusions” section that explicitly identifies the species with the highest bioactive properties based on the studied attributes. This new paragraph provides a clear summary of the findings and addresses your comment directly by identifying the standout species. (See page 15, lines 859–867).

 

Authors very much appreciated the encouraging, critical, and constructive comments on this manuscript by the Reviewer. The comments have been very thorough and useful in improving the manuscript.

We would like to thank the Reviewer again for taking the time to review our manuscript.

 

Note from Assistant Editor

Comments:

There are some sections that have significant overlap with previously published articles, and we would therefore request rephrasing during the revision. We have highlighted these sentences and attached the file to this email.

Response:

Thank you very much for your observation. The sentences have been rephrased accordingly to remove the significant overlap with previously published articles. However, the names of institutions, manufacturers and analytical techniques could not be modified. (See pages 3–6, lines 317–321, 330–333, 356, 402–404, 429–432, 473–482 & 507–510).

 

We have also introduced other additions/modifications that we hope will improve the quality of the manuscript:

▪ Tables 1–5 have been renumbered accordingly: Tables 1–3, S1 and S2.

▪ Figures 5–11 have been renumbered accordingly: Figures 5–9, S1 and S2.

▪ One new citation has been introduced: Ref. [11] (Royer et al., 2011).

▪ The Reference list has been entirely checked and renumbered accordingly.

▪ All abbreviations have been defined the first time they appear in the text.

▪ Some grammar, stylistic or spelling errors have been corrected.

 

Kind regards,

Ludovic Everard BEJENARU, Associate Professor, PhD

Corresponding Author

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Results included in Table 2 are repeated in Figures 2 and 3, it is recommended to remove the figures. 

Authors must not describe the numeric results that are already shown in tables and Figures, it is redundant. Authors must focused on highlighting which sample has more or less, and must use % or fold-changes to describe these differences.

Results shown in Table 3 are also included in Figure 5, it is recommended to eliminate this latter, since it is very difficult to understand the results in this format. 

Table S1 is unnecessary since those results are already included in Figure 1.

Author Response

Dear Reviewer,

First of all, we would like to address you many thanks for your accurate observations and valuable comments. We used all these and improved the paper accordingly.

All changes in the revised manuscript were marked up using the “Track Changes” function.

The following changes have been made for the Manuscript (ID: applsci-3423460):

 

Reviewer #2 questions/comments

Comments 1:

Results included in Table 2 are repeated in Figures 2 and 3, it is recommended to remove the figures.

Response 1:

Thank you very much for your valuable comment. Figures 2 and 3 have been removed from the manuscript. (See pages 7 & 8, lines 559 & 595).

The remaining Figures have been renumbered accordingly.

 

Comments 2:

Authors must not describe the numeric results that are already shown in tables and Figures, it is redundant. Authors must focused on highlighting which sample has more or less, and must use % or fold-changes to describe these differences.

Response 2:

Thank you very much for pointing this out.

 

Comments 3:

Results shown in Table 3 are also included in Figure 5, it is recommended to eliminate this latter, since it is very difficult to understand the results in this format.

Response 3:

Thank you very much for your suggestion. Figure 5 has been removed from the manuscript. (See page 9, line 644).

The remaining Figures have been renumbered accordingly.

 

Comments 4:

Table S1 is unnecessary since those results are already included in Figure 1.

Response 4:

Thank you very much for bringing this to our attention. Table S1 has been removed from “Supplementary Material” section. Also, “Table S2” had been renumbered as “Table S1”. (See pages 7, 8 & 14, lines 559, 608 & 884–885).

 

Authors very much appreciated the encouraging, critical, and constructive comments on this manuscript by the Reviewer. The comments have been very thorough and useful in improving the manuscript.

We would like to thank the Reviewer again for taking the time to review our manuscript.

 

Kind regards,

Ludovic Everard BEJENARU, Associate Professor, PhD

Corresponding Author

Author Response File: Author Response.pdf