Review Reports - Phenolic Profile, Antioxidant Activity, and Wound-Healing Potential of Oenothera rosea and Taxodium mucronatum Extracts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript evaluated the phenolic composition, antioxidant activity and in vitro wound-healing capacity of Oenothera rosea and Taxodium mucronatum extracts. The topic is relevant, and the experimental design is reasonable. The results support the traditional use of these two plants for wound healing. However, several points require revision before acceptance.

1.The identification of compound is a core and essential part of this phytochemical study. In the current manuscript, all constituents were tentatively identified merely by matching experimental MS/MS data with public databases. However, a single mass-to-charge ratio can correspond to multiple elemental compositions and candidate molecular formulas, which may lead to ambiguous or inaccurate compound annotation. To improve the reliability and accuracy of qualitative identification, the authors are strongly recommended to employ authentic reference standards for further verification by comparing their retention times, precursor ions, and fragment ion profiles under identical chromatographic and mass spectrometric conditions.

In addition, the chromatographic separation of the first sample is not satisfactory, and peak resolution needs further improvement. Typical examples include isoquercetin and hyperoside, two structurally analogous flavonoids with highly similar fragmentation patterns. Under many chromatographic conditions, they are difficult to separate and elute nearly simultaneously; moreover, their precursor and fragment ions in mass spectrometry are almost identical. Therefore, optimization of the current UHPLC separation conditions is necessary to achieve better peak separation. Combined with standard substance verification, such optimization will significantly enhance the accuracy and credibility of the phytochemical identification results.

2. The correlation between phenolic components and biological activities is insufficiently analyzed

The study identified distinct chemical characteristics of the two extracts: O. rosea is rich in phenolic acids, tannins and flavonoids, while T. mucronatum mainly contains bioflavonoids and diterpenes. However, the authors did not reasonably explain the differential bioactivity between the two extracts based on component characteristics. For example, why only T. mucronatum significantly inhibited nitric oxide production, while O. rosea had no significant anti-inflammatory effect; and why the 50:50 mixture exerted a better wound-healing effect under high-glucose conditions. It is necessary to supplement the structure-activity relationship analysis of differential characteristic components and corresponding antioxidant, anti-inflammatory and migration-promoting activities.

3. Lack of necessary positive control of activity evaluation.

In antioxidant activity evaluation (DPPH, ABTS) and cell activity experiments, the manuscript only set negative control groups without corresponding positive controls (such as vitamin C for antioxidant detection).

4. Formatting standardization

The positional prefixes such as o-, m-, p- and linkage markers such as O- in phenolic compound names should be italicized uniformly to conform to phytochemical nomenclature specifications. In line 180, p-coumaric acid, and in line 192, quercetin-3-O-vicianoside: the letters p and O should be italicized.

In addition, the statistical symbol P value should be standardized to italic form to meet academic publication requirements.

In Table 1, numerical subscripts within compound molecular formulas require proper subscript formatting. The numeral “2” in MS² Fragments should be presented as a superscript. Two entries in Table 1 are incorrectly assigned the same serial number 15.

5. Figure 2: The chromatogram is of low quality. Please supply a high-resolution version of this chromatogram.

6. Detailed information regarding the reagents used in the experiments should be supplemented, including their purity, manufacturer, and other relevant specifications.

7. It is recommended toadd compound numbers to the mass spectra in the supplementary materials to correspond to the data in Tables 4 and 5.

Author Response

We sincerely thank Reviewer 1 for the careful evaluation of our manuscript and for the constructive comments and recommendations. We appreciate the time and effort devoted to improving the quality of this work. All comments have been carefully considered and addressed in the revised manuscript. Detailed point-by-point responses are provided below. Corresponding revisions in the manuscript have been highlighted in blue for ease of review.

1. The identification of compound is a core and essential part of this phytochemical study. In the current manuscript, all constituents were tentatively identified merely by matching experimental MS/MS data with public databases. However, a single mass-to-charge ratio can correspond to multiple elemental compositions and candidate molecular formulas, which may lead to ambiguous or inaccurate compound annotation. To improve the reliability and accuracy of qualitative identification, the authors are strongly recommended to employ authentic reference standards for further verification by comparing their retention times, precursor ions, and fragment ion profiles under identical chromatographic and mass spectrometric conditions.

We thank the reviewer for this important comment and for highlighting the need for greater rigor and transparency in the metabolite annotation process. In response, we conducted a comprehensive re-evaluation of all annotated features reported in Tables 4 and 5 and revised both the annotation workflow and confidence assignments accordingly.

The LC–MS/MS analyses were performed at a specialized metabolomics facility with access to an in-house mzRT library containing 416 reference standards characterized using the same analytical platform and acquisition conditions applied in this work. Therefore, all metabolite annotations were re-evaluated by matching precursor m/z measurements and retention time against authentic standards from the in-house mzRT library. In addition, MS/MS spectra were compared against the NIST23 spectral library, which served as the primary reference database due to its curated collection of experimentally acquired spectra from authentic standards. As a result of this re-analysis, several metabolite annotations and confidence assignments were revised, and the metabolite identification methodology was expanded in the Materials and Methods section (Lines 523-537) to provide a clearer description of the criteria used for each confidence level.

Regarding the chromatograms presented in the manuscript, we would like to clarify that these figures were included primarily to illustrate the overall chromatographic profiles of the extracts. Because the displayed chromatograms correspond to UV detection, their resolution may not be sufficient to confidently distinguish structurally related metabolites in complex plant matrices. Extracted ion chromatograms generated from high-resolution MS data provide compound-specific signals based on accurate m/z values, allowing features to be differentiated even when UV chromatographic peaks partially overlap. Consequently, compound assignments relied on the integrated LC-MS/MS workflow, including accurate mass measurements, retention time matching when authentic standards were available, and MS/MS spectral matching against reference libraries. We have revised the manuscript to better emphasize that compound assignments were based on this integrated LC-MS/MS approach rather than solely on the resolution observed in the UV chromatograms.

We also carefully reconsidered the specific example raised by the reviewer concerning the distinction between isoquercetin and hyperoside. While the MS/MS data supported annotation as a quercetin hexoside, an authentic hyperoside standard was not available in the in-house mzRT library, preventing unambiguous discrimination between these isomeric compounds through retention time comparison. To better reflect this uncertainty, and following the confidence framework proposed by Schymanski et al., the annotation has been revised to indicate unresolved structure (“u.s.”) and its confidence level has been downgraded from MSI level 2 to MSI level 3. Similar revisions were applied throughout the dataset wherever positional or stereochemical ambiguities could not be confidently resolved.

We believe these revisions significantly strengthen the reliability and transparency of the metabolite annotation process and adequately address the reviewer's concerns regarding compound identification.

2. The correlation between phenolic components and biological activities is insufficiently analyzed

We thank the reviewer for this valuable observation. We agree that a clearer discussion linking phytochemical composition with biological activity would strengthen the manuscript. Accordingly, the Discussion section has been expanded to better correlate the characteristic metabolites identified in each extract with their observed antioxidant, anti-inflammatory, and migration activities. Additional discussion was included regarding the potential contributions of phenolic acids, flavonoid glycosides, biflavonoids, and diterpenoids to the differential biological responses observed. The corresponding changes have been highlighted in blue in the revised manuscript.

As the present work was conceived as an exploratory study aimed at identifying phytochemical features associated with bioactivity rather than establishing definitive structure-activity relationships through compound isolation and mechanistic characterization, these interpretations are presented as literature-supported correlations. We have also stated in the manuscript that future studies focused on compound isolation, quantification, and mechanistic evaluation will be necessary to confirm the specific metabolites and pathways responsible for the observed effects.

3. Lack of necessary positive control of activity evaluation.

We thank the reviewer for this important observation. We agree that the description of the controls used in the different assays could be clarified. Accordingly, we have revised the manuscript to better explain the role of the standards and controls employed throughout the study.

For the chemical antioxidant assays, calibration curves were prepared using well-established reference compounds under the same experimental conditions and in the same microplates as the samples. Gallic acid was used for the Folin-Ciocalteu assay, while Trolox was used for both the DPPH and ABTS assays. These standards are commonly employed to express results relative to a known antioxidant reference, facilitating comparisons among studies and serving as a validation of assay performance.

For the Cellular Antioxidant Activity (CAA) assay, the experimental design followed the methodology described by Wan et al. In this assay, antioxidant activity is determined by the inhibition of AAPH-induced intracellular oxidation. Therefore, the positive control consisted of cells treated with DCFH-DA and AAPH in the absence of extract, representing the maximum oxidation condition. The negative control consisted of cells treated with DCFH-DA without AAPH or extract, while blank wells contained cells without DCFH-DA and AAPH. These controls allow confirmation that the oxidative challenge is effectively induced and provide the reference points required to quantify inhibition of cellular oxidation.

Similarly, in the nitric oxide inhibition assay, the biological response of interest was the reduction of nitrite production following LPS stimulation. Consequently, the positive control corresponded to LPS-stimulated cells without extract treatment, representing maximal nitric oxide production, whereas non-stimulated cells served as the negative control. Additional controls included cells treated with extract in the absence of LPS to account for any effects unrelated to the inflammatory stimulus.

For the fibroblast migration assay, centella asiatica extract was included as a reference treatment because of its well-documented wound-healing and cell migration-promoting properties, providing a suitable comparator for evaluating the effects of the tested extracts.

It is important to note that the present study was exploratory in nature and aimed to characterize the antioxidant and cellular activities of complex plant extracts under defined experimental conditions rather than establish their relative potency against purified antioxidant or anti-inflammatory compounds. We acknowledge that inclusion of additional reference compounds would provide an extra level of comparison and facilitate broader contextualization of the results. However, this objective was beyond the scope of the current investigation, which was primarily intended to identify promising biological activities and generate a foundation for future mechanistic studies. To address the reviewer's concern, we have clarified the controls employed in each assay and better defined the scope of the study, such changes have been highlighted in blue in the revised manuscript (lines: 81-84, 546-549, 561-567).

4. Formatting standardization

In addition, the statistical symbol P value should be standardized to italic form to meet academic publication requirements.

We thank the reviewer for this observation. The manuscript has been carefully revised to correct and standardize formatting throughout. Positional prefixes and linkage descriptors (e.g., p- and O-), as well as the statistical symbol p, have been italicized where appropriate. In addition, subscripts and superscripts were corrected in chemical formulas and MS² notation. Regarding the duplicate entry numbered 15 in Table 4, a re-evaluation of the data indicated insufficient evidence to support the annotation of two distinct features; therefore, one of the entries was removed and the table was updated accordingly. These changes have been implemented throughout the manuscript to ensure consistency with phytochemical nomenclature and journal formatting standards. The requested modifications were implemented throughout the manuscript and are highlighted in blue for ease of review.

5. Figure 2: The chromatogram is of low quality. Please supply a high-resolution version of this chromatogram.

We thank the reviewer for this suggestion. A high-resolution version of Figure 2 has been provided in the revised manuscript to improve clarity and facilitate visualization of the chromatographic profile.

6. Detailed information regarding the reagents used in the experiments should be supplemented, including their purity, manufacturer, and other relevant specifications.

We thank the reviewer for this suggestion. To improve the reproducibility and methodological transparency of the study, a dedicated subsection describing the reagents and materials used has been added to the Materials and Methods section with the corresponding changes (lines 405-420) highlighted in blue. This section now includes the supplier, purity, concentration, and other relevant specifications for all reagents employed in the experimental procedures. We believe this addition provides the level of detail necessary for accurate interpretation and replication of the study.

7. It is recommended to add compound numbers to the mass spectra in the supplementary materials to correspond to the data in Tables 4 and 5.

We thank the reviewer for this suggestion. In the revised supplementary material, compound/feature numbers have been added to the MS/MS spectra to facilitate direct correspondence with the metabolites reported in the tables. Additionally, as part of a broader revision to improve data presentation and respond to related reviewer comments, detailed fragmentation information was moved from the main manuscript tables to the Supplementary Material. A complementary table containing feature numbers, metabolite names, and diagnostic fragment ions has been included in the same document as the spectra, allowing readers to more easily match each feature with its corresponding MS/MS data and annotation. We believe these changes improve both the clarity and accessibility of the supporting information.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates the extraction, phytochemical composition, antioxidant activity, nitric oxide inhibition, and in vitro wound-healing effects of two traditionally used medicinal plants Oenothera rosea and Taxodium mucronatum. The manuscript reports that 70% ethanol extraction at 60 °C for 24 h produced extracts with high antioxidant activity and that O. rosea and T. mucronatum showed different biological responses. However, several methodological, statistical, analytical, and interpretational issues need to be addressed.

There are inconsistencies in the factorial experimental design; as per methods, it is 2 × 2 × 2 design with 8 treatment combinations. However, actually it appears 2 × 3 × 2 design. What was the exact design, how many treatment combinations and independent replication were there? Ther is no mention of 40 degree C in methods!

There is unclear statistical methodology. The section states that ANOVA was used to determine the effects of temperature, solvent, and extraction time, followed by post-hoc tests. Which Anova, which post hoc test?

In results authors claim : “There were no statistically significant differences (p < 0.05) observed between both extract treatments.” It is contradictory!. Correct.

Extraction deal should be given in mg/g instead of μg/g. Provide details of yield calculation.

Another concern is use of fresh material of one species and dried of other. It should be consistent in view of comparison.

Provide exact details and clarify solvent concs.

Authors have not used standards so metabolite confirmation should be used cautiously. Provide details!

As regards, desirability-function optimization, what was the desirability scale? Provide details

Synergistic action as claimed is not demonstrated.

The wound-healing assay does not establish wound healing in vivo. Correct accordingly.

Why T mucronatum material 30:70? Justification and rationale

Units of DPPH and ABTS are not consistent throughout the ms.

Provide details of voucher authentication!

Discussion be improved

Author Response

We sincerely thank Reviewer 2 for the thorough assessment of our manuscript and for the valuable feedback provided. The comments have helped us improve the clarity, rigor, and presentation of the study. All comments have been carefully addressed, and detailed responses are provided below. Changes made in the revised manuscript have been highlighted in blue for ease of evaluation.

1. There are inconsistencies in the factorial experimental design; as per methods, it is 2 × 2 × 2 design with 8 treatment combinations. However, actually it appears 2 × 3 × 2 design. What was the exact design, how many treatment combinations and independent replication were there? Ther is no mention of 40 degree C in methods!

We thank the reviewer for identifying this inconsistency. We agree that the omission of the 40 °C extraction temperature level in the Materials and Methods section made the experimental design unclear. The methodology has been corrected to include all extraction temperatures evaluated (25, 40, and 60°C), corresponding to a 2 × 3 × 2 factorial design comprising 12 treatment combinations for each species. We have also clarified that each treatment combination was performed in triplicate using independent extractions. These revisions ensure consistency between the experimental design described in the Methods section and the results presented in the manuscript. The corresponding additions have been highlighted in blue in the revised version of the manuscript (lines 440-443, 591).

2. There is unclear statistical methodology. The section states that ANOVA was used to determine the effects of temperature, solvent, and extraction time, followed by post-hoc tests. Which Anova, which post hoc test?

We thank the reviewer for this observation. We agree that the original manuscript did not provide sufficient detail regarding the statistical analyses performed. To improve clarity and reproducibility, the Statistical Analysis section has been revised to explicitly specify the statistical tests used.

The revised methodology now states:

“Data were obtained from at least three independent extractions. Statistical analyses were performed using JMP software. A three-way analysis of variance (ANOVA) was used to evaluate the effects of temperature, solvent type, and extraction time on extraction yield. Differences in desirability scores among experimental conditions were evaluated using one-way ANOVA, followed by Tukey’s honestly significant difference (HSD) post hoc test to identify significant differences between combinations of extraction conditions. Results were considered statistically significant at p < 0.05.”

These additions clarify both the ANOVA models employed and the post hoc procedure used for multiple comparisons. The corresponding changes have been highlighted in blue in the revised version of the manuscript (lines 591-598).

3. In results authors claim : “There were no statistically significant differences (p < 0.05) observed between both extract treatments.” It is contradictory!. Correct.

We thank the reviewer for identifying this ambiguity. We agree that the original wording could be misinterpreted as indicating that neither extract differed from the control treatment. Our intention was to convey that both O. rosea and T. mucronatum significantly reduced AAPH-induced oxidative stress relative to the AAPH-stimulated control, while no statistically significant differences were observed between the two extract treatments themselves. To clarify this point, the text has been revised to explicitly distinguish the comparison against the stimulated control from the comparison between extracts (lines 241-245, highlighted in blue). We believe this revision more accurately reflects the statistical analysis and avoids potential misinterpretation of the results.

4. Extraction yield should be given in mg/g instead of μg/g. Provide details of yield calculation.

We thank the reviewer for this observation. After reviewing the original data, we confirmed that the extraction yields were incorrectly reported as µg/g. The correct units are mg/g, and this has been corrected throughout the manuscript. This was a reporting error in the unit designation and did not require recalculation of the yield values. We appreciate the reviewer bringing this error to our attention, which has improved the accuracy of the manuscript. Additionally, the Materials and Methods section was revised to clarify how extraction yield was calculated. Details can be found highlighted in blue in lines 450-453.

5. Another concern is use of fresh material of one species and dried of other. It should be consistent in view of comparison.

We thank the reviewer for this observation. We agree that differences in the initial condition of the plant materials should be clearly described to avoid confusion when interpreting comparisons between species. As clarified in the revised manuscript, Taxodium mucronatum was collected as fresh plant material, whereas Oenothera rosea was obtained commercially in dried form. To ensure consistency prior to extraction, both materials underwent the same pretreatment procedure, including freeze-drying. The term “fresh” has also been removed from the manuscript where appropriate to avoid ambiguity, and the Materials and Methods section has been revised accordingly and changes have been highlighted in blue (line 432).

6. Provide exact details and clarify solvent concs.

We thank the reviewer for this suggestion. As part of revisions made in response to a related reviewer comment, we added a dedicated subsection in the Materials and Methods section describing the reagents and materials used throughout the study, with the corresponding changes (lines 405-420) highlighted in blue. This section now includes detailed information on solvent compositions, reagent concentrations, purities, and suppliers. The extraction solvents and their concentrations are now explicitly stated to improve methodological transparency and reproducibility. We believe these additions adequately address the reviewer's concern.

7. Authors have not used standards so metabolite confirmation should be used cautiously. Provide details!

We thank the reviewer for this important observation. This concern was also raised by other reviewers and prompted a major revision of the metabolite annotation workflow throughout the manuscript. We fully acknowledge that the absence of authentic standards for all detected metabolites precludes definitive confirmation of every compound. Accordingly, we have revised the manuscript to clearly distinguish between confirmed and tentatively annotated metabolites.

Compared with the previous version of the manuscript, the annotation workflow has been strengthened by incorporating retention time matching against an in-house mzRT library generated under the same chromatographic and mass spectrometric conditions used in this study. This additional level of evidence was combined with accurate precursor m/z measurements and MS/MS spectral matching against curated reference libraries, primarily NIST23, as well as public databases when appropriate.

To ensure a cautious interpretation of the data, all annotations were re-evaluated according to the confidence framework proposed by Schymanski et al. Only metabolites supported by the appropriate level of evidence were assigned higher confidence levels, whereas compounds lacking standard confirmation or presenting unresolved structural ambiguity were retained as tentative annotations and assigned lower confidence levels accordingly. In several instances, annotations were downgraded, revised, or reported as unresolved structures to more accurately reflect the available evidence.

The metabolite identification methodology has been substantially expanded in the Materials and Methods section, and complete MS/MS spectra together with fragmentation data have been provided in the Supplementary Material. We believe these revisions provide a more rigorous and transparent annotation strategy while appropriately acknowledging the tentative nature of compounds that could not be unequivocally confirmed.

8. As regards, desirability-function optimization, what was the desirability scale? Provide details

We thank the reviewer for this observation. The desirability function approach used in this study follows the methodology described by Derringer and Suich and is a well-established multi-response optimization technique. In this approach, both the individual desirability values and the composite desirability index range from 0 to 1, where 0 represents a completely undesirable outcome and 1 represents the most desirable outcome.

To improve clarity for readers unfamiliar with this methodology, we have added a brief explanation of the desirability scale to the Materials and Methods section (lines 494-496, highlighted in blue), indicating the interpretation of desirability values and the composite desirability index. We believe this addition provides sufficient context while maintaining the concise description of the optimization procedure.

9. Synergistic action as claimed is not demonstrated.

We thank the reviewer for this observation. We agree that a synergistic effect cannot be concluded from the experiments performed in the present study, as formal synergy analyses were not conducted. Accordingly, the term "synergistic" has been removed from the manuscript and replaced with more appropriate terminology such as "complementary" activity. The Discussion section was also revised to clarify that the improved performance of the O50:T50 treatment is interpreted as a potential consequence of the complementary phytochemical composition and biological activities of both extracts rather than evidence of synergy. The corresponding changes are highlighted in blue in the revised manuscript (lines 389-399).

10. The wound-healing assay does not establish wound healing in vivo. Correct accordingly.

We thank the reviewer for this observation. We agree that the assay employed in this study does not directly demonstrate wound healing in vivo. While the term “in vitro wound-healing assay” is frequently used in the literature to describe assays that evaluate monolayer gap closure, we recognize that this terminology may lead to overinterpretation of the findings.

To avoid ambiguity and more accurately reflect the biological endpoint evaluated, the assay has been consistently renamed as fibroblast migration assay throughout the manuscript, with changes being highlighted in blue. The corresponding results and discussion sections have also been revised accordingly. We believe this terminology better represents the scope of the experiment and avoids implying direct evidence of wound healing in vivo.

11. Why T mucronatum material 30:70? Justification and rationale

We thank the reviewer for this observation. We believe the original wording may have led to a misunderstanding regarding the selection of the 30:70 ratio. This proportion was not specific to T. mucronatum nor was it chosen arbitrarily. Rather, after separating the aerial tissues of both species, we determined that the relative proportions of leaves/flowers and stems/branches were comparable between the collected T. mucronatum material and the commercially obtained O. rosea material. Therefore, a fixed 30:70 proportion was used for both species to ensure consistency during extraction and subsequent analyses. To clarify this point, the Materials and Methods section has been revised to better describe the origin of the ratio and how it was determined (lines 434-437, highlighted in blue). We believe this modification eliminates the ambiguity present in the original version and better justifies the sample preparation strategy employed in the study.

12. Units of DPPH and ABTS are not consistent throughout the ms.

We thank the reviewer for this observation. We agree that the units used for reporting antioxidant activity were not presented consistently throughout the manuscript. This has been corrected in the revised version to ensure uniformity across the text, tables, and figures. The corresponding changes are highlighted in blue in the revised manuscript.

Specifically, total phenolic content is now consistently reported as mg GAE/g dry weight of extract, whereas DPPH and ABTS antioxidant activities are consistently expressed as µmol TE/g dry weight of extract. We believe these revisions improve the clarity and comparability of the reported results.

13. Provide details of voucher authentication!

We thank the reviewer for this suggestion. Voucher specimens were not deposited as part of the present study. Plant identification was performed using standard taxonomic keys and authoritative floristic references for both Taxodium mucronatum and Oenothera rosea. To improve transparency regarding plant authentication, this information has been incorporated into the Materials and Methods section (lines 422-431, highlighted in blue). For O. rosea, the material was obtained from a licensed herbal supplier and its identity was further supported by examination of its morphological characteristics. For T. mucronatum, identification was based on established botanical descriptions and diagnostic morphological traits. As a widespread and emblematic species in Mexico, its taxonomy and morphological characteristics are well documented, which facilitates botanical recognition in the field. As part of the revision process, we reviewed recent literature involving T. mucronatum and observed that several studies have reported the use of plant material authenticated through conventional botanical identification, field observations, or documented provenance (Refs. 1-4). To further document the plant material used in this study and facilitate traceability, representative photographs of the original samples have been included in the Supplementary Material.

1. Gao et al. Plants 2025, 14, 3295. https://doi.org/10.3390/plants14213295

2. Siaz Torres et al. Plants 2024, 13, 2754. https://doi.org/10.3390/plants13192754

3. Wu et al. Plants 2022, 11, 894. https://doi.org/10.3390/plants11070894

4. Gao et al. Front. Plant Sci. 2026, 17, 1686191. https://doi.org/10.3389/fpls.2026.1686191

14. Discussion be improved

We thank the reviewer for this comment. The Discussion section has been revised and expanded. Greater emphasis was placed on correlating the identified phytochemical classes with the biological responses observed, while acknowledging the exploratory nature of the study and the limitations associated with the lack of metabolite quantification and mechanistic analyses. Additional discussion regarding the differential anti-inflammatory effects of the extracts and the enhanced wound-healing response of the O50:T50 treatment has also been incorporated. We believe these revisions provide a more comprehensive and balanced interpretation of the results.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript under the title “Phenolic Profile, Antioxidant Activity, and Wound-Healing Potential of Oenothera rosea and Taxodium mucronatum Extracts” is properly structured with detail characterized their phenolic profiles by high resolution mass spectrometry methods: UHPLC-PDA-Orbitrap-MS/MS and the

antioxidant activity was preciously studied by adequate methods. The supplementary materials confirmed the adequate identification and purities of every elucidated structure. The obtained results allow recommending the manuscript to publication for wide research the potential of both species “as sources of bioactive compounds for wound management”.

Author Response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the overall quality of the study. We appreciate the reviewer's comments regarding the characterization of the phenolic profiles by UHPLC-PDA-Orbitrap-MS/MS, the assessment of antioxidant activity, and the supporting information provided in the Supplementary Materials. We are grateful for the reviewer's assessment that the results contribute to the understanding of the bioactive potential of Oenothera rosea and Taxodium mucronatum and may support further research into their possible applications in wound management.

Reviewer 4 Report

Comments and Suggestions for Authors

Phenolic Profile, Antioxidant Activity, and Wound-Healing Potential of Oenothera rosea and Taxodium mucronatum Extracts is a nicely presented research paper. Experimental setup, results and discussion are presented in a clear and scientifically sound manner. All of my comments are given es embedded in the PDF file uploaded, please open it in the PDF viewer in order to see all of them properly.

Beside them there are few important points for discussion:

-there is no quantification of detected compounds. This was not planned in the research, but it should be discussed in this manner. Authors presented differences in the profile, but without any quantification it is very hard to talk about the contribution of the detected compounds. Standards are hard to find, but this should be the next step in the research. Also, sampling accuracy is also important (%leaves and branches in both extracts, some explanation should follow for the commercially obtained material). And, as said, separation could be better, especially if you go for the quantification. even with the MS detector, in my opinion.

- for the change in the title suggestion, you decide to focus on hydroethanolic extracts. Completely different effect you can observe with water extraction, possibly... So, we need to be sure what is the focus of the research. This is just my opinion; decision is surely up to the Authors.

-calculation of the yield is not completely clearly presented, or I'm missing something, a minor point

-In Supplementary results, you have one extra blank page, and it would be nice to insert the compound name in each mass spectra plot. Also, annotate the masses second to the base peaks in intensity. Sometimes they are OK, sometimes it is missing, like for Rutin (base peak at 609 m/z). And, please, put m/z always in italic...

After all of the comments, I recommend that this research can be published after addressing to the comments given in this review.

Comments for author File: Comments.pdf

Author Response

We thank Reviewer 4 for the revision of our work and for the constructive comments and recommendations. Detailed point-by-point responses to the comments submitted in the review report are presented below. Furthermore, all observations and annotations included in the reviewed PDF document have been addressed through corresponding revisions to the manuscript. These changes have been highlighted in blue in the revised version.

1. There is no quantification of detected compounds. This was not planned in the research, but it should be discussed in this manner. Authors presented differences in the profile, but without any quantification it is very hard to talk about the contribution of the detected compounds. Standards are hard to find, but this should be the next step in the research. Also, sampling accuracy is also important (%leaves and branches in both extracts, some explanation should follow for the commercially obtained material). And, as said, separation could be better, especially if you go for the quantification. even with the MS detector, in my opinion.

We thank the reviewer for these valuable comments. We agree that the absence of quantitative data limits the ability to directly assess the contribution of individual metabolites to the observed biological activities. To acknowledge this limitation, the Discussion section has been revised to clarify that the proposed relationships between metabolite profiles and biological activities should be interpreted cautiously, as they are based on metabolite occurrence rather than measured concentrations. However, the present study was designed as an exploratory investigation, with the primary objective of identifying metabolites associated with the antioxidant and cellular activities of O. rosea and T. mucronatum and generating sufficient information to guide future mechanistic studies.

Metabolite annotation relied primarily on high-resolution MS and MS/MS data rather than UV chromatographic resolution, which was included mainly to illustrate overall chromatographic profiles. We agree that targeted quantification of key compounds, further chromatographic optimization, and confirmation with authentic standards represent important next steps. However, these objectives fall beyond the scope of the present exploratory work and are planned for future studies integrating targeted metabolite quantification, molecular docking, and omics-based approaches to investigate the mechanisms underlying the biological activities reported here.

Regarding sample composition, the Materials and Methods section has been revised to clarify that both species were processed using the same standardized 30:70 weight ratio of leaf/flower to stem/branch fractions, which reflects the comparable relative proportions observed in the plant materials prior to extraction. This information has been added to improve transparency and reproducibility (lines 434-437, highlighted in blue).

2. For the change in the title suggestion, you decide to focus on hydroethanolic extracts. Completely different effect you can observe with water extraction, possibly... So, we need to be sure what is the focus of the research. This is just my opinion; decision is surely up to the Authors.

We thank the reviewer for this thoughtful suggestion. We considered modifying the title to specifically refer to hydroethanolic extracts. However, we elected to retain the original title because the study was not limited to hydroethanolic extracts. Rather, both aqueous and hydroethanolic extracts were evaluated during the extraction condition selection process, and the subsequent phytochemical and biological analyses were performed using the conditions identified as most suitable for each species.

We agree that extraction solvent can substantially influence phytochemical composition and biological activity. However, we believe that specifying “hydroethanolic extracts” in the title would not fully reflect the scope and experimental design of the study. Therefore, we consider the current title to be a more accurate representation of the work presented. We appreciate the reviewer's comment and the opportunity to clarify this point.

3. Calculation of the yield is not completely clearly presented, or I'm missing something, a minor point

We thank the reviewer for this observation. We agree that the description of the extraction yield calculation could be improved. In response, the Materials and Methods section has been revised to explicitly describe how extraction yield was determined and reported. The revised text now states:

“Extraction yield was calculated gravimetrically as the ratio of the mass of the dried extract recovered after solvent removal to the initial mass of plant material used for extraction. The yield was expressed as mg of dried extract per g of plant material (mg/g).”

This modification has been highlighted in blue in the revised manuscript (lines 450-453). We believe this clarification improves the transparency of the methodology and eliminates any ambiguity regarding yield calculation.

4. In Supplementary results, you have one extra blank page, and it would be nice to insert the compound name in each mass spectra plot. Also, annotate the masses second to the base peaks in intensity. Sometimes they are OK, sometimes it is missing, like for Rutin (base peak at 609m/z). And, please, putm/z always in italic...

We thank the reviewer for these valuable suggestions. In the revised supplementary material, the extra blank page has been removed, and feature numbers have been added to the MS/MS spectra to facilitate direct correspondence with the metabolites reported in the manuscript.

Following the reviewer's recommendation and related comments from other reviewers, the metabolite annotation tables were substantially reorganized. Rather than transferring the original tables in their entirety, the main metabolite table was simplified to improve readability and now includes chemical structures for the highest-confidence annotations. Detailed fragmentation information, which previously appeared in the main table, was moved to a dedicated supplementary table together with the corresponding MS/MS spectra. This organization allows readers to directly match feature numbers, compound names, fragment ions, and spectra within a single document.

In addition, the supplementary information was carefully reviewed, and peak labeling and spectral annotations were added for the principal fragment ions. Also, the notation m/z has been standardized and italicized throughout the manuscript and supplementary material. We believe these revisions substantially improve the clarity, organization, and accessibility of the metabolite identification data.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been significantly improved following the major revision. All previous concerns have been satisfactorily addressed. The study is timely, the data are robust, and the conclusions are well justified. I recommend acceptance in present form with no further changes required.