Review Reports - Isothiocyanate-Rich Essential Oil of <i>Morisonia flexuosa</i> L. Exhibits Anxiolytic-like Effects That May Involve Serotonergic Pathways in Zebrafish

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript evaluates the anxiolytic-like effects of an isothiocyanate-rich essential oil from Cynophalla flexuosa in adult zebrafish through behavioral, pharmacological, and chemometric analyses. The study addresses an interesting topic with potential neuropharmacological relevance. However, several aspects related to figure clarity, methodological details, and the organization of the Results and Discussion sections should be improved

Section 2

Please revise and standardize the numbering format of the subsections.
Figure 2: The meaning of the numbers 4, 20, and 40 shown in the figure is unclear. Please clarify their significance.
Figure 2 is not referenced within the main text. Please include the corresponding citation in the manuscript.
Figure 2: Please specify the meaning of the asterisks shown in the figure.
Figure 2: The Y-axis is not clearly defined. Please specify whether it represents time (minutes), number of crossings, or another parameter.
Figure 2: Please clarify the meaning of the abbreviation “OF” in the figure. The information included in parentheses is already described in the figure caption.
The results presented in Figure 2 should be described and discussed in greater detail.
Figure 3: The meaning of the numbers 4, 20, and 40 shown in the figure is unclear. Please clarify their significance.
Figure 3: The Y-axis is not clearly defined. Please specify whether it represents time (minutes), number of entries into the light zone, or another parameter.
Figure 3: Please clarify the meaning of the abbreviation “OF” in the figure. The information included in parentheses is already explained in the figure caption.
The results presented in Figure 3 should be explained in greater detail.
Lines 181–185 correspond to discussion rather than results. Please relocate this text to the appropriate section.
Please provide a more detailed explanation of the results shown in Figure 4.
Figure 4: The Y-axis is not clearly defined. Please specify the parameter represented.
Figure 4: Please clarify the meaning of the abbreviation “OF” in the figure. The information in parentheses is already explained in the caption.
Figure 4: Please improve the clarity of the figure legend and series labels, particularly “OF” and “FMZ+OF”.
Figure 4: Please indicate the meaning of the asterisks shown in the figure.
Figure 5: The same observations indicated for Figure 4 apply to this figure.
In the Discussion section, please carefully review which paragraphs correspond to Results and which belong to the Discussion.
Care should be taken to avoid presenting results within the Discussion section.

Section 4

It would be advisable to include representative images of the experimental methodology in the Materials and Methods section, such as the gridded Petri dish used for the open field test and the light/dark apparatus containing the zebrafish specimens. These images would improve methodological clarity and facilitate reproducibility of the experimental procedures.

Section 4.5

Please indicate the temperature at which the animals were anesthetized prior to treatment administration.

Section 4.6

The temperature used for fish anesthesia was not specified. Please include this information.

Section 4.6.1

The authors indicate that six fish per group were used for the toxicity assay; however, the OECD guideline specifies the use of ten organisms. Please justify this discrepancy or revise accordingly.

The specific OECD toxicity test was not indicated.

Section 4.6.5

Please indicate the duration of the assay.

Comments on the Quality of English Language

N / A

Author Response

Comments and Suggestions for Authors

Section 2

Please revise and standardize the numbering format of the subsections.

Response: Done.

Figure 2: The meaning of the numbers 4, 20, and 40 shown in the figure is unclear. Please clarify their significance.

Response: Dear reviewer, thank you for your feedback. The meaning of the numbers shown in the graph is explained in the information below the graph, where it shows (mg/Kg; 20 uL; i.p.).

Figure 2 is not referenced within the main text. Please include the corresponding citation in the manuscript.

Response: Done.

Figure 2: Please specify the meaning of the asterisks shown in the figure.

Response: Done.

Figure 2: The Y-axis is not clearly defined. Please specify whether it represents time (minutes), number of crossings, or another parameter.

Response: Done.

Figure 2: Please clarify the meaning of the abbreviation “OF” in the figure. The information included in parentheses is already described in the figure caption.

Response: Done.

The results presented in Figure 2 should be described and discussed in greater detail.

Response: Done.

Figure 3: The meaning of the numbers 4, 20, and 40 shown in the figure is unclear. Please clarify their significance.

Response: Dear reviewer, thank you for your feedback. The meaning of the numbers shown in the graph is explained in the information below the graph, where it shows (mg/Kg; 20 uL; i.p.).

Figure 3: The Y-axis is not clearly defined. Please specify whether it represents time (minutes), number of entries into the light zone, or another parameter.

Response: Dear reviewer, thank you for your feedback. On the Y-axis, the text in parentheses (s) indicates the time spent in the light zone in seconds.

Figure 3: Please clarify the meaning of the abbreviation “OF” in the figure. The information included in parentheses is already explained in the figure caption.

Response: Done.

The results presented in Figure 3 should be explained in greater detail.

Response: Done.

Lines 181–185 correspond to discussion rather than results. Please relocate this text to the appropriate section.

Response: Done.

Please provide a more detailed explanation of the results shown in Figure 4.

Response: Done.

Figure 4: The Y-axis is not clearly defined. Please specify the parameter represented.

Response: Dear reviewer, thank you for your feedback On the Y-axis, it describes the time spent in the light zone (s), indicating the time spent in the light zone in seconds.

Figure 4: Please clarify the meaning of the abbreviation “OF” in the figure. The information in parentheses is already explained in the caption.

Response: Done.

Figure 4: Please improve the clarity of the figure legend and series labels, particularly “OF” and “FMZ+OF”.

Response: Done.

Figure 4: Please indicate the meaning of the asterisks shown in the figure.

Response: Done.

Figure 5: The same observations indicated for Figure 4 apply to this figure.

Response: Done.

In the Discussion section, please carefully review which paragraphs correspond to Results and which belong to the Discussion.

Response: Done.

Care should be taken to avoid presenting results within the Discussion section.

Response: Done.

Section 4

It would be advisable to include representative images of the experimental methodology in the Materials and Methods section, such as the gridded Petri dish used for the open field test and the light/dark apparatus containing the zebrafish specimens. These images would improve methodological clarity and facilitate reproducibility of the experimental procedures.

Response: Done.

Section 4.5

Please indicate the temperature at which the animals were anesthetized prior to treatment administration.

Response: Done.

Section 4.6

The temperature used for fish anesthesia was not specified. Please include this information.

Response: Done.

Section 4.6.1

The authors indicate that six fish per group were used for the toxicity assay; however, the OECD guideline specifies the use of ten organisms. Please justify this discrepancy or revise accordingly.

Response: Dear reviewer, thank you for your feedback The sample size cited by the OECD indicates the n for studies involving embryos and larvae. The sample size of n = 6/group was selected based on previous studies with zebrafish, in which this number proved sufficient to detect statistically significant differences in behavioral tests between doses and control groups (Magalhães et al., 2017; Ferreira et al., 2023). This follows established practices in the field and aligns with the ethical guidelines of CONCEA (protocol CEUA/UECE nº 04983945/2021) to minimize the use of animals without compromising statistical robustness.

References:

-F. E. A. Magalhães, C. Á. P. B. de Sousa, S. A. A. R. Santos, R. B. Menezes, F. L. A. Batista, Â. O. Abreu, M. V. de Oliveira, L. F. W. G. Moura, R. S. Raposo, A. Rolim, ‘Adult Zebrafish (Danio rerio): An Alternative Behavioral Model of Formalin-Induced Nociception’, Zebrafish 2017, 14, 422–429.

-M. K. A. Ferreira, W. P. O Freitas, I. M. Barbosa, M. N. da Rocha, A. W. da Silva, E. L. Rebouças, F. R. S. Mendes, C. R. Alves, P. I. G. Nunes,M. M. Marinho, R. F. Furtado, F. A. Santos, E. S., Marinho, J. E. S. A. de Menezes, H. S. dos Santos, ‘Heterocyclic chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one derived from a natural product with antinociceptive, anti-inflammatory, and hypoglycemic effect in adult zebrafish’ Biotech 2023, 13, 1–21.

The specific OECD toxicity test was not indicated.

Response: Done.

Section 4.6.5

Please indicate the duration of the assay.

Response: Done.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

According to WFO "World Flora on Line" the accepted name of the plant is Morisonia flexuosa L. while the used name by the authors Cynophalla flexuosa (L.) J.Presl is a synonym. Please change the name in all parts in the manuscript, write also the plant family.
The authors claimed serotonergic-mediated anxiolytic-like effects but they did not measure serotonin levels, receptor expression, receptor binding, receptor activation. The observed effect could be a possible serotonergic involvement but not a confirmed serotonergic mechanism. It is recommended changing the wording: “EOCF may involve serotonergic pathways” Avoid saying “mediated” unless direct receptor-level evidence is added.
The authors used to inject 20 µL for the zebra fish, this quantity is high "the usual dose is 10 µL, so the authors should justify this double dose.
The manuscript uses one-way ANOVA followed by Tukey’s test with only n = 6 animals per group. However, the statistical reporting is incomplete, as exact p-values, effect sizes, and confidence intervals are not provided. With such small group sizes, tests for normality and homogeneity may have limited reliability. Therefore, the authors are encouraged to strengthen the statistical reporting by including exact p-values, effect sizes, confidence intervals, and individual data points in the figures. In addition, the sample size should be justified, and non-parametric tests should be considered if the assumptions required for ANOVA are not adequately met
2 authors have the same initials which are Fázia Fernandes Galvão Rodrigues and Fabiola Fernandes Galvão Rodrigues, please distinguish between them the authors contribution section.

Author Response

Comments and Suggestions for Authors

According to WFO "World Flora on Line" the accepted name of the plant is Morisonia flexuosa L. while the used name by the authors Cynophalla flexuosa (L.) J.Presl is a synonym. Please change the name in all parts in the manuscript, write also the plant family.

Response: Done.

The authors claimed serotonergic-mediated anxiolytic-like effects but they did not measure serotonin levels, receptor expression, receptor binding, receptor activation. The observed effect could be a possible serotonergic involvement but not a confirmed serotonergic mechanism. It is recommended changing the wording: “EOCF may involve serotonergic pathways” Avoid saying “mediated” unless direct receptor-level evidence is added.

Response: Done.

The authors used to inject 20 µL for the zebra fish, this quantity is high "the usual dose is 10 µL, so the authors should justify this double dose.

Response: Dear reviewer, thank you for your feedback. The volume of 20 µL was selected based on previous studies conducted in adult zebrafish, in which this administration volume was shown to be safe and effective for intraperitoneal injection without causing mortality or behavioral impairment. In addition, the tested compounds required adequate solubilization and precise dosing, making the use of 20 µL necessary to ensure proper administration. It is important to note that no signs of toxicity, abnormal swimming, or stress-related behaviors were observed following injection, indicating that the selected volume was well tolerated by the animals.

The manuscript uses one-way ANOVA followed by Tukey’s test with only n = 6 animals per group. However, the statistical reporting is incomplete, as exact p-values, effect sizes, and confidence intervals are not provided. With such small group sizes, tests for normality and homogeneity may have limited reliability. Therefore, the authors are encouraged to strengthen the statistical reporting by including exact p-values, effect sizes, confidence intervals, and individual data points in the figures. In addition, the sample size should be justified, and non-parametric tests should be considered if the assumptions required for ANOVA are not adequately met.

Response: Dear reviewer, thank you for your feedback. The sample size of n = 6/group was selected based on previous studies with zebrafish, in which this number proved sufficient to detect statistically significant differences in behavioral tests between doses and control groups (Magalhães et al., 2017; Ferreira et al., 2023). This follows established practices in the field and aligns with the ethical guidelines of CONCEA (protocol CEUA/UECE nº 04983945/2021) to minimize the use of animals without compromising statistical robustness.

References:

2 authors have the same initials which are Fázia Fernandes Galvão Rodrigues and Fabiola Fernandes Galvão Rodrigues, please distinguish between them the authors contribution section.

Response: Done

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, the manuscript presents an interesting preliminary investigation into the anxiolytic-like effects of the essential oil of Cynophalla flexuosa in adult zebrafish, combining phytochemical characterization with behavioral pharmacology and exploratory chemometric analyses. The topic is relevant and the study contributes valuable initial data regarding the neuropharmacological potential of isothiocyanate-rich essential oils. However, several aspects of the manuscript require substantial revision to improve scientific rigor, interpretative balance, and methodological transparency.

A major concern throughout the manuscript is the tendency toward mechanistic overinterpretation. Repeated references to oxidative stress modulation, Nrf2 activation, redox-sensitive signaling, neuronal homeostasis, and indirect serotonergic modulation are not experimentally supported by the presented data, since no molecular, biochemical, or neurochemical analyses were performed. These mechanisms should therefore be discussed more cautiously and clearly distinguished from experimentally demonstrated findings. Similarly, expressions such as “mechanistic pathway,” “multi-target modulator,” and “therapeutic window” appear premature considering that the study is primarily based on behavioral assays and pharmacological antagonism tests.

The distinction between anxiolytic-like activity and nonspecific sedative effects also requires further clarification. Although locomotor reduction was less pronounced than that observed with diazepam, decreased exploratory activity may still influence performance in the light/dark test. Inclusion of additional behavioral endpoints, complementary anxiety paradigms, or a more cautious interpretation of the behavioral findings would strengthen the manuscript.

Another important limitation concerns the chemometric analyses. Because the global bioactivity values of the essential oil were mathematically replicated across all compounds according to relative abundance, the resulting PCA, HCA, and weighted contribution analyses largely reflect abundance-driven statistical associations rather than experimentally validated compound-specific biological activities. While the authors acknowledge the exploratory nature of these analyses, the biological relevance of the obtained associations should not be overstated. The manuscript would benefit from a clearer presentation of these limitations and from avoiding mechanistic conclusions based primarily on exploratory multivariate statistics.

In addition, the presentation of the behavioral data relies heavily on graphical representation. The inclusion of detailed tables containing exact numerical values, statistical parameters, confidence intervals, and effect sizes would improve transparency and facilitate critical evaluation of the results.

Finally, the Materials and Methods section should clarify whether behavioral scoring and data analysis were conducted in a blinded manner to reduce potential observational bias.

Overall, the manuscript has potential and may become suitable for publication after major revision. The study would be substantially strengthened by moderating mechanistic and translational claims, improving methodological transparency, clarifying the limitations of the chemometric approach, and presenting the behavioral findings in a more cautious and balanced manner.

Author Response

Comments and Suggestions for Authors

Response:

We thank the reviewer for this important and constructive observation. We agree that the chemometric analyses presented in the manuscript are exploratory and that the statistical associations obtained are strongly influenced by the proportional weighting strategy used in the constructed matrix. In response to this comment, we revised the text throughout the Results and Discussion sections to more clearly acknowledge these methodological limitations and to avoid overinterpretation of the multivariate analyses.

Specifically, we reinforced that the replicated bioactivity values across compounds were mathematically distributed according to relative abundance, meaning that PCA, HCA, and weighted contribution analyses predominantly reflect abundance-driven statistical structure rather than experimentally validated compound-specific pharmacological activity. Accordingly, we revised the PCA interpretation to explicitly state that the high variance explained by PC1 primarily reflects the dominant influence of compound abundance in organizing the dataset.

In the HCA section, we clarified that clustering reflects chemical similarity and proportional contributions within the model framework, rather than true biological independence of the compounds. Similarly, in the heatmap interpretation, we emphasized that the observed correlations derive from the mathematical dependence inherent to the weighted model and therefore should not be interpreted as causal biological relationships.

In addition, the Conclusions section was carefully revised to avoid mechanistic overstatements. The previous wording suggesting direct associations between major isothiocyanates and behavioral endpoints was softened to indicate only statistical associations under the adopted exploratory framework. We also explicitly stated that direct molecular interactions and compound-specific contributions remain to be experimentally confirmed through testing isolated compounds and mechanistic studies.

Response: New graphic representations were inserted.

Finally, the Materials and Methods section should clarify whether behavioral scoring and data analysis were conducted in a blind manner to reduce potential observational bias.

Response: Done.

We believe that after the suggestions and corrections were made, the manuscript has been strengthened. We are very grateful to the reviewers for their considerations.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Please specify the OECD toxicity test employed in this study, including whether the assay was conducted under static, semi-static, or continuous exposure conditions.
Please carefully review and standardize the reference formatting throughout the manuscript. For example, inconsistencies were identified in Section 4.6.1.
Line 449: The citation format does not appear to comply with the journal's reference style. Please revise accordingly.
The discussion section would benefit from a more in-depth analysis of the results. Several findings are presented without sufficient interpretation or comparison with previously published studies, which limit the scientific significance of the observations.

Author Response

Response to Reviewer 1

Comments and Suggestions for Authors

Please specify the OECD toxicity test employed in this study, including whether the assay was conducted under static, semi-static, or continuous exposure conditions.

Response: Thank you for your observation. In our study, the essential oil was administered via a single intraperitoneal (i.p.) injection, and not dissolved in the aquarium water. Therefore, OECD Guideline 203 (Fish, Acute Toxicity Testing), which focuses on water exposure, was not directly applicable. Instead, we performed an acute toxicity assessment after a single i.p. dose, with the animals kept under standard housing conditions. The water was renewed every 24 hours solely for animal welfare and maintenance of water quality, and not as part of the exposure protocol, according to the methodological adaptation made by Patrícia B.D.C. et al., 2020. The manuscript has been revised to avoid any misinterpretation.

Please carefully review and standardize the reference formatting throughout the manuscript. For example, inconsistencies were identified in Section 4.6.1.

Response: We thank the reviewer for their careful review. The entire manuscript has been revised to standardize the reference format, including correcting inconsistencies identified in section 4.6.1. Citations in the text and entries in the reference list now uniformly follow the style required by the journal, ensuring consistency across all sections.

Line 449: The citation format does not appear to comply with the journal's reference style. Please revise accordingly.

Response: We thank the reviewer for their observation. The citation on line 449 has been reviewed and adjusted to conform to the journal's required referencing style.

The discussion section would benefit from a more in-depth analysis of the results. Several findings are presented without sufficient interpretation or comparison with previously published studies, which limit the scientific significance of the observations.

Response: We thank the reviewer for their valuable suggestion. In response to this comment, we have substantially improved the Discussion section, incorporating more in-depth analyses and comparisons with previously published studies. All changes are duly indicated in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have thoroughly and successfully addressed the reviewers’ comments and significantly improved the quality of the manuscript. The revised version is clear, well-structured, and scientifically sound. Therefore, I recommend the manuscript entitled “Isothiocyanate-rich essential oil of Morisonia flexuosa L. exhibits serotonergic-involved anxiolytic-like effects in zebrafish” for publication in Plants.

Author Response

Comments and Suggestions for Authors

Response: Thank you for the positive feedback and recommendation for publication.

Author Response File: Author Response.docx