Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Combining natural product isolation, structure elucidation, semi-synthesis, and bioactivity assessment of Inula helenium metabolites and derivatives against Varroa destructor, the manuscript offers a worthwhile and ambitious study. The project offers valuable insights into the structural pharmacophore requirements of acaricidal agents, and the dual approach "from nature to synthesis and vice versa" is conceptually sound.

However, several aspects warrant clarification or improvement before the manuscript can be considered for publication.

The introduction provides substantial background on Inula species, but the novelty of the study should be more explicitly articulated: 

1. What knowledge gap does this work fill relative to previous work on costic acid and its analogs?

A more explicit comparison with earlier reports (e.g.Georgiladaki et al., 2020) would strengthen the justification.

Two different extraction methods are presented, but their choice, purpose, and comparative advantages are not clearly articulated.

Why were two methods used?

Did both yield the same major compounds?

The authors should clarify whether both methods were necessary or if one was used only to obtain antioxidant extracts. 

Structural Characterization

Although the NMR data tables are comprehensive, the structure elucidation section would benefit from:

• A brief comparative discussion showing how the present data aligns with or differs from literature values.
• A clearer explanation of how each proton/carbon assignment was validated, especially for the more complex 3a and 4a derivatives.

The numbering scheme in Figure 2 should match exactly the numbering used in the NMR tables (some inconsistencies are present).

Diastereomeric Ratios and Stereochemical Assignments

The stereochemical reasoning is sound but overly verbose. Consider summarizing with: A table listing all derivatives, their diastereomeric ratios, stereochemical outcomes, and method of determination.

The authors should also discuss potential epimerization issues under strongly basic conditions more clearly, as this is central to understanding the altered diastereomeric ratios.

Biological Assays

The rationale for evaluating antioxidant activity alongside acaricidal activity requires further clarification. The biochemical mechanisms underlying radical-scavenging processes (SET/HAT pathways) are fundamentally different from those expected to mediate toxicity against Varroa destructor. Could the authors explain how the assessment of antioxidant capacity contributes to the overall understanding of the bioactive profile of these eudesmanolides and their derivatives? Is there a mechanistic hypothesis suggesting a connection between redox properties and acaricidal activity, or was the antioxidant assay conducted independently to provide a broader characterization of the compounds?

Antioxidant Activity

The finding that lactones 3 and 4 lack strong activity, whereas the extracts exhibit high scavenging activity, is important.

The discussion should connect this with known antioxidant constituents of Inula helenium.

Inclusion of phytochemical profiling of extracts (even briefly) would enhance interpretability.

Acaricidal Activity

The acaricidal results are promising but require clarification:

• Authors should provide statistical significance, especially for comparisons between analogs and parent compounds.
• The discussion should be expanded to explain why amine derivatives lose activity, in relation to potential active-site binding.

Mechanistic Interpretation

The conclusion suggests a pharmacophore containing a free carboxylate moiety, but this should be strengthened with:

• A graphical summary or simplified pharmacophore model.
• A brief comparison to known acaricides targeting Varroa destructor.

Clarity and Writing

• The manuscript is generally well written, but several paragraphs contain overly long sentences that could be streamlined.
• Minor grammatical adjustments are needed in the Introduction and Results sections.
• Avoid repeating general botanical information that does not directly support the study’s objectives.

Recommendation: Major Revision Required

The study has scientific merit and strong analytical foundations. However, improvements are necessary in the clarity of methodological description, biological data interpretation, structural commentary, and overall. 

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is well-researched and constitutes an excellent research paper. The issues I am concerned about are as follows:

1. The manuscript lacks positive controls in the activity evaluation of natural products, making it impossible to accurately assess the relative activity of natural products and their derivatives, and also difficult to judge whether they have practical application value. Add the comparison with the efficacy of existing acaricides.
2. In the manuscript, the free carboxyl group is the key to activity, but this has not been verified through structural modification experiments (e.g., converting the carboxyl group of costic acid into an ester or amide and testing the changes in activity). The existing data only show an indirect correlation and cannot rule out interference from other structural factors. Add the experiment to verify whether the efficacy remains after masking the carboxyl group.

3.Add the active site of action of this class of acaricides.

4. There is a lack of significance analysis in Figure 5.
5. The GC-MS spectrum should bepresented in the main text.

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed all reviewers’ comments. The revised manuscript is clear, well structured, and scientifically sound. The methodology and discussion have been significantly improved, and the results are well supported by the literature. This work represents a valuable contribution to the field, and I recommend the manuscript for acceptance for publication.