Ovicidal Effect on Haemonchus contortus of Extract Partitions Shrubby Plants of the Tropical Dry Forest and Potentially Active Compounds Identification by UHPLC-Q/Orbitrap/MS/MS

Round 1

Reviewer 1 Report

Line 23. “AA” An explanation of the abbreviation is required.

Line 27. Missprint. “organ”. It is worth using the following word «organic».

Lines 47, 357. “anthelmintic activity (AA)” The transcription of the abbreviation from line 39 is repeated.

Lines 82-87. It is necessary to describe the extraction, namely how much dried plant material was extracted with what volumes of extractants.

Line 109. “was centrifuged at 459 × g” Could you explain what that means?

Line 119. Problems with the degree symbol.

Lines 173-175.” (Universidad Nacional de Córdoba-Córdoba, Argentina) …(University of Auckland-Auckland, New Zealand)” You need to replace hyphens with commas.

It is not necessary to cite Figure 8, because the structures are common knowledge, and the connection of these structures with AA is not shown.

I would like to see the authors' comments about the toxicological data of their extracts. Indeed, inhibitory concentrations of extracts are important, but only in relation to their toxicity. For example, benzene will definitely inhibit any life, but that does not make it a promising drug because of its toxicity.

Author Response

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Reviewer 2 Report

The article by Romero et al. presents a well-designed study investigating the anthelmintic potential of extract particles from Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce. The study has significant implications for the development of novel anthelmintic treatments and serves as a foundation for future investigations in this field. The authors apply a robust methodology using the inhibition of egg hatching (IEH) assay to evaluate the anthelmintic activity of different extract partitions from the three forage species and provide a comprehensive data analysis to explain the observed effects.

The identification of bioactive compounds such as flavonoids, fatty acid esters, hydroxycinnamic acids, oxygenated organic compounds, benzene derivatives, phenolic glycosides, and phenols with potential anthelmintic activity is an important finding. The large amount of flavonoids in the three plants underscores their possible importance in the observed anthelmintic effects. The study suggests that flavonoids can penetrate the cuticle of H. contortus eggs, which provides a basis for further investigation of their mechanisms of action. The potential synergistic effects among the various compounds identified also warrant further research to understand metabolite interactions in tropical forage plants with nutraceutical potential.

This study is scientifically important because it demonstrates the in vitro ovicidal activity of extracts of Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce against the parasitic nematode Haemonchus contortus. The research is valuable for several reasons: it identifies potential anthelmintic compounds, validates ethnobotanical knowledge, provides a sustainable and natural source of anthelmintics, and lays the groundwork for further research.

However, limitations of the study include the fact that it relies on in vitro experiments that may not accurately reflect efficacy in vivo; insufficient exploration of the underlying mechanisms behind the relationships between concentration, polarity, and shrub species; the lack of detailed analysis of specific functions or interactions between different flavonoids; the limited investigation of synergistic activity among identified compounds; the exclusive use of the IEH assay, which may not capture the full spectrum of anthelmintic effects; and the preliminary identification of compounds by UHPLC-Q/Orbitrap/ MS without additional confirmatory methods. Despite these limitations, the study contributes to the understanding of the anthelmintic properties of the plant species studied and their potential bioactive compounds, which supports the development of new treatments for parasite infections and encourages further research in this area. I recommend this work for publication.

Author Response

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Reviewer 3 Report

The manuscript title “Anthelmintic Effect of Extract partitions Obtained from Gliricidia sepium, Leucaena leucocephala, Pithecellobium dulce on Haemonchus contortus Eggs, analysis by UHPLC-Q/Or bitrap/MS/MS” has scientific worth but it need several modification and requires major revisions.

Reviewer Comments:

1- The abstract needs significant modification! Don’t directly start the abstract with objectives, please write 2-3 lines of background of this study! What was the worth, scientific gap, that needs to be filled…….

2- Line 19-21: Don’t use so many abbreviations in abstract.

3- All scientific names should be in italics.

4- Line 21 and 26: why you have explained the IEH abbreviation twice??

5- Introduction section should be split into three paragraphs.

6- In almost all figures why authors write this “Means with the same letter do not present significant differences (p > 0.05).” the authors should write and focus on the significant data and explain the significant data. Since this article have several significant results, so, I suggest to focus and discuss the significant results.

7- In each figure the authors should explain what does the different letter represents “a, b, c”?what was the level of significance??? which statistical software was used??? which statistical method was used??? these all information should be explained in each figure.

8- In table 1 and 2: what authors used “3,0035” , in between decimals???? authors should used ‘.’

9- Figure 8 is not useful! It is better to provide the compounds detection details, standards, retention time, etc….

Minor editing of English language required

Author Response

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Round 2

Reviewer 3 Report

Accepted.

Its fine.