PREreview of Antiplasmodial Potential of Compounds from the Bark of Mitragyna inermis (Rubiaceae): In Silico and In Vitro Studies Against LDH, PKG Enzymes and 3D7/Dd2 Strains

Reviewer-1 Comments

1. Is the research question and objective of the study clear?

Ans: Yes, the research question and objective of the study are clearly stated.

2. Is the study design and methodology appropriate for addressing the research question?

Ans: No. The objective of the manuscript is to evaluate the antiplasmodial activity of compounds from the bark of Mitragyna inermis. Only the antiplasmodial activity of the test compounds was evaluated against two strains (chloroquine-sensitive and chloroquine-resistant), but no cytotoxicity test was performed in host cells.

3. Are the methods and data analysis sufficiently rigorous and reliable?

Ans: The methods and data analysis are rigorous. However, a host cell cytotoxicity test and calculation of the Selectivity Index (SI) are essential to determine whether any of the compounds can be considered promising therapeutic candidates.

4. Are the results clearly presented and adequately support the main findings?

Ans: Yes, the results are clearly presented. However, the authors are advised to include host cell cytotoxicity data and SI values.

5. Are the interpretations and conclusions supported by the data?

Ans: There are insufficient data in the “Antiplasmodial Activity” section. Host cell cytotoxicity data and SI values are essential to justify the test compounds as potential antiplasmodial therapeutics.

6. Does the study provide a meaningful contribution to the field, and are its limitations adequately addressed?

Ans: Overall, the study would provide a meaningful contribution to the field. However, no limitations of the study have been stated in the manuscript.

7. Overall, do you consider the preprint scientifically sound and suitable for dissemination in its current form?

Ans: No. Additional experimental data are needed to justify the objective of the study.

Additional comments (optional): Please provide any specific suggestions or concerns that you feel should be addressed.

1. The plant’s scientific name should be italicized. In the Introduction section, please change the plant’s scientific name to italics.

2. Add host cell cytotoxicity data and calculate the Selectivity Index (SI).

3. Add limitations of the study.

Reviewer-2 comments

1. Is the research question and objective of the study clear?

Yes the research question and objective of the study is very clear.

2. Is the study design and methodology appropriate for addressing the research question?

The authors have done a commendable job addressing several important research questions in this manuscript. However, some key aspects remain unaddressed. Notably, no cytotoxicity studies were planned or performed for the compounds under investigation. In silico analysis alone is insufficient to establish safety, as it cannot substitute for empirical determination of the cytotoxic concentration (CC50). Without this value, the selectivity index (SI = CC50/IC50) cannot be calculated.

Another aspect in the design is how the authors landed on to select PfLDH and PfPKG as the potential targets of the compounds. Either the compounds show have some degree of structural similarity to the known inhibitors of these targets or these family of compounds have shown to bind to these enzymes in Plasmodium or some other organisms.

3. Are the methods and data analysis sufficiently rigorous and reliable?

A rigorous amount of in silico analysis was employed to address the research questions posed in this study, and the structural biology component stands out as a particular strength of the manuscript. That said, the methodology section could be simplified to improve accessibility for readers with limited or no background in this area of research.

4. Are the results clearly presented and adequately support the main findings?

The results are clearly presented in the manuscript. However, Section 3.2 would benefit from a line graph illustrating the percentage of parasite death across varying compound concentrations. Such a dose-response curve would provide a clearer visual representation of compound potency and facilitate easier comparison of efficacy across the tested concentrations.

5. Are the interpretations and conclusions supported by the data?

The interpretations and conclusions are generally supported by the data; however, the depth of interpretation could be strengthened. For instance, rather than relying solely on a single set of drug-likeness criteria, the authors could have also applied complementary filters such as the Ghosh and Weber rules. Incorporating multiple drug-likeness frameworks would have allowed for a more comprehensive and nuanced assessment of the compounds' pharmaceutical potential, lending greater depth to the overall interpretation of the results.

6. Does the study provide a meaningful contribution to the field, and are its limitations adequately addressed?

Given the significant global health burden of Plasmodium infections, investigations into novel therapeutic strategies against the parasite are of considerable importance. This study makes a meaningful contribution to the field; however, its limitations have not been adequately addressed. Several key gaps remain unaddressed in the discussion: the absence of in vitro evaluation of host cytotoxicity, the lack of reference to previously reported antimalarial activity of Mitragyna inermis, and the omission of structural comparisons between the identified hits and known PfLDH and PfPKG inhibitors. Furthermore, compound 3 (one of the top hits) violates three points of Lipinski's Rules, yet this drug-likeness concern is not discussed or contextualized by the authors.

7. Overall, do you consider the preprint scientifically sound and suitable for dissemination in its current form?

The preprint is scientifically sound however it requires some changes in order to be accepted for publication.

Additional comments (optional): Please provide any specific suggestions or concerns that you feel should be addressed.

SI index, a critical parameter for evaluating the compound's therapeutic window and distinguishing genuine antimalarial activity from general cellular toxicity. I recommend the authors could have included in vitro cytotoxicity assays (e.g., MTT or similar viability assays) on relevant cell lines to determine the CC50 and report the corresponding SI, which would substantially strengthen the translational relevance of their findings.

The further downstream genes directly associated with proposed target protein could’ve been quantified for their expression levels post exposure to the compound of interest.

Reviewer-3 comments

1. Is the research question and objective of the study clear?

Yes, Introduction states the aim explicitly - to isolate and characterize phytoconstituents from M. inermis bark, assess their interaction with PfLDH and PfPKG via docking/MD, and evaluate in vitro antiplasmodial activity against 3D7/Dd2 strains - and this is well motivated by the roles of these two enzymes in parasite survival and the plant's ethnomedical use.

2. Is the study design and methodology appropriate for addressing the research question?

Yes. The study follows a standard study design, combining isolation/structure elucidation, in vitro 3D7/Dd2 assays, molecular docking, ADMET prediction, and 100 ns MD/MM-PBSA is an appropriate multi-tiered design for natural-product lead discovery.

3. Are the methods and data analysis sufficiently rigorous and reliable?

I. The manuscript should provide additional information and experimental data supporting the reported antiplasmodial activity. Although an appropriate reference method is cited, the manuscript should provide sufficient dose–response data to demonstrate the effect of compound treatment. In addition to reporting IC₅₀ values in µg/mL, please provide the corresponding IC₅₀ values in µM, particularly because the molecular masses of the isolated compounds differ substantially. Reporting both units would allow a more meaningful comparison of the intrinsic potency of the compounds.

II. In the molecular docking and MD simulation sections, the authors should include known/reference ligands for PfLDH and PfPKG, where such ligands are available. If appropriate target-specific reference ligands are not available, the authors should consider including suitable reference proteins/related targets to provide a comparative framework. Since the proposed targets appear to have been selected with limited experimental target-validation information, inclusion of at least two relevant reference targets would strengthen the relative interpretation of the docking results. Docking scores are generally more informative when interpreted comparatively rather than as absolute measures of binding affinity. This is particularly important in the present study because no direct enzyme inhibition assays for PfLDH or PfPKG were performed. The authors should therefore provide appropriate computational controls and moderate their mechanistic conclusions accordingly.

III. No cytotoxicity assay against a normal mammalian cell line (so no selectivity index can be calculated)

IV. The detailed structural characterization of compounds 1–5 could be moved to the Supplementary Information to improve the readability and flow of the main manuscript. The authors should provide comprehensive supporting spectroscopic and analytical evidence in the Supplementary Information, including relevant ¹H/¹³C NMR, 2D-NMR, MS/HRMS data, spectra, and key correlations supporting the proposed structures. The main manuscript can retain a concise summary of the structural identification and refer readers to the corresponding supplementary data.

4. Are the results clearly presented and adequately support the main findings?

Figure 4f's axes lack the unit labeling used consistently in panels (a)-(e) (no explicit "Time (ns)" or energy-unit label), making that panel harder to interpret alongside the rest of Figure 4.

5. Are the interpretations and conclusions supported by the data?

Partially. The results are generally clearly presented, but they do not fully support some of the stronger claims.

6. Does the study provide a meaningful contribution to the field, and are its limitations adequately addressed?

Yes, there is a genuine contribution: compounds 4 and 5 are reported for the first time in the genus Mitragyna, and this appears to be the first study testing isolated quinovic acid glycosides from M. inermis directly against P. falciparum together with a docking/MD-based mechanistic hypothesis.

Limitations are under-addressed, there is no discussion of the missing cytotoxicity/selectivity data, no acknowledgement that artesunate and chloroquine (used as docking reference standards) do not act primarily through direct PfLDH/PfPKG active-site binding in vivo. A dedicated limitations paragraph in the Discussion or Conclusions would strengthen the manuscript.

7. Overall, do you consider the preprint scientifically sound and suitable for dissemination in its current form?

Recommend many improvements for improve rigor. If done it will be significant contribution to the filed.

Additional comments:

· Check for text mislabeling.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they used generative AI to come up with new ideas for their review.