Identification of Anastatica hierochuntica L. Methanolic-Leaf-Extract-Derived Metabolites Exhibiting Xanthine Oxidase Inhibitory Activities: In Vitro and In Silico Approaches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a study on the metabolic profile of Anastatica (A.) hierochuntica L. with a focus on identifying potential xanthine oxidase inhibitors. The researchers hypothesizes that metabolites in this plant may have the ability to inhibit xanthine oxide, an enzyme implicated in gout development. The manuscript adheres to generally accepted scientific standards. It contains all necessary sections and the material is logically organized, ensuring accessibility and clarity. The introduction provides all relevant information. The majority of cited literature is recent, published within the past five years, and there is no excessive self-citation.

The experimental design is scientifically sound, utilizing modern promising techniques such as molecular docking to predict the inhibitory activity of xanthine oxidase on metabolites. Additionally, the study predicts pharmacokinetic properties, toxicity, oral bioavailability, and potential endocrine disorders using appropriate web-based tools such as SwissADMET, PASS, ProTox-II, and the Endocrine Disruptome.

Research methods are adequately described, ensuring transparency and reproducibility.The article includes a sufficient amount of graphical material, which enhances its visual appeal and facilitates information comprehension. The captions accompanying the figures are clear and easy to understand.

The conclusions drawn from the study are consistent with the findings.In this regard, the manuscript is recommended for publication.

Author Response

Thank you for your positive comments. Really appreciated, indeed.

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Article “Identification of Anastatica hierochuntica L. methanolic 2 leaves extract-derived metabolites exhibiting xanthine 3 oxidase inhibitory activities: In vitro and in silico ap- 4 proaches” is well done study  about a new type of natural inhibitor. Excellent work has been done in purifying, separating, isolating and identifying chemical structures.  Molecular docking analysis has been thoroughly performed with modeling. The dependence of inhibitory activity on the concentration of metabolites was shown. The pharmaceutical oral bioavailability of the drug is shown. It has been shown that private metabolites can lead to carcinoma. However, much of the work is purely predictive in nature, which is only a preface to the extensive work of actually testing the subject in vitro and in vivo studies.

- The subject matter of the paper is entirely consistent with that of the journal. 

- The references used by the authors are up-to-date and appropriate. However, there are problems with their design. Some of the links have doi, and some do not. Also the page style is different . 71(4), pp.674-698 or 2019, 15, 68‐70.. Please make it uniform

- The text is well structured. Also it would be nice to add meddle conclusion after each section in the results part. Now it's hard to see where the general conclusion comes from

- Figures and tables are well organized. Picture 2, please improve the quality if possible

 

- Section 2.4 Missing outputs

Author Response

- The references used by the authors are up-to-date and appropriate. However, there are problems with their design. Some of the links have doi, and some do not. Also the page style is different . 71(4), pp.674-698 or 2019, 15, 68‐70.. Please make it uniform

Thank you for your comment. The references are now uniform.

- The text is well structured. Also it would be nice to add meddle conclusion after each section in the results part. Now it's hard to see where the general conclusion comes from

Thank you for your suggestion. Brief conclusions after each result part have been added, which clarify the different stages of the investigation leading to the overall conclusion.

- Figures and tables are well organized. Picture 2, please improve the quality if possible

As requested, the Figure 2 has been improved.

 - Section 2.4 Missing outputs

Thank you for your comment but after careful checks, we can confirm that there are no missing outputs.

Please see the attachment. Thank you

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors!

The research carried out by your team made a very favorable impression. The problem to which you apply your scientific efforts is truly worth achieving positive results. Indeed, the potential of medicinal plants for the treatment of many diseases is far from being studied; it has high potential and requires the closest scientific attention.

The scientific results show the presence of rich sources of 12 secondary metabolites in the methanol leaves extract of A. hierochuntica. Rutin inhibited the XO active site, as revealed by molecular docking studies and was further validated in vitro through an XO inhibition assay. Moreover, the in silicon prediction model strongly suggests the safety and drug-like-ness of these metabolites, providing a promising approach to treat hyperuricemia and may serve as good candidates for selective screening of anticancer leads. Thus, the current study aims to introduce a new paradigm for treating human malignancies, supported by future investigations of metabolites in in vitro studies.

Without any doubt, this scientific article deserves to be published in such a respected scientific journal. But first you need to make some edits and changes to the layout of the article and the text.

1. Line 142. Authors write: The recursive analysis workflow was extracted by screening detected nodes at various retention times per minute, with a minimum intensity of 6,000 counts and aligned with previously detected compounds( Table 1) considering adducts ([M+K]+ and [M-H]-).

It's mistake? [M+H]+

2. Authors write: Rutin demonstrated the best docking score (-12.39) with multiple inter-actions including hydrogen bonds (H bond) with GLU 802, GLU 879, SER 876, HIS 875 residues, a pi (π)-cation interaction with LYS 771 and a π-π stacking interaction with PHE 1013. 

It is necessary to provide an explanation in the text of why the polyphenolic compound rutin shows such a high molecular docking score. And why such powerful antioxidants as quercetin and luteolin lag behind it. Is it possible to cite other studies in the same area to clarify the issue?

3. Table 7 is uninformative and very difficult to understand and draw the main conclusions. I suggest that the team of authors transform this table into a Venn diagram (if possible).

 

 

Author Response

Without any doubt, this scientific article deserves to be published in such a respected scientific journal. But first you need to make some edits and changes to the layout of the article and the text.

1. Line 142. Authors write: The recursive analysis workflow was extracted by screening detected nodes at various retention times per minute, with a minimum intensity of 6,000 counts and aligned with previously detected compounds( Table 1) considering adducts ([M+K]+ and [M-H]-).

It's mistake? [M+H]+

That is correct. Thank you for noticing this mistake. It has been corrected.

2. Authors write: Rutin demonstrated the best docking score (-12.39) with multiple inter-actions including hydrogen bonds (H bond) with GLU 802, GLU 879, SER 876, HIS 875 residues, a pi (π)-cation interaction with LYS 771 and a π-π stacking interaction with PHE 1013. 

It is necessary to provide an explanation in the text of why the polyphenolic compound rutin shows such a high molecular docking score. And why such powerful antioxidants as quercetin and luteolin lag behind it. Is it possible to cite other studies in the same area to clarify the issue?

Thank you for your comment. As suggested, further explanation has been included in this additional part in the Discussion as follows:

In addition to the common mechanisms of XO inhibition by polyphenols, such as active site binding, substrate blocking, and induction of rearrangements with conformational changes in XO secondary structures, rutin was predicted to significantly increase binding affinity and interaction energies compared to other metabolites, probably due to critical binding interactions of specific amino acids and chemical structure [28, 31, 32].

Furthermore, Rutin, quercetin, and luteolin are all polyphenolic compounds known for their antioxidant properties, but rutin often shows higher antioxidant activity (Antioxidants, 2019). This difference in antioxidant capacity can be attributed to several factors related to their chemical structures and the presence of specific functional groups. Specifically, Rutin is a glycoside of quercetin, meaning it has a sugar moiety (rhamnose and glucose) attached to its quercetin backbone (Žugić et al., 2014). This glycosylation enhances its water solubility, bioavailability, and stability, which can contribute to its higher antioxidant capacity in biological systems. However, Quercetin and Luteolin(Yang et al., 2008). Compounds are aglycones (non-glycosylated forms), which might be less soluble and less stable under physiological conditions compared to rutin. Quercetin and luteolin both lack the sugar moiety that rutin possesses, which could affect their overall antioxidant effectiveness (Škerget et al., 2005).

 

 

References

Antioxidants. (2019). BoD – Books on Demand.

Škerget, M., Kotnik, P., Hadolin, M., Hraš, A. R., Simonič, M., & Knez, Ž. (2005). Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chemistry, 89(2), 191–198. https://doi.org/10.1016/j.foodchem.2004.02.025

Yang, J., Guo, J., & Yuan, J. (2008). In vitro antioxidant properties of rutin. LWT - Food Science and Technology, 41(6), 1060–1066. https://doi.org/10.1016/j.lwt.2007.06.010

Žugić, A., Đorđević, S., Arsić, I., Marković, G., Živković, J., Jovanović, S., & Tadić, V. (2014). Antioxidant activity and phenolic compounds in 10 selected herbs from Vrujci Spa, Serbia. Industrial Crops and Products, 52, 519–527. https://doi.org/10.1016/j.indcrop.2013.11.027

 

3. Table 7 is uninformative and very difficult to understand and draw the main conclusions. I suggest that the team of authors transform this table into a Venn diagram (if possible).

Thank you for suggesting this easier way to understand these data. A Figure 6 showing the suggested Venn diagram has been added.

Please see the attachment. Thank you

Author Response File: Author Response.docx