Inhibition Kinetics and Theoretical Studies on Zanthoxylum chalybeum Engl. Dual Inhibitors of α-Glucosidase and α-Amylase

Round 1

Reviewer 1 Report

The article presents a study about inhibition kinetics and theoretical studies of eleven alkaloids obtained from Zanthoxylum chalybeum Engl., which present inhibitory activity of amylase and glucosidase enzymatic action. The methodology is based on Lineweaver-Burk/Dixon plot analysis, ADMET prediction and molecular docking calculations. The results showed some compounds to have mixed inhibition against both enzimes, a–glucosidase and a–amylase. One compound presented competitive inhibition, and other compounds presented non-competitive and uncompetitive inhibition. The results were compared with experimental data of the reference compound acarbose. Using the technique the molecular docking good binding affinities and significant interactions were found.

I consider original and relevant the topic included in the manuscript. The alkaloid compounds are interesting and they could be promising pharmacological agents against diabetes disease through the modulation of the a–glucosidase and a–amylase enzymes.

Based on before mentioned, I recommend the publication of the manuscript in the Journal of Xenobiotics, of the MDPI editorial, after some improvements. Therefore, my recommendation is Accept after Minor revisions.

Minor revisions:

In Abstract section

Page 1, line 39, change: “Kcal/mol” by “kcal/mol”. Please correct in all the manuscript.

Page 1, line 40, use the Greek symbol “p” instead of “pi”. Please correct in all the manuscript.

In Materials and Methods section

Page 5, in 2.3.1 Ligands preparation sub-section, any compound was optimized using an ab initio or DFT method in addition to the MMFF94x calculations to prove the reliability of the geometries?

Page 6, in 2.3.5 Docking simulation, the scoring function and related parameters should be more specifically described in this section of the manuscript.

In Results and Discussion section

Page 11, line 335, in 3.3. ADME/Tox Prediction section, the BBB term should be defined.

Page 12, line 417, in 3.4. Molecular docking section, in Table 4, use the Greek symbol “p” instead of “pi”.

Page 15, line 433, Figures 3 and 4 contain 2D graphs very small, they contain indistinguishable information. I recommend include the most representative cases and including the rest in the Supplementary Materials section.

Page 16, lines 440 and 463, Figure 5 and Figure 6 contain a lot of information, which is not well distinguished for the reader. I suggest include the most representative cases and including the rest in the Supplementary Materials section.

Pages 13-19, the description of the molecular docking results of the each compound is repetitive, and in most cases it does not provide information other than that contained in Table 4. I suggest summarizing the discussion including the most relevant cases compared with the control compound.

Page 18, line 463, Figure 6 contains a lot of information, which is not well distinguished for the reader. I suggest include the most representative cases and including the rest in the Supplementary Materials section.

Finally, the manuscript should be carefully revised to correct some mistakes of punctuation and typos, blank spaces, etc.

Author Response

Dear Sri/ Madam,

Please see the attachment. 

Thank you!

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Inhibition kinetics and theoretical studies on Zanthoxylum chalybeum Engl. dual inhibitors of α-glucosidase and α-amylase” by Kimani and coworkers describes experimentally determined kinetic parameters as well as computational analysis of eleven natural active compounds as enzyme inhibitors. The work is presented in the correct form and the study is divided into logical sections, although the English language and style require some corrections. The level of originality and novelty is moderate. I would like to make some suggestions. The authors discuss the pharmacokinetic properties of the compounds, among others the BBB penetration and gastrointestinal absorption, therefore the BOILED-Egg plot could be provided. I would also recommend to compare theoretically obtained values such as lipophilicity or affinity to human plasma proteins with experimentally determined.

To sum up, I support the paper for publication in the Journal of Xenobiotics after minor revisions.

Author Response

Dear Sir/Madam,

Please see the attachment.

Thank you!

Author Response File: Author Response.docx

Reviewer 3 Report

The paper entitled "Inhibition kinetics and theoretical studies on Zanthoxylum 2 chalybeum Engl. dual inhibitors of α-glucosidase and α-amyl-3-ase" by Kimani and co-workers describes the binding of several phytochemicals from the Knob Wood family on α-glucosidase and α-amylase activity. The authors have provided both in vitro biological data as well as computational binding assays. I think the paper is not currently ready for publication, and offer the following points to the authors:

 

1.       The methods describing the Kinetic Analyses could be clearer. I do not see a supporting information file in the submission packet, so perhaps this data could be added there if not directly to the manuscript.

2.       The compounds used in the analysis are presumably isolated from natural sources, however, the manuscript makes no mention of where they came from. The authors should state the specific source of these compounds (if commercial) or the procedure for their isolation if not, with the purity noted where known.

3.       In Figure 2 the authors attempt to validate their docking, but these structures have significant deviations from the crystal structures in the I and II rings of the tetrasaccharide. I do not see a clear discussion or quantification of the overlaid structures in Figure 2, but this should be done. How much do the calculated energies deviate by?

4.       The molecular docking section implies that the docking was performed with a static protein and only the individual ligands were given flexibility. If that is the case this section needs to be revised to more clearly state that. In addition, I would encourage the authors to consider the validity of their calculations in that case – the induced fit model would suggest that both protein and ligand will move to the lowest energy conformation, and in that case calculating energies solely giving freedom to the ligand may miss significant interactions that can occur if the protein active site is allowed to change conformation around the ligands.

Author Response

Dear Sir/ Madam

Please see the attachment.

Thank you!

Author Response File: Author Response.docx

Reviewer 4 Report

Please refer to the attachment

Comments for author File: Comments.pdf

Author Response

Dear Sir/ Madam,

Thank you for your valuable time. We have reviewed the sections highlighted and acted as appropriate.

Warm regards

Round 2

Reviewer 3 Report

 

1.         The methods describing the Kinetic Analyses could be clearer. I do not see a supporting information file in the submission packet, so perhaps this data could be added there if not directly to the manuscript.

A supplementary file has been added with all the supporting information.

This has been added, no further comments.

2.       The compounds used in the analysis are presumably isolated from natural sources, however, the manuscript makes no mention of where they came from. The authors should state the specific source of these compounds (if commercial) or the procedure for their isolation if not, with the purity noted where known.

The compounds were isolated from the root barks of Z. chalybeum.   A short desecription have been added in section 2. More details can be found on this published work Ochieng, C.O.; Nyongesa, D.W.; Yamo, K.O.; Onyango, J.O.; Langat, M.K.; Manguro, L.A.O. Fitoterapia 2020, 146, doi:10.1016/j.fitote.2020.104719, which has been cited accordingly.

The authors should add the characterization data which confirms the purity of the compounds.

3.       In Figure 2 the authors attempt to validate their docking, but these structures have significant deviations from the crystal structures in the I and II rings of the tetrasaccharide. I do not see a clear discussion or quantification of the overlaid structures in Figure 2, but this should be done. How much do the calculated energies deviate by?

More details have been provided under section 2.3.5 and it's now clear to the reader.

I appreciate that the authors have added to the discussion, but I am not convinced (as a non-theoretician) by it. THE RMSD is an average over the entire docked structure. There are significant portion of the model that do align well, but what would be the RMSD of just the 3rd ring in A, and the 3rd and 4th ring in B? Notably, the 3rd ring has significant deviations in both structures - can the authors better explain this?

4.       The molecular docking section implies that the docking was performed with a static protein and only the individual ligands were given flexibility. If that is the case this section needs to be revised to more clearly state that. In addition, I would encourage the authors to consider the validity of their calculations in that case – the induced fit model would suggest that both protein and ligand will move to the lowest energy conformation, and in that case calculating energies solely giving freedom to the ligand may miss significant interactions that can occur if the protein active site is allowed to change conformation around the ligands.

The suggestions have been considered and a detailed description is included in section 2.3.5. This section is now clearer and more concisely written.

I'm not sure that the authors have successfully improved the discussion on this, but as a non-theoretician I do not have any useful suggestion about how to improve it.

 

Author Response

Dear reviewer,

Thank you for your feedback.

Please see the attachment

 

Author Response File: Author Response.docx

Reviewer 4 Report

N/A

Author Response

Dear Reviewer,

Thank you for taking the time to review our submission and providing your comments. We appreciate your review and the valuable feedback you have provided to us. We are pleased to hear that you have no further comments to make and we are grateful for your positive assessment of our manuscript. 

Thank you once again for your time and efforts in reviewing our manuscript. We look forward to the next steps in the publication process.

Best Regards,