Evaluation of Ruthenium(II) N-Heterocyclic Carbene Complexes as Enzymatic Inhibitory Agents with Antioxidant, Antimicrobial, Antiparasitical and Antiproliferative Activity

Reviewer Number

Original comments of the reviewer

Reply by the author(s)

#1

The results are clearly presented however the techniques for determining the MIC  for bacteria lack completely. This part should include : where the strains come from, the medium used, the condition of growth, (temperature, incubation time). 

An outline for the methods was added to the text according to your valuable comments and observation, all the details are presented or may be the reference mentioned contain all the details you asked about. Between page 2 – 7

The diameters must have been measured on agar plate (to be detailed) but how MIC values were determined:  if on agar plates which reference has been used to rely inhibition diameter to product concentration. If done in liquid medium,  what were the concentrations of the product solutions used ? Are the dilutions the same that the ones used for parasites. Indicate also the number of replicates, this not only for the samples but also for the antibiotics used for comparison.

Details were found in the methods section of the text. The results were taken from 3 replicates for all the experiments.

Why antibiotics are not present in the figure 1. In the figure 2 what is the yellow square here for?

Antibiotics with well-known results against all used strains, so we used them only in the MIC

The yellow square is omitted

How authors explain the great discrepancies between the indicated inhibition diameters and MIC results. The discussion should be a little developed. Is the proposed compound have an advantage when compared to other metallic compounds and if yes, why.

When we take the IZ results 3d and 3b gave the higher IZ diameter (over 25 mm), the same results were found when MIC was used for evaluation 3d and 3b gave the lowest values for the MIC (that indicate no discrepancies present in the results). only 3 bacteria were  for MIC evaluation as supporting indicators for the values of IZ.

Third minor notices. What was the mM concentration of the solution of the different compounds, Were they adjusted at the same level? Indicate the meaning of the abbreviation SI. It is unusual to have the Material an Methods part not included in the text. At least an ouline may be given...

Actually we use serial concentration of compounds, when we put outlines of methods we give the references used in our previously published data.

All the names of bacteria have also to be written in italics in full at their first apparition in the text and abbreviated afterwards (e.g. Escherichia coli and E.coli) .

Was done for all microorganisms and parasites

#2

1. In the manuscript, the author combines the result with the discussion, and it is recommended that the author separate the discussion from the result.

Was done. The discussion is separated from results in a specific section

2. The title of the article should be more precise and the word "evaluation" was inappropriate

The title is approved from the committee of Scientific research at Qassim University, so we cannot change it.

3. L44, L58: the reference should be placed before the full stop, please check the full text.

Was done

4. Materials is not found in the article and data analysis method, please add them in detail.

An outline for the methods was added to the text according to your valuable comments and observation

5. L11-112: What substance is evaluated by biological evaluation? How is it evaluated?

We mean the selected compounds

6. To find out how the IC50 values are measured, what is the significance of their values?

All these were mentioned in the section of the methods, that added to the articles

7. The callout should be placed below the picture.

Done for all figures

8. The article pointed out that 3b and 3d were the most effective inhibitors of AChE, and why the conclusion only suggested further research on 3d.

Due to its activity against other microorganisms, cancer cells and parasites. Also it is less toxic against vero cells 1.8 while 3b 1.3 uM.

9. Line 164-166: ruthenium(II) complex 3a can induce apoptosis. Is there any literature or data supporting this？

The sentence shifted from results to discussion and recommendation part.

10.The overall discussion of the article is not deep enough and the layout is confusing. 2 is the result and discussion, 3-5 is only the result, and the author is suggested to further adjust.

Each activity was discussed in separate but some compounds sharing many good activities, that is why we recommend 3d in the last.

#3

The biological functions reported here seem to be interesting. However, the authors show only results without discussion in the text, and this manuscript is simply regarded as a technical report, not a scientific paper. The authors are requested to show their research concept or molecular design and dictate the substituent effects of the carbene ligands in detail in the discussion part.

An outline of methods was added in a separate part, also the discussion is separated from the results according to the valuable comments of all review. We recommend in the future research for mode of action detection to elaborate the exact activity reasons.

In addition, the authors have published several papers on the similar compounds. The authors should show the relevance of the following papers and the present paper at least.

L. Boubakri, A. Chakchouk-Mtiba, O. Naouali, L. Mellouli, L. Mansour, I. Özdemir, et al.: Ruthenium(II) complexes bearing benzimidazole-based N-heterocyclic carbene (NHC) ligands as potential antimicrobial, antioxidant, enzyme inhibition, and antiproliferative agents. J. Coordin. Chem. 75, 2022 - Issue 5-6

.

Was done, in the methods and discussion

Actually these papers were prepared in the same time, and this paper published later after this article was written. Thank you for this valuable observation. 

Abstract: Many symbols and abbreviated names are used here. They must be shown in full names.

There are many abbreviations that are not well defined and making the manuscript less understandable. For example:

P. 2, line no. 65: “acetylcholinesterase (AChE) and tyrosinase (TyrE)”

Was done

In the methods the used enzymes were explained

P. 11, The references numbered [57] and [58] are absent

Were added

Reviewer Number

Original comments of the reviewer

Reply by the author(s)

#1

The results are clearly presented however the techniques for determining the MIC  for bacteria lack completely. This part should include : where the strains come from, the medium used, the condition of growth, (temperature, incubation time). 

An outline for the methods was added to the text according to your valuable comments and observation, all the details are presented or may be the reference mentioned contain all the details you asked about. Between page 2 – 7

The diameters must have been measured on agar plate (to be detailed) but how MIC values were determined:  if on agar plates which reference has been used to rely inhibition diameter to product concentration. If done in liquid medium,  what were the concentrations of the product solutions used ? Are the dilutions the same that the ones used for parasites. Indicate also the number of replicates, this not only for the samples but also for the antibiotics used for comparison.

Details were found in the methods section of the text. The results were taken from 3 replicates for all the experiments.

Why antibiotics are not present in the figure 1. In the figure 2 what is the yellow square here for?

Antibiotics with well-known results against all used strains, so we used them only in the MIC

The yellow square is omitted

How authors explain the great discrepancies between the indicated inhibition diameters and MIC results. The discussion should be a little developed. Is the proposed compound have an advantage when compared to other metallic compounds and if yes, why.

When we take the IZ results 3d and 3b gave the higher IZ diameter (over 25 mm), the same results were found when MIC was used for evaluation 3d and 3b gave the lowest values for the MIC (that indicate no discrepancies present in the results). only 3 bacteria were  for MIC evaluation as supporting indicators for the values of IZ.

Third minor notices. What was the mM concentration of the solution of the different compounds, Were they adjusted at the same level? Indicate the meaning of the abbreviation SI. It is unusual to have the Material an Methods part not included in the text. At least an ouline may be given...

Actually we use serial concentration of compounds, when we put outlines of methods we give the references used in our previously published data.

All the names of bacteria have also to be written in italics in full at their first apparition in the text and abbreviated afterwards (e.g. Escherichia coli and E.coli) .

Was done for all microorganisms and parasites

#2

1. In the manuscript, the author combines the result with the discussion, and it is recommended that the author separate the discussion from the result.

Was done. The discussion is separated from results in a specific section

2. The title of the article should be more precise and the word "evaluation" was inappropriate

The title is approved from the committee of Scientific research at Qassim University, so we cannot change it.

3. L44, L58: the reference should be placed before the full stop, please check the full text.

Was done

4. Materials is not found in the article and data analysis method, please add them in detail.

An outline for the methods was added to the text according to your valuable comments and observation

5. L11-112: What substance is evaluated by biological evaluation? How is it evaluated?

We mean the selected compounds

6. To find out how the IC50 values are measured, what is the significance of their values?

All these were mentioned in the section of the methods, that added to the articles

7. The callout should be placed below the picture.

Done for all figures

8. The article pointed out that 3b and 3d were the most effective inhibitors of AChE, and why the conclusion only suggested further research on 3d.

Due to its activity against other microorganisms, cancer cells and parasites. Also it is less toxic against vero cells 1.8 while 3b 1.3 uM.

9. Line 164-166: ruthenium(II) complex 3a can induce apoptosis. Is there any literature or data supporting this？

The sentence shifted from results to discussion and recommendation part.

10.The overall discussion of the article is not deep enough and the layout is confusing. 2 is the result and discussion, 3-5 is only the result, and the author is suggested to further adjust.

Each activity was discussed in separate but some compounds sharing many good activities, that is why we recommend 3d in the last.

#3

The biological functions reported here seem to be interesting. However, the authors show only results without discussion in the text, and this manuscript is simply regarded as a technical report, not a scientific paper. The authors are requested to show their research concept or molecular design and dictate the substituent effects of the carbene ligands in detail in the discussion part.

An outline of methods was added in a separate part, also the discussion is separated from the results according to the valuable comments of all review. We recommend in the future research for mode of action detection to elaborate the exact activity reasons.

In addition, the authors have published several papers on the similar compounds. The authors should show the relevance of the following papers and the present paper at least.

L. Boubakri, A. Chakchouk-Mtiba, O. Naouali, L. Mellouli, L. Mansour, I. Özdemir, et al.: Ruthenium(II) complexes bearing benzimidazole-based N-heterocyclic carbene (NHC) ligands as potential antimicrobial, antioxidant, enzyme inhibition, and antiproliferative agents. J. Coordin. Chem. 75, 2022 - Issue 5-6

.

Was done, in the methods and discussion

Actually these papers were prepared in the same time, and this paper published later after this article was written. Thank you for this valuable observation. 

Abstract: Many symbols and abbreviated names are used here. They must be shown in full names.

There are many abbreviations that are not well defined and making the manuscript less understandable. For example:

P. 2, line no. 65: “acetylcholinesterase (AChE) and tyrosinase (TyrE)”

Was done

In the methods the used enzymes were explained

P. 11, The references numbered [57] and [58] are absent

Were added

Reviewer Number

Original comments of the reviewer

Reply by the author(s)

#1

The results are clearly presented however the techniques for determining the MIC  for bacteria lack completely. This part should include : where the strains come from, the medium used, the condition of growth, (temperature, incubation time). 

An outline for the methods was added to the text according to your valuable comments and observation, all the details are presented or may be the reference mentioned contain all the details you asked about. Between page 2 – 7

The diameters must have been measured on agar plate (to be detailed) but how MIC values were determined:  if on agar plates which reference has been used to rely inhibition diameter to product concentration. If done in liquid medium,  what were the concentrations of the product solutions used ? Are the dilutions the same that the ones used for parasites. Indicate also the number of replicates, this not only for the samples but also for the antibiotics used for comparison.

Details were found in the methods section of the text. The results were taken from 3 replicates for all the experiments.

Why antibiotics are not present in the figure 1. In the figure 2 what is the yellow square here for?

Antibiotics with well-known results against all used strains, so we used them only in the MIC

The yellow square is omitted

How authors explain the great discrepancies between the indicated inhibition diameters and MIC results. The discussion should be a little developed. Is the proposed compound have an advantage when compared to other metallic compounds and if yes, why.

When we take the IZ results 3d and 3b gave the higher IZ diameter (over 25 mm), the same results were found when MIC was used for evaluation 3d and 3b gave the lowest values for the MIC (that indicate no discrepancies present in the results). only 3 bacteria were  for MIC evaluation as supporting indicators for the values of IZ.

Third minor notices. What was the mM concentration of the solution of the different compounds, Were they adjusted at the same level? Indicate the meaning of the abbreviation SI. It is unusual to have the Material an Methods part not included in the text. At least an ouline may be given...

Actually we use serial concentration of compounds, when we put outlines of methods we give the references used in our previously published data.

All the names of bacteria have also to be written in italics in full at their first apparition in the text and abbreviated afterwards (e.g. Escherichia coli and E.coli) .

Was done for all microorganisms and parasites

#2

1. In the manuscript, the author combines the result with the discussion, and it is recommended that the author separate the discussion from the result.

Was done. The discussion is separated from results in a specific section

2. The title of the article should be more precise and the word "evaluation" was inappropriate

The title is approved from the committee of Scientific research at Qassim University, so we cannot change it.

3. L44, L58: the reference should be placed before the full stop, please check the full text.

Was done

4. Materials is not found in the article and data analysis method, please add them in detail.

An outline for the methods was added to the text according to your valuable comments and observation

5. L11-112: What substance is evaluated by biological evaluation? How is it evaluated?

We mean the selected compounds

6. To find out how the IC50 values are measured, what is the significance of their values?

All these were mentioned in the section of the methods, that added to the articles

7. The callout should be placed below the picture.

Done for all figures

8. The article pointed out that 3b and 3d were the most effective inhibitors of AChE, and why the conclusion only suggested further research on 3d.

Due to its activity against other microorganisms, cancer cells and parasites. Also it is less toxic against vero cells 1.8 while 3b 1.3 uM.

9. Line 164-166: ruthenium(II) complex 3a can induce apoptosis. Is there any literature or data supporting this？

The sentence shifted from results to discussion and recommendation part.

10.The overall discussion of the article is not deep enough and the layout is confusing. 2 is the result and discussion, 3-5 is only the result, and the author is suggested to further adjust.

Each activity was discussed in separate but some compounds sharing many good activities, that is why we recommend 3d in the last.

#3

The biological functions reported here seem to be interesting. However, the authors show only results without discussion in the text, and this manuscript is simply regarded as a technical report, not a scientific paper. The authors are requested to show their research concept or molecular design and dictate the substituent effects of the carbene ligands in detail in the discussion part.

An outline of methods was added in a separate part, also the discussion is separated from the results according to the valuable comments of all review. We recommend in the future research for mode of action detection to elaborate the exact activity reasons.

In addition, the authors have published several papers on the similar compounds. The authors should show the relevance of the following papers and the present paper at least.

L. Boubakri, A. Chakchouk-Mtiba, O. Naouali, L. Mellouli, L. Mansour, I. Özdemir, et al.: Ruthenium(II) complexes bearing benzimidazole-based N-heterocyclic carbene (NHC) ligands as potential antimicrobial, antioxidant, enzyme inhibition, and antiproliferative agents. J. Coordin. Chem. 75, 2022 - Issue 5-6

.

Was done, in the methods and discussion

Actually these papers were prepared in the same time, and this paper published later after this article was written. Thank you for this valuable observation. 

Abstract: Many symbols and abbreviated names are used here. They must be shown in full names.

There are many abbreviations that are not well defined and making the manuscript less understandable. For example:

P. 2, line no. 65: “acetylcholinesterase (AChE) and tyrosinase (TyrE)”

Was done

In the methods the used enzymes were explained

P. 11, The references numbered [57] and [58] are absent

Were added