Anastatin Derivatives Alleviate Myocardial Ischemia-Reperfusion Injury via Antioxidative Properties

Round 1

Reviewer 1 Report

This study characterized a list of anastatin derivatives as antioxidative agents for alleviating myocardial ischemia-reperfusion injury in vitro and in a rat model.

Major point:

The authors claimed that 24 anastatin derivatives were newly designed. However, all the compounds have been disclosed and tested for antioxidative activity in Pan et al, EJMC, 2017. The authors only provided the structure information of compounds without citation, and all the designation numbers used in this study are different from those used in the other one. Although the biological evaluation is comprehensive, this study is lack of novelty in term of the chemistry part.

Other points:

1. The introduction should have updated information on other anti-oxidative agents that are currently used in the clinic or under-investigation, and what are their disadvantages or side effects.
2. The authors should indicate how many biological and technical repeats had been done and calculated in all experiments.
3. Fig. 1 is not cited in the text.
4. The key cell-based assays should be also tested in human cell lines.
5. Fig. 2. The authors should explain more clearly why they choose 2 hours, but not 1 hour, for both H and R. I could not see any difference between these conditions.
6. Fig. 2-4. Some values are pretty close in different figures. For example, the fourth bar of Fig. 2B v.s. the second bar of Fig. 2C. Are they obtained from the same experiment? If yes, the data should be combined into the same figure.
7. Fig. 2D. The legend needs to be corrected.
8. Fig. 4. It is unclear if the compounds protected cells from the oxidative stress resulted from H/R or reduced the toxicity from hypoxia. The authors should test the effect of compound treatments on the toxicity of Na₂S₂O₄ without reoxygenation.
9. Line 100, page 3. Should be 82.68%, not 62.38%.
10. Fig. 4. The positive control resveratrol was the agent with the strongest protective effect, not 1-20. Should be corrected.
11. Should provide information on the time for the pretreatment of compounds.
12. Page 4, 2.3. Should give some references for explaining the importance of the markers used for evaluation of oxidative stress.
13. Page 5, 2.4. Should at least briefly explain the aim and principle of FRAP, ABTS, and DPPH assays.
14. Fig. 6. What is Vc?
15. Fig. 7. What is MDA?

 

Based on these reasons, I do not recommend this manuscript to be published in the Journal.

 

 

Author Response

Response to Reviewer 1 Comments

Dear reviewer,

Manuscript ID: molecules-1278605

TITLE: Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties

We are most grateful to you and the reviewers for the helpful comments on the original version of our manuscript entitled “Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties”. These comments are all valuable and very helpful for revising and improving my paper, as well as the important guiding significance to our researches. We have taken all comments into account and submit, herewith, a revised version of our paper. We marked newly added content in the paper as green and marked modified place as red.

We hope that the revised version of our paper is now suitable for publication in the Molecules and we look forward to hearing from you at your earliest convenience.

 

                                                                        Yours sincerely,

  

                                                                           Yuou Teng, Ph.D.

 

 

The responds to the reviewer’s comments are as follows:

Major point:

The authors claimed that 24 anastatin derivatives were newly designed. However, all the compounds have been disclosed and tested for antioxidative activity in Pan et al, EJMC, 2017. The authors only provided the structure information of compounds without citation, and all the designation numbers used in this study are different from those used in the other one. Although the biological evaluation is comprehensive, this study is lack of novelty in term of the chemistry part.

Dear reviewer, regarding your question, our laboratory had synthesized 24 derivatives of anastatin in the early stage and published them on EJMC (European Journal of Medicinal Chemistry, 2017, 138, 577-589, reference 13). The compounds are synthesized by our laboratory and have applied for patent (ZL201610325752.4). The main work of this research focuses on the biological evaluation of 1-20 of the 24 derivatives with the best activity, which is equivalent to the continuation of the previous work.

The inaccurate description of anastatin derivatives in the paper has been modified. And We have changed the designation numbers of compounds in this paper to make it consistent with the designation numbers in EJMC, which were marked red.

Thanks for your professional advice.

Other points:

1. The introduction should have updated information on other anti-oxidative agents that are currently used in the clinic or under-investigation, and what are their disadvantages or side effects.

Thank you for your suggestion. Your advice can make the introduction of the paper more complete. And therefore, we added some examples and their disadvantages to the introduction to make it more informative, which were marked green.

2. The authors should indicate how many biological and technical repeats had been done and calculated in all experiments.

The repetition times of the experiment have been added in the methods and materials (line 433). Thank you.

3. Fig. 1 is not cited in the text.

Thanks you for pointing out that. We have changed Figure 1 to Figure 2 in order of sequence and added it in the corresponding position.

4. The key cell-based assays should be also tested in human cell lines.

The corresponding experiments in human cell lines and mechanism studies of the compounds are indeed the direction of our next phase of efforts. This point has also been updated in the discussion. Thank you for your suggestions.

5. Fig. 2. The authors should explain more clearly why they choose 2 hours, but not 1 hour, for both H and R. I could not see any difference between these conditions.

When the hypoxia / reoxygenation time was screened, the cell survival rate was higher than 50% at 0.5 hours of hypoxia, but when the time reached 1 hour to 4 hours, the cell survival rate was all below 50%, hypoxia time of 1 hour at the inflection point where the survival rate is higher than 50% or less than 50%, in order to ensure a stable model, we finally choose 2 hours as experimental time.

6. Fig. 2-4. Some values are pretty close in different figures. For example, the fourth bar of Fig. 2B v.s. the second bar of Fig. 2C. Are they obtained from the same experiment? If yes, the data should be combined into the same figure.

The fourth bar of Figure 2B and the second bar of Figure 2C are not data obtained from the same experiment. The former was an exploration of the optimal hypoxia time, and the latter was an exploration of the optimal reoxygenation time after the hypoxia time was determined. There are two different experiments, but the two experiments overlap in the experimental conditions.

7. Fig. 2D. The legend needs to be corrected.

We are so sorry for the mistake. The labeling order of Figure 2D does not match the order of the pictures, and has been changed in the legend. Thanks for your advice.

8. Fig. 4. It is unclear if the compounds protected cells from the oxidative stress resulted from H/R or reduced the toxicity from hypoxia. The authors should test the effect of compound treatments on the toxicity of Na₂S₂O₄ without reoxygenation.

We refer to a lot of literature to establish hypoxia/reoxygenation cell model, but the design may not be very thorough. Your suggestion will be applied to the subsequent establishment of the hypoxia/reoxygenation model on human cell lines. Thanks for your advice again.

9. Line 100, page 3. Should be 82.68%, not 62.38%.

The problem you pointed out is careless of our writing and has been changed in the article. Thank you for your comments.

10. Fig. 4. The positive control resveratrol was the agent with the strongest protective effect, not 1-20. Should be corrected.

Thanks for your advice. Our expression in that is not rigorous and has been revised in the corresponding position (line 125-126).

11. Should provide information on the time for the pretreatment of compounds.

We can’t agree you anymore. The pretreatment time of compounds has been marked in section 4.3.2.

12. Page 4, 2.3. Should give some references for explaining the importance of the markers used for evaluation of oxidative stress.

Thanks for your suggestion. The addition of the explaining of markers can make the article more organized. The corresponding content has been added in section 2.3.

13. Page 5, 2.4. Should at least briefly explain the aim and principle of FRAP, ABTS, and DPPH assays.

The aim and principle of the assays have been added in the corresponding position. Thanks for your advice.

14. Fig. 6. What is Vc?

Thank you for your comment, Vc (Vitamin C). Regarding whether the first English abbreviation has an explanation, we have checked the whole article and marked it in the corresponding position. Sorry again for our negligence.

15. Fig. 7. What is MDA?

Malondialdehyde (MDA), we have modified it in the corresponding position. Thanks for your comment.

 

 

Reviewer 2 Report

In the present manuscript the authors have investigated the cardioprotective and antioxidative effects of Anastatins A and B flavonoids as well as their di- and 13 tri-substituted newly synthesized derivatives.  The cardioprotective effects of the anastatins and their derivatives were evaluated in H9c2 cells. The most potent compound among all derivatives was the 1-20 compound. This compound was used for further examinations. The antioxidative activity of the 1-20 compound was measured using DPPH and ABTS assays. It has been shown that the investigated compound possesses higher antioxidative activity than resveratrol and ascorbic acid. Moreover, the effect of 1-20 compound on myocardial I/R injury in rats was investigated. It has been observed that compound 1-20 significantly reduced infarcted areas and improved histopathological and electrocardiogram changes in rats with myocardial ischemia-reperfusion (I/R) injury. Moreover, compound 1-20 decreased the leakage rates of serum lactate dehydrogenase, creatine kinase, and malonyldialdehyde from rat myocardial tissues and increased the level of glutathione and superoxide dismutase activities following myocardial I/R injury in rats. Considering all the abovementioned facts, the authors concluded that compound 1-20 had potent cardioprotective effects against myocardial I/R injury both in vitro and in vivo owing to its extensive antioxidant activity.

Although the present manuscript has the potential to be published in the Molecules, some crucial parts of the manuscript are missing. The manuscript could be reconsidered for publishing after providing those details.

In the aims of the present study, as well as in the abstract, it has been stated that 24 derivatives of anastatins A and B were synthesized. In addition, in the abstract, it has been stated that their structures were confirmed by 1H NMR and 13C NMR as well as LC-MS. However, no details about the synthesis or structural characterization of the synthesized compounds were provided. The authors should provide all those details.

Additional comments:

Keywords: If a certain word appears in the title, it should not be in the keyword section. Please replace the repeating keywords.

Figure 1. The authors provided Figure 1 (A and B). However, they do not refer to this figure. Please correct this.

Lines 103-105. The authors claim: “Therefore, compound 1-20 (Fig.1C) had strongest protective effect among resveratrol and other anastatins derivatives from H/R injury and was used in the subsequent experiments.” From the results in Table S2 it can be seen that resveratrol increased the cell survival rate to 82.68%, which is more than the increase produced by the anastatin A (71.49%) and compounds 1-44b (71.49%), 1-44c (71.00%), 1-38b (76.5%), 1-38c (73.24%), 1-20 (80.82%), and 1-30 (77.30%). Considering this fact, is it correct to conclude that 1-20 compound has a stronger protective effect than resveratrol? In addition, could the authors provide some structure-activity relationship insights to explain why compound 1-20 possesses greater activity than the other investigated compounds?

Lines 244-252, as well as 253-264 suit better in the Introduction part.

Lines 274-276. The sentences are not clear enough. Please be more precise.

I suggest lines 287-302 to be placed as a separate paragraph to be more transparent.

Lines 297-302: What is the connection between tournefolic acid B and information provided in selected lines with the current study? Please be more precise.

In Table S3 in Supporting Information what the ** and *** signs mean?

All abbreviations should be explained when used for the first time throughout the manuscript.

In addition, correct typos throughout the manuscript.

Author Response

Response to Reviewer 2 Comments

Dear reviewer,

Manuscript ID: molecules-1278605

TITLE: Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties

We are most grateful to you and the reviewers for the helpful comments on the original version of our manuscript entitled “Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties”. These comments are all valuable and very helpful for revising and improving my paper, as well as the important guiding significance to our researches. We have taken all comments into account and submit, herewith, a revised version of our paper. We marked newly added content in the paper as green and marked modified place as red.

We hope that the revised version of our paper is now suitable for publication in the Molecules and we look forward to hearing from you at your earliest convenience.

 

                                                                        Yours sincerely,

  

                                                                           Yuou Teng, Ph.D.

 

 

The responds to the reviewer’s comments are as follows:

 

In the aims of the present study, as well as in the abstract, it has been stated that 24 derivatives of anastatins A and B were synthesized. In addition, in the abstract, it has been stated that their structures were confirmed by 1H NMR and 13C NMR as well as LC-MS. However, no details about the synthesis or structural characterization of the synthesized compounds were provided. The authors should provide all those details.

Thank you for your advice. The 24 derivatives are compounds synthesized by our laboratory in the early stage. The chemical characterization results had been published in EJMC (European Journal of Medicinal Chemistry, reference 13). This point was specially explained in the discussion. The main work of this research focuses on the biological evaluation of the 24 derivatives with the best activity, which is equivalent to the continuation of the previous work.

 

Keywords: If a certain word appears in the title, it should not be in the keyword section. Please replace the repeating keywords.

The keywords have been modified. Thank you for your reminder.

Figure 1. The authors provided Figure 1 (A and B). However, they do not refer to this figure. Please correct this.

Thank you for the carefulness. We have added the reference of Figure 1 (A and B).

 Lines 103-105. The authors claim: “Therefore, compound 1-20 (Fig.1C) had strongest protective effect among resveratrol and other anastatins derivatives from H/R injury and was used in the subsequent experiments.” From the results in Table S2 it can be seen that resveratrol increased the cell survival rate to 82.68%, which is more than the increase produced by the anastatin A (71.49%) and compounds 1-44b (71.49%), 1-44c (71.00%), 1-38b (76.5%), 1-38c (73.24%), 1-20 (80.82%), and 1-30 (77.30%). Considering this fact, is it correct to conclude that 1-20 compound has a stronger protective effect than resveratrol? In addition, could the authors provide some structure-activity relationship insights to explain why compound 1-20 possesses greater activity than the other investigated compounds?

Thanks you for pointing out that. It should be 1-20 has the strongest protective effect among anastatins derivatives and has been changed in the corresponding position. Thank you for your rigor and seriousness.

Lines 244-252, as well as 253-264 suit better in the Introduction part.

We can’t agree you anymore, and the corresponding content has been transferred to the introduction, thanks for your advice.

Lines 274-276. The sentences are not clear enough. Please be more precise.

Thank you, we noticed the expression problems, and reorganized the sentences shown in the discussion which were marked green.

I suggest lines 287-302 to be placed as a separate paragraph to be more transparent.

Thank you for your comments. Lines 287-302 as a separate paragraph will make the ideas of discussion clearer. We have modified the corresponding part.

Lines 297-302: What is the connection between tournefolic acid B and information provided in selected lines with the current study? Please be more precise.

The compound mentioned in lines 297-302 is compound that has been reported to protect against myocardial ischemia/reperfusion injury. The study of its mechanism has instructive significance for the next step of this research to study the mechanism of 1-20 to exert protective effects.

We added content about the relationship between tournefolic acid B and information provided in selected lines and our future research at the end of the discussion section. Thank you for your advice.

In Table S3 in Supporting Information what the ** and *** signs mean?

Thank you for your suggestion. The corresponding significant difference analysis has been added below the Table S3.

All abbreviations should be explained when used for the first time throughout the manuscript.

Thank you for your comments. Regarding whether the first English abbreviation has an explanation, we have checked the whole article and modified it in the corresponding position.

In addition, correct typos throughout the manuscript.

Thank you for your advice. We have conducted a thorough check of typos.

In addition, we have changed the designation numbers of compounds in this paper to make it consistent with the designation numbers in European Journal of Medicinal Chemistry, which is the journal published our chemistry-related results. 1-20 to 13, I hope it won't cause any trouble to your review again.

Reviewer 3 Report

Work with flavonoids always would be an interesting area. The authors propose a cardioprotective strategy based on an Anastatin derivative and have excellent results that may open up an interesting area of study.

For to ensure a quality manuscript  the Authors must may the following changes:

1. Figure 1 it is not correct localized (they must be the Figure 3 and be explained in line 103-104.
2. Figures 8 and 9 must be compressed into only one figure.
3. Please remake the graphs to ensure that all the bar scales have the same format.
4. In discussion explain the possible mechanism of antioxidant and cardioprotective (molecular and cellular).
5. How the authors choose the doses of 1-20 compound?
6. The bioethical declaration and committee approval are missing
7. What was the statistical software used for the analysis?

Author Response

Response to Reviewer 1 Comments

Dear reviewer,

Manuscript ID: molecules-1278605

TITLE: Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties

We are most grateful to you and the reviewers for the helpful comments on the original version of our manuscript entitled “Anastatin derivatives alleviate myocardial ischemia-reperfusion injury via antioxidative properties”. These comments are all valuable and very helpful for revising and improving my paper, as well as the important guiding significance to our researches. We have taken all comments into account and submit, herewith, a revised version of our paper. We marked newly added content in the paper as green and marked modified place as red.

We hope that the revised version of our paper is now suitable for publication in the Molecules and we look forward to hearing from you at your earliest convenience.

 

                                                                        Yours sincerely,

  

                                                                           Yuou Teng, Ph.D.

 

 

The responds to the reviewer’s comments are as follows:

 

1. Figure 1 it is not correct localized (they must be the Figure 3 and be explained in line 103-104.

Thank you for your suggestion. Because Figure 3 and Figure 4 have a comparative relationship, we changed Figure 1 to Figure 2 and the corresponding positions in the article have been changed and marked as red.

2. Figures 8 and 9 must be compressed into only one figure.

Figure 8 shows the typical segments related to the electrocardiogram appearing in the experimental results. To make it easier for readers to read electrocardiogram. Figure 9 shows the effect of the compound on the rats with myocardial I/R injury. At present, the two cannot be compressed into one graph. We can change the content of Figure 8 or delete it. We look forward to your advice.

3. Please remake the graphs to ensure that all the bar scales have the same format.

Thank you for your suggestions on the format of the bar. We have modified the format of the bar. Thank you again for your comments.

4. In discussion explain the possible mechanism of antioxidant and cardioprotective (molecular and cellular).

Your opinion is very important for the completeness of the discussion section, we added the possible cellular mechanisms and pointed out the mechanism research content to be done in the next step, which marked as green in the discussion.

5. How the authors choose the doses of 1-20 compound?

The dose of 1-20 is confirmed based on the dose commonly used in the literature for the positive drug resveratrol.

6. The bioethical declaration and committee approval are missing.

The content of bioethical declaration and committee approval have been added in the methods and materials (line 397-400). Thank you for your rigor and conscientiousness.

7. What was the statistical software used for the analysis?

The software for analyzing statistics has been added in the methods and materials (line 433-434). Thank you for pointing out the loopholes in the article.

 

 

In addition, we have changed the designation numbers of compounds in this paper to make it consistent with the designation numbers in European Journal of Medicinal Chemistry, which is the journal published our chemistry-related results. 1-20 to 13, I hope it won't cause any trouble to your review again.

 

 

Round 2

Reviewer 1 Report

Thanks for the authors' response. Overall, I am satisfied with most changes. However, the abstract has to be further modified to avoid confusion with the same question. My suggestion is as below:
(±)-Anastatins A and B are flavonoids isolated from Anastatica hierochuntica. These two compounds are reported to have hepatoprotective effects. In the present study, we evaluated the cardioprotective effects of the anastatins and twenty-four di- and tri-substituted derivatives in H9c2 cells that had been subjected to mimic hypoxia/reoxygenation treatment………

In addition, the reference [13] should be cited in the first sentence of the Result section 2.2 (line 115), the title of Table S1, and at the end of the first sentence of the Discussion section. The sentence “The detailed chemical synthesis and characterization results have been published in the journal European Journal of Medicinal Chemistry (EJMC)” can be removed (page 8, line 274).

Additional suggestion: It’s not necessary to have the key word “in vitro and in vivo”.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is much better now. The authors implemented most of the suggestions. I would recommend this manuscript for publication after minor revision.

In the response, the authors stated that the 24 derivatives were synthesized and characterised in their previous publication (European Journal of Medicinal Chemistry, reference 13) and that they provided an explanation of this point in the Discussion part. However, in the abstract, the following confusing sentences are present: "In the present study, twenty-four di- and tri- 13 substituted novel derivatives of anastatins were designed and synthesized in 5–6 steps with 25–45% 14 overall yields. Their structures were confirmed by 1H NMR and 13C NMR as well as LC-MS." 

Please, change the selected sentences to clarify that the compounds were synthesised and characterized in the previous study.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The new improved manuscript is ok for me.

In this answer, please be sure that it is correctly referenced the elected doses.

5. How did the authors choose the doses of 1-20 compounds?

The dose of 1-20 is confirmed based on the dose commonly used in the literature for the positive drug resveratrol.

Author Response

Please see the attachment.

Author Response File: Author Response.docx