PONE-D-21-29456Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotectionPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

Reviewer #5: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: After reviewing this manuscript entitled: “Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotection”. I consider that this article is very interesting but I have some concerns which could be improved in order to generate a major impact in readers:

1. In your study, you include an experimental model of hepatoprotection administrating two extracts. Thereby, I consider that your title should be modify as follows:

• Nonlinear Molecular Dynamics of Quercetin determined in Gynocardia odorata and Diospyros malabarica fruits: Its mechanistic role in hepatoprotection.

• However, you could include a similar title that highlights the two fruits involved in your study.

2. Please, include the main objective in your abstract.

3. In page 9 (line 60-61): Scientific names must be italicized. Please, check the whole manuscript.

4. Page 9; Lines 62-64: Authors refers silymarin, which is not the main metabolite of interest in this study. Please, verify your references and only explain the role of quercetin in hepatoprotection.

5. In regard to your introduction: I suggest the followings.

• Try to include only medicinal plants which quercetin has been the responsible effect in the hepatoprotective effect.

• Another paragraph explaining the mechanism of quercetin on the main targets involved in hepatoprotections with updated references.

• Include the main objective in the final paragraph of your introduction and/or secondary objectives.

In material and methods:

1. This sentence should be excluded of the Chemicals section (Molecular interaction and Molecular Dynamics studies were carried out in HP Workstation having (core i7, 3.9 GHZ 85 processor), 32GB RAM, 2TB HDD, NVIDIA Geforce GTX 1650ti graphics processor.)

2. In collection plant: include the GPS data, period of collection, months, where were fruits identified? Any herbarium.

3. In animals’ section: please, include the ethical approval of your institutional committee and reference any international guide for use of experimental animals.

4. In animals’ section: Try to improve your experimental design, how many males and females per group?

5. Type of food or balanced diet?

6. I cannot observe the methodology of the antioxidant activity in your main file. I consider that both antioxidant and histopathological studies should be included in the methodology and referenced.

7. For your antioxidant activity, you could use this reference: Hossain M. S, Uddin M. S, Kabir M. T, Begum M. M, Koushal P, Herrera-Calderon O, Akter R, Asaduzzaman M, Abdel-Daim M. M. In Vitro Screening for Phytochemicals and Antioxidant Activities of Syngonium Podophyllum L.: an Incredible Therapeutic Plant. Biomed Pharmacol J 2017;10(3). https://dx.doi.org/10.13005/bpj/1229

In your results, I cannot observe the table of liver function parameters and biochemical analysis (Table 2) in the main file and supplementary material.

The docking and dynamic analysis is well structured and written.

Your discussion is well planned.

General comments: Authors must correct my comments to improve some aspects such as: Order your methodology. Correct scientific names. Order your figures and tables according to your results. Please, verify and correct the references according to Plos One guide.

Reviewer #2: The manuscript entitled “Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotection”

The liver performs number critical functions in the body. Accumulation of free radicals in liver may eventually cause damage, fibrosis, chirrhosis and cancer. Carbon tetrachloride (CCl4) belongs to hepatotoxin is converted to a highly reactive free radical by cytochrome P450 enzymes that causes liver damage. Plant extracts derived quercetin has substantial role in hepatoprotection. HPLC analysis revealed the abundance of quercetin in the fruit extracts of Gynocardia odorata and Diospyros malabarica, were isolated, purified and subjected to liver function analysis on Wistar rats. Post quercetin treatment improved liver function parameters in the hepatotoxic Wistar rats by augmenting bilirubin content, SGOT and SGPT activity. Gene expression profile of quercetin treated rats revealed down regulation of HGF, TIMP1and MMP2 expressed during CCl4 induction. In silico molecular mechanism prediction suggested that quercetin has a high affinity for cell signaling pathway proteins BCL, JAK and Cytochrome P450 CYP2E1, which all play a significant role in CCl4 induced

hepatotoxicity. In silico molecular docking and molecular dynamics simulation have shown that quercetin has a plausible affinity for major signaling proteins in liver. MMGBSA studies have revealed high binding of quercetin (ΔG) -41.48±11.02, -43.53±6.55 and -39.89±5.78 kcal/mol, with BCL-2 , JAK2 and Cyp2E1, respectively which led to better stability of the quercetin bound protein complexes. Therefore, quercetin can act as potent inhibitor against CCl4 induced hepatic injury by regulating BCL, JAK and Cyp2E1.

Manuscript written very well and extensive studies were done. Instead of having positive points I have seen few silly points which should be rectified before been accepted for publication. My review comments are provided below

1. BCL-2 and JAK2 naming are not uniform throughout the manuscript, uniformity should be maintained throughout the manuscript

2. CCL4, 4 should be in subscript

3. Line 82-83 Himedia India Pvt. Ltd should be incorporated

4. Line 167 Cyp2E1 should be CYP2E1, uniformity must be maintained.

5. Line 241-242 G.odorata and D. malabarica should be in italics as well as in line 248

6. After minute observation I have found in many places few words became conjugated together and that create problematic to read e.g. line 258 “D. malabaricaand” throughout the manuscript. Must be reviewed thoroughly and rectify accordingly.

Reviewer #3: The manuscript entitled “Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotection” has engrossed extensive work and well planned manuscript. The results are presented well and authors have well executed each result. Apart from many pros I have seen some major concern which should be rectified or more deeply explained for better understanding. The manuscript can be accepted after the proper answers of the major and minor comments provided by the author. The comments are as follows:

Major Comments:

1. In, molecular docking studies, the major methodological segment must include the population size, selection of best pose based on RMSD clustering and the RMSD tolerance values. The rationale of selecting best dock poses having lowest binding energy score must be mentioned.

Minor Comments:

1. Beginning from abstract, BCL-2 and JAK2 nomenclature have discrepancies throughout the manuscript, somewhere BCL and JAK, in somewhere BCL-2 and JAK2 or in italics, uniformity should be maintained throughout the manuscript.

2. Line 202, MMGBSA equation should be properly aligned in single line

3. In line 241-242, 248 scientific names should be in italics and uniformity should maintain throughout the manuscript.

Reviewer #4: In this manuscript, Ghosh et al has studied the hepatoprotective potential of the Gynocardia odorata and Diospyros malabarica plant extracts derived quercetin. Authors have used qRT-PCR, in silico molecular docking, molecular dynamics simulation and histological methods to elaborate the hepatoprotection potential of the quercetin. However, there are some dots which need to be connected to make the study cleaner and clearer.

1- Line 19- # contributed equally, must be clear with whom author has contributed equally.

2- Line 20 in Abstract- The liver performs number critical functions in the body. Authors should correct this very first line of the manuscript.

3- There are many mistakes like in line 105- (SYS-LC-138, Systronics, India)with the mobile phase. Authors should revise the manuscript extensively to correct the syntax and grammar.

4- In experimental design, on page 11. “D. malabarica (body weight 200 mg/kg), G. odorata (body weight 200 mg/kg), and Silymarin (200 mg/kg body weight) were given orally to these groups of animals at 2, 24 and 48 h interval after the

administration of the last dose of CCl4 (Table 1)”. Authors must ensure that these intermittent doses must fall in the range of human effective dose to follow terms of translational medicine.

5- Quercetin, is very well established for its osteogenic role. Therefore, authors must ensure that there are not any heterotopic ossification or calcification concern, with the administration of the plants extracts, in the vital organs (heart and kidney).

6- In the Fig S2, Authors must keep the scale bar and it would better if authors replace the figures with higher magnification or more clear ones, to see better at cellular morphology.

7- Authors have used frozen liver tissue to isolate the RNA and perform the qRT-PCR. Authors must show the quantification and quality of the isolated RNA.

Reviewer #5: Dear Author

Thanks for the efforts that are put in this work on Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotection. This work is a combination of in vivo and in silico CADD which is a welcome development in drug discovery process. The study has revealed that quercetin can act as potent inhibitor against CCl4 induced hepatic injury by regulating BCL, JAK and Cyp2E confirming it antioxidant potentials as a flavonoid

1. There are few issues that require revision:

Line 20 need to be revised to read; the liver performs a number of critical functions in the body

2. Line 21 and 22 need to also be revised for proper comprehension

1. How was the purity of the extracted quercetin measured?

2. Line 147 should be clarified. The authors reported molecular redocking, but there was no record of any docking in previous sections

3. Line 148-154 is not necessary, the commentary should be taken to discussion section

4. The diagrams are eye catching and interesting

5. Over all, it was a great study and should be accepted after minor revisions are made on the manuscript

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: MD BASHIR UDDIN

Reviewer #4: No

Reviewer #5: Yes: Daniel Ejim Uti PhD. Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medical Sciences, Federal University of Health Sciences, Otukpo, Benue State Nigeria

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Nonlinear Molecular Dynamics of Quercetin in Gynocardia odorata and Diospyros malabarica fruits : Its mechanistic role in hepatoprotection

PONE-D-21-29456R1

Dear Dr. Baishya,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ghulam Md Ashraf, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed all the comments and the manuscript is now acceptable for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled "Nonlinear Molecular Dynamics of Quercetin in Gynocardia odorata and Diospyros

malabarica fruits : Its mechanistic role in hepatoprotection" has been improved according to my comments and could be accepted in its current form.

Reviewer #2: All comments were properly addressed and clarified. So, the manuscript may be accepted in this Journal

Reviewer #3: All comments are well addressed and could be Accepted for publication. Editing manuscript Language also well improved.

Reviewer #4: Ghosh et. al, have beautifuly addressed, point by point, all my comments and corrected the manuscript accordingly.

Reviewer #5: Dear Author

Thanks for the efforts that are put in this work on Nonlinear Molecular Dynamics of quercetin: Its mechanistic role in hepatoprotection. This work is a combination of in vivo and in silico CADD which is a welcome development in drug discovery process. The study has revealed that quercetin can act as potent inhibitor against CCl4 induced hepatic injury by regulating BCL, JAK and Cyp2E confirming it antioxidant potentials as a flavonoid. The minor issues raised in the earlier manuscript were well addressed in this revised copy. Over all, it was a great study and should be accepted for publication

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes: Daniel Ejim Uti (PhD.)