PONE-D-20-17909

An evidenced based efficacy and safety assessment of the antivenom ethnobiologicals used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PLOS ONE

Dear Dr. Malik,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The detailed reports of the reviewers are enclosed above. All questions deserve attention, especially the improvement of English language use and the discussion section's reformulation.

Please submit your revised manuscript by Aug 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

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2. To ensure that you obtained ethics approval before your study began, please upload the ethics approval document issued by West Bengal Biodiversity Board approval (1.1.2008-22.5.2009) [Memo No. 5k(bio)-2/2007].

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

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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

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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: No

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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: The article “An evidenced based efficacy and safety assessment of the antivenom ethnobiologicals used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects.” presents a survey conducted with traditional healers from a specific region in India, aiming to catalogue traditional formulations used as treatments against animal bites and stings and assess their efficacy and safety using experimental assays with the reported ethnobotanicals. It is an interesting work that addresses an important matter because not only the traditional knowledge can drive further investigations on the pharmacological potential of the natural products, leading to new drugs, but it is also important to characterize possible toxic effects of such formulations in order to protect the population making use of them. Considering the neglected issue of envenoming and the scarcity of available treatments, and the fact that the populations living in poor and isolated districts are mostly affected by these problems, the presented article has merit.

I would recommend this work for publication in PLOSOne, but it must be submitted to a major revision, in order to attend the journal’s quality standards. First of all, the manuscript is to long. Information should be presented in a more concise and direct language, highlighting the main findings. For this, English language, although intelligible, must also be revised. Other issues are listed below:

� Title: the work title mentions “antivenom ethnobiologicals”, but dog/cat/hyena bites, which do not contain venom, are listed as ailments for the traditional formulations. Whether the title should be modified or these particular conditions must be taken out from the results.

� The interviewees are mentioned as “traditional healers” (TH) and “traditional medicine man” (TMM) along the text. This can cause confusion, so only one of the terms should be chosen.

� Material and Methods: in the “Study area and aboriginals” session, the authors make narrative description that, although interesting, should be better placed in the discussion session. The Material and Methods should deliminate the study subject in more direct form.

� The period of time when the survey was conducted differs between the ‘Abstract’ (2008-2009 and 2010-2017) and ‘Materials and Methods’. Which one is correct?

� Table 1 would be better placed in the ‘Results session’.

� In the Results session, it would be interesting to have the demographic profile of the interviewees (sex/age/profession/district).

� The second session in ‘Results’ is named ‘Preparations, application and dose-dependence of TFs’, but there is no mention of the dose-dependence in the results.

� Table 2 should me made horizontally, to increase readability and should be placed in the ‘Quantitative ethnobiology’ session. Also, in this table, what does “voucher number’ stands for? And the numbers that appear after the description of ‘parts used’? This information should be removed or explained in the table’s legend. Information on abbreviations is also lacking in table 5.

� As a suggestion, data presented in table 6 would be more clearly visualized as a Graph.

� Table 7 would also be better read horizontally.

� The Discussion session, unlike the other session, is rather succinct. I believe the other parts of the manuscript could be more concise and Discussion should be further explored, comparing the found information with other similar articles, performed in other regions.

� In line 419, authors state that ‘Most of the snake bites are either dry bites or from non-Poisonous snakes. Still death occurs. There are many instances where patients die only because of fear and psychological shock.’ This seemed odd to me. Are there any references to support this statement?

� In line 494 of the ‘Conclusion’, authors claim that ‘Various conservation strategies have also been described as adopted by the indigenous people in order to use plant resources sustainably.’. I did not find the description of these strategies throughout the text.

Reviewer #2: A broad review regarding the English language, punctuation and spacing should be made.

Several references are mentioned incorrectly during the text, such as seen on lines 102, 105 and 109, for example.

During introduction, authors stress that PLA2 enzymes are responsible for the inflammatory effects of snake bites, and that plant extracts detain some sort of PLA2 neutralizing capacity. However, activity of PLA2 of both human and porcine origin are also evaluated. These subgroups must be more clearly determined in methodology and results and discussion.

Authors should classify the different Authors also claim to evaluate genotoxicity and cytotoxicity, and that should be clear as an aim of the present study as well. The aim of the study is not clearly outlined by the authors.

On Table 1, TF4 is explained as masticated by hand, a term with dubious meaning.

Table 2 is impossible to read and contains some of the main results from the experiment that should be clearly available to the reader.

Table 7 also needs formatting.

Discussion is very superficial, mainly replicating results obtained. It should be rewritten, stressing what each plant extract detains from a biochemical or pharmacological level that corroborates with a lesser or more prominent effect regarding PLA2 neutralization.

Since no aim of the study was properly established, conclusion is also in need of revision.

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6. 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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-17909R1

An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PLOS ONE

Dear Dr. Malik,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 04 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

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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

**********

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

Reviewer #1: N/A

**********

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

**********

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

**********

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: Although the manuscript has been consistently improved from the first submission, there are still some points that should be further addressed prior to publication:

--> In Table 1, there are still unexplained numbers, placed after "Disease/ Disorder" and after some words like "ointment

(1070000)". These numbers should be further explained in the legend or footnote. In addition, the units abbreviations described in the TF Composition (eg: Seowra (Streblus asper) leaves: 4-5 in no.) should also be described in the table's footnotes.

--> Likewise, for Table 2, numbers placed after "Parts used" (eg: root (12040000)), and "Ailment treated" are not explained neither in the table's legend nor in the footnote.

--> The session "Use of ethnobotanicals: toxicity, conservation status and Economic Botany Data Standard" is still a bit confusing, as different subjects are discussed in the same paragraph, without an evident connection between them.

--> I am still concerned about the statement in lines 428-429: "There are many instances where patients die only because of fear and psychological shock." I believe that, indeed, the stress caused by a snakebite can cause some harm but not be the responsible for the victim's death. I did not find support to this statement in the respective references. Therefore, I would recommend to rephrase the statement or to remove it from the discussion.

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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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

An evidence based efficacy and safety assessment of the ethnobiologicals against poisonous and non-poisonous bites used by the tribals of three westernmost districts of West Bengal, India: Anti-phospholipase A2 and genotoxic effects

PONE-D-20-17909R2

Dear Dr. Malik,

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,

Benito Soto-Blanco, DVM, MSc, PhD

Academic Editor

PLOS ONE

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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: (No Response)

**********

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