PONE-D-23-03097Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. in domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysisPLOS ONE

Dear Dr. Kar,

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.

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Reviewers have now commented on your paper. You will see that they are advising that you revise your manuscript. If you are prepared to undertake the work required, I would be pleased to reconsider my decision. Moreover, the writing must be seriously improved. In several occasions phrases seems not have connection with the following phrase, besides lack of scientific accuracy in several parts of manuscript and typographic mistakes.

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: 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: Comments to the authors of the article:

1- It is recommended that in the introduction part of the article, a short explanation about the properties of chemical compounds and active ingredients of Holarrhena pubescens plant extract is given.

2- Raillietina worms isolated from the intestines of birds survived for how many hours in the culture media and laboratory conditions, and by what index is the boundary between the paralysis of the worms and the complete death of the worms determined?

3- It is better for the authors to use more recent references (after 2015) in the article. For example, it is recommended that from the article [Garedaghi yagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

4- It is recommended that in the materials and methods section of the article, the exact geographical location (for example, the exact latitude and longitude) of the sampling location of the Holarrhena pubescens plant, which is in the region of West Bengal, India, and also the sampling year should be mentioned.

Reviewer #2: The manuscript “Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. in domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis” it is interesting and presents good execution in the methodology. However, I would like the authors to consider the following comments:

- In section: Introduction

The introduction is poor. I suggest describing biological, morphological characters, zoonotic and veterinary importance of Raillietin spp. once infections are highly pathogenic in domestic chickens (Gallus gallus domesticus), for example.

In addition, reporting the role of man as an accidental host in Raillietina spp infections. Human infection is rare, but has been reported from Central and South America, Cuba, Iran, Japan, Southeast Asia, the Philippines, French Polynesia, Australia, and the United States (Hawaii).

Please specify the real objective of this study. Why was Raillietina spp chosen as a study model?

-In section: Materials and Methods- Experiments- Efficacy Testing, line 86.

Write more details as parasites were exposed to extracts. Petri dish? Culture plates? medium of culture?

-In section: Results

Fig. 1. The control group cause Mortality of Raillietina spp. ???

Please insert subtitles on the Y axis of the graph (mortality in hours)

-In section: Discussion

The discussion is poor. I suggest discussing the importance of morphological changes in Raillietina spp. presented in SEM and MET, on the influence on the parasite- host relationship.

In addition, emphasize the need to develop new therapeutic alternatives in the veterinary parasitology field, since Raillietina spp. it represents such importance.

Reviewer #3: The reviewed manuscript describes the obtention and study of anthelmintic properties of ethanolic crude extract and their fractions obtained from different solvents from steam bark of Holarrhena pubescens against Raillietina spp. The manuscript affirms the efficacy of extracts base on comparison with Praziquantel (time of paralysis and time of death), besides observation of effects in parasites by Scanning Electron Microscopy, Transmission Electron Microscopy for instance.

The addition of chromatographic profile, dereplication or any other real information about extract and active fractions would improve the manuscript since it does not afford any information about real composition of evaluated extract and fractions.

The manuscript needs considerably improve in terms of scientific accuracy and correction of several typographic mistakes.

The manuscript in the present format is acceptable for publishing in Plos One since the recommendations below should be taken in consideration or answered.

Major corrections:

1. The authors must carefully and judiciously review the writing of manuscript by complete. There are several points that must be rewrite to text become clearer and present scientific accuracy that is needed.

Minor corrections:

1. It would be possible add consistent information of composition of extracts and active fractions of plant species, besides literature data cited?

2. Plant species can vary easily the chemical composition depending on period of year. Add the date of obtention of plant species.

3. Identification of plant species is important to attest its the real identity? Please afford the A the nunber of voucher specimen and Herbarium where it was deposited.

4. (Line17, 18): The authors say that manuscript focuses on ethanolic extract and ethyl acetate fraction, however fractions obtained from different solvents were evaluated in the efficacy test. As result, EtOH and AcOEt presented more efficacy and then this extracts were subject to following procedures. Please could you explain why n-BuOH was not selected for following tests, since the values are quite similar with EtOH extract (Table 1)?

5. (Line 70) Could you explain the observation “(without chlorophyll)”? What does this mean? Any method to remove chlorophyll was used or just to mention that this part of plant is free of chlorophyll?

6. (line 72) “The dried bark was processed…”. What exactly is processed? Please be clear. This phase as others presents in the manuscript lack of scientific accuracy. Probably, processed means “submitted to extraction”. However, the extraction method is described at lines 75, 76, 77 and 78. The authors must rewrite this section aiming a better description of complete process (since obtention of plant material to fraction obtention).

7. (line72) The extraction process took 15-20 days? Could you explain why and describes in detail the complete methodology?

8. (line 248) In discussion section authors bring to manuscript information about phytochemistry aspects of studied plant species base on literature. However, all these points raised by authors necessarily don’t reflect any considerably discussion to paper in this section. These points must be in introduction to present bioactive compounds of studied species. If authors had performed LC-MS/MS analysis of ETOH extract and/or active fractions and dereplication of compounds, a discussion could explored in this section.

9. (line 306) The section conclusion should be rewrite, since points mentioned don’t bring the idea of conclusion of presented work. The ideas should be expanded. I recommend the authors don’t use sentences as “The result of various experiments has shown”.

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

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Reviewer #1: Yes: Yagoob Garedaghi

Department of Parasitology, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran.

Email: Yagoob.garedaghi@gmail.com

https://orcid.org/0000-0003-2976-2706

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-23-03097_review-er-revised.pdf

PONE-D-23-03097R1Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysisPLOS ONE

Dear Dr. Kar,

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 authors have made significant improvements to the manuscript compared to the first version. However, there are still some typographical errors and sentences that need to be rephrased to enhance the text's conciseness and fluency, which are crucial for a scientific article. I recommend that the authors thoroughly revise the text to enhance its English quality. Utilizing the services of a native English speaker would be beneficial in this regard.

Please submit your revised manuscript by August 15, 2023. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Josué de Moraes, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

I recommend that the authors thoroughly revise the text to enhance its English quality. Utilizing the services of a native English speaker would be beneficial in this regard.

[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: All comments have been addressed

Reviewer #3: (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

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #3: Yes

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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 #3: Yes

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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 #3: 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: it is recommended that from the article [Garedaghi yagoob, Khaki, A., Raza, S.H.A., (...), Abdelgayed, S.S., Kakar, M.U. Epidemiological and pathological studies on the helminthic parasites in native chickens of Tabriz city, Iran. Genetics and Molecular Research. 2017; 16(4),gmr16039824. DOI http://dx.doi.org/10.4238/gmr16039824. ] to use in them article references.

Reviewer #3: The authors improved the manuscript compared to the first version. However, it is necessary several adjustments in the text taking into consideration typographic mistakes and phrases that must be rewritten to let the text concise and fluid which is necessary for a scientific paper. Unfortunately, the section that describes the extraction procedure, as well as the obtention of organic phases, remains without details. Even though the focus of the work is the evaluation of anthelmintic properties of extract and organic phases of H. pubescens, details of the obtention of these materials are required and essential. As a final consideration, it would be helpful to submit the text to a scientific-technical translation company to adjust the manuscript to be published.

Bellow follows specific comments and considerations to the authors.

Minor corrections:

1. Please, maintain a solvent nomenclature standardized. Use the first letter capitalized for all of them or none of them (except at the beginning of a phrase, for instance);

2. Use italics for “n” before “-butanol”.

3. Please, pay attention to spaces between characters, punctuation, and hyphen. Several points must be reviewed.

4. The same aspect is seen for units after numbers. Please follow a pattern and check spaces between numbers and units.

5. Lines 143 and 144 – Unfortunately the procedure described is not clear enough. Please be specific as much as you can.

6. Lines 143 and 144 – It is necessary to inform the right procedure of liquid-liquid extraction as requested previously. Be specific in terms of the used mass of crude extract, how this crude extract was solubilized (what solvent), or partially solubilized, the addition order of solvents, quantities of used solvents the final mass of each phase obtained, for instance.

7. Is not completely clear how and which procedures (liquid-liquid or fractional distillation) were employed or in which stage each of them was used.

8. Line 144 – Could you explain how fractional distillation was employed to obtain the organic phases?.

9. Despite information about the chemistry of plant species was included in the introduction, the text must be reviewed from lines 100 to 122 to become more concise and let the text fluid. The whole idea of this part is impaired by the repetition of ideas and phrases that should appear in a better position to bring consistency to the text.

10. Line 111 – Please, replace “Kutaja” with the scientific name of the plant species.

11. Lines 114 to 117 – Please, include the reference about alkaloids in H. pubescens.

12. From lines 134 to 137 – This part of the text needs to be rewritten to bring fluidity.

13. In many parts of the manuscript, the authors don’t let clear the amount of material or time of procedure used. For example, lines 137, 138, 141, and other parts. Why is not shown the exact amount of material and time used?.

14. Line 141 – As a suggestion replace “stored in a container at” with “stored under refrigeration at”.

15. Line 155 mentions a 20 mg/mL concentration in the row of samples tested for efficacy testing. However, it is not seen this concentration in Table 1. Please add the value in the table or remove the information (line 155).

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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: Yes: Yagoob Garedaghi

1. Department of Parasitology, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran.

https://orcid.org/0000-0003-2976-2706

Reviewer #3: No

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[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.

Anthelmintic efficacy of Holarrhena pubescens against Raillietina spp. of domestic fowl through ultrastructural, histochemical, biochemical and GLCM analysis

PONE-D-23-03097R2

Dear Dr. Kumar Kar,

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,

Josué de Moraes, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: 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 #3: Yes

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

Reviewer #3: 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 #3: 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 #3: 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 #3: (No Response)

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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 #3: No

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