Dear Dr. Shittu,

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.

ACADEMIC EDITOR:  Major Revision

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We look forward to receiving your revised manuscript.

Kind regards,

Salem S. Salem

Academic Editor

PLOS 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?

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The study is equally contributed in antibacterial activity, so re-consider to change the title accordingly.

Moringa oleifera leaf extracts possess biocompounds with antioxidant and antibacterial activities (24).: Better include the major bioactive compounds in M.oleifera leaf extracts.

Quantitative RT-PCR Assay for antiviral activity: Primer details missing

Fig 1: Explain the small insert in caption. If possible provide spectra of extracts and AgNPs alone as control.

Fig 2.: Very poor resolution. Image is not clear.

Fig 3.: Provide the major components in table or list in caption.

Fig 4.: Provide the image for NC. Also, need to include positive control using commercial antibiotics as reference.

Fig 5.: Did you test the cytotoxicity of leaf extracts?

Fig 6.: Do not provide Ct value, its not actual expression of genes, Change to "Relative expression to HKG or percentage expression"

FIg.6: should investigate major genes such as ACE2 (Angiotensin-Converting Enzyme 2), TMPRSS2 (Transmembrane Serine Protease 2), etc....

The discussion should include a more in-depth comparison of the findings with previous studies, supported by appropriate references to earlier works. Currently, the discussion is concise and would benefit from elaboration and citations to strengthen its context and relevance.

Reviewer #2: Title:

Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water

Dear editor

Greetings

Thank you for giving me the opportunity to review this manuscript. Thanks to the valuable efforts of the authors. This article is very well-organized. However, my comments on this manuscript are as follows.

*The introduction provides a good background but lacks depth in reviewing recent literature. Incorporate more recent studies to contextualize the research within the current state of knowledge.

*The method requires more precise details. Please use the reference below in the method section.

https://www.scopus.com/record/display.uri?eid=2-s2.0-85148103559&origin=inward&txGid=beff2bfbdbe858b60a401178ebbf0d89

*Please summarize the discussion section by presenting key findings and comparisons with recent studies (last 3 years).

*Please explain what are the limitations of this study? The discussion currently does not address the limitations of the study. Including a section that outlines any potential biases, methodological constraints, or external factors that may have influenced the results is essential for transparency and helps contextualize the findings.

*It would be beneficial to suggest specific areas for future research based on the findings and limitations of this study. This can guide subsequent studies and contribute to the ongoing discourse.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes:  Jeevithan Elango

Reviewer #2: 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.

Dear Dr. Shittu,

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 Aug 23 2025 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.

• 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,

Salem S. Salem

Academic Editor

PLOS ONE

Comments from PLOS Editorial Office:

We note that one or more reviewers has recommended that you cite specific previously published works in an earlier round of revision. As always, we recommend that you please review and evaluate the requested works to determine whether they are relevant and should be cited. It is not a requirement to cite these works and you may remove them before the manuscript proceeds to publication. We appreciate your attention to this request.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Comments:

the antiviral activity of the AgNPmo on the SARS-CoV-2 virus: provide the evidance how did the authors claim this effect in abstract.

its efficacy against the bacterial isolates: How it was demonstrated by this study? provide the results, how it was confirmed.

AgNPmo also showed low toxicity on the Vero cells: Evidence.

The IC50 from the cytotoxicity assay demonstrated : Provide IC50 value.

synthesized was cost-effective: Whats the proof compared to commercial one?

These findings suggest that the nanoparticles could be a promising alternative: So the abstract concludes specifically on NPs not moringa, should describe the benefits of moringa and NPs in a distinct way for combating SARS-CoV-2.

Abstract needs to revise in a proper manner to describe the novel results findings of this study.

AgNPmo has the potential to be used for purification or treatment of SARS-CoV-2 contaminated water without exhibiting toxicity: this study did not investigate the objective directly in SARS-CoV-2 contaminated waste-water treatment. How does (in which way) the AgNPmo use in real water treatment.

The silver nanoparticles (AgNPmo) were synthesized ...X-ray spectroscopy (EDX), and X-ray diffraction....using qPCR assay in a dose and time dependent manner. : can be deleted this general statement as already explained before.

color change of the reaction mixture, changing from yellowish to dark brown within 5 minutes. : Its better to provide some images to visualize to the readers.

CCK-8 assay revealed an IC50 value of 38 µg/µl for the 10mM stock concentration: But in Fig.5, the value was mentioned as "µg/ml" not µg/µl. Check the unit.

Analysis of antimicrobial activity, Cytotoxicity assay , Quantitative RT-PCR Analysis: In all these experiments, the authors need to explain why AgNPmo showed beneficial effect? What was the major active compound from Moringa aqueous leaf extract involved? How combining AgNP with moringa affects these activities? Their major signaling pathways.

until reddish-brown coloration was observed. : Its interesting to see this in picture.

The Cell viability test of the synthesized AgNPmo was analyzed through a Cell Counting Kit-8 (CCK-8) on Vero cells.: Why not with SARS-CoV-2 ?

Statistical analysis : Its advised to use some reliable statistical analysis tool to conduct proper statistical significance, using excel 2016 version is not reliable.

Fig 1. Spectrophotometry analysis of the biosynthesized silver nanoparticles Fourier transform infrared spectroscopy (FTIR) : Why the spectra was observed from 350 to 500 nm, however the auhtor stated in method: AgNPmo nanoparticles within the spectral range of 3500 to 500 cm-1. ? What was the small insert? Mark the major peaks in spectra. Did the spectra done for major active compound from Moringa aqueous leaf extract ?

Fig 2.:Scanning Electron Microscopy showing the particle size: The images showed just round red circle, not exact particle size. Its advised to conduct XRD spectra combined with SEM to know the NP size and distribution. At present, XRD analysis of the AgNPmo reported only diffractogram peaks at crystallographic planes (27.91°, 32.37°, and 38.30°. 44.41°, 46.20°), but not NP size and distribution.

Here how did the authors make sure that the red circle is the one corresponding to the NPs?

Fig 3.: Authors should provide the particle size and distrubtion from XRD spectra not just diffractogram peaks corresponds to peaks crystallographic planes. Label Y axis.

Fig 4. : Negative control (NC) and positive control (PC) were missing in Fig 4 A and B. In Fig C and D: explain what was NC in legends and also PC is missing in C and D. Provide statistical significance of test groups compared to controls (P value) in all graphs.

Fig 5.: How many days the cells were cultured, mentioned in legends. The findings were contradictory to the claim by auhtors in abstract "AgNPmo also showed low toxicity on the Vero cells", but the dose of 78.13 ug/ml had high toxicity in Vero cells. Why the values were in negative after 1250 ug/ml dose?

Also its better to provide AgNPmo treated Vero cell images in a seperated Figure as an evidence to show the morphological changes in all these doses.

Fig 6 and 7. Statistical significance on bar

Fig 1.after the synthesis (red line), and analysis after 12 months of synthesis (blue line).: What was the necessity and significance between these two peaks

Include S1 Fig. Negative control of DMSO in Fig.4 as commented above.

S1. Spectrophotometry readings and S2 Table. Spectrophotometry readings: add these peaks directly into corresponding Figure peaks. At least the major peaks can be added in Figs.

S3 Table: The reason for negative value (high cytotoxicity) of doses 312.5 to 5000 μg/μl should be discussed in discussion.

Reviewer #2: Remaining comment:

Comment: The method requires more precise details. Please use the reference below in the method section. https://www.scopus.com/record/display.uri?eid=2-s2.0- 85148103559&origin=inward&txGid=beff2bfbdbe858b60a401178ebbf0d89

Response: the reference (Barjoee et al., 2020) has been cited in the method section.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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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:  Jeevithan Elango

Reviewer #2: 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.

<p>Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water

PONE-D-25-03763R2

Dear Dr. Shittu,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Salem S. Salem

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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

Reviewer #2: Yes

**********

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

Reviewer #2: (No Response)

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

The PLOS Data policy

Reviewer #2: (No Response)

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

Reviewer #2: (No Response)

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Reviewer #2: Dear Authors,

Thank you for your revised manuscript. The revisions have addressed all prior concerns, and the paper now meets the journal's standards for publication.

Congratulations on your work.

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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 #2: No

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