-->PONE-D-25-54833-->-->In vitro and in vivo antibacterial activities of 80% methanol crude Ehretia cymosa leaf extract (Boraginaceae)-->-->PLOS One

Dear Dr. Gashaw,

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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?

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: 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: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

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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

Reviewer #3: No

Reviewer #4: No

**********

-->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: 1. The Introduction: Add a short paragraph comparing your work to recent related studies (2020–2024) to emphasize relevance and current contribution.

Extraction yield: The manuscript describes the extraction method but does not report the yield (% w/w). Include this value for reproducibility.

2. Justification of doses: Clarify why 5% and 10% ointment concentrations were selected and how they relate to MIC/MBC data.

3. Controls: The use of ciprofloxacin and nitrofurazone is appropriate but explicitly mention the rationale for their selection (e.g., broad-spectrum antibiotic and standard topical antibacterial agent).

4. Sample size and power: Include the number of replicates (n) for each assay and justify animal group sizes (e.g., n=5) according to statistical power or ethical guidelines.

5. Ensure all p-values and post-hoc comparisons are clearly stated. Some tables (e.g., Tables 2, 5, 6) have multiple superscripts and comparisons that are difficult to interpret. Simplify with consistent notation and include legends explaining all comparisons.

6. Specify the software version and statistical tests (SPSS 27, one-way ANOVA with Tukey’s post-hoc) in the Methods section only once, avoiding repetition in Results.

7. Consider including effect sizes or confidence intervals where possible.

8. Figures 2 and 3 (burn wound healing images): Add clear labels (A–E) for each treatment group, include a scale bar, and improve resolution.

9. Table formatting: Ensure uniform decimal places and consistent units (e.g., mg/mL).

10. MIC/MBC tables: Include reference antibiotic data for comparison (e.g., ciprofloxacin MICs).

11. Graphical summary: Adding a schematic diagram summarizing experimental design or main findings would greatly improve readability.

12. The wound contraction results are promising, but interpretation should avoid overclaiming. Rephrase statements like “demonstrated greater effectiveness than controls” to “demonstrated significantly faster wound contraction compared to control groups.”

13. Discuss possible mechanisms (e.g., presence of flavonoids, phenolics) more critically and cite specific references linking these compounds to antibacterial and wound-healing effects.

14. Provide visual quantitative comparison (e.g., line graph of % wound contraction over time).

15. Relating MIC/MBC data to previously published values.

16. Explaining why S. aureus was more susceptible than P. aeruginosa (e.g., cell wall differences).

17. Adding a Limitations paragraph — e.g., crude extract used instead of purified compounds, lack of toxicity profiling, small sample size, absence of histopathology.

Reviewer #2: Please consider the following comments:

1- The resolution of the figures should be adjusted as it is very poor.

2- The wound are should be presented in a figure

3- where is the characterization of the prepared ointment?

4- Where is the characterization of the plant extract?

5- There are numerous English errors in the manuscript that should be revised.

6- The instruments section should be removed and each instrument should be clarified in its position in the manuscript

7- A graphical abstract should be added

8- More recent references should be cited

9- Where is the histological and immunohistochemical analysis of wound tissues?

Reviewer #3: Review Report

MS No.: PONE-D-25-54833

Title: In vitro and in vivo antibacterial activities of 80% methanol crude Ehretia cymosa leaf extract (Boraginaceae)

Date: Dec. 12, 2025

The manuscript investigates the antibacterial activity of Ehretia cymosa leaf extract through both in vitro assays and an in vivo infected-wound model, an important area of study given rising antimicrobial resistance and growing interest in medicinal plants. The work is generally well organized, provides adequate methodological detail, and has several strengths, including the combined use of laboratory and animal models, clear descriptions of plant processing and animal handling, and strong relevance to Ethiopian traditional medicine. However, substantial revisions are needed, particularly regarding experimental design, statistical analysis, clarity of writing, and interpretation of the findings, before the manuscript can be considered suitable for publication.

Thank you!

________________________________________

I. Major Comments

1. Lack of Phytochemical Characterization

The study uses a crude methanolic extract but does not include any qualitative or quantitative phytochemical analysis. This omission limits interpretation, as the antibacterial activity can vary greatly among different constituents, and it also makes comparison with previous studies difficult. I recommend that the authors include at least a preliminary phytochemical screening (e.g., phenolics, flavonoids, tannins, alkaloids, and saponins). Ideally, they should also report the total phenolic and flavonoid contents.

2. Choice of Concentrations in Agar Diffusion

The manuscript employs very high extract concentrations (100–300 mg/mL), which may lead to non-specific inhibition simply due to the large mass of extract used, and may also introduce diffusion limitations that complicate comparisons with ciprofloxacin. The authors should justify the need for such high concentrations and discuss the inherent limitations of comparing crude plant extracts with purified antibiotics.

3. Missing MIC and MBC Clarity

The reported MIC values are relatively high (6.25–75 mg/mL), and bactericidal activity is observed only for two organisms at very high concentrations (200 mg/mL). However, the manuscript does not compare these MIC values to standard interpretive criteria, nor does it explain the absence of MBC values for S. aureus, S. pyogenes, and K. pneumoniae. The authors should provide a clearer interpretation of whether these results indicate strong, moderate, or weak antibacterial activity

4. Burn Model Description Needs Clarification

The burn-creation procedure raises methodological concerns. The stated temperature of 100 °C is considerably lower than the typical 150–300 °C used in standardized rodent burn models, while the reported contact duration of 10 seconds is unusually long and may result in excessive tissue damage. The authors should provide appropriate citations supporting this method and justify the selected temperature and exposure time.

5. Absence of Microbial Load Quantification

The in vivo study lacks quantitative assessment of bacterial load before and after treatment, and it does not include microbiological confirmation that extract-treated wounds exhibit a reduced bacterial burden. This gap limits the ability to conclude whether the observed effects are truly antibacterial rather than solely wound-healing. The authors should include CFU quantification if feasible, or acknowledge this omission as a limitation.

6. Randomization, Blinding, and Sample Size

The manuscript does not clarify whether treatment group allocation was randomized, whether wound measurements were performed in a blinded manner, or how the chosen sample size (n = 5) was statistically justified. The authors should add statements addressing randomization, blinding, and any power estimation used to support the selected group size.

7. Statistical Analysis Issues

The tables use multiple superscript letters (a, b, c, d, e) that are not always clearly defined or applied consistently, and several results refer to “significant differences” without reporting the corresponding p-values. Moreover, effect sizes and confidence intervals are not provided. The authors should improve statistical transparency and refine the presentation of the results.

8. Writing and Clarity Problems

Several sections of the manuscript contain grammatical errors, repetition, and overly long sentences that affect clarity.

Examples (from Abstract and Introduction):

• “E. Cymose 80% methanol leaf extract” → E. cymosa (typo)

• “There is some evidence of its antibacterial activity…” but no citation support.

• In addition, many paragraphs in the Introduction are excessively long and combine unrelated ideas.

Comprehensive language editing is required throughout the manuscript.

9. Interpretation and Overstatement

The manuscript’s conclusion that the extract exhibits “significant antibacterial activity” appears overstated. The MIC values are very high, indicating low potency; several important pathogens showed no bactericidal response; and the observed in vivo improvements may be attributable to wound-healing effects rather than direct antibacterial action. The authors should revise the conclusions to present a more cautious and accurate interpretation of the findings.

II. Minor Comments

1. Include Latin binomial in italics throughout (Ehretia cymosa).

2. Clarify units (mg/ml vs mg/mL).

3. Avoid unnecessary repetition in Methods.

4. Replace low-quality figures with high-resolution ones.

5. Ensure all abbreviations are defined upon first use.

Recommendation

Major Revision

The study has scientific merit but requires substantial methodological, analytical, and editorial improvements.

Reviewer #4: I was hardly following the narrations of tables in the results section. Narration was not well written. Please describe the main findings explicitly, and mention the significance level when comparing the tests.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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Attachments

Attachment

Submitted filename: Review Report-Daniel.docx

Attachment

Submitted filename: PONE-D-25-54833 comment.pdf

-->PONE-D-25-54833R1-->-->In vitro and in vivo antibacterial activities with phytochemical screening of 80% methanol crude Ehretia cymosa leaf extract (Boraginaceae)-->-->PLOS One

Dear Dr. Gashaw,

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 May 13 2026 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 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Jorddy Neves Cruz

Academic Editor

PLOS One

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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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: All comments have been addressed

Reviewer #4: (No Response)

**********

-->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: Partly

Reviewer #4: Partly

**********

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

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: No

Reviewer #4: No

**********

-->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 authors have satisfactorily addressed the reviewers’ comments and revised the manuscript accordingly. The revisions have improved the clarity of the methodology, statistical presentation, figures, and discussion. The manuscript is now clearer and the interpretation of the findings is more balanced. Overall, the study presents useful data on the antibacterial activity of Ehretia cymosa leaf extract.

Reviewer #3: Reviewer Report for PONE-D-25-54833_R1

The manuscript titled “ In vitro and in vivo antibacterial activities with phytochemical screening of 80% methanol crude Ehretia cymosa leaf extract (Boraginaceae)” investigates the biological activity and preliminary phytochemical screening of the leaf extract. The topic is scientifically relevant, particularly within the fields of natural product chemistry, pharmacology, and antimicrobial research.

While the study presents experimental work and some potentially useful findings, the manuscript requires substantial revision before it can meet the standards of clarity and scientific rigor expected for publication in PLoS One. Several aspects of the manuscript—including the clarity of the research objective, statistical analysis, presentation of results, and integration of findings with existing literature—require improvement.

Comments

1. Title

The title generally reflects the topic of the study; however, it could be improved for greater clarity. Therefore, the title may be revised as: “In Vitro and In Vivo Antibacterial Activities and Phytochemical Screening of 80% Methanol Extract of Ehretia cymosa Leaves.”

2. Abstract

The abstract provides a general overview of the study; however, it lacks sufficient clarity. In particular, the last two sentences should be revised and improved to enhance their clarity and readability: “The 80% methanol extract of E. cymosa leaves showed moderate antibacterial activity. These findings may partially support its traditional medicinal use and suggest that the plant could serve as a potential source of natural antimicrobial agents and other bioactive secondary metabolites. However, further studies are recommended, including the isolation and characterization of active compounds, microbiological investigations to quantify bacterial load, and pharmacological evaluation using wound-healing models.”

3. Introduction

The introduction provides some background information; however, it needs a clearer and stronger statement of the research gap. The manuscript states that “Experimental studies have shown that crude extracts and solvent fractions of E. cymosa exhibit a range of biological activities, including antioxidant, antihyperglycemic, antidiarrheal, paralysis-relieving, antiepileptic, and antimicrobial effects (11).” Since the antimicrobial activity of the plant has already been reported, the rationale for investigating its antibacterial activity again is not clearly justified. Therefore, the authors should explicitly explain the specific research gap and clarify how the present study differs from or adds to the existing knowledge.

4. Materials and Methods

The Methods section has been significantly improved, but some sentences still lack clarity and could benefit from language refinement.

5. Results

The statistical reporting in the tables (e.g., Tables 2 and 5) is quite complex due to the use of superscript letters and asterisks, which makes it difficult to understand. Please consider simplifying the presentation so that the results can be interpreted more easily.

In Table 2, 5 μg (Cip) is reported. However, 5 μg represents only the amount, not the concentration. Please report Cip in terms of concentration instead.

Tables 2 and 3 could be merged to improve clarity and the flow of data.

In Tables 3 and 4, it is not necessary to report the test concentration since it has already been described in the Methods section.

6. Discussion

The discussion interprets the results but is somewhat limited and does not adequately compare the findings with previous studies.

In some sections, the discussion overinterprets the results, with conclusions appearing stronger than what the data actually support.

I therefore recommend: Major Revision

Reviewer #4: The plant extract was previously evaluated for antibacterial activity, so this study appears to be a duplication of effort. The narration of tables in the results section didn't follow standard English or scientific article writing standards. Additionally, the conclusion is not in line with the data presented in the manuscript.

**********

-->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.

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Reviewer #1: Yes: Voravuth Somsak

Reviewer #3: No

Reviewer #4: Yes: Wubayehu Kahaliw (PhD, Associate Professor of Pharmacology), Department of Pharmacology, School of Pharmacy, College of Medicine and Health Sciences, University of Gondar.

**********

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Attachments

Attachment

Submitted filename: Review Report-PONE-D-25-54833_R1.docx

Attachment

Submitted filename: Plose One comment.pdf

-->PONE-D-25-54833R2-->-->In Vitro and In Vivo Antibacterial Activities and Phytochemical Screening of 80% Methanol Extract from Ehretia cymosa Leaves-->-->PLOS One

Dear Dr. Gashaw,

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:  -->

• Dear Authors, In this new round of manuscript revision, please make all requested changes carefully and accurately..

-->Please submit your revised manuscript by Jul 10 2026 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 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Jorddy Neves Cruz

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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

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In vitro and in vivo antibacterial activities and phytochemical screening of 80% methanol extract from Ehretia cymosa leaves.

PONE-D-25-54833R3

Dear Dr. Gashaw,

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