Synergistic antifungal effects of botanical extracts against Candida albicans

Eunjin Cho; Kenneth Acosta; Joshua Henkin; Rinat Abzalimov; Ilya Raskin

doi:10.1371/journal.pone.0340665

Peer Review History

Original SubmissionJuly 14, 2025
29 Aug 2025 Decision Letter - Theerapong Krajaejun, Editor Candida albicans Dear Dr. Cho, 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 Oct 13 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'. 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We look forward to receiving your revised manuscript. Kind regards, Theerapong Krajaejun, M.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf . 2. Thank you for stating in your Funding Statement: “This work was partially supported by NIH / ODS / NCCIH Botanical Center Grant (P50 AT002776 to IR), and the NJ Agricultural Experiment Station of Rutgers, The State University of NJ. Funding support for KA and JH was provided by the National Center for Complementary and Integrative Health through training grant 5T32AT004094.” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 3. Thank you for stating the following financial disclosure: “This work was partially supported by NIH / ODS / NCCIH Botanical Center Grant (P50 AT002776 to IR), and the NJ Agricultural Experiment Station of Rutgers, The State University of NJ. Funding support for KA and JH was provided by the National Center for Complementary and Integrative Health through training grant 5T32AT004094.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.] Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): Two experts in the field have reviewed your manuscript. They provided valuable feedback and suggestions for improvement, which I have reviewed and agree with. Please revise your manuscript in accordance with their comments and submit the revised version by the specified deadline to facilitate the continued evaluation of your work. [Note: HTML markup is below. Please do not edit.] 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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The manuscript "Synergistic antifungal effects of botanical extracts against Candida albicans" is a very interesting manuscript which explained quite detail about the interactions of many active compounds. There are some comments below. Line 32: P. granatum should be written completely Punica granatum since it is stated for the first time in the manuscript. Line 60: Please change word “antibiotic” to “antifungals”. Line 88: Please verify the species name Matricularia chamomilla or Matricaria chamomilla (in Table 1). Table 1: In my opinion, it is better to put Table 1 in Materials and Methods rather than in Introduction. Line 97: Please write “10 µL” in words since it is in the beginning of a sentence. Line 136, 138, 141, 142 : Please correct typo “Sigma-Aldritch” to “Sigma-Aldrich”. Line 146: Please write “200 mg” in words since it is in the beginning of a sentence. Line 178: “minimum inhibitory concentration” should be written for the first time in Line 80 “lower minimum inhibitory concentration (MIC)”. Line 191: Please change word “antibiotic” to “antifungal”. Line 198: Please write “100 µL” in words since it is in the beginning of a sentence. Table 3: Why was Humulus lupulus not tested in checkerboard analysis?. In Line 222-226, it seems that H. lupulus was not excluded. It has low MIC50 (i.e., 0.625 mg/mL). Line 236: The lowest FICI score is 0.039 (highly synergistic). Range of FICI is 0.039 to 4.0, based on data shown in Table 3. Line 241-242: Please verify the econazole concentration as positive control. In Line 241-242: econazole concentration is 2 mg/mL, whereas in Line 191 econazole 30 µg/mL. Figure 2: Please check again the colour in figure 2 with the data in Table 3, and consistent with the criteria of FICI (i.e., Synergistic = FICI < 0.5, additive = 0.5 ≤ FICI < 1, neutral = 1 ≤ FICI < 2, antagonistic = FICI ≥ 2). Below are some inconsistencies: • Figure 2A. H. canadensis/P. granatum MIC90 0.50 is additive (Table 3). Therefore, the colour should be light green. • Figure 2A. P. granatum/E. globulus MIC90 1.0 is neutral (Table 3). Therefore, the colour should be pink. • Figure 2B. H. canadensis/P. amurense MIC90 0.50 is additive (Table 3). Therefore, the colour should be light green. • and other cells Figure 3: Please verify the MIC50 of H. canadensis. In table 2, MIC50 of H. canadensis is 2.5 mg/mL. However in Figure 3, in 1 mg/mL H. canadensis have >50% inhibition. Line 348: The lowest FICI value of H. canadensis is 0.039, combined with E. globulus. Therefore, the value is “0.52 to 0.039”. Reviewer #2: The manuscript titled “Synergistic antifungal effects of botanical extracts against Candida albicans” by Cho et al. investigated the combined action between 86 ethanolic extracts of nine plant previously reported to have strong antifungal activity on prevailing fungal pathogen C. albicans. In this detailed study, the authors used checkerboard microdilution assays to find the synergistic, additive or antagonistic interactions. As resistance of C. albicans to available antifungals increases, the search for the alternative therapies is of high importance. I recommend the publication of this manuscript. Minor comments: Please, explain the reason to choose the C. albicans strain 10231. ****** 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 Attachments Attachment Submitted filename: 250804_Reviewer_HY.docx https://doi.org/10.1371/journal.pone.0340665.r001
Revision 1
2 Nov 2025 Author Response Point-by-point Responses: Revised Funding Statement: This work was supported by NIH / ODS / NCCIH Botanical Center Grant (P50 AT002776 to IR), and the NJ Agricultural Experiment Station of Rutgers, The State University of NJ. Funding support for KA and JH was provided by the National Center for Complementary and Integrative Health through training grant 5T32AT004094. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1. Line 32: P. granatum should be written completely Punica granatum since it is stated for the first time in the manuscript. We have changed the formatting for this as suggested. 2. Line 60: Please change word “antibiotic” to “antifungals”. We have changed the wording as suggested. 3. Line 88: Please verify the species name Matricularia chamomilla or Matricaria chamomilla (in Table 1). We verify that the species used for this research is Matricaria chamomilla, or German chamomile, as written in the manuscript in Table 1, and have changed the typo. 4. Table 1: In my opinion, it is better to put Table 1 in Materials and Methods rather than in Introduction. Thank you for the suggestion, however we believe that Table 1 is best suited for the Introduction as it summarizes previous literature on bioactive compounds in the studied plants and provides context to why they were selected for the synergy testing. It also introduces important conceptual information rather than procedural information. 5. Line 97: Please write “10 µL” in words since it is in the beginning of a sentence. Changed the structure of the sentence. 6. Line 136, 138, 141, 142 : Please correct typo “Sigma-Aldritch” to “Sigma-Aldrich”. Changed as suggested. 7. Line 146: Please write “200 mg” in words since it is in the beginning of a sentence. Changed the structure of the sentence. 8. Line 178: “minimum inhibitory concentration” should be written for the first time in Line 80 “lower minimum inhibitory concentration (MIC)”. Changed as suggested. 9. Line 191: Please change word “antibiotic” to “antifungal”. Changed as suggested. 10. Line 198: Please write “100 µL” in words since it is in the beginning of a sentence. Changed the structure of the sentence. 11. Table 3: Why was Humulus lupulus not tested in checkerboard analysis?. In Line 222-226, it seems that H. lupulus was not excluded. It has low MIC50 (i.e., 0.625 mg/mL). H. lupulus was not tested because our criteria for determining extracts for candidates of synergy testing was focused on higher overall percent inhibition. Although H. lupulus displayed a low MIC50, on the other hand for MIC90, it had overall poorer results. For example, although H. lupulus and P. granatum are both depicted as MIC90 ≥20, at 10 mg/mL H. lupulus has only a 50.92% inhibition whereas P. granatum has 88.10% inhibition against C. albicans. This data in Table 3 does not capture this nuance, but additional supplementary information has been added in S1_File that reflects this. Line 236: The lowest FICI score is 0.039 (highly synergistic). Range of FICI is 0.039 to 4.0, based on data shown in Table 3. Changed as suggested. Line 241-242: Please verify the econazole concentration as positive control. In Line 241-242: econazole concentration is 2 mg/mL, whereas in Line 191 econazole 30 µg/mL. Adjusted to fit most recent data collections – 30 µg/mL. Figure 2: Please check again the colour in figure 2 with the data in Table 3, and consistent with the criteria of FICI (i.e., Synergistic = FICI < 0.5, additive = 0.5 ≤ FICI < 1, neutral = 1 ≤ FICI < 2, antagonistic = FICI ≥ 2). Below are some inconsistencies: • Figure 2A. H. canadensis/P. granatum MIC90 0.50 is additive (Table 3). Therefore, the colour should be light green. • Figure 2A. P. granatum/E. globulus MIC90 1.0 is neutral (Table 3). Therefore, the colour should be pink. • Figure 2B. H. canadensis/P. amurense MIC90 0.50 is additive (Table 3). Therefore, the colour should be light green. • and other cells Changed the ranges on Figure 2 so values at the thresholds (i.e. 0.500, 1.000, etc.) have appropriate colors for their synergy designation. Figure 3: Please verify the MIC50 of H. canadensis. In table 2, MIC50 of H. canadensis is 2.5 mg/mL. However in Figure 3, in 1 mg/mL H. canadensis have >50% inhibition. MIC50 is 2.5 mg/mL. The original value of % inhibition at 1 mg/mL in Figure 3 was a mistake and has been fixed. Line 348: The lowest FICI value of H. canadensis is 0.039, combined with E. globulus. Therefore, the value is “0.52 to 0.039”. Changed as suggested. Minor comments: Please, explain the reason to choose the C. albicans strain 10231. We chose to use C. albicans strain 10231 because it is widely studied and characterized and is a designated strain for QC standards for various assays by CLSI and EUCAST, whose methods were used to base our own study methodology on. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0340665.r002
5 Nov 2025 Decision Letter - Theerapong Krajaejun, Editor Candida albicans Dear Dr. Cho, 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 20 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 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, Theerapong Krajaejun, M.D. 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. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: - Regarding Table 1, while your intention to summarize prior literature and provide conceptual context is noted, relocation to the Materials and Methods section is recommended, consistent with the reviewer’s suggestion and standard scientific conventions. The Introduction should remain a narrative overview supported by citations, rather than a place for structured data. Placing the table in the Methods section, where the selection of plant species is described, will improve clarity, enhance manuscript flow, and allow for more appropriate referencing in the Results and Discussion when interpreting outcomes related to the extracts. - Please include a brief rationale in the Methods section for selecting Candida albicans strain ATCC 10231. This will help clarify its relevance and support reproducibility. [Note: HTML markup is below. Please do not edit.] [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0340665.r003
Revision 2
20 Dec 2025 Author Response We thank the Editor and the Reviewers for their careful evaluation of our manuscript and for providing insightful comments that have helped us improve the quality and clarity of our work. Below, we provide a detailed, point-by-point response to all comments. Changes made in the revised manuscript are indicated by tracked changes. Point-by-point Responses: 1. Regarding Table 1, while your intention to summarize prior literature and provide conceptual context is noted, relocation to the Materials and Methods section is recommended, consistent with the reviewer’s suggestion and standard scientific conventions. The Introduction should remain a narrative overview supported by citations, rather than a place for structured data. Placing the table in the Methods section, where the selection of plant species is described, will improve clarity, enhance manuscript flow, and allow for more appropriate referencing in the Results and Discussion when interpreting outcomes related to the extracts. Moved Table 1 to the “Plant materials” subsection in Materials and Methods, as recommended, with additional brief context on their selection. 2. Please include a brief rationale in the Methods section for selecting Candida albicans strain ATCC 10231. This will help clarify its relevance and support reproducibility. Provided a brief explanation of ATCC 10231 in “Reagents, chemicals and fungal strain” subsection in Materials and Methods as recommended. Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0340665.r004
26 Dec 2025 Decision Letter - Theerapong Krajaejun, Editor Synergistic antifungal effects of botanical extracts against Candida albicans PONE-D-25-38285R2 Dear Dr. Cho, 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, Theerapong Krajaejun, M.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0340665.r005
Formally Accepted
Acceptance Letter - Theerapong Krajaejun, Editor PONE-D-25-38285R2 PLOS One Dear Dr. Cho, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Theerapong Krajaejun Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0340665.r006

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