-->PONE-D-26-02620-->-->Exfoliating effect of β-glycyrrhetinic acid on marginal gingivitis-inducing plaque biofilms: comparison with cetylpyridinium chloride-->-->PLOS One-->-->

Dear Dr. Yoshida,-->-->

Thank you for submitting your manuscript entitled “Exfoliating effect of β-glycyrrhetinic acid on marginal gingivitis-inducing plaque biofilms: comparison with cetylpyridinium chloride” to PLOS ONE.

Your manuscript has now been evaluated by three independent reviewers. Two reviewers consider the study to be of interest and potentially valuable, whereas the third reviewer has raised substantive concerns regarding the adequacy of experimental controls and the clarity of the proposed mechanism of action, and has recommended rejection. After careful consideration of all reports, the Editorial assessment is that the work demonstrates potential merit; however, substantial revisions are required to address the identified methodological and conceptual gaps.

Accordingly, we invite you to submit a Major Revision of your manuscript.

Below is a synthesis of the principal issues that must be addressed in your revised submission and detailed rebuttal.

1. Study Scope and Conceptual Framing

The study characterizes the non-conventional antimicrobial profile of β-glycyrrhetinic acid (BGA) in preformed 24-hour supragingival biofilms. A central contribution of the work is the distinction between the bactericidal activity of cetylpyridinium chloride (CPC) and the apparent physical “exfoliating” or “embrittling” effect attributed to BGA. This conceptual distinction is potentially important for plaque management strategies that do not rely exclusively on direct bacterial lethality.

However, the mechanistic interpretation and terminology require greater precision and consistency throughout the manuscript.

2. Major Methodological Limitations

The reviewers identified several limitations that currently constrain the translational and scientific impact of the study:

• Static versus dynamic biofilm conditions: The use of a static 24-hour biofilm model grown on polystyrene does not adequately reflect the shear forces present in the oral cavity. The limitations of this model must be explicitly acknowledged and discussed.

• Exposure duration: A 6-hour treatment exposure does not reflect clinically relevant mouthwash contact times. The manuscript must address how BGA would be expected to perform under shorter, clinically realistic exposure intervals (e.g., approximately 60 seconds), either experimentally or through a reasoned discussion supported by literature.

• Pooling of plaque samples: Although plaque was collected from 12 volunteers, pooling may obscure inter-individual variability in microbial composition and treatment response. This methodological choice and its implications should be clearly justified and discussed.

3. Critical Scientific Concerns

The following issues must be resolved to meet publication standards:

a. Distinction between “killing” and “removal”

The manuscript reports a reduction in CFUs following BGA treatment while indicating no significant change in the live/dead ratio. This suggests that bacterial cells may be detached from the biofilm surface rather than killed. However, the manuscript at times conflates “disinfection” with “exfoliation.” The terminology must be rigorously standardized throughout to clearly distinguish bactericidal effects from physical removal or structural disruption.

b. Insufficient mechanistic evidence

The claim that BGA induces “embrittlement” of biofilms is not currently supported by direct biochemical or structural evidence demonstrating interaction with the extracellular polymeric substances (EPS) matrix. Without such evidence, the proposed mechanism remains speculative. Additional data or a substantially strengthened mechanistic rationale is required.

4. Experimental Gaps and Required Controls

To address the major concerns raised, the following points must be considered:

• Positive control for biofilm dispersal: The study lacks a comparator known to induce matrix degradation or biofilm dispersal. Inclusion of a positive control (e.g., a matrix-degrading enzyme or a well-characterized surfactant) would allow benchmarking of the exfoliating effect attributed to BGA.

• Negative and vehicle controls: The manuscript must clearly confirm that DMSO concentrations used as vehicle controls do not independently affect biofilm integrity. Inclusion of an additional water or saline control is strongly recommended if not already performed.

• EPS-specific visualization: To substantiate the claim of scaffold disruption, incorporation of EPS-targeted staining (e.g., lectin-based probes) in CLSM analysis is recommended. Direct visualization of matrix alteration would significantly strengthen the mechanistic argument.

5. Revision Guidance

In preparing your revised manuscript, please:

• Reframe the Discussion to emphasize biofilm structural modification rather than general antimicrobial activity, unless bactericidal effects are conclusively demonstrated.

• Address all reviewer comments in a detailed, point-by-point response document.

• Explicitly respond to the concerns that led to the recommendation for rejection and explain how each has been resolved.

Please submit your revised manuscript by  Apr 08 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.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelwahab Omri, Pharm B, Ph.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 the following in the Competing Interests section:

“I have read the journal's policy and the authors of this manuscript have the following competing interests: Kayo Sato and Aya Okumura are paid employees of Kao Corporation, Tokyo, Japan. This research was funded by Kao Corporation. The authors declare no conflicts of interest.”

We note that one or more of the authors are employed by a commercial company: Kao Corporation

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

4. 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.]

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: Partly

Reviewer #2: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

-->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

**********

-->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

**********

-->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: This manuscript shows the effects of β-glycyrrhetinic acid (BGA) on supragingival plaque biofilms and compares these effects with those of cetylpyridinium chloride (CPC). The use of complex plaque biofilms is meaningful for clinical application. However, the conclusion that BGA shows a “superior exfoliating effect” appears to be somewhat overstated based on presented results. After appropriate revisions in response to the points, the suitability for publication should be reconsidered.

1. The authors conclude that BGA showes a stronger biofilm-exfoliating effect than CPC. However, an increase in turbidity may reflect not only detached biofilms but also cell debris or aggregates of dead bacteria. Therefore, I think that a greater turbidity does not necessarily indicate a superior exfoliating effect. The authors should clearly describe these limitations and discuss the results more cautiously, avoiding overinterpretation.

2. Iimportant finding of this study is that BGA treatment induces detachment of biofilm components containing viable bacteria. However, in vivo setting, such bacteria may retain the potential for re-adhesion to make biofilms. Therefore, biofilm detachment alone may not reflect a clinical benefit. In addition, the potential application of BGA in oral care products may require more cautious discussion. This point should be addressed in the Discussion.

3. The concentrations of BGA (128 µg/mL) and CPC (40 µg/mL) were determined based on their respective MIC values, which is scientifically reasonable. However, it remains unclear whether these concentrations are clinically relevant and whether comparisons at the same MIC appropriately reflect differences in biofilm-exfoliating activity. The Discussion should clarify the rationale and limitations of the concentration settings and the interpretation of MIC-based comparisons.

4. The discussion of quorum sensing is based on previous studies and is not directly supported by the present results. Some statements may imply quorum sensing involvement and should be more clearly presented as speculation.

5. In Fig. 6, arrows or brief annotations indicating detached biofilm aggregates would improve clarity.

6. In Fig. 5B, the detection limit of the CFU assay should be clearly indicated.

Reviewer #2: The following corrections are required

1. Lines 224–226: Revise all claims of “bactericidal action” through out manuscript because MIC/MBC ratios and LIVE/DEAD data show BGA is mainly bacteriostatic, not bactericidal.

2. Lines 227: Correct the misreporting of bacterial strains (e.g., JP2 is not Prevotella denticola according to Table 1) 3. Line 232: The author has declared that all the data is available but here mentioned -"Data not shown". Please justify not including the data, update the claim or provide the data if possible and relevant for the article.

4. Line 394-395: Tone down translational claims (e.g., prevention of periodontal disease) because in vitro biofilm data alone do not justify clinical implications.

5. Line199-201: Clarify whether assumptions for parametric tests ANOVA and t-test (normality, homogeneity) were tested.

6. A few typos and grammatical errors need corrections, for ex- Line 232: "date not shown"...here its date or data, Line 245: viable bacterial, Line 288: "biofilms biomass" should be replaced with 'biofilm biomass', Line 306: CFU unmeasurable should be replaced with -'CFU were below the limit of detection' etc.

Reviewer #3: This study investigated the in vitro effects of BGA on preformed supragingival plaque biofilms and compared its efficacy that with of CPC. The authors reported that BGA reduced viable bacteria and exhibited superior biofilm exfoliation compared to CPC, suggesting a distinct mechanism of action. However, the reviewer has several concerns regarding this manuscript.

1. The title contains ‘marginal gingivitis’. What classification was used for ‘marginal gingivitis’?

2. Introduction: In P3L43-44, there is a statement “The disruption of this homeostatic host-bacteria relationship occurs during gingivitis, marking the initiation of periodontitis [6,7].”. However, is there any evidence in Refs. 6 and 7 to support the idea that the progression of gingivitis causes periodontitis?

3. Introduction: The concern regarding the ‘emergence of CPC-resistant oral bacteria’ is emphasized repeatedly. Is there no concern regarding the emergence of BGA-resistant oral bacteria?

4. Materials and methods: Supragingival plaque was collected from 12 healthy participants. Why were samples not collected from patients with gingivitis?

5. Materials and methods: Minimum bactericidal concentrations (MBCs) were determined using the microdilution method, in which BGA was adjusted to 0, 8, 16, 32, 64, 128, 256, 512, and 1024 μg/mL. How was the MBC of 2048 μg/mL measured in Table 1?

6. Materials and methods: The amount of exfoliated biofilm was determined by measuring the absorbance of the supernatants at OD600 after incubation in the test media for 6 h. However, OD600 was affected by bacterial growth in the supernatants. How do the authors justify the method to consider the absorbance of the supernatants at OD600 as the amount of exfoliated biofilm?

7. Materials and methods: How were the 24h/6h incubation periods determined?

8. Materials and methods: P14L227, ‘Prevotella denticola JP2’ or ‘Aggregatibacter actinomycetemcomitans JP2’?

9. Materials and methods: P14L228, ‘Prevotella denticola ATCC25611’ or ‘Prevotella intermedia ATCC25611’?

10. Results: P15L242, What does “which was significantly (97.8 %) lower (P<0.001, Fig. 1) than that of BGA exposed to DMSO alone.” mean? Was it a comparison with the BGA or DMSO group?

11. Results: P18L294-295, It was stated that “both CPC and BGA reduced biofilm formation to the same degree”; however, Fig 4 shows that there is a significant difference between these two groups.

12. Discussion: P22L368-369: There is a statement that “BGA demonstrated greater effectiveness than CPC in exfoliating biofilms.”. However, OD600 should be affected by the bactericidal and bacteriostatic properties of the test media. How were these bactericidal and bacteriostatic properties excluded from turbidity?

13. The discussion acknowledges the in vitro nature of the study and the unknown exfoliating effects in vivo. However, it could be more comprehensive by discussing other limitations, such as the specific concentrations used, fixed incubation times, or generalizability of results from polystyrene plates to complex oral surfaces.

14. English editing is required throughout the manuscript. The following are examples:

P6L99. ‘Aggraegatibacter’ should be ‘Aggregatibacter’.

P8L120. ‘Flanklin’ should be ‘Franklin’.

P14L232. ‘(date not shown)’ should be ‘data not shown)’.

**********

-->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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.]

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.

Exfoliating effect of β-glycyrrhetinic acid on plaque inducing gingivitis: comparison with cetylpyridinium chloride

PONE-D-26-02620R1

Dear Dr. Akihiro Yoshida,

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,

Abdelwahab Omri, Pharm B, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Accept

Reviewers' comments: