Combined Cytotoxic Effects of Carvacrol-Based Essential Oil Formulations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

After reading the article Synergistic Anticancer Effects of Carvacrol-Based Essential Oil Formulations, it is important to mention that the study addresses a relevant topic: the use of synergistic formulations of essential oils (Vacrol and S-Mix) against triple-negative breast cancer (TNBC), a pathology with limited therapeutic options. However, I have several comments:

1. Why didn’t you use a positive control in your study?
2. Separate the histology technique into a materials and methods section
3. The interpretation of synergy is based on chemical composition, but no quantitative methods are applied (e.g., isobolographic analysis).
4. Improve the graphical presentation.
5. Mention the limitations of the study.

Author Response

Comment 1: “Why didn’t you use a positive control in your study?”
Response: We agree that a positive control is necessary. Carvacrol (the major component of both formulations) was included as a positive control, and we revised the manuscript to ensure these results are explicitly reported and appropriately discussed. 

Comment 2: “Separate the histology technique into a materials and methods section.”
Response: Done. We moved the full histology workflow into a dedicated subsection under Materials and Methods to improve clarity and reproducibility.

Comment 3: “Synergy interpretation is based on chemical composition; no quantitative methods (e.g., isobolographic analysis).”
Response: We agree. Because we did not test isolated individual components (beyond carvacrol) in defined combinations, we cannot claim quantitatively validated synergy (CI/isobologram). We therefore revised the manuscript throughout to avoid overstatement and to present the observed effects as combined formulation effects, while positioning any synergy discussion as a hypothesis supported by composition/context, and clearly listing this as a limitation.

Comment 4: “Improve the graphical presentation.”
Response: Done. We improved figure quality, labeling, scaling, and readability across all figures.

Comment 5: “Mention limitations of the study.”
Response: Done. We added a clear limitations statement, including (i) lack of quantitative synergy validation and (ii) absence of mechanistic apoptosis/ROS/pathway assays in the current work.

Reviewer 2 Report

Comments and Suggestions for Authors

My recommendations

Abstract

1. In the abstract, include specific results, including the ratios in which synergism was observed, and clearly describe the novelty of this study.

The introduction is excessively long and contains epidemiological content beyond the manuscript's scope; it could be significantly shortened. Some sections repeat similar literature findings; consolidation would improve flow. The general section on cancer can only be summarized in one sentence. Information on breast cancer is also redundant, with some repetition regarding diagnostics and other general information. The negative aspects of cancer also are well known; the introduction should be revised.The authors should describe breast cancer in the context of the activity of this oil or carvacrol, with a short introduction.

2. In the introduction, the authors write that "This high concentration increases the antimicrobial and antioxidant capacity of the plant and simultaneously enhances its potential cytotoxic effects." The text should explain how these activities are related, and why the presence of antimicrobial and antioxidant activity will contribute to the anticancer effect.
3. When mentioning any type of activity in the introduction, IC50 data should be provided.
4. Give examples of plants rich in thyme oil.
5. Line 64. Indicate the type of cell (cell death)
6. Indicate the concentration range for line 72.
7. Has the anticancer activity of carvacrol or vacrol been previously studied?

Results Section

1.What is C-Mix (must be explained immediately). What is its composition, ratio, etc.?

2. Need for characterization of synergy. The manuscript repeatedly refers to "synergistic effects," but no synergy quantification (e.g., Chou–Talalay CI, isobolograms) is presented.

3.In the Results Section, add a description of this table and provide the structural formulas of the components predominantly contained in these extracts.

4.Figures 2 and 3 should be improved in quality and presentation. They are difficult to interpret without clearer dose scaling and labeling.

Figures 6 and 7 require scale bars, more standardized lighting, and clearer representation of sample numbers. A vehicle control (e.g., ethanol, DMSO) should be explicitly stated, as essential oils require solvents for solubilization.

5.The physiological relevance of the chosen concentrations (1–10 mM) should be discussed, as these are extremely high for essential-oil components and raise concerns about non-specific toxicity.

6. Statements about pathways (NF-κB, TRPM7, PI3K/AKT) are speculative; no pathway analysis (Western blot, PCR, ROS, mitochondrial potential) was performed.
7. Claims of “selectivity” toward cancer cells need more rigor. Only one normal cell line was tested with single time point and limited replicates. Reframe mechanistic claims as hypotheses and reduce assertive language.
8. For MCF-12A experiments, only one biological replicate was performed—not sufficient to support conclusions about safety or selectivity.
9. Statistical comparisons between Vacrol and S-Mix at identical concentrations/time points are not clearly presented.
10. How the authors determined compound purity (GC-MS analysis) should be included in the supplementary materials.
11. CAM tumor volume analysis lacks statistical tests (ANOVA or Kruskal–Wallis).
12. Please add full statistical analysis for all datasets and report exact p-values.
13. Typographic errors are present in multiple places (“Mm” instead of “mM,” inconsistent spacing).

 

Major revision

Comments for author File: Comments.pdf

Author Response

Comment 1: “Abstract should include specific results and novelty; include ratios where synergism was observed.”
Response: We revised the Abstract to include the key quantitative outcomes and novelty (combined in vitro + in ovo + histopathological evaluation of these commercial formulations). Because synergy was not quantified by CI/isobolograms, we removed wording that implies confirmed “synergism” and avoided reporting “synergy ratios” as definitive outcomes.

Comment 2: “Introduction is too long and contains redundant epidemiological/general cancer text; focus on carvacrol/essential oil context; clarify how antimicrobial/antioxidant relates to anticancer.”
Response: We substantially shortened and restructured the Introduction to focus on TNBC clinical challenges, the rationale for plant-derived essential oil constituents (particularly carvacrol-rich profiles), and the motivation for testing Vacrol and S-Mix. We also revised the wording to avoid mechanistic leaps from antimicrobial/antioxidant activity to anticancer claims unless supported by specific literature and appropriately framed.

Comment 3: “When mentioning activity in the introduction, IC50 data should be provided.”
Response: We revised the relevant statements to include quantitative values when available in cited literature, and removed non-quantitative claims where appropriate.

Comment 4: “Give examples of plants rich in thyme oil.”
Response: Added in the revised Introduction.

Comment 5: “What is C-Mix? Must be explained immediately; composition/ratio.”
Response: We removed ambiguous terminology and ensured the formulations are consistently referred to as Vacrol and S-Mix, with composition summarized in the GC–MS table.

Comment 6: “Need characterization/quantification of synergy (CI/isobolograms).”
Response: We agree. We did not perform combination-index style synergy quantification; therefore, we revised the manuscript to avoid stating synergy as a proven finding, and we identify this explicitly as a limitation and a priority for future work.

Comment 7: “Add description of the GC–MS table; provide structural formulas for predominant components.”
Response: We added a brief description of the table in the Results and included a figure showing structural formulas of the major constituents.

Comment 8: “Figures 2 and 3 quality/presentation should be improved; clearer dose scaling/labeling.”
Response: Done. We revised axis scaling, labeling, and resolution to improve interpretability.

Comment 9: “Figures 6 and 7 require scale bars, standardized lighting; state vehicle control explicitly.”
Response: We added scale bars and clarified image acquisition consistency. We also clarified controls: for in ovo applications, formulations were diluted in HBSS and HBSS served as the vehicle control; no additional organic solvents (e.g., DMSO/ethanol) were introduced in the CAM experiments.

Comment 10: “Discuss physiological relevance of high concentrations; concern for non-specific toxicity.”
Response: We revised the Discussion to contextualize the tested concentration range, to focus interpretation on the concentration window where effects emerge, and to avoid overinterpreting high-dose effects as specific mechanism without supporting mechanistic assays.

Comment 11: “Pathway statements (NF-κB, TRPM7, PI3K/AKT) are speculative without pathway assays.”
Response: We agree and revised the Discussion to clearly distinguish literature context from our findings. We reframed mechanistic statements as hypotheses and removed overly assertive pathway claims.

Comment 12: “Selectivity claims need more rigor; only one normal line; reframe and reduce assertive language.”
Response: We revised the wording to avoid strong “selectivity” claims and limited conclusions to what the data directly support.

Comment 13: “For MCF-12A experiments, only one biological replicate; insufficient.”
Response: Addressed by ensuring MCF-12A data are based on appropriate biological replication, and the revised figure/legend reports replicate structure clearly.

Comment 14: “Statistical comparisons between Vacrol and S-Mix at identical concentrations/time points not clear.”
Response: We clarified the comparisons in the Results text and figure legends and ensured the statistical approach is described transparently.

Comment 15: “CAM tumor volume lacks statistical tests (ANOVA or Kruskal–Wallis). Add full statistics; report exact p-values.”
Response: We added the CAM tumor volume statistical testing approach (Kruskal–Wallis for multi-group comparisons with appropriate post-hoc testing) and revised reporting to improve transparency. Where the software output provides thresholds (e.g., p < 0.0001), we report them as such; otherwise, we report exact values.

Comment 16: “Typographic errors (Mm vs mM; spacing).”
Response: Corrected throughout.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript evaluating the anticancer effects of carvacrol-based essential oil formulations (Vacrol and S-Mix) against triple-negative breast cancer MDA-MB-231 cells is of interest. However, several major concerns should be addressed to strengthen the manuscript's impact and scientific rigor

1. Excessive Length and Structure of the Introduction:
   The Introduction spans 4 pages (approximately 35% of the manuscript) with 22 paragraphs, which is disproportionately long. It contains repetitive information and lacks a focused structure, with some content more suitable for the Discussion section. We recommend condensing it to approximately 1.5–2.0 pages. Consider restructuring it along a clearer logical pathway, for example:
   • "Clinical challenges of TNBC → Limitations of carvacrol monotherapy → The synergistic theory as a solution → Rationale behind the Vacrol/S-Mix formulations → Aims of the present study"; or
   • "Therapeutic potential of natural products and essential oils → Anticancer mechanisms and limitations of carvacrol → Synergistic strategy: from single compounds to formulations → Research challenges in TNBC → Study significance and objectives."

 

2. Lack of Key Phenotypic Data on Migration and Invasion:
   As correctly noted in the manuscript, the aggressive and highly metastatic nature of TNBC is a major therapeutic challenge. Therefore, incorporating cell migration (e.g., wound healing assay) and invasion (e.g., Matrigel Transwell assay) experiments would significantly enhance the relevance and completeness of the study by directly assessing the potential anti-metastatic effects of the formulations.

 

3. Insufficient Evidence for Pro-apoptotic Effects:
   The evidence for apoptosis induction is currently limited to the description of "pro-apoptotic morphology." To provide direct and quantitative evidence, it is essential to supplement the findings with:
   • Apoptosis detection(e.g., Annexin V/PI staining analyzed by flow cytometry).
   • Cell cycle analysisto confirm cell cycle arrest.
   • If possible, Western blot analysisof key apoptosis-related proteins (e.g., Cleaved Caspase-3, Bax, Bcl-2) would strongly support the proposed mechanistic insights into apoptosis.

 

4. Inadequate Validation of Synergistic Mechanisms:
   The core innovation of this work lies in the proposed synergistic anticancer effects of the essential oil formulations. However, the current support for synergy relies primarily on speculative discussion (lines 294-297，313-315,361-365) based on the chemical composition (Table 1), which is insufficient. To robustly demonstrate synergy, it is crucial to include experimental validation, such as:
   • Comparing the effects of individual major components (e.g., carvacrol, α-pinene, eugenol) alone versus in combination, using assays like cell viability and/or apoptosis.
   • Calculating combination indices (e.g., using the Chou-Talalay method) to quantitatively confirm synergistic interactions.

 

5. Technical and Presentation Issues:
   • Table 1:The numerical identifiers for decimal points appear inconsistent (commas are used). Please use periods for decimal points consistently. Furthermore, the method used for quantifying the essential oil components by GC-MS must be explicitly stated (e.g., internal standard method, external standard method, or area normalization).
   • Figure 3:There is an inconsistency. The concentration axis is labeled with actual values at evenly spaced intervals, yet the figure legend states data are presented as "log10 values." The figure should either use a proper logarithmic scale on the x-axis or the legend should be corrected to clarify how the data were transformed and plotted.
   • Figure 7:The 40x magnification image for the Control group is missing from the panel of histopathological examination images. Please ensure all groups are represented at both magnifications.
   • References:
     • Citations for common knowledge (e.g., chemical structures in Ref 9) are generally unnecessary.
     • Citations should be checked, e.g., Refs 10-11
     • Several references (e.g., R1, R5, R6, R7, R13, R15) lack page numbers or article IDs.
     • Latin binomials for species names must be italicized (e.g., Origanum vulgarein R1; Origanum onites in R4-7).

Author Response

Comment 1: “Introduction excessively long; restructure and condense.”
Response: Done. We condensed and restructured the Introduction along a clearer rationale-driven narrative.

Comment 2: “Include migration/invasion assays (wound healing/Transwell).”
Response: We agree these would strengthen the work; however, they are outside the current experimental scope. We added this explicitly as a limitation and a planned follow-up.

Comment 3: “Apoptosis evidence insufficient; add Annexin V/PI, cell cycle, WB (cleaved caspase-3, Bax/Bcl-2).”
Response: We agree. These mechanistic assays were not performed in the present study and are now clearly identified as limitations and priorities for future work. We also revised the text to avoid implying confirmed apoptosis where only histomorphological features were observed.

Comment 4: “Synergistic mechanism validation inadequate; compare individual components and calculate CI.”
Response: We agree and revised the manuscript to avoid claiming validated synergy. We present the formulation effects as combined effects and explicitly state that CI/isobologram-based synergy validation requires future experiments with isolated components and defined combinations.

Comment 5: “Table 1 decimals inconsistent; use periods.”
Response: Corrected.

Comment 6: “GC–MS quantification method must be stated (internal/external standard or area normalization).”
Response: We clarified the GC–MS method information provided by the service and included supporting documentation as supplementary material where allowed.

Comment 7: “Figure 3 log10 inconsistency.”
Response: Corrected by aligning the axis scaling and legend description.

Comment 8: “Figure 7 missing control 40× image.”
Response: Added the missing panel so that all groups are represented consistently.

Comment 9: “References: remove unnecessary common-knowledge citations; fix formatting; italicize Latin binomials.”
Response: Corrected.

Reviewer 4 Report

Comments and Suggestions for Authors

Plants (Manuscript ID: plants-4015281), Comments to the Authors:

 

Title: Synergistic Anticancer Effects of Carvacrol-Based Essential Oil

 

Comments

 

The submitted paper focused on studying synergistic formulations such as Vacrol and S-Mix, enriched with carvacrol and complementary essential oil compounds, may enhance therapeutic efficacy while reducing toxicity. Essential oil components were analyzed via GC-MS. Cell viability was assessed using the sulforhodamine B (SRB) assay at different concentrations and incubation periods. An in ovo chorioallantoic membrane (CAM) assay was performed to investigate tumor volume changes and histopathological alterations. Vacrol and S-Mix demonstrated concentration- and time-dependent cytotoxic effects in MDA-MB-231 cells, with significant reductions in viability at higher concentrations (100 µM–1 mM). In ovo, S-Mix induced ~40% reduction in tumor volume and promoted apoptotic morphology compared to controls. Synergistic effects of carvacrol with α-pinene, eugenol, and β-terpineol likely contributed to enhanced bioactivity. Vacrol and S-Mix exhibit promising antiproliferative and pro-apoptotic activity against triple-negative breast cancer models.

 

I think the submitted paper can be accepted after the authors respond to the following comments:

1. The authors should evaluate pure carvacrol. They should include dose–response curves for pure carvacrol in parallel with Vacrol and S-Mix in the same experiments. Without this, the statement that other terpenes “enhance” carvacrol activity is speculation.
2. Did the authors evaluate the apoptosis markers (e.g., caspase-3/7 activation, Annexin V staining, Bax/Bcl-2 ratio).
3. Did the authors evaluate ROS production, mitochondrial membrane potential, or relevant signaling pathways (e.g., PI3K/AKT, NF-κB, TRPM7).
4. The authors showed that S-Mix contains significantly less carvacrol (24%) than Vacrol (50.1%), yet it demonstrated greater potency. The authors attribute this to synergy. However, they did not provide dose-normalized comparisons based on carvacrol-equivalent concentrations. The authors should validate synergy using combination index (CI) analysis. They should compare formulations against pure carvacrol or other individual components (e.g., cinnamaldehyde, menthol).
5. The authors should test the most effective formulation (S-Mix) on at least one other breast cancer cell line, preferably another TNBC line (e.g., BT-549 or HCC1806) or a different subtype (e.g., an ER+ line like MCF-7) to assess the specificity of the effect.
6. In Table 1, the authors should include retention index.
7. The authors should test other TNBC lines (e.g., MDA-MB-468, BT-549, HCC1937) or non-TNBC lines.

 

 

Author Response

Comment 1: “Evaluate pure carvacrol; include dose–response curves in parallel with Vacrol and S-Mix.”
Response: Agreed. Carvacrol was included as a positive control; we ensured its dose–response data are clearly presented and directly compared with Vacrol and S-Mix.

Comment 2: “Did you evaluate apoptosis markers/ROS/mitochondrial potential/pathways?”
Response: These mechanistic endpoints were not evaluated in the present work. We revised the manuscript to avoid mechanistic overclaims and added a clear limitations/future directions statement specifying these assays as next steps.

Comment 3: “S-Mix has less carvacrol yet greater potency; provide carvacrol-equivalent comparisons.”
Response: We agree. We revised the comparisons and discussion to ensure interpretations are consistent with carvacrol content and the carvacrol control data, and we avoided presenting untested component interactions as confirmed synergy.

Comment 4: “Validate synergy using CI analysis; compare formulations vs individual components.”
Response: We agree that CI/isobologram validation requires defined combinations of isolated constituents. Since those experiments are not part of the current dataset, we revised the manuscript to avoid claiming validated synergy and listed this explicitly as a limitation and planned follow-up.

Comment 5: “Test S-Mix on additional breast cancer cell line(s).”
Response: We agree this would strengthen generalizability. Given the proof-of-concept scope, we did not add additional cell lines in this revision; we explicitly acknowledge this limitation and indicate it as a next step.

Comment 6: “Table 1 should include retention index.”
Response: We agree that retention indices would improve comparability. However, the GC–MS service report did not provide retention index values, and we do not have access to the raw data required to compute reliable indices post hoc. We therefore revised the manuscript to avoid stating that retention indices were calculated, and instead report retention times and confirm major compounds by co-injection with appropriate standards.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made the suggestions

Author Response

Comment: The authors made the suggestions.

Response: We sincerely thank the reviewer for the assessment and for the helpful suggestions provided in the previous round. We have implemented additional revisions for clarity and methodological transparency across the manuscript, with changes highlighted in yellow.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks to the authors for the revised manuscript. I think that the authors have adequately addressed the comments and most of my concerns. The research paper can be accepted for publication in this journal.

Author Response

Comment: The revised manuscript adequately addressed the comments; recommended acceptance.

Response: We thank the reviewer for the positive evaluation and for confirming that the revised manuscript adequately addresses the previous concerns. We appreciate the supportive recommendation.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have undertaken significant revisions to the manuscript, primarily addressing and incorporating changes related to textual, formatting, and clarity issues raised in the previous round. The title has also been revised to reflect a more cautious stance, demonstrating the authors' commitment to improving the quality of the work.

However, concerning the core scientific issues raised—specifically, comment 2 (anti-metastatic phenotype assays such as migration/invasion), comment  3 (evidence and mechanism of apoptosis), and comment 4 (validation of synergistic mechanisms)—the authors have not resolved these through the addition of new experimental data in this revision. Instead, they have chosen to list them as "limitations" of the study. While candidly acknowledging the boundaries of the research is a sign of scientific rigor, this approach results in a significant gap between the scientific claims made and the supporting evidence, thereby weakening the overall impact and completeness of the study.

Furthermore, the use of molar concentration (μM) to report the IC₅₀ values for the complex essential oil mixtures Vacrol and S-Mix is methodologically problematic. The IC₅₀  (μM)  metric is typically applied to well-defined single compounds. For botanical essential oil formulations, where bioactivity results from the combined effects of multiple constituents with vastly different molecular weights, it is standard practice to use mass-based units such as µg/mL. Additionally, for accurate IC₅₀ determination, the concentration points used for the calculation should be strategically placed around the preliminary estimate. The algorithm or concentration range used to generate the curves in Figure 3 appears suboptimal for obtaining a precise IC₅₀ value.

Author Response

Comment 1: Core scientific issues (migration/invasion; apoptosis mechanism; validation of synergistic mechanisms) were not resolved by new data and were moved to limitations.

Response: We understand and agree that migration/invasion assays, mechanistic apoptosis assays, and formal synergy quantification would substantially strengthen the biological depth of the study. However, these experiments require additional time, optimization, and resources beyond the scope of the current submission, and we have therefore taken a more conservative, evidence-aligned approach in the revised manuscript.

Specifically, we made the following adjustments to ensure that claims do not exceed the presented data:

We added PI staining as an additional cell-death–related readout and described it cautiously as an indicator of loss of membrane integrity/terminal cell death, while explicitly stating that this does not establish an apoptotic mechanism on its own.

We maintained histopathology-based descriptions as “apoptosis-like morphology” (e.g., nuclear condensation/fragmentation) and explicitly noted when apoptosis is not quantified, thereby preventing overinterpretation.

Regarding synergy: we explicitly state that formal synergy quantification (CI/isobologram/Chou–Talalay) was not performed and is planned as future work; accordingly, we frame the current findings as combined/formulation-driven effects supported by chemical profiling and functional outcomes across in vitro and in ovo models.

We believe this revision resolves the reviewer’s concern about a gap between claims and evidence by bringing the interpretation in line with what is directly supported.

 

Comment 2: Reporting IC₅₀ for complex mixtures in μM/mM is problematic; should be in μg/mL; IC₅₀ is typically for single compounds.

Response: We appreciate this methodological concern and agree that for botanical extracts with undefined or variable composition, mass-based reporting (e.g., µg/mL) is often the most appropriate convention.

However, Vacrol and S-Mix in our study are not crude extracts; they are standardized essential-oil formulations with defined composition, and we provide the major/minor constituents and their relative abundance via GC–MS profiling (Table 1). This compositional definition is precisely what allows reproducible formulation-level dosing and meaningful cross-comparison between Vacrol and S-Mix within a controlled experimental design. (In the revised Methods, we also clarify the stock standardization approach and concentration series used for the SRB assay.)

Accordingly, we retained IC₅₀ reporting in molarity as an operational concentration unit for the formulation stock dilutions, while ensuring that the manuscript clearly presents:

The full concentration range tested, including intermediate points around the IC₅₀ region (1 mM, 2 mM, 2.5 mM), to support robust nonlinear regression-based IC₅₀ estimation.

The composition profiling that enables readers to interpret the formulation-level IC₅₀ in the context of the known constituent percentages.

In other words: we are not treating Vacrol or S-Mix as single molecules, but rather reporting IC₅₀ at the formulation level under defined compositional conditions. We also acknowledge that alternative reporting formats exist, and we have strengthened the manuscript to ensure that readers can interpret, reproduce, and—if desired—re-express concentrations using the composition table.

 

Comment 3: Concentration points around IC₅₀ appear suboptimal; curve generation algorithm/range may not yield precise IC₅₀.

Response: We addressed this concern by ensuring that the tested concentration series includes multiple points bracketing the IC₅₀ region, rather than relying on a sparse or widely spaced range. The SRB assay concentration series includes 1 mM, 2 mM, and 2.5 mM in addition to higher and lower log steps, and IC₅₀ was calculated using nonlinear regression (log-transformed concentration series).

Additional clarification (Figure 5): We also note that Figure 5 contains panels with different experimental purposes: the viability panels (a–b) and the PI imaging panel (c) are not intended to represent an identical dosing series. This is now reflected in the Methods where PI staining uses a targeted exposure scheme (72 h; defined formulation concentrations) rather than the full SRB dose–response series.

Reviewer 4 Report

Comments and Suggestions for Authors

Plants (Manuscript ID: plants-4015281), Comments to the Authors:

 

Title: Synergistic Anticancer Effects of Carvacrol-Based Essential Oil

 

Comments

Having reviewed the authors' responses to my comments, I find that the replies to points 2, 3, 4, 5, and 6 remain inadequate and do not sufficiently address the concerns raised. Consequently, I cannot recommend acceptance of the manuscript for publication in its current form.

Author Response

Comment: Replies to points 2–6 remain inadequate; cannot recommend acceptance.

Response: We respectfully acknowledge Reviewer 4’s continuing concerns. While we do not have the reviewer’s full text in the decision summary, the remaining issues appear to center on methodological clarity/rigor and the strength of mechanistic claims. We therefore performed a focused revision aimed at (i) tightening claims to the evidence, and (ii) improving methodological transparency and reproducibility, including:

Chemical characterization rigor: We clarified GC–MS identification and RI determination and described confirmation of major compounds by co-injection, improving traceability of the formulation composition.

Clear formulation dosing framework: We explicitly list the SRB concentration series (including intermediate points around IC₅₀), describe the use of carvacrol as a positive control in the relevant cell lines, and specify how formulation stocks were prepared/standardized.

Cell-death readout added with conservative interpretation: We included PI staining as an additional supportive assay and explicitly state its interpretational limitations regarding apoptosis mechanism.

In ovo relevance and histopathology workflow clarified: We detail CAM xenograft handling and histopathology processing to strengthen reproducibility and justify the in ovo interpretation.

Claims toned down where mechanistic validation is not available: We explicitly state that formal synergy quantification and molecular pathway validation were beyond the study scope, and we frame findings as formulation-level combined effects consistent with component-driven activity.

We hope these revisions address Reviewer 4’s concerns by improving rigor and aligning conclusions with the presented evidence.

 

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors The authors have not addressed the core scientific issues raised in the previous two rounds of review—namely, the study's depth and completeness regarding anti-metastasis, apoptosis mechanism, and validation of synergistic effects—by supplementing key experimental data. In essence, the authors' responses and revisions have recategorized these unresolved issues from "uncompleted work" into "acknowledged limitations." While this improves the transparency of the manuscript, it does not enhance the weight of its scientific findings. The work therefore remains a welldesigned but preliminary study with limited conclusions. Furthermore, the insistence on reporting IC₅₀ values for the complex mixtures in “mM” rather than in massbased units such as “µg/mL” or “mg/mL” constitutes a methodological flaw that may affect the reproducibility and comparability of the research. Ultimately, the decision on whether to accept the manuscript rests with the editor and the journal’s specific scope and standards.

Author Response

Comment 1: The authors have not addressed… anti-metastasis, apoptosis mechanism, and validation of synergistic effects… Instead… listed as ‘limitations’… the work remains preliminary…

Response: We understand the reviewer’s expectation for deeper mechanistic validation and agree that migration/invasion assays, dedicated apoptosis assays, and formal synergy quantification would substantially strengthen the study.

However, in this revision round, we were not in a position to generate new experimental datasets. Therefore, we took the scientifically conservative route: we tightened the scope and conclusions to ensure that every statement is supported by the data currently presented, and we explicitly clarified what our data can—and cannot—demonstrate.

Concretely:

Apoptosis mechanism: We now explicitly state that PI staining supports terminal cell death/ loss of membrane integrity, but does not discriminate apoptosis from other death modes, and that mechanistic apoptosis conclusions require dedicated assays (e.g., Annexin V / cleaved caspases).

Similarly, in the in ovo section, we retain “apoptosis-like morphology” as a descriptive histologic observation but clearly note that apoptosis was not quantified.

Synergy validation: We agree that formal synergy validation requires CI/isobologram-type analyses. We therefore avoid presenting “synergy” as a definitive mechanistic conclusion and explicitly note the limitation that isobologram analyses were not performed.

In the Conclusions, we transparently state that formal synergy quantification and molecular pathway validation were beyond scope, and frame our findings as providing rationale for follow-up synergy-focused experiments rather than claiming completion of them.

Anti-metastatic phenotype: We acknowledge that migration/invasion assays were suggested in prior rounds. In the current manuscript, we do not claim an anti-metastatic phenotype. Our in ovo readouts are limited to tumor volume change and histopathologic descriptions, with appropriate caution about interpretation.

We also note that the CAM model is widely used in contexts including invasion/angiogenesis, but we do not present invasion endpoints as outcomes of this work.

In summary, we accept the reviewer’s characterization that the study is well-designed but preliminary with respect to mechanistic depth, and we revised the manuscript accordingly so that the claims match the evidence.

 

Comment 2: The insistence on reporting IC₅₀ values… in mM rather than mass-based units… constitutes a methodological flaw…

Response: We respectfully disagree that reporting in molar units is a methodological flaw for this specific case, and we would like to clarify the rationale.

Vacrol and S-Mix are standardized commercial formulations with defined composition, not undefined extracts. They are treated as standardized, chemically profiled formulations (GC–MS characterization and standard confirmation are provided), and the dosing framework in the manuscript is built on a molar reference derived from Trolox-equivalent based preparation of stock solutions.

In contrast, µg/mL is indeed common for poorly defined botanical extracts/essential oils where an interpretable molar reference cannot be provided. That is not the situation here.

Reproducibility is supported by composition and defined preparation. Beyond the unit label, reproducibility depends on whether another lab can recreate the tested material and dosing approach. We provide the composition profiling and describe how solutions were prepared; thus, another group can reproduce the formulation-based concentrations as used here.

IC₅₀ determination used concentrations bracketing the expected range. We also respectfully disagree that the concentration points are “suboptimal.” The tested range includes multiple points around the observed IC₅₀ values (e.g., 1, 2, 2.5, 5, 10 mM), and IC₅₀ was determined by nonlinear regression in GraphPad Prism.

Reviewer 4 Report

Comments and Suggestions for Authors

Plants (Manuscript ID: plants-4015281), Comments to the Authors:

 

Title: Synergistic Anticancer Effects of Carvacrol-Based Essential Oil

 

Comments

After reading the comments of the authors, I think the revised paper can be accepted for publication after the authors change “Anticancer activity” into “cytotoxic activity” in the title and the rest of the paper. “Anticancer activity” should only be used for drugs or compounds tested on human patients otherwise the term “cytotoxic activity” should be used.

Author Response

Comment: After reading the comments of the authors, I think the revised paper can be accepted for publication after the authors change ‘anticancer activity’ into ‘cytotoxic activity’ in the title and the rest of the paper.

Response: We thank the reviewer for this clear recommendation and fully agree. We revised the title and updated the manuscript wording to describe our experimental findings as cytotoxicity / viability-suppressing activity, rather than “anticancer activity.”

For clarity, we did not alter the terminology when summarizing previously published studies where authors explicitly use “anticancer,” because those statements refer to cited literature rather than our own claims.