Taxonomic Patterns in Euphorbiaceae Seed Tocopherol and Tocotrienol Profile: Contribution of Tocochromanols to Antioxidant Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

(1) A few species have only one biological replicate. It is suggested to clearly explain the impact of single replicate on data reliability in the discussion, and suggest expanding the sample size of key species in future research.

(2) Check the full names and first occurrence marks of abbreviations such as T, T3, UAEE, RPLC‑FLD, DPPH to ensure consistency and standardization.

(3) It is suggested to add the application potential of high‑tocochromanol Euphorbiaceae species in functional food, cosmetics, and plant protection at the end of the conclusion to enhance the practical value of the research.

(4) The difference (2% vs. near 100% dominance) needs a more nuanced discussion. Is the 2% figure derived by comparing DPPH values between saponified and ethanolic extracts? If so, please clarify the calculation method in section 2.5, as the current description is ambiguous.

(5) “β-T3 is the rarest tocochromanol found in plants”, cite a reference or soften this claim.

(6) The discussion of methyltransferase activity as an explanation for taxonomic patterns is speculative without enzyme activity data. Regard it as hypothesis.

(7) Section 2.3, what was the basis for selecting the six validation species? Were they chosen to represent different tocochromanol profiles?

(8) Line 349, R. communis contained similar or larger amounts of γ-T, compared to what? Clarify.

(9) Lines 437-442, The discussion of cross-pollination as a confounding factor is highly speculative without evidence. Move to a Limitations paragraph rather than presenting as a serious hypothesis.

(10) The comparison with coffee phenolic acids is interesting. Consider removing.

(11) How many replicates per sample in DPPH assay?

(12) The UAEE recoveries ranged from 57% to 99%. While the authors justify this by noting that poor recovery affected minor components, the 57% recovery in some species is concerning. Please provide species-specific recovery data rather than pooled means

(13) PC1 and PC2 explain what percentage of total variance? Add in the main text.

Author Response

We express our gratitude for your insightful comments, remarks, and suggestions, which have essentially elevated the manuscript`s scientific quality and overall excellence. The English language of the manuscript, the quality of the figures, and the tables have been improved. Provided changes are marked in red font. Literature references were managed using reference management software, changes are not highlighted.

 Comments and Suggestions for Authors

(1) A few species have only one biological replicate. It is suggested to clearly explain the impact of single replicate on data reliability in the discussion, and suggest expanding the sample size of key species in future research.

Thank you for your comment. The discussion has been strengthened with the following statement: „This limitation should be interpreted with caution, as the inclusion of species represented by a single biological replicate may reduce the robustness of species-level variability assessment; therefore, future studies should prioritize increasing the number of replicates for key taxa to strengthen statistical reliability and improve the generalizability of the findings.”

(2) Check the full names and first occurrence marks of abbreviations such as T, T3, UAEE, RPLC‑FLD, DPPH to ensure consistency and standardization.

Thank you for your suggestion. The revision has been made in accordance with the recommendation.

(3) It is suggested to add the application potential of high‑tocochromanol Euphorbiaceae species in functional food, cosmetics, and plant protection at the end of the conclusion to enhance the practical value of the research.

Thank you for your suggestion. The revision has been made in accordance with the recommendation.

 (4) The difference (2% vs. near 100% dominance) needs a more nuanced discussion. Is the 2% figure derived by comparing DPPH values between saponified and ethanolic extracts? If so, please clarify the calculation method in section 2.5, as the current description is ambiguous.

Yes, the 2% figure derived by comparing DPPH values between saponified and ethanolic extracts. The calculation method was suplemented in section 2.5.3. In the discussion, we clearly emphasize that “By contrast, ethanolic extracts—particularly when assisted by ultrasonication—solubilize both lipophilic and hydrophilic phytochemicals from plant material, potentially reshaping the antioxidant landscape”.

 (5) “β-T3 is the rarest tocochromanol found in plants”, cite a reference or soften this claim.

The claim has been softened and reference was provided.

(6) The discussion of methyltransferase activity as an explanation for taxonomic patterns is speculative without enzyme activity data. Regard it as hypothesis.

The statement has been rewritten as a clearer hypothesis.

(7) Section 2.3, what was the basis for selecting the six validation species? Were they chosen to represent different tocochromanol profiles?

The explanation was provided in the section 2.3.3.

(8) Line 349, R. communis contained similar or larger amounts of γ-T, compared to what? Clarify.

The sentence has been clarified.

(9) Lines 437-442, The discussion of cross-pollination as a confounding factor is highly speculative without evidence. Move to a Limitations paragraph rather than presenting as a serious hypothesis.

The discussion of cross-pollination was moved to new paragraph “3.3. Limitations and future study directions”

(10) The comparison with coffee phenolic acids is interesting. Consider removing.

The discussion about coffee phenolic acids was removed.

(11) How many replicates per sample in DPPH assay?

The information was supplemented in paragraph 2.5.3. “(n=2)

(12) The UAEE recoveries ranged from 57% to 99%. While the authors justify this by noting that poor recovery affected minor components, the 57% recovery in some species is concerning. Please provide species-specific recovery data rather than pooled means

Pooled means were removed and the species names with the lowest and highest recovery were provided.

(13) PC1 and PC2 explain what percentage of total variance? Add in the main text.

The percentage of explained variance (PC 1, PC2, and total) has been added to the main text.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive and valuable dataset on seed tocochromanol composition across the Euphorbiaceae, addressing an under-explored taxonomic group. The scope of sampling is impressive, and the analytical work provides novel information with potential chemotaxonomic and applied relevance. The findings pertaining to intergeneric variation in tocopherol/tocotrienol profiles, the correlation between total tocochromanol content and antioxidant activity, and the evaluation of ultrasound-assisted ethanolic extraction as a screening method are all of clear interest and suitable for the readership of the journal.

The submitted manuscript demonstrates merit and publishable potential; however, substantial revision is required prior to consideration for acceptance.

The primary concern pertains to the conceptual framing. The manuscript under scrutiny places significant emphasis on the concept of "phylogenetic patterns" in its title and throughout the text. However, the analyses presented primarily support taxonomic or lineage-associated differences, rather than providing unequivocal evidence for the presence of a phylogenetic signal in a formal sense. In the absence of phylogenetic comparative methods or trait mapping, it is recommended that the wording be softened to refer to "taxonomic patterns" or "patterns across Euphorbiaceae lineages." This approach would ensure a greater congruence between the conclusions and the evidence presented.

A secondary yet pivotal concern pertains to the configuration of the manuscript. At present, three related but partially independent objectives are combined: The primary objective of the study was a large-scale screening of Euphorbiaceae seed tocochromanols. The secondary objectives were methodological validation of ultrasound-assisted extraction versus saponification and the antioxidant contribution of tocochromanols in selected ethanolic extracts. While each of these elements possesses its own inherent value, the integration between them is not always readily apparent. The manuscript would benefit from clearer prioritisation of the primary research question, with the validation and antioxidant analyses positioned more explicitly as supporting or secondary objectives.

The limitations of the sampling method also require further elucidation. The study incorporates material obtained from various botanical collections over several years, with several taxa represented by a single accession. Whilst this approach is deemed acceptable for exploratory chemotaxonomic screening, it does have the effect of limiting broader biological inference. It is recommended that authors acknowledge more directly the potential influence of provenance, environmental effects, storage conditions, and within-species variability.

Several internal inconsistencies should also be resolved carefully before resubmission, particularly:

• number of species reported (68 vs. 70 in different sections),
• terminology related to taxa and hierarchical classification,
• spelling of species names,
• consistency in abbreviations and method descriptions.

In conclusion, the manuscript would benefit from thorough language editing. The dataset is robust, and the results are intriguing; however, grammatical and structural elements occasionally compromise clarity. A meticulous revision of English usage, in conjunction with the streamlining of numerous protracted or repetitive sections (particularly in the Introduction), would markedly enhance readability.

This study is noteworthy for its novel approach and the clarity with which it presents its findings. Following substantial revision for the purpose of clarification of the framing, resolution of inconsistencies, and improvement of the language, it is anticipated that the work will make a useful contribution to the existing literature on seed phytochemistry and Euphorbiaceae diversity.

Comments on the Quality of English Language

The English is generally comprehensible, but the manuscript would benefit from moderate language editing before publication. The primary concerns pertain to grammatical errors, sentence structure, sporadic unclear phrasing, inconsistent terminology, and minor typographical or formatting errors. While these do not substantively impact the scientific content, they can compromise readability and occasionally impede interpretation. A thorough language revision and greater consistency in terminology throughout the manuscript would enhance clarity and the overall presentation.

Author Response

We express our gratitude for your insightful comments, remarks, and suggestions, which have essentially elevated the manuscript`s scientific quality and overall excellence. The English language of the manuscript, the quality of the figures, and the tables have been improved. Provided changes are marked in red font. Literature references were managed using reference management software, changes are not highlighted.

 Comments and Suggestions for Authors

This manuscript presents a comprehensive and valuable dataset on seed tocochromanol composition across the Euphorbiaceae, addressing an under-explored taxonomic group. The scope of sampling is impressive, and the analytical work provides novel information with potential chemotaxonomic and applied relevance. The findings pertaining to intergeneric variation in tocopherol/tocotrienol profiles, the correlation between total tocochromanol content and antioxidant activity, and the evaluation of ultrasound-assisted ethanolic extraction as a screening method are all of clear interest and suitable for the readership of the journal.

The submitted manuscript demonstrates merit and publishable potential; however, substantial revision is required prior to consideration for acceptance.

The primary concern pertains to the conceptual framing. The manuscript under scrutiny places significant emphasis on the concept of "phylogenetic patterns" in its title and throughout the text. However, the analyses presented primarily support taxonomic or lineage-associated differences, rather than providing unequivocal evidence for the presence of a phylogenetic signal in a formal sense. In the absence of phylogenetic comparative methods or trait mapping, it is recommended that the wording be softened to refer to "taxonomic patterns" or "patterns across Euphorbiaceae lineages." This approach would ensure a greater congruence between the conclusions and the evidence presented.

We agree. References to phylogenetic patterns in the text have been edited to taxonomic patterns.

A secondary yet pivotal concern pertains to the configuration of the manuscript. At present, three related but partially independent objectives are combined: The primary objective of the study was a large-scale screening of Euphorbiaceae seed tocochromanols. The secondary objectives were methodological validation of ultrasound-assisted extraction versus saponification and the antioxidant contribution of tocochromanols in selected ethanolic extracts. While each of these elements possesses its own inherent value, the integration between them is not always readily apparent. The manuscript would benefit from clearer prioritisation of the primary research question, with the validation and antioxidant analyses positioned more explicitly as supporting or secondary objectives.

We agree. The results related to the primary objective of the study are described in as much detail as is useful, but mostly presented in a table and accompanying figures, which are better for presenting medium and large datasets. Additional statistical analyses were considered, but could not be done due to the low number of replicates and limited information on harvested plant material.

The limitations of the sampling method also require further elucidation. The study incorporates material obtained from various botanical collections over several years, with several taxa represented by a single accession. Whilst this approach is deemed acceptable for exploratory chemotaxonomic screening, it does have the effect of limiting broader biological inference. It is recommended that authors acknowledge more directly the potential influence of provenance, environmental effects, storage conditions, and within-species variability.

A short discussion on these limitations has been added to the manuscript.

Several internal inconsistencies should also be resolved carefully before resubmission, particularly:

• number of species reported (68 vs. 70 in different sections),
• terminology related to taxa and hierarchical classification,
• spelling of species names,
• consistency in abbreviations and method descriptions.

The number of investigated species has been updated to the correct 68.

In conclusion, the manuscript would benefit from thorough language editing. The dataset is robust, and the results are intriguing; however, grammatical and structural elements occasionally compromise clarity. A meticulous revision of English usage, in conjunction with the streamlining of numerous protracted or repetitive sections (particularly in the Introduction), would markedly enhance readability.

This study is noteworthy for its novel approach and the clarity with which it presents its findings. Following substantial revision for the purpose of clarification of the framing, resolution of inconsistencies, and improvement of the language, it is anticipated that the work will make a useful contribution to the existing literature on seed phytochemistry and Euphorbiaceae diversity.

 

Comments on the Quality of English Language

The English is generally comprehensible, but the manuscript would benefit from moderate language editing before publication. The primary concerns pertain to grammatical errors, sentence structure, sporadic unclear phrasing, inconsistent terminology, and minor typographical or formatting errors. While these do not substantively impact the scientific content, they can compromise readability and occasionally impede interpretation. A thorough language revision and greater consistency in terminology throughout the manuscript would enhance clarity and the overall presentation.

English language has been improved.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a potentially valuable database on tocopherols and tocotrienols in Euphorbiaceae seeds, with broad taxonomic coverage, application of an ethanol extraction protocol, and attempt to connect tocochromanol composition with antioxidant capacity. However, there are substantial problems that prevent acceptance in this form: numerical and taxonomic inconsistencies (68 vs. 70 species; "3 genera" instead of subfamilies; repeated or missing species), phylogenetic claims without formal phylogenetic analysis, statistics limited by many n=1, insufficient description of quality control/validation, overinterpretation of DPPH correlations, internal contradictions in the results, and numerous grammatical flaws that affect clarity.

 

Title and Abstract

 

Lines 2-4 ("Phylogenetic Patterns in Euphorbiaceae Seeds Tocopherol..."): The title is grammatically inappropriate. Suggestion: "Taxonomic Patterns in Tocopherol and Tocotrienol Profiles of Euphorbiaceae Seeds: Contribution of Tocochromanols to Antioxidant Potential". Also, if no formal phylogenetic analysis is performed, replace "Phylogenetic" with "Taxonomic" or include an explicit phylogenetic analysis.

Lines 11-13 ("Therefore, Euphorbiaceae seeds, in total, 68 species..."): The sentence is truncated and there is inconsistency with lines 324-326, which indicate 70 species. Rewrite and harmonize the number of species throughout the manuscript. Suggestion: "Therefore, seeds of X Euphorbiaceae species from 13 genera were investigated for their tocochromanol profiles."

Lines 14-18 ("some species had elevated δ-tocopherol content..."): Correct the unit and punctuation: "mg 100 g−1 dw" or "mg/100 g dw"; It remains to close parenthesis after "DW". It is also necessary to clarify whether 50.96 mg 100 g−1 dw in Triadica cochinchinensis corresponds to the total tocotrienols, the total tocochromanols, or γ-tocotrienol.

Lines 20-25 ("This is supported by a strong correlation..."): The wording suggests causality. Correlations between tocochromanol content and DPPH do not demonstrate, by themselves, a causal contribution. I recommend: "is consistent with" instead of "is supported by", and indicate n=20 when referring to antioxidant assays. Also harmonize the p-values between the summary and section 3.3.

 

Introduction

 

Lines 32-35 ("contains about 7500 species..."): Avoid using iNaturalist as the main source for taxonomic family circumscription. I recommend citing more appropriate taxonomic bases or works, such as World Flora Online/POWO/APG, and keeping iNaturalist only for supplementary observations, if necessary.

Lines 41-49 ("can be found on all green continents..."): "green continents" is an unscientific expression. Suggestion: "on all continents except Antarctica" or "across tropical and temperate regions worldwide", as the case may be. Correct "cassava contain" to "cassava contains". The section on toxicity also needs specific reference.

Lines 63-65 ("another reports γ-T and γ-T3 in cassava (J. curcas) seeds..."): There is factual/nomenclatural error: Jatropha curcas is not cassava; cassava is Manihot esculenta. Correct the species, common name, and associated citation.

Lines 95-100 ("evaluate whether more convenient and sustainable technique..."): The goal is extensive and contains an unproven safety claim for "topical use or consumption". The study does not carry out toxicological or safety evaluation. Tip: limit the claim to "a more convenient, less hazardous and high-throughput extraction technique for analytical screening".

 

Materials and Methods

 

Lines 117-124 ("The species verification of provided plant material..."): Identification of species by botanical garden staff alone is insufficient to support robust taxonomic/phylogenetic conclusions. Inform vouchers, access numbers, origin of each lot, identification criteria and, if possible, validation by herbarium or molecular barcode. The phrase "While this reduces Synonymic species..." is incomplete and should be rewritten.

Lines 125-127 ("Typical growing aerials..."): "growing aerials" seems to be an error for "growing areas" or "native ranges". The use of iNaturalist observations to infer distribution should be described with caution as it reflects occurrence records and not necessarily the full native distribution.

Rows 128-137 ("Seeds were obtained and analyzed between 2019 and 2024..."): The age of the lots, previous storage conditions and degree of maturity of the seeds can affect the profiles of tocochromanols. Since the manuscript itself discusses maturity as a possible explanatory factor, authors should inform how maturity was controlled or explicitly acknowledge this limitation.

Lines 135–137 ("Lyophilized seeds contained of 3–7% moisture..."): Correct grammar ("contained 3–7% moisture"). Quantitatively justify the use of 5% as a constant value, as the variation of 3–7% can affect the calculation on a dry basis. Consider presenting sensitivity analysis or reporting the uncertainty introduced.

Lines 140-141 ("for selected five samples..."): There is inconsistency with lines 166-169 and 274-276, which describe six species. Correct "five" to "six" or explain exactly which samples were used.

Lines 149-153 ("The organic solvent were evaporated..."): Correct to "The organic solvents were evaporated". In addition, indicate whether there was an internal standard, recovery control, analyte stability and matrix validation. The reference to "produced earlier" calibrations should be supplemented with evidence that the calibration remained valid for this sample/instrument set.

Lines 155-162 ("placed in 15 mL tube..."): Detail the UAEE better: type of tube, exact mass, sample/solvent ratio, position in the ultrasonic bath, control of the actual temperature of the sample, effective power or power density, number of extractions, filtration and any losses. These parameters are important for reproducibility.

Lines 164-169 ("Recovery (%) tests..."): The comparison with saponification provides relative recovery/relative extractability, not absolute recovery. Avoid treating saponification as "100% recovery" without independent evidence. I recommend using "relative recovery compared with saponification" and ideally include addition/recovery assays with standards or internal standard.

Lines 187-192 ("we randomly selected 20 Euphorbiaceae seed species..."): Specify the randomization procedure. The list contains E. myrsinites twice, "Jatropa curcas" is misspelled, and E. lambii appears in the antioxidant assays, but is not found in Table 1. Correct the list, confirm the number of unique species and harmonize with supplementary materials.

Lines 203-210 ("total phenols and other oxidation substrates..."): The approach correctly recognizes the lack of specificity of Folin-Ciocalteu, but the subsection should report calibration curve, linear interval, blanks, analytical replicates, and form of correction for interferents. Consistently use "FCR"; on line 205 it appears "FRC".

Lines 218-224 ("The chromatograms were not used for qualitative identification..."): If chromatograms were not used for identification or quantification, inferences about other phytochemicals should be presented as indirect evidence. I recommend using "putative UV-absorbing constituents" and avoiding unsupported compositional conclusions.

Lines 227-237 ("The antioxidant activity was evaluated..."): The DPPH formula is poorly formatted and should be revised. Inform sample blank controls, correction of absorbance of the extracts, linear range of response, final extract concentration, replicates and final unit. Percentage of inhibition at single concentration is limited to compare matrices; consider Trolox equivalents, EC50/IC50 or serial dilutions.

Lines 238-249 ("Statistical Analysis..."): The statistical section is insufficient. Indicate how ND values were treated, whether the data were centered/scaled in the PCA, which post hoc tests were used after Kruskal-Wallis, whether there was correction for multiple comparisons, which variables were included in the PCA, and how p-values/correlations were calculated. The large number of species with n=1 strongly limits statistical inferences and should be treated as a central limitation.

 

Results and Discussion

 

Lines 252-266 ("The same protocol has already been applied..."): The phrase "cranberry grape (Vitis spp.) seeds [33] and (Vaccinium macrocarpon) [30]" is confused. Rewrite separating grape seeds and cranberry seeds. The current wording incorrectly suggests "cranberry grape."

Lines 274-277 ("six selected species from four genera..."): Correct "prepared using of both" to "prepared using both". Also review the consistency between "selected five samples", "six selected species" and the number of replicates described in the method.

Lines 305-310 ("Since the discrepancies between the two protocols..."): The sentence is grammatically incorrect ("and due to the detection and quantification of..." and "yielded, on average, demonstrated"). Rewrite it in two sentences. Also, avoid inferring esterified/glycosylated forms without direct structural analysis.

Lines 324-327 ("In total, 70 species' seeds..."): This is a critical inconsistency. The abstract indicates 68 species, while the results indicate 70. Also, "13 genera, 8 tribes and 3 genera" should be "13 genera, 8 tribes and 3 subfamilies". Correct before publication.

Lines 327-330 and Table 1 ("Data is presented as species means±standard deviation..."): Correct "Data are". The table shows several cases of n>1 with a standard deviation of 0, which needs explanation: is it rounding, identical values, or compilation error? It is suggested to move part of the table to the add-in and keep a summary table with totals, proportions and number of samples in the text.

Lines 331-339 ("In most Euphorbioideae subfamily samples..."): Correct "The highest number if representatives" to "of representatives". There is a taxonomic error: J. curcas belongs to the subfamily Crotonoideae, not to Euphorbioideae. Also correct the spelling "T. cochinensis" to "T. cochinchinensis".

Lines 345-351 ("Existing research shows δ-T is the primary..."): The paragraph on Ricinus communis is difficult to interpret and seems contradictory. Clarify whether the comparison is between seed oil and whole seeds, and clearly differentiate data from this study and data from the literature. Correct "dependant" to "dependent".

Lines 352-364 ("While most of the species were tocotrienol-dominated..."): Correct "other contained" to "others contained". The conclusion of dominance by genus or subfamily should be smoothed out, because many genera have only one species and many species have n=1.

Lines 371-378 ("While all E. canariensis and E. marginata samples..."): The sentence "only one seed sample was analysed from the latter species" conflicts with Table 1, where E. marginata appears with n=4. Correct the number of samples or the interpretation.

Lines 385-389 ("potential intra-specific variation is exemplified Euphorbia nicaeensis..."): The comparison between E. nicaeensis and its glareous subspecies, both with n=1, does not demonstrate intraspecific variation. Reformulate as a preliminary observation and avoid statistical inference.

Lines 399-412 ("The Kruskal-Wallis test identified little difference..."): Provide complete statistics from the Kruskal-Wallis test (H, degrees of freedom, p) and the post hoc test. The PCA interpretation also needs to be reconciled with the dominances described above; in the current way, the association of the groups with the vectors of γ-T/γ-T3 seems confusing or possibly inverted.

Lines 417-443 ("The observed variability is unlikely to be explained entirely..."): This discussion is interesting, but speculative. Since maturity, germination, cross-pollination, and environment were not measured, present these factors explicitly as hypotheses/limitations, not as probable explanations.

Lines 444-453 ("different tocochromanol precursor or the activity of enzymes..."): The biosynthetic explanation needs technical review. " "Pyruvate ring" seems incorrect in this context; Check the biochemical terminology of the Cromanol ring/precursor and the enzymes involved. The phrase "different precursor tocochromanol" must also be plural or specified.

Lines 473-480 ("Distinctly tocotrienol-dominated species..."): There is an important contradiction. The species listed as "tocotrienol-dominated" (E. leuconeura, E. canariensis, E. hirsuta, E. maculata, E. prostrata) were described in lines 371-376 as predominantly tocopherol-dominated. Then, species with high tocotrienol content (E. salicifolia, E. fischeriana, E. lathyris) appear as "almost exclusively tocopherols". Review this passage with the data in Table 1; it compromises the credibility of the results.

Lines 482-497 ("statistical analysis of tocochromanol content based on these factors was not feasible..."): I agree that ecological analysis is not feasible with these data, but the discussion of C3/C4/CAM and shikimate/GGPP pathways is excessively speculative without study data. Consider reducing and moving to "future directions".

Lines 511-513 ("unequivocally supports the conclusion..."): Replace "unequivocally supports" with more cautious language. The strong correlation is compatible with the hypothesis that tocochromanols contribute in an important way, but the unsaponifiable fraction contains other lipophilic antioxidants and the correlation does not prove causation.

Lines 516-519 ("determined by RPLC-DAD..."): The legend in Figure 5 states that the total tocochromanol content was determined by RPLC-DAD, but the main method describes RPLC-FLD. Correct to RPLC-FLD, if applicable.

Lines 527-535 ("we did not attempt a qualitative or quantitative characterization..."): As there was no identification/quantification of the compounds detected by DAD, avoid directly relating the number/area of peaks with "total reducing phytochemicals". The argument should be presented as complementary and exploratory evidence.

Lines 551-552 ("r = 0.774, p < 0.00001..."): Check the p-value. For n=20, r=0.774 tends to produce very significant p, but probably not as low as p<0.00001, depending on the test used. Enter the correlation method (Pearson/Spearman), n and exact p.

Lines 559-565 ("contribute 10–12 times high antioxidant activity..."): Correct to "10–12 times higher antioxidant activity". In addition, explain exactly how the percentage contribution of tocochromanols was calculated and whether the extracts were compared on the same basis of mass, volume and concentration.

 

Conclusions

 

Lines 572-580 ("with some relation to plant taxonomical classification..."): Correct "taxonomical" to "taxonomic". The conclusion should acknowledge the limitations of sampling, n=1 for many species and the absence of formal phylogenetic testing. The sentence "which either had higher tocopherol concentration, or it was typical for the genus" is unclear and should be rewritten.

Lines 581-598 ("These results further highlight the value of UAEE..."): The conclusion on UAEE is promising, but it should be tempered: the validation was done in six species and does not demonstrate absolute recovery. Suggestion: "UAEE appears suitable for high-throughput screening, provided that matrix-specific validation is performed for new or unusual seed types."

 

References, Citations and Style

 

Lines 56-58 and references 2-3 ("δ-T3 was first isolated..."): The historical statement is not aligned with the references: reference [2] is from 1966, while [3] is from 1965. Review the order and wording to avoid chronological error.

Lines 621-762 ("References..."): Review the list of references for typographical and formatting errors. Examples: reference [4] contains "potenices"; reference [19] seems to lack punctuation between the title and "Molecules"; Reference [23] contains irregular capitalization ("champ. Ex benth. And"). Also check if the 2026 references are published, "in press" or preprints, and indicate DOI when available.

Throughout the manuscript ("analysed/analyzed", "RPLC/RP-HPLC", scientific units and names): Standardize British or American English, abbreviations, use of italics for scientific names, spelling of "tocochromanol/tocotrienol/tocopherol", and format of units. Several sentences are grammatically problematic and impair interpretation; I recommend professional linguistic review before resubmission.

Throughout the manuscript (claims about phylogeny): The manuscript uses phylogeny and phylogenetic patterns, but the analyses presented are essentially taxonomic/descriptive and PCA. To sustain the term "phylogenetic", authors must incorporate a phylogenetic tree and appropriate tests, such as phylogenetic signal or comparative models. Otherwise, I recommend renaming the approach "taxonomic patterns".

Throughout the manuscript (interpretation of antioxidant assays): The contribution of tocochromanols to antioxidant capacity should be presented with caution. DPPH is a chemical measure of radical-scavenging/reducing capacity, not a direct measure of biological antioxidant activity. Avoid extrapolations for nutritional functionality, topical application, or safety without additional assays.

Author Response

We express our gratitude for your insightful comments, remarks, and suggestions, which have essentially elevated the manuscript`s scientific quality and overall excellence. The English language of the manuscript, the quality of the figures, and the tables have been improved. Provided changes are marked in red font. Literature references were managed using reference management software, changes are not highlighted.

 Comments and Suggestions for Authors

The manuscript presents a potentially valuable database on tocopherols and tocotrienols in Euphorbiaceae seeds, with broad taxonomic coverage, application of an ethanol extraction protocol, and attempt to connect tocochromanol composition with antioxidant capacity. However, there are substantial problems that prevent acceptance in this form: numerical and taxonomic inconsistencies (68 vs. 70 species; "3 genera" instead of subfamilies; repeated or missing species), phylogenetic claims without formal phylogenetic analysis, statistics limited by many n=1, insufficient description of quality control/validation, overinterpretation of DPPH correlations, internal contradictions in the results, and numerous grammatical flaws that affect clarity.

Thank you for highlighting the weakness in our manuscript. All of them have been improved.

Title and Abstract

 Lines 2-4 ("Phylogenetic Patterns in Euphorbiaceae Seeds Tocopherol..."): The title is grammatically inappropriate. Suggestion: "Taxonomic Patterns in Tocopherol and Tocotrienol Profiles of Euphorbiaceae Seeds: Contribution of Tocochromanols to Antioxidant Potential". Also, if no formal phylogenetic analysis is performed, replace "Phylogenetic" with "Taxonomic" or include an explicit phylogenetic analysis.

The title has been corrected and references to phylogeny have been replaced unless referencing phylogeny studies.

Lines 11-13 ("Therefore, Euphorbiaceae seeds, in total, 68 species..."): The sentence is truncated and there is inconsistency with lines 324-326, which indicate 70 species. Rewrite and harmonize the number of species throughout the manuscript. Suggestion: "Therefore, seeds of X Euphorbiaceae species from 13 genera were investigated for their tocochromanol profiles."

The sentence has been rewritten.

Lines 14-18 ("some species had elevated δ-tocopherol content..."): Correct the unit and punctuation: "mg 100 g−1 dw" or "mg/100 g dw"; It remains to close parenthesis after "DW". It is also necessary to clarify whether 50.96 mg 100 g−1 dw in Triadica cochinchinensis corresponds to the total tocotrienols, the total tocochromanols, or γ-tocotrienol.

The sentence has been rewritten so it is clearer.

Lines 20-25 ("This is supported by a strong correlation..."): The wording suggests causality. Correlations between tocochromanol content and DPPH do not demonstrate, by themselves, a causal contribution. I recommend: "is consistent with" instead of "is supported by", and indicate n=20 when referring to antioxidant assays. Also harmonize the p-values between the summary and section 3.3.

The sentence has been changed according to suggestions.

 Introduction

Lines 32-35 ("contains about 7500 species..."): Avoid using iNaturalist as the main source for taxonomic family circumscription. I recommend citing more appropriate taxonomic bases or works, such as World Flora Online/POWO/APG, and keeping iNaturalist only for supplementary observations, if necessary.

Yes, iNaturalist was only used as a supplementary source. Correct accepted species name, tribe, subfamily and family were double-checked using Kew science and World Flora Online.

Lines 41-49 ("can be found on all green continents..."): "green continents" is an unscientific expression. Suggestion: "on all continents except Antarctica" or "across tropical and temperate regions worldwide", as the case may be. Correct "cassava contain" to "cassava contains". The section on toxicity also needs specific reference.

The text has been corrected and updated with relevant references on toxicity.

Lines 63-65 ("another reports γ-T and γ-T3 in cassava (J. curcas) seeds..."): There is factual/nomenclatural error: Jatropha curcas is not cassava; cassava is Manihot esculenta. Correct the species, common name, and associated citation.

The error has been corrected.

Lines 95-100 ("evaluate whether more convenient and sustainable technique..."): The goal is extensive and contains an unproven safety claim for "topical use or consumption". The study does not carry out toxicological or safety evaluation. Tip: limit the claim to "a more convenient, less hazardous and high-throughput extraction technique for analytical screening".

Thank you for the tip. We have implemented the change as suggested. 

Materials and Methods

 Lines 117-124 ("The species verification of provided plant material..."): Identification of species by botanical garden staff alone is insufficient to support robust taxonomic/phylogenetic conclusions. Inform vouchers, access numbers, origin of each lot, identification criteria and, if possible, validation by herbarium or molecular barcode. The phrase "While this reduces Synonymic species..." is incomplete and should be rewritten.

Thank you. We are fully aware of certain limitations inherent in the present study. Indeed, assembling over 10,000 seed samples from various botanical gardens was unfeasible for numerous reasons. To mitigate the risk of human error in identification, the majority of species were sourced from at least two different locations. Furthermore, to explicitly address the scope of our study, a “3.3. Limitations and future study directions” section has been added to the manuscript. The description in the Materials and Methods section has been revised accordingly. 

Lines 125-127 ("Typical growing aerials..."): "growing aerials" seems to be an error for "growing areas" or "native ranges". The use of iNaturalist observations to infer distribution should be described with caution as it reflects occurrence records and not necessarily the full native distribution.

Thank you. The reference “iNaturalist” was exchanged by https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:30375178-2.

Rows 128-137 ("Seeds were obtained and analyzed between 2019 and 2024..."): The age of the lots, previous storage conditions and degree of maturity of the seeds can affect the profiles of tocochromanols. Since the manuscript itself discusses maturity as a possible explanatory factor, authors should inform how maturity was controlled or explicitly acknowledge this limitation.

Seed maturity could not be controlled during the study. The harvested fruits may only be characterized as “ripe”.

Lines 135–137 ("Lyophilized seeds contained of 3–7% moisture..."): Correct grammar ("contained 3–7% moisture"). Quantitatively justify the use of 5% as a constant value, as the variation of 3–7% can affect the calculation on a dry basis. Consider presenting sensitivity analysis or reporting the uncertainty introduced.

The sentence has been improved. About moisture, to the paragraph “3.3. Limitations and future study directions” we add the following discussion. “Owing to constraints in sample availability, seed moisture content could not be quantified for every accession. Accordingly, data normalization was performed using a uniform moisture content value of 5%, which reflects the average of the measured range (3–7%) observed among the evaluated samples. Although this assumption facilitates dataset standardization, it represents an inherent simplification and may introduce a small systematic deviation, estimated at up to 2%.”

Lines 140-141 ("for selected five samples..."): There is inconsistency with lines 166-169 and 274-276, which describe six species. Correct "five" to "six" or explain exactly which samples were used.

Thank you for highlighting this issue. Six is the correct one. The names of samples are provided.

Lines 149-153 ("The organic solvent were evaporated..."): Correct to "The organic solvents were evaporated". In addition, indicate whether there was an internal standard, recovery control, analyte stability and matrix validation. The reference to "produced earlier" calibrations should be supplemented with evidence that the calibration remained valid for this sample/instrument set.

The description of the methodology has been condensed following the Editor’s request to reduce redundant methodological sections previously detailed in our earlier publications on tocochromanols. The analytical method employed has been standard practice in our research group and others for over two decades. Supplementary Material, specifically the ‘Recovery_&_Precision’ sheet, provides comprehensive data on measurements conducted for six species tested across both extraction protocols. Only the key information were added. The internal standard, recovery control, analyte stability and matrix validation for tested samples in the present study was not tested. Each sample can be specific and can require separate tests. To ensure that this limitation is transparent to the potential reader, a paragraph concerning the saponification procedure and analyte recovery has been incorporated into Section 3.1.

Lines 155-162 ("placed in 15 mL tube..."): Detail the UAEE better: type of tube, exact mass, sample/solvent ratio, position in the ultrasonic bath, control of the actual temperature of the sample, effective power or power density, number of extractions, filtration and any losses. These parameters are important for reproducibility.

The description of the methodology has been condensed following the Editor’s request to reduce redundant methodological sections previously detailed in our earlier publications on tocochromanols. However, all requested details were supplemented.

Lines 164-169 ("Recovery (%) tests..."): The comparison with saponification provides relative recovery/relative extractability, not absolute recovery. Avoid treating saponification as "100% recovery" without independent evidence. I recommend using "relative recovery compared with saponification" and ideally include addition/recovery assays with standards or internal standard.

The terminology has been updated to ‘relative recovery’ throughout the entire manuscript. Furthermore, a discussion regarding the recovery of tocochromanols during the saponification protocol has been incorporated into section 3.1.

Lines 187-192 ("we randomly selected 20 Euphorbiaceae seed species..."): Specify the randomization procedure. The list contains E. myrsinites twice, "Jatropa curcas" is misspelled, and E. lambii appears in the antioxidant assays, but is not found in Table 1. Correct the list, confirm the number of unique species and harmonize with supplementary materials.

Thank you. The randomization was specified. Spelling was corrected. Yes, E. lambii appears in the antioxidant assays, but is not found in Table 1. We do not think that it is a problem since antioxidant assays and Table 1 are not connected. The antioxidant assays was considered only several months when topic of taxonomy was described, while seeds of E. lambii we received after that period, therefore are not included in Table 1 and taxonomy evaluation.

Lines 203-210 ("total phenols and other oxidation substrates..."): The approach correctly recognizes the lack of specificity of Folin-Ciocalteu, but the subsection should report calibration curve, linear interval, blanks, analytical replicates, and form of correction for interferents. Consistently use "FCR"; on line 205 it appears "FRC".

Thank you. The correction of “FRC” to “FCR” was done. The calibration curve was supplemented in the Supplementary Materials. For each sample were performed two measurements (n=2) – the information was supplemented. About potential interferences, we describe clearly about the weaknesses of this method in section 2.5. we stating: “total reducing phytochemicals (phenols and other oxidation substrates and antioxidants) using the Folin–Ciocalteu assay (FCR)”.

Lines 218-224 ("The chromatograms were not used for qualitative identification..."): If chromatograms were not used for identification or quantification, inferences about other phytochemicals should be presented as indirect evidence. I recommend using "putative UV-absorbing constituents" and avoiding unsupported compositional conclusions.

Thank you. Any unsubstantiated conclusions regarding the chemical composition have been excluded from the manuscript.

Lines 227-237 ("The antioxidant activity was evaluated..."): The DPPH formula is poorly formatted and should be revised. Inform sample blank controls, correction of absorbance of the extracts, linear range of response, final extract concentration, replicates and final unit. Percentage of inhibition at single concentration is limited to compare matrices; consider Trolox equivalents, EC50/IC50 or serial dilutions.

Thank you. The methodological description has been corrected accordingly, and the Supplementary Materials now provide details concerning the blank measurements. Under standard conditions, linearity is generally expected within an absorbance range of 0.05–1.0. Owing to the heterogeneous nature of the matrices investigated, establishing a universally applicable linear calibration based on a single standard compound would not be appropriate. Antioxidant activity is reported as percentage DPPH radical scavenging (% DPPH scavenging). We recognize that inhibition values obtained at a single concentration offer limited discriminatory power for comparisons across diverse matrices. Nevertheless, the primary aim of this work was to evaluate the overall contribution of tocochromanols, assessed in saponified samples, relative to that of crude ethanolic extracts. The objective was not to characterize individual species in detail, but rather to identify general trends across members of the Euphorbiaceae family.

Lines 238-249 ("Statistical Analysis..."): The statistical section is insufficient. Indicate how ND values were treated, whether the data were centered/scaled in the PCA, which post hoc tests were used after Kruskal-Wallis, whether there was correction for multiple comparisons, which variables were included in the PCA, and how p-values/correlations were calculated. The large number of species with n=1 strongly limits statistical inferences and should be treated as a central limitation.

The statistical analysis section has been updated with requested information. The limitation has been acknowledged in the section.

 Results and Discussion

Lines 252-266 ("The same protocol has already been applied..."): The phrase "cranberry grape (Vitis spp.) seeds [33] and (Vaccinium macrocarpon) [30]" is confused. Rewrite separating grape seeds and cranberry seeds. The current wording incorrectly suggests "cranberry grape."

Thank you. Correction has been done.

Lines 274-277 ("six selected species from four genera..."): Correct "prepared using of both" to "prepared using both". Also review the consistency between "selected five samples", "six selected species" and the number of replicates described in the method.

Thank you. Corrections have been done.

Lines 305-310 ("Since the discrepancies between the two protocols..."): The sentence is grammatically incorrect ("and due to the detection and quantification of..." and "yielded, on average, demonstrated"). Rewrite it in two sentences. Also, avoid inferring esterified/glycosylated forms without direct structural analysis.

Thank you. Corrections have been done.

Lines 324-327 ("In total, 70 species' seeds..."): This is a critical inconsistency. The abstract indicates 68 species, while the results indicate 70. Also, "13 genera, 8 tribes and 3 genera" should be "13 genera, 8 tribes and 3 subfamilies". Correct before publication.

Thank you. Corrections have been done.

Lines 327-330 and Table 1 ("Data is presented as species means±standard deviation..."): Correct "Data are". The table shows several cases of n>1 with a standard deviation of 0, which needs explanation: is it rounding, identical values, or compilation error? It is suggested to move part of the table to the add-in and keep a summary table with totals, proportions and number of samples in the text.

It is a case of deviation being less than 0.005, rounding out to 0. The table has been left in the manuscript for easiest reading, and the full dataset is available in the supplementary material. We feel that a smaller table only containing the mean values could misrepresent heterogenous subsets.

Lines 331-339 ("In most Euphorbioideae subfamily samples..."): Correct "The highest number if representatives" to "of representatives". There is a taxonomic error: J. curcas belongs to the subfamily Crotonoideae, not to Euphorbioideae. Also correct the spelling "T. cochinensis" to "T. cochinchinensis".

The errors have been corrected.

Lines 345-351 ("Existing research shows δ-T is the primary..."): The paragraph on Ricinus communis is difficult to interpret and seems contradictory. Clarify whether the comparison is between seed oil and whole seeds, and clearly differentiate data from this study and data from the literature. Correct "dependant" to "dependent".

The paragraph has been adjusted accordingly.

Lines 352-364 ("While most of the species were tocotrienol-dominated..."): Correct "other contained" to "others contained". The conclusion of dominance by genus or subfamily should be smoothed out, because many genera have only one species and many species have n=1.

The sentences have been clarified to more accurately represent the results.

Lines 371-378 ("While all E. canariensis and E. marginata samples..."): The sentence "only one seed sample was analysed from the latter species" conflicts with Table 1, where E. marginata appears with n=4. Correct the number of samples or the interpretation.

The sentence has been clarified.

Lines 385-389 ("potential intra-specific variation is exemplified Euphorbia nicaeensis..."): The comparison between E. nicaeensis and its glareous subspecies, both with n=1, does not demonstrate intraspecific variation. Reformulate as a preliminary observation and avoid statistical inference.

The sentence has been reformulated.

Lines 399-412 ("The Kruskal-Wallis test identified little difference..."): Provide complete statistics from the Kruskal-Wallis test (H, degrees of freedom, p) and the post hoc test. The PCA interpretation also needs to be reconciled with the dominances described above; in the current way, the association of the groups with the vectors of γ-T/γ-T3 seems confusing or possibly inverted.

The statistical analysis description has been supplemented. The PCA plots are correct. This was the clearest version we were able to produce.

Lines 417-443 ("The observed variability is unlikely to be explained entirely..."): This discussion is interesting, but speculative. Since maturity, germination, cross-pollination, and environment were not measured, present these factors explicitly as hypotheses/limitations, not as probable explanations.

The paragraphs have been adjusted.

Lines 444-453 ("different tocochromanol precursor or the activity of enzymes..."): The biosynthetic explanation needs technical review. " "Pyruvate ring" seems incorrect in this context; Check the biochemical terminology of the Cromanol ring/precursor and the enzymes involved. The phrase "different precursor tocochromanol" must also be plural or specified.

The paragraph has been partly rewritten for clarity and avoiding overblown claims, as suggested by the reviewers.

Lines 473-480 ("Distinctly tocotrienol-dominated species..."): There is an important contradiction. The species listed as "tocotrienol-dominated" (E. leuconeura, E. canariensis, E. hirsuta, E. maculata, E. prostrata) were described in lines 371-376 as predominantly tocopherol-dominated. Then, species with high tocotrienol content (E. salicifolia, E. fischeriana, E. lathyris) appear as "almost exclusively tocopherols". Review this passage with the data in Table 1; it compromises the credibility of the results.

The error has been corrected.

Lines 482-497 ("statistical analysis of tocochromanol content based on these factors was not feasible..."): I agree that ecological analysis is not feasible with these data, but the discussion of C3/C4/CAM and shikimate/GGPP pathways is excessively speculative without study data. Consider reducing and moving to "future directions".

It was intended as a potential future study direction. The paragraph has been moved to future directions.

Lines 511-513 ("unequivocally supports the conclusion..."): Replace "unequivocally supports" with more cautious language. The strong correlation is compatible with the hypothesis that tocochromanols contribute in an important way, but the unsaponifiable fraction contains other lipophilic antioxidants and the correlation does not prove causation.

Thank you. Corrections have been done.

Lines 516-519 ("determined by RPLC-DAD..."): The legend in Figure 5 states that the total tocochromanol content was determined by RPLC-DAD, but the main method describes RPLC-FLD. Correct to RPLC-FLD, if applicable.

Thank you. Corrections have been done.

Lines 527-535 ("we did not attempt a qualitative or quantitative characterization..."): As there was no identification/quantification of the compounds detected by DAD, avoid directly relating the number/area of peaks with "total reducing phytochemicals". The argument should be presented as complementary and exploratory evidence.

Thank you. Corrections have been done.

Lines 551-552 ("r = 0.774, p < 0.00001..."): Check the p-value. For n=20, r=0.774 tends to produce very significant p, but probably not as low as p<0.00001, depending on the test used. Enter the correlation method (Pearson/Spearman), n and exact p.

Thank you. Pearson correlation. The p value is correct (please see SM sheet “DPPH_&_FCR”.

Lines 559-565 ("contribute 10–12 times high antioxidant activity..."): Correct to "10–12 times higher antioxidant activity". In addition, explain exactly how the percentage contribution of tocochromanols was calculated and whether the extracts were compared on the same basis of mass, volume and concentration.

 Thank you. Corrections have been done. To be all clear the additional details have been provided in the section 2.5. While SM sheet “DPPH_&_FCR” is transparent.

Conclusions

 Lines 572-580 ("with some relation to plant taxonomical classification..."): Correct "taxonomical" to "taxonomic". The conclusion should acknowledge the limitations of sampling, n=1 for many species and the absence of formal phylogenetic testing. The sentence "which either had higher tocopherol concentration, or it was typical for the genus" is unclear and should be rewritten.

The limitation has been acknowledged more clearly. The excerpt has been removed.

Lines 581-598 ("These results further highlight the value of UAEE..."): The conclusion on UAEE is promising, but it should be tempered: the validation was done in six species and does not demonstrate absolute recovery. Suggestion: "UAEE appears suitable for high-throughput screening, provided that matrix-specific validation is performed for new or unusual seed types."

Thank you. Corrections have been done.

References, Citations and Style

 Lines 56-58 and references 2-3 ("δ-T3 was first isolated..."): The historical statement is not aligned with the references: reference [2] is from 1966, while [3] is from 1965. Review the order and wording to avoid chronological error.

Thank you. Corrections have been done.

Lines 621-762 ("References..."): Review the list of references for typographical and formatting errors. Examples: reference [4] contains "potenices"; reference [19] seems to lack punctuation between the title and "Molecules"; Reference [23] contains irregular capitalization ("champ. Ex benth. And"). Also check if the 2026 references are published, "in press" or preprints, and indicate DOI when available.

Thank you. Corrections have been done.

Throughout the manuscript ("analysed/analyzed", "RPLC/RP-HPLC", scientific units and names): Standardize British or American English, abbreviations, use of italics for scientific names, spelling of "tocochromanol/tocotrienol/tocopherol", and format of units. Several sentences are grammatically problematic and impair interpretation; I recommend professional linguistic review before resubmission.

The text has been reviewed.

Throughout the manuscript (claims about phylogeny): The manuscript uses phylogeny and phylogenetic patterns, but the analyses presented are essentially taxonomic/descriptive and PCA. To sustain the term "phylogenetic", authors must incorporate a phylogenetic tree and appropriate tests, such as phylogenetic signal or comparative models. Otherwise, I recommend renaming the approach "taxonomic patterns".

References to phylogeny have been changed to taxonomy unless specifically referring to a study investigating phylogenetics.

Throughout the manuscript (interpretation of antioxidant assays): The contribution of tocochromanols to antioxidant capacity should be presented with caution. DPPH is a chemical measure of radical-scavenging/reducing capacity, not a direct measure of biological antioxidant activity. Avoid extrapolations for nutritional functionality, topical application, or safety without additional assays.

Thank you. We completely agree. The antioxidant assays in present study plays just secondary role to demonstrate tocochromanols participation in antioxidant activity of ethanolic extracts. We do not advise to use non purified ethanol extracts obtained from Euphorbiaceae seeds in food.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author's response have addressed my concerns.

Author Response

Thank you.

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript provides a valuable dataset on tocochromanol composition in 68 Euphorbiaceae species and offers novel insights into their taxonomic patterns and antioxidant potential. The authors have adequately addressed the main concerns raised during the previous review. However, it is imperative to exercise caution when interpreting the conclusions, given the limited number of replicates for numerous species and the potential influence of factors such as sample origin, environmental conditions, and storage. In addition, it would be beneficial to emphasise the exploratory nature of some statistical analyses, continue to highlight methodological limitations, and streamline parts of the Introduction and Discussion. The study's scientific merit is evident, and it provides significant contributions to the fields of seed phytochemistry and Euphorbiaceae diversity. Following minor revisions aimed at enhancing clarity, conciseness, and the judicious interpretation of results, the study is deemed suitable for publication.

Comments on the Quality of English Language

The English is generally comprehensible, but the manuscript would benefit from moderate language editing before publication. The primary concerns pertain to grammatical errors, sentence structure, sporadic unclear phrasing, inconsistent terminology, and minor typographical or formatting errors. While these do not substantively impact the scientific content, they can compromise readability and occasionally impede interpretation. A thorough language revision and greater consistency in terminology throughout the manuscript would enhance clarity and the overall presentation.

Author Response

Thank you for this valuable comment. We agree that linguistic clarity and consistency are essential for effective scientific communication.

In response to the reviewer's suggestion, the manuscript has undergone a comprehensive language revision. We carefully reviewed the entire text and corrected grammatical errors, improved sentence structure, refined unclear or awkward phrasing, standardized terminology, and addressed typographical and formatting inconsistencies. Particular attention was given to ensuring consistency in the use of technical terms and to improving the overall readability of the manuscript.

We believe that these revisions have substantially improved the clarity and presentation of the work. Nevertheless, language assessment is inherently subjective, and we acknowledge that some issues may still remain unnoticed. Therefore, if the reviewer considers that specific sections or sentences continue to require improvement, we would be grateful if they could indicate a few representative examples. Such guidance would allow us to address any remaining concerns more effectively and ensure that the manuscript fully meets the expected linguistic standard.

We sincerely appreciate the reviewer's constructive feedback, which has helped us improve the quality of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript has been corrected. 

Author Response

Thank you.