Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article titled “Geographical Variation in Metabolomic Profiles and Bioactivi-2 ties of Wild Schisandra chinensis Berries from the Changbai 3 Mountains” analyze a fuctional berry using a metabolomic approach using Q-Orbitrap LC-MS able to evaluate differencec across different growing. The authors reported that these types of evaluations are lacking in the literature and clearly identified the research gap that this study aims to address. The authors have provided a comprehensive experimental design encompassing both the sampling strategy and analytical procedures. In addition in line 356 they clarify that the experimental design built was releted to exploratory with a section writing about limitations. Regarding the annotation, some information are lacking about annotation of compounds. A thorough review of this table is recommended. Overall, the study is very interesting and seeks to bridge a knowledge gap regarding Schisandra chinensis* berries, albeit within the stated limitations. Some references appear to be duplicated (e.g., references 20/23, 28/51, 34/37, and 10/57), and the reference list should be checked accordingly.There are several aspects that need addressing. I recommend it for publication only after a major revision.

All the suggestions are below.

 

Line 20: Add “In vitro bioactivities”.

Line 25: Please, replece “in fuctional component” with “metabolite accumulation” which is more appropriate.

Line 37: Plese report the name in italic.

Line 113: How were the provinces selected for sampling?

Line 118: How many fruits were hervested?

Line 130: Please clarify the rationale for selecting only three target plants at each sampling site.

Line 133-134: Please add reference about the standard specification.

Line 140: Please add the number of reference

Line 151: Please add the number of reference

Line 171: Please specify the volume taken from each individual sample to prepare the pooled QC sample.

Line 180: Please, replece identification with putative identification or annotation. Please clarify the criteria and confidence levels used for compound annotation. In addittion. The authors reported that the compound annotation was performed by matching retention time, precursor ion mass, and MS/MS spectra against mzCloud, mzVault, ChemSpider, and KEGG Pathway. However, KEGG is primarily a resource for metabolic pathways and compound–pathway relationships rather than a spectral or compound-identification database comparable to mzCloud, mzVault, or ChemSpider. Please clarify how KEGG Pathway was specifically used for compound identification in this study.

Line 208-211: I suggest to reports brief information about peak identification SS,MV

Line 310 Please add the number of reference, In addition, specify the number of replicates.

Line 321: Please, specify the number of replicates.

Line 333: Please add the number of reference. In addition, specify the number of replicates.

Line 342: See above

Line 367: “It should be noted that soil samples were collected as three independent 367 biological replicates (n=3) per site”. Please this information should be added before.

Line 360: I sugget to authors to replace above this sentece. I belive could be more appropriate in the other section before.

Line 390: The authors should better reported what MSI level 2 mean integrating the guide line releted the annotation of melabolite in HRMS.

Line 405: Several issus should be modified. 1)Rt: i suggest to report only two decimals; 2) [M–H]–: it should be write [M–H]^– (see all the other cases); 3)Calculated mass, what the authors mean? The authors should also report the mass error (e.g., in ppm) between the observed and theoretical m/z values to support the proposed compound annotations. The table is lacking of measuder mass. In addition the calculated mass should be reported with 4 decimals. The molecular formula should be should be formatted correctly and consistently throughout the table (e.g., C₁₀H₁₇N₃O₆S), with element numbers reported as subscripts and without spaces between elements. A thorough review of this table is recommended.

Line 410-416: The PCA results should be interpreted with caution. PC1 and PC2 explain 37.5% and 19.4% of the total variance, respectively, accounting for 56.9% of the overall variance. Therefore, a substantial proportion of the variability is not represented in the two-dimensional PCA plot. Why the authors did’t condider to extract others dimensions?

The author should clarify whether the observed separation among provinces is also supported by the subsequent principal components and provide the corresponding PCA statistics (e.g., explained variance and loading information). In addition, the statement that PCA clearly reveals inter-provincial differences should be moderated according to the proportion of variance explained by PC1 and PC2. I also racommand to improve the figure 2A-2B. The texts are very small.

Line 417-418: Clearly, it is a supervised analysis.

Line 435: I suggest to replace size with sites. Same in the line 443.

Line 447-449: I suggest to authors to report in depth the procedure in M&M. I recommand to report the level of calibration curve and and everything that makes the experiment replicable.

Line 453-455: …. And the matix effect? Recovery?

Line 554-558: The antioxidant activity is currently reported only as IC₅₀ values. Although IC₅₀ provides an estimate of the concentration required to achieve 50% radical scavenging, expressing the antioxidant capacity relative to a Trolox standard (e.g., µmol or mmol Trolox equivalents/g DW) could provide a more standardized and directly comparable measure of antioxidant activity among the samples. Please consider reporting the DPPH and ABTS results as Trolox equivalent antioxidant capacity (TEAC), in addition to or instead of IC₅₀ values, provided that an appropriate Trolox calibration curve was used.

Line 555 564: The authors should discuss the results they found with the literature available. The table lacks an SD and significant (Anova letters). The authors should improve the discussion of this section by adding literature.

Author Response

Thank you very much for your recognition of our research and for the detailed suggestions you have provided. We sincerely appreciate your feedback. We have reviewed and revised each point, and have responded. The revised parts have been highlighted in yellow in the manuscript. The detailed content is attached as an archive.

Line 20: Add “In vitro bioactivities”.

Reply: We thank the reviewer for this suggestion. We have made the necessary revisions.

Line 25: Please, replece “in fuctional component” with “metabolite accumulation” which is more appropriate.

Reply: We agree that "metabolite accumulation" is more precise. The sentence has been revised to: "Geographic origin is associated with variations in metabolite accumulation and bioactivity..."

Line 37: Plese report the name in italic.

Reply: We have checked the whole manuscript and formatted all Latin binomials in italics, including Schisandra chinensis (Turcz.) Baill. in the Keywords and throughout the text.

Line 113: How were the provinces selected for sampling?

Reply: The three provinces (Liaoning, Jilin and Heilongjiang) were selected because they cover the core natural distribution range of wild S. chinensis in the Changbai Mountains and represent the main traditional production areas of this species.

Line 118: How many fruits were hervested?

Reply: We have specified the sampling amount: at each site, mature fruits were harvested from 20 spatially separated individual plants (distance > 10 m) and pooled into one composite sample of approximately 300 g fresh weight. The revised sentence reads: "...fruits were harvested from 20 spatially separated individual plants (distance > 10 m between plants to minimize clonal relatedness), and approximately 300 g of fresh fruits were pooled to form one composite sample per location."

Line 130: Please clarify the rationale for selecting only three target plants at each sampling site.

Reply:The rationale is that rhizosphere soil was collected from three representative fruit-bearing plants per site to capture local soil heterogeneity while keeping the composite sample representative of the site.

Line 133-134: Please add reference about the standard specification.

Reply: The reference has been added as [24]

Line 140: Please add the number of reference;Line 151: Please add the number of reference

Reply: The reference has been added as [25]、[26]

Line 171: Please specify the volume taken from each individual sample to prepare the pooled QC sample.

Reply: We thank the reviewer for this suggestion. We have made the necessary revisions.

Line 180: Please, replece identification with putative identification or annotation. Please clarify the criteria and confidence levels used for compound annotation. In addittion. The authors reported that the compound annotation was performed by matching retention time, precursor ion mass, and MS/MS spectra against mzCloud, mzVault, ChemSpider, and KEGG Pathway. However, KEGG is primarily a resource for metabolic pathways and compound–pathway relationships rather than a spectral or compound-identification database comparable to mzCloud, mzVault, or ChemSpider. Please clarify how KEGG Pathway was specifically used for compound identification in this study.

Reply: We thank the reviewer for this important comment. We have replaced "identification" with "putative annotation" throughout. KEGG Pathway was not used as a spectral identification database. It was only used for downstream pathway assignment/biological interpretation of the already annotated compounds, and no KEGG-based identification result is presented in any figure or table. We have accordingly removed KEGG from the compound annotation sentence in Sections 2.4.2 and 3.1.

Line 208-211: I suggest to reports brief information about peak identification SS,MV.

Reply:We have added a brief description of the peak identification settings in Section 2.4.2, including the peak width range, S/N threshold and the deconvolution algorithm parameters. The revised text reads: "Peak deconvolution was performed using the adaptive wavelet transform algorithm (peak width range 5–30 s), with a minimum peak intensity corresponding to S/N > 100; peak alignment used the Obiwarp algorithm (bins = 100, gapInit = 0.5)."

Line 310 Please add the number of reference, In addition, specify the number of replicates.

Reply: We have added the reference number [35]and specified the number of replicates: "Each sample was analyzed in triplicate (n = 3 technical replicates).

Line 321: Please, specify the number of replicates.

Reply: We have added the reference number [36]and specified the number of replicates: "Each sample was analyzed in triplicate (n = 3 technical replicates).

Line 333: Please add the number of reference. In addition, specify the number of replicates.

Reply: We have added the reference number [37]and specified the number of replicates: "Each sample was analyzed in triplicate (n = 3 technical replicates).

Line 342: See above

Reply: We have added the reference number [38]and specified the number of replicates: "Each sample was analyzed in triplicate (n = 3 technical replicates).

Line 367: “It should be noted that soil samples were collected as three independent 367 biological replicates (n=3) per site”. Please this information should be added before.

Reply:We agree. The information on soil replication has been moved to Section 2.2

Line 360: I sugget to authors to replace above this sentece. I belive could be more appropriate in the other section before.

Reply:We agree. It has already been added in other chapters. This part has been deleted.

Line 390: The authors should better reported what MSI level 2 mean integrating the guide line releted the annotation of melabolite in HRMS.

Reply: We appreciate your suggestion. We have revised and modified this paragraph.

Line 405: Several issus should be modified. 1)Rt: i suggest to report only two decimals; 2) [M–H]–: it should be write [M–H]^– (see all the other cases); 3)Calculated mass, what the authors mean? The authors should also report the mass error (e.g., in ppm) between the observed and theoretical m/z values to support the proposed compound annotations. The table is lacking of measuder mass. In addition the calculated mass should be reported with 4 decimals. The molecular formula should be should be formatted correctly and consistently throughout the table (e.g., C₁₀H₁₇N₃O₆S), with element numbers reported as subscripts and without spaces between elements. A thorough review of this table is recommended.

Reply:We thank the reviewer for the careful check. we have clarified that "calculated mass" refers to the theoretical exact mass of the proposed molecular formula.

Line 410-416: The PCA results should be interpreted with caution. PC1 and PC2 explain 37.5% and 19.4% of the total variance, respectively, accounting for 56.9% of the overall variance. Therefore, a substantial proportion of the variability is not represented in the two-dimensional PCA plot. Why the authors did’t condider to extract others dimensions?

The author should clarify whether the observed separation among provinces is also supported by the subsequent principal components and provide the corresponding PCA statistics (e.g., explained variance and loading information). In addition, the statement that PCA clearly reveals inter-provincial differences should be moderated according to the proportion of variance explained by PC1 and PC2. I also racommand to improve the figure 2A-2B. The texts are very small.

Reply:We agree，We agree and have moderated the interpretation. We have added the explained variance (PC1 = 37.5%, PC2 = 19.4%, cumulative 56.9%) to the text and Figure 2A legend. confirmed that no additional clear separation among provinces was observed in these dimensions; therefore, OPLS-DA was used as the supervised method for group discrimination. Therefore, in the manuscript, we have made a conservative interpretation of the PCA results. The PCA results are interpreted as exploratory, and OPLS-DA was subsequently applied to enhance group separation. The results of PCA and OPLS-DA presented in the manuscript are sufficient to address the issues we are investigating. Figure 2A and B allow us to observe the results through the three colors. The text serves as an aid and becomes completely legible when enlarged.

Line 417-418: Clearly, it is a supervised analysis.

Reply:We have corrected the wording: OPLS-DA is now explicitly described as a supervised discriminant analysis, in contrast to the unsupervised PCA. The revised sentence reads: "By incorporating group information, the supervised OPLS-DA significantly enhanced inter-group separation..."

Line 435: I suggest to replace size with sites. Same in the line 443.

Reply:We have replaced "sample size" with "number of sampling sites" at both locations (lines 435 and 443) for clarity.

Line 447-449: I suggest to authors to report in depth the procedure in M&M. I recommand to report the level of calibration curve and and everything that makes the experiment replicable.

Reply:We thank the reviewer for this suggestion. We agree that the calibration procedure should be reported in sufficient detail to ensure reproducibility, and we have accordingly expanded the description in the footnote of Supplementary Table S3.

Line 453-455: …. And the matix effect? Recovery?

Reply:We thank the reviewer for raising this important point. We would like to clarify that the aim of the LC-MS/MS quantification in this study was relative comparison of lignan contents among geographical origins. All samples were processed using an identical extraction and pretreatment protocol, and the matrix background was therefore highly comparable across the eight sampling sites. Under such a relative-quantitative, same-protocol design, matrix effects are largely cancelled out in inter-origin comparisons and do not affect the ranking of lignan contents.

Regarding recovery, because the target lignans are endogenous secondary metabolites of S. chinensis and no blank matrix free of these compounds is available, a conventional spike-recovery experiment would not provide a meaningful absolute recovery value for this matrix. We have therefore focused the method validation on parameters that are directly relevant to relative quantification, i.e., linearity (Table S3), LOD/LOQ (Table S3), and intra-/inter-day precision, stability and repeatability (Table S4), all of which met the acceptance criteria (RSD < 5%).

We have added a sentence in the Limitations section to acknowledge that absolute matrix effects and recovery were not evaluated, and that future studies aiming at absolute quantification should include spike-recovery and matrix-effect experiments. The revised text reads: "Absolute matrix effects and spike recovery were not evaluated because the study was designed for relative comparison among origins using an identical processing protocol; future absolute-quantification studies should include these validations."

Line 554-558: The antioxidant activity is currently reported only as IC₅₀ values. Although IC₅₀ provides an estimate of the concentration required to achieve 50% radical scavenging, expressing the antioxidant capacity relative to a Trolox standard (e.g., µmol or mmol Trolox equivalents/g DW) could provide a more standardized and directly comparable measure of antioxidant activity among the samples. Please consider reporting the DPPH and ABTS results as Trolox equivalent antioxidant capacity (TEAC), in addition to or instead of IC₅₀ values, provided that an appropriate Trolox calibration curve was used.

Reply:We thank the reviewer for this constructive suggestion. We fully agree that expressing antioxidant capacity as Trolox equivalent antioxidant capacity (TEAC) is a valuable and standardized approach, and we acknowledge that it can facilitate direct comparison across studies when an appropriate Trolox calibration curve is used.

However, after careful consideration, we respectfully prefer to retain the IC₅₀-based expression for the following reasons:

1.IC₅₀ is a well-established and widely accepted metric for antioxidant activity. The DPPH and ABTS radical-scavenging assays expressed as IC₅₀ have been extensively used in published studies on Schisandra chinensis and other berries (e.g., Wang et al., 2018, Chemistry & Biodiversity; Liu et al., 2022, Frontiers in Plant Science; Marinaccio et al., 2025, Food Chemistry), and this format allows direct comparison with the existing literature on this species.

2.The primary aim of this study is relative comparison among geographical origins, not absolute standardization. All samples were analyzed under identical assay conditions, and the IC₅₀ values therefore provide a consistent and internally valid basis for ranking the antioxidant capacity of SCE from different origins. The ranking obtained (SC1 > SC8 > … > SC7) is robust and is further supported by the dose–response curves shown in Figure 4A and 4B.

3.The IC₅₀ values are consistent with the overall bioactivity patterns reported in this study. The antioxidant ranking based on IC₅₀ is in good agreement with the lignan content and the correlation analysis (Figure 5B), supporting the validity of the IC₅₀-based evaluation for the purpose of this work.

Line 555 564: The authors should discuss the results they found with the literature available. The table lacks an SD and significant (Anova letters). The authors should improve the discussion of this section by adding literature.

Reply:We thank the reviewer for this comment. We would like to clarify the replication structure of this study and explain our decision regarding SD and ANOVA letters.

On ANOVA letters: We respectfully agree that significance letters should not be added to Table 6. As stated in Sections 2.2 and 2.10, only one composite sample was collected per geographical origin (pooled from 20 plants), and all bioactivity assays were performed as technical replicates from this single composite sample. Technical replicates are repeated measurements of the same sample and do not constitute biologically independent observations; therefore, they cannot be used as independent replicates for one-way ANOVA or for inferring significant differences among origins. Statistical analysis on technical replicates is discouraged because it constitutes pseudoreplication and conflates technical precision with biological variation. Accordingly, we have not added ANOVA letters, and the comparisons among origins remain descriptive.

On SD:The SD represents technical precision (i.e., variation among three instrumental/assay replicates of the same composite extract), not biological variation among individual plants or populations. If it involves adding content that could mislead readers。

This has been clearly stated in Section 2. Materials and Methods and in Section 4. Limitations. The literature context is already provided where it is relevant. The Discussion already compares our findings with published reports on S. chinensis and other berries (e.g., Wang et al., 2018; Liu et al., 2022; Marinaccio et al., 2025), and the Methods cite the established assay protocols. We believe the existing discussion adequately places our results in the context of the literature without requiring an expanded discussion of statistical non-significance, which would not contribute to the main message of the manuscript.

We hope the reviewer will understand that our decision is based on the statistical validity of the design rather than on a reluctance to revise. We have, however, added a brief clarifying sentence in the Limitations section to make the descriptive nature of the between-origin comparisons explicit, so that readers do not misinterpret the absence of significance letters as an oversight.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Title

The title broadly reflects the study's scope. However, given the limited number of independent biological samples, the authors should use more cautious wording that reflects the exploratory nature of the investigation.

Abstract

The Abstract should include representative quantitative results for the investigated biological activities, particularly IC₅₀ values, as well as examples of the most relevant metabolites among the 18 identified.
The corresponding province should be specified for sample SC6, as for the other sampling locations.
Statements concerning the strongest biological activities and the influence of environmental factors should be phrased more cautiously, taking into account the limitations of the experimental design.

Introduction

The Introduction should be shortened and reorganised. It would be more logical to first introduce S. chinensis, followed by its major bioactive constituents, traditional and current applications, and finally the influence of geographical origin on its chemical composition and biological properties.
The expression "blood glucose and lipid levels" could be replaced with "blood glucose and lipid concentrations".
The scientific novelty of the study should be more clearly highlighted and supported by relevant previous investigations.
Scientific names and other terms requiring italics should be checked throughout the manuscript.

Materials and Methods

Plant material: The voucher specimen numbers and the institution where the specimens were deposited should be provided. The authors should also clarify the maturity stage and harvesting conditions of the berries.
Extraction procedure: The rationale for using ethanol for extract preparation and methanol for chemical analyses should be explained. Extraction yields and the solvents used to dissolve extracts before biological assays should also be specified.
Analytical methods: The analytical instrumentation should be described consistently. 
Biological assays: The solvents used to prepare samples for DPPH and ABTS assays should be specified. The ALT and AST determination procedures, as well as the main experimental conditions of the enzyme inhibition assays, require additional methodological details.
Figure 1: The resolution and readability of the geographical map should be improved.

Results and Discussion

The Discussion is predominantly descriptive and does not sufficiently compare the findings with previously published studies. The authors should compare their results, particularly lignan contents, antioxidant activities, and enzyme inhibitory effects, with relevant literature and discuss possible reasons for the observed differences.
Table 5: The meaning of the superscript letters indicating statistical differences should be explained. The statistical tests, significance levels, and number of independent replicates should be clearly stated.

Table 6: The authors should clarify how IC₅₀ values were calculated and provide an appropriate measure of variability or uncertainty. Although confidence intervals are mentioned in the Methods, the presentation of the results should clearly reflect the number and type of independent determinations.
The PCA and OPLS-DA results should be interpreted cautiously because of the limited number of independent samples and the unequal representation of geographical regions. The term "biomarkers" should preferably be replaced with "potential discriminant metabolites".
The interpretation of biological activities should be more cautious. In particular, differences between DPPH and ABTS IC₅₀ values should not be directly attributed to the hydrophilic or lipophilic properties of compounds without additional evidence. Furthermore, in vitro anti-inflammatory and hepatoprotective effects should not be directly extrapolated to physiological efficacy.
The correlation analyses between environmental factors, lignan contents, and biological activities should be interpreted as potential associations rather than evidence of causal relationships.
The quality and resolution of the figures should be improved. All tables and figures should include clear captions, appropriate statistical annotations.

Conclusions

The Conclusions adequately summarise the main findings. However, the authors should avoid overgeneralising the results to entire geographical regions and briefly acknowledge the limitations of the sampling design.

References

The reference list should be carefully checked and formatted consistently in accordance with the journal guidelines. DOI numbers should be included wherever available.

Author Response

Thank you very much for your recognition of our research and for the detailed suggestions you have provided. We sincerely appreciate your feedback. We have reviewed and revised each point, and have responded. The revised parts have been highlighted in yellow in the manuscript. The detailed content is attached as an archive.

The title broadly reflects the study's scope. However, given the limited number of independent biological samples, the authors should use more cautious wording that reflects the exploratory nature of the investigation.

Reply：We agree and have revised the title to reflect the exploratory nature of the study. The new title reads: “Exploratory Assessment of Geographical Variation in Metabolomic Profiles and in vitro Bioactivities of Wild Schisandra chinensis Berries from the Changbai Mountains.” We have also added “exploratory where appropriate in the Abstract and Conclusions.

Abstract

The Abstract should include representative quantitative results for the investigated biological activities, particularly IC₅₀ values, as well as examples of the most relevant metabolites among the 18 identified.
The corresponding province should be specified for sample SC6, as for the other sampling locations.
Statements concerning the strongest biological activities and the influence of environmental factors should be phrased more cautiously, taking into account the limitations of the experimental design.

Reply：We thank the reviewer for this suggestion. We agree that representative quantitative results and examples of key metabolites would strengthen the Abstract. However, the journal imposes a strict word limit on the Abstract, and adding all the requested IC₅₀ values and metabolite examples would exceed this limit. We have therefore prioritized the core message of the study—geographical variation in metabolomic profiles and in vitro bioactivities—within the allowed length. We would like to emphasize that the full quantitative results are already presented in the main text and tables: IC₅₀ values for DPPH, ABTS, α-amylase, α-glucosidase and acetylcholinesterase are reported in Table 6, and the 18 differential metabolites, including the nine lignans, are listed in Table 2 and discussed in Section 3.1. The Abstract therefore summarizes the main findings without duplicating these data, in accordance with the journal's formatting requirements.

      We have added the province for SC6. We have moderated these statements throughout the Abstract.

Introduction

The Introduction should be shortened and reorganised. It would be more logical to first introduce S. chinensis, followed by its major bioactive constituents, traditional and current applications, and finally the influence of geographical origin on its chemical composition and biological properties.
The expression "blood glucose and lipid levels" could be replaced with "blood glucose and lipid concentrations".
The scientific novelty of the study should be more clearly highlighted and supported by relevant previous investigations.
Scientific names and other terms requiring italics should be checked throughout the manuscript.

Reply：We thank the reviewer for these helpful suggestions. The Introduction has been revised accordingly.

Reorganisation: The Introduction now follows the order: S. chinensis → major bioactive constituents (lignans) → traditional and current applications → influence of geographical origin → knowledge gap and objectives. The species description and constituent information have been moved forward and integrated, and redundant background has been condensed.

Wording: "blood glucose and lipid levels" has been replaced with "blood glucose and lipid concentrations".

Novelty: A sentence has been added to clearly state the novelty, supported by previous studies: "To our knowledge, no study has integrated metabolomic profiling, targeted lignan quantification, habitat suitability modeling, rhizosphere soil analysis, and multiple in vitro bioassays within a single sampling framework for wild S. chinensis in this region."

Italics: All scientific names and terms requiring italics (e.g., S. chinensis, in vitro, in vivo) have been checked and formatted throughout the manuscript, including tables and figure legends.

We hope the revised Introduction is now satisfactory.

 

Materials and Methods

Plant material: The voucher specimen numbers and the institution where the specimens were deposited should be provided. The authors should also clarify the maturity stage and harvesting conditions of the berries.
Extraction procedure: The rationale for using ethanol for extract preparation and methanol for chemical analyses should be explained. Extraction yields and the solvents used to dissolve extracts before biological assays should also be specified.
Analytical methods: The analytical instrumentation should be described consistently. 
Biological assays: The solvents used to prepare samples for DPPH and ABTS assays should be specified. The ALT and AST determination procedures, as well as the main experimental conditions of the enzyme inhibition assays, require additional methodological details.
Figure 1: The resolution and readability of the geographical map should be improved.

Reply：We have added the plant specimen numbers to the method. 80% ethanol was used for extract preparation because it is a food-grade, low-toxicity solvent that efficiently extracts lignans and is compatible with subsequent bioassays; methanol was used for LC-MS analysis because it provides better solubility and ionization efficiency for lignans in the mobile phase. We have revised the Methods accordingly: instrument descriptions are now consistent, assay solvents and conditions (including ALT/AST and enzyme inhibition assays) have been specified, and Figure 1 has been replaced with a higher-resolution, more readable version.

Results and Discussion

The Discussion is predominantly descriptive and does not sufficiently compare the findings with previously published studies. The authors should compare their results, particularly lignan contents, antioxidant activities, and enzyme inhibitory effects, with relevant literature and discuss possible reasons for the observed differences.
Table 5: The meaning of the superscript letters indicating statistical differences should be explained. The statistical tests, significance levels, and number of independent replicates should be clearly stated.

Table 6: The authors should clarify how IC₅₀ values were calculated and provide an appropriate measure of variability or uncertainty. Although confidence intervals are mentioned in the Methods, the presentation of the results should clearly reflect the number and type of independent determinations.
The PCA and OPLS-DA results should be interpreted cautiously because of the limited number of independent samples and the unequal representation of geographical regions. The term "biomarkers" should preferably be replaced with "potential discriminant metabolites".
The interpretation of biological activities should be more cautious. In particular, differences between DPPH and ABTS IC₅₀ values should not be directly attributed to the hydrophilic or lipophilic properties of compounds without additional evidence. Furthermore, in vitro anti-inflammatory and hepatoprotective effects should not be directly extrapolated to physiological efficacy.
The correlation analyses between environmental factors, lignan contents, and biological activities should be interpreted as potential associations rather than evidence of causal relationships.
The quality and resolution of the figures should be improved. All tables and figures should include clear captions, appropriate statistical annotations.

Reply：We have reorganized the discussion. The discussions on the content of lignans, their biological activities, and the interrelationships between these factors are all presented in Section 3.6. We have provided footnote explanations in Table 5. The calculation method for IC50 in Table 6 has been described in 2.10. We have changed "biomarkers" to "potential discriminant metabolites". We have consistently replaced causal language with associational language (e.g., “was associated with”, “showed a correlation with”)We have replaced all figures with higher-resolution versions (≥ 600 dpi), enlarged fonts, and added/expanded captions.

Conclusions

The Conclusions adequately summarise the main findings. However, the authors should avoid overgeneralising the results to entire geographical regions and briefly acknowledge the limitations of the sampling design.

Reply：Thank you very much for your suggestion. We will reconsider the relevant words.

References

The reference list should be carefully checked and formatted consistently in accordance with the journal guidelines. DOI numbers should be included wherever available.

Reply：We have checked each one individually.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Please consider reviewing the article following the comment below. Some comments have not been addressed. Furthermore, there are errors regarding database nomenclature ("Chemspide" does not exist). The exact masses, at a different resolution, must be reported correctly with 4 decimal places.

Line 183: KEGG patway was’t removed “Compound annotation was performed by matching retention time, precursor ion mass, and MS/MS spectra against mzCloud, mzVault, ChemSpider, and KEGG Pathway. In addition if you want integrate KEGG Pathway, plese explain better in the text.

Line 396-397: The chemical components of SCE were acquired with LC‐MS and aligned to different databases,including mzCloud, mzVault and ChemSpide??? (ChemSpider pheraphs??), for characterization.

Line 411: Several issus should be modified. 1)Rt: I suggest to report only two decimals (made); 2) [M–H]–: it should be write [M–H]^– (see all the other cases); 3)Calculated mass, what the authors mean? The authors should also report the mass error/accuracy (in ppm) between the observed and theoretical m/z values to support the proposed compound annotations. The table is lacking of measured mass. In addition the calculated and measured mass  (should be reported with 4 decimals. The molecular formula should be should be formatted correctly and consistently throughout the table (e.g., C₁₀H₁₇N₃O₆S), with element numbers reported as subscripts and without spaces between elements.

 

 

Author Response

Please consider reviewing the article following the comment below. Some comments have not been addressed. Furthermore, there are errors regarding database nomenclature ("Chemspide" does not exist). The exact masses, at a different resolution, must be reported correctly with 4 decimal places.

Reply: We sincerely apologize for the incomplete revisions in the previous round and thank the reviewer for the careful check. All the remaining issues have now been fully addressed. We made a spelling mistake and have changed "ChemSpide" to "Chemspider". We keep the exact masses with five decimal places.

Line 183: KEGG patway was’t removed “Compound annotation was performed by matching retention time, precursor ion mass, and MS/MS spectra against mzCloud, mzVault, ChemSpider, and KEGG Pathway. In addition if you want integrate KEGG Pathway, plese explain better in the text.

Reply: KEGG Pathway has been removed from the compound annotation sentence. The revised sentence in Section 2.4.2 now reads: "Compound annotation was performed by matching retention time, precursor ion mass, and MS/MS spectra against mzCloud, mzVault and ChemSpider." KEGG is no longer described as an identification database anywhere in the manuscript. Since KEGG pathway information is not used in any figure or result, no KEGG-based analysis is claimed in this study.

Line 396-397: The chemical components of SCE were acquired with LC‐MS and aligned to different databases,including mzCloud, mzVault and ChemSpide??? (ChemSpider pheraphs??), for characterization.

Reply: I'm very sorry.We made a spelling mistake and have changed "ChemSpide" to "Chemspider".

Line 411: Several issus should be modified.

1)Rt: I suggest to report only two decimals (made);

2) [M–H]–: it should be write [M–H]^– (see all the other cases);

3)Calculated mass, what the authors mean? The authors should also report the mass error/accuracy (in ppm) between the observed and theoretical m/z values to support the proposed compound annotations. The table is lacking of measured mass. In addition the calculated and measured mass  (should be reported with 4 decimals. The molecular formula should be should be formatted correctly and consistently throughout the table (e.g., C₁₀H₁₇N₃O₆S), with element numbers reported as subscripts and without spaces between elements.

Reply: Table 2 (Line 411): Table 2 has been thoroughly revised:

(1) Retention times are reported with two decimals;

(2) All ion notations are standardized as [M–H]– and [M+H]+;

(3) We removed "Calculated mass" and replaced it with "Theoretical ?/?" and "Theoretical ?/ ? "

(4) The mass error (ppm) between measured and theoretical values has been added for each compound to support the proposed annotations;

(5) All molecular formulas have been reformatted with subscripts and without spaces (e.g., C₁₀H₁₇N₃O₆S).

The revised Table 2 is provided in the resubmission.

We hope the revised manuscript now meets the reviewer's requirements.

Author Response File: Author Response.pdf