Review Reports

Comments 1: Introduction line 52-53. The existing quality control methods cannot entirely control the quality of SR. Which methods quality control methods; this is confusion. please include these methods including their drawbacks with literature references.

Response 1: We sincerely thank the Reviewer for pointing out this lack of clarity. We agree that the statement regarding the existing quality control methods for SR was too general in the original manuscript.

We have therefore revised the Introduction to explicitly describe the currently quality evaluation approaches and their limitations, with appropriate literature references. These revisions can be found in the revised manuscript– Introduction section, lines 56–81.

Comments 2: The protocol used in the study is novel or the authors have used previous protocol for experiments with some modification. Add some literature references in the materials and method section.

Response 2: We sincerely thank the Reviewer for this valuable suggestion.

The analytical procedure used in the present study was established with reference to previously reported methods for the analysis of volatile compounds, with adjustments according to the characteristics of SR samples and the instrumentation used in the present study. The relevant references have been provided in the original manuscript([13-16]).

We have added a statement in the revised Materials and Methods section clarifying that the analytical conditions were established with reference to previously reported HS-GC-MS methods for volatile compound analysis in medicinal plants, with appropriate adjustments according to the characteristics of SR samples and the instrumentation used in the present study. The corresponding revisions in Section 2.3 (Lines 140–143).

Comments 3: Some figures are very small in size. Please increase their size without disturbing their quality and resolution.

Response 3: Thank you. The sizes and resolutions of the figures have been carefully checked and improved in the revised manuscript to enhance readability without compromising image quality.

Comments 4: The discussion section should be further improved by relevant literature finding. Further compare the results with updated and relevant literature study.

Response 4: Thank you for this valuable suggestion. We agree that further comparison with recent and relevant literature would strengthen the interpretation of our findings.

Accordingly, we have added a new Discussion section to the revised manuscript and compared our results with recent studies on the chemical variation and comprehensive quality evaluation of Saposhnikoviae Radix. In particular, we discussed the findings of Fuchino et al. regarding compositional differences among cultivated, wild, and commercial SR, and Xu et al. regarding the differences in chemical constituents and pharmacological activities associated with different growth patterns. We also incorporated a recent study on multidimensional biological–chemical integrative quality evaluation of SR to further discuss the practical significance of comprehensive quality assessment.

The revised Discussion highlights the consistency and differences between previous studies and our findings, particularly regarding the contribution of growth pattern to chemical variation. Moreover, we emphasize that the present study extends previous work by comparatively evaluating the effects of growth pattern, cultivation duration, and geographical origin on the volatile chemical profiles of SR within a single HS-GC-MS/MS-based analytical framework.

These revisions have been incorporated into the newly added Discussion section 4, pages 18-19, and lines 433-472

Comments 5-6: 5-There are some minor mistakes in English grammar of the text. Some of the sentences are very long. Moreover, awkward sentences should be reviewed and thoroughly checked for correction without any repetition of the phrases in the text.

6-The manuscripts need intensive English language editing.

Response 5-6: Thank you for these comments. The entire manuscript has been carefully revised to improve grammar, sentence structure, clarity, and readability. Repetitive and overly long sentences have been shortened or rewritten. In addition, the manuscript has undergone comprehensive English-language editing to improve grammar, terminology, style, and overall readability.

Comments 7: The number of references for the manuscript are 31; Please increase the number of references up to 45-50; it will strengthen and support your study findings.

Response 7: We sincerely thank the Reviewer for this valuable suggestion. In accordance with the Reviewer's recommendation, we have substantially expanded the reference list. The number of references has been increased from 31 to 47 in the revised manuscript. The newly added references include [6-9, 21-23, 35-42, 47].

Comments 8: The units of concentration in the paper should be rechecked according to the SI standard. Please carefully review all the concentration units in the whole text.

Response 8: We apologize for the errors in the concentration units in the original manuscript. All units and numerical formats throughout the manuscript have been carefully checked and standardized according to the journal's formatting requirements and SI conventions.

Comments 9: Some like TNF, IL1B, ALB, EGFR, and CASP3 create misunderstanding when they are appearing first in the text. A list of abbreviation is necessary. It should be placed before references.

Response 9: We appreciate this suggestion. We fully agree that providing a comprehensive list of abbreviations, including gene symbols, will improve the readability of the manuscript for readers from diverse backgrounds.

Although the gene symbols used in this study are standard HGNC-approved gene symbols and are internationally recognized abbreviations, we have added the core target genes (TNF, IL1B, ALB, EGFR, and CASP3) to the Abbreviations section to facilitate reader understanding. In addition, all gene symbols in the main text have been formatted in italic, uppercase letters to clearly distinguish them from ordinary text and to comply with HGNC formatting guidelines. Furthermore, we have also included all pathway abbreviations (e.g., PI3K-Akt, HIF-1) in the Abbreviations section with their full names provided, ensuring that readers from all backgrounds can easily understand all abbreviated terms used in the manuscript.

All abbreviations are now defined at their first appearance in the text, and the Abbreviations section has been placed before the References, as recommended. 

Comments 1: In the "Introduction" section, the author provides an extensive review of the literature, but there is no clear statement of the purpose and objectives of the study. At the end of the introduction, it is advisable to explicitly indicate the main hypothesis of the work and specific research questions that the authors seek to answer, which will allow the reader to better understand the logic of subsequent experiments and discussions.

Response 1: We sincerely thank the Reviewer for this insightful suggestion. We agree that the purpose, objectives, and research questions of the study were not sufficiently explicit in the original Introduction. We have therefore substantially revised the final paragraph of the Introduction.

The revised Introduction now explicitly presents “the main hypothesis that the volatile chemical profile of SR is influenced by growth pattern, cultivation duration, and geographical origin, and that volatile compounds identified as differential components may represent candidate quality-associated compounds with potential relevance to the anti-inflammatory and analgesic activities of SR.” “Accordingly, the specific objectives of this study are clearly stated, including the characterization of SR volatile profiles using HS-GC-MS/MS, the evaluation of the effects of the three quality-associated factors using multivariate statistical analysis, and the screening candidate compounds followed by network pharmacology and molecular docking analyses to predict their potential anti-inflammatory and analgesic mechanisms.” (page 3, lines 93–102)

The revised introduction, combined with subsequent experiments, can clearly answer the main research questions concerning whether the volatile profiles of SR differ according to growth pattern, cultivation duration, and geographical origin, which volatile compounds are identified as differential compounds and may serve as candidate quality-associated markers, and whether these compounds may be associated with the anti-inflammatory and analgesic activities of SR.

Comments 2: In paragraph 2.1. Plant materials, the author mentions that detailed information on 140 samples is provided in Table S1 in additional materials. However, the main text should briefly describe the key parameters of the sample (range of geographical coordinates, cultivation conditions, identification criteria), since the reproducibility of the study largely depends on the completeness of the description of the source material. This will increase the informative content of the article for readers who do not have access to additional files.

Response 2: We thank the Reviewer for this valuable suggestion. We agree that key information regarding the source materials should be briefly described in the main text to improve the reproducibility and informative value of the study. We have revised Section 2.1 to provide a clearer description of the sample sources, growth patterns, cultivation methods, cultivation durations and identification procedure in the main text. Detailed information regarding the sample sources, collection location, growth patterns, cultivation methods cultivation duration and collection date of each sample is provided in Table S1.

Regarding geographical coordinates, we acknowledge that precise GPS coordinates of the individual sampling sites were not recorded at the time of collection. Therefore, to avoid introducing potentially inaccurate information, we have not retrospectively assigned estimated coordinates. Instead, the geographical origins of all samples are reported at the locality or administrative-region level according to the original collection records, and the detailed information is provided in Table S1.

Comments 3: The section “2.2. Sample Preparation” provides sample preparation conditions, but there is no information on the number of parallel measurements (biological and analytical repeatability), which is necessary to assess the reproducibility and statistical reliability of the data obtained. It is recommended to specify the number of repetitions for each sample and the method of processing emissions.

Response 3: We thank the Reviewer for highlighting the importance of clearly describing biological and analytical replication.

However, we emphasize that for each collection site, multiple individual plants were harvested from the same field plot and randomly pooled, and randomly divided into five or ten portions. These portions originated from the same field collection and therefore were not treated as independent biological replicates. In addition, each analytical sample was prepared once and subjected to one HS-GC-MS/MS measurement. To ensure the reliability of the analytical data, a pooled quality-control (QC) sample was analyzed after every 10 injections throughout the entire analytical run, which can confirm the stability and repeatability of the instrument. No data points were excluded from the analysis based on statistical outlier removal. 

Furthermore, we clarified the identification procedure in the revised manuscript. “All samples were identified by Prof. Feng Wei (Institute for National Institutes for Food and Drug Control, Beijing, China), and meet the requirements of the 2020 edition of China Pharmacopoeia.” (Section 2.1, lines 122-124)

We acknowledge that the absence of independent parallel analytical measurements is a limitation of the present study. The inclusion of multiple biological replicates in future studies would further enhance the statistical robustness of the findings.

Comments 4: In the conclusions, the authors list the key compounds identified for each differentiation factor, but the biological significance and chemical stability of these markers, as well as possible mechanisms of their accumulation, depending on the growth conditions, are not sufficiently discussed. It is recommended to expand this section with an emphasis on the practical significance of the results obtained for pharmacopoeial control.

Response 4: Thank you for this insightful comment. We agree that the biological significance, potential mechanisms underlying the accumulation of the differential volatile compounds, and their practical implications for quality control required further discussion.

Accordingly, the relevant interpretation has been expanded in both the newly added Discussion section and the revised Conclusions. We now discuss that the observed variation in volatile profiles may be associated with differences in plant developmental status, cultivation practices, and environmental adaptation. We also clarify that the mechanisms underlying the differential accumulation of these volatile compounds remain to be further investigated through targeted chemical, physiological, and biosynthetic studies.

In addition, we have emphasized the practical significance of the findings for quality evaluation. The identified volatile compounds are now more cautiously described as candidate quality-associated volatile markers, rather than established quality control markers. We further acknowledge that their analytical reproducibility, chemical stability during processing and storage, batch-to-batch consistency, and relationships with pharmacological efficacy require independent validation before potential incorporation into routine quality control or future pharmacopoeial applications.

These revisions have been incorporated into Sections 4 (Discussion) and 5 (Conclusions) of the revised manuscript.