Review Reports - Seasonal and Organ-Specific Variations of Alkaloids in <i>Buxus obtusifolia</i> (Mildbr.) Hutch: A Multivariate LC/MS Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive investigation of seasonal and organ-specific variation in aminosteroid alkaloids in Buxus obtusifolia using UHPLC–QqTOF-MS/MS combined with multivariate data analysis. The study addresses an underexplored species of ethnopharmacological relevance and contributes novel insights into phytochemical variability, with potential implications for optimized harvesting strategies and natural product discovery. The study design is generally appropriate to address the stated research question. The authors employ a longitudinal sampling strategy over a full annual cycle, combined with organ differentiation (leaves vs. twigs), which is well aligned with the objective of assessing temporal and spatial metabolite variability. The use of replicated LC/MS measurements, inclusion of quality control samples, and normalization to an internal standard enhance analytical robustness. The application of principal component analysis and volcano plot analysis is suitable for handling high-dimensional metabolomics data and supports the identification of discriminant metabolites. Overall, the conclusions regarding organ-specific differences are well supported by the data. However, the claim of “minimal seasonal trends” requires more cautious phrasing, as some clustering and compound-specific variation are evident, particularly during the warmest months. The interpretation of abiotic factors influencing metabolite accumulation is plausible but remains correlative and should be framed accordingly to avoid overstatement.

The methods are described in substantial detail and are largely sufficient to allow reproducibility. The LC/MS conditions, extraction procedures, and data preprocessing parameters are clearly outlined, including thresholds used for feature extraction and statistical filtering. The description of PCA scaling approaches and cross-validation strengthens methodological transparency. Nevertheless, some clarifications would improve reproducibility further. The rationale for selecting specific preprocessing thresholds (e.g., feature occurrence ≥20/84) should be justified. Additionally, the statistical treatment of replicate measurements could be described more rigorously, particularly regarding how technical versus biological variability is handled. The use of Microsoft Excel for statistical testing (t-test) in volcano plot analysis is acceptable but may raise concerns regarding reproducibility and robustness; consideration of specialized statistical software would strengthen the methodological framework.

The manuscript would benefit from clearer guidance on moderating overstated claims. In particular, statements suggesting that the study identifies “optimal harvest periods” should be tempered, as these conclusions are based on relative signal intensities rather than absolute quantification or validation across multiple populations. Similarly, the linkage between environmental stress (temperature and drought) and alkaloid accumulation is discussed in a mechanistic manner that implies causality, whereas the presented data only demonstrate correlation. The authors should revise such statements to reflect associative rather than causal relationships unless additional experimental evidence is provided.

From a scientific perspective, the manuscript is strong in its analytical approach and data richness, but some aspects of interpretation could be improved. The discussion would benefit from deeper integration with existing literature on Buxaceae alkaloids and metabolomic seasonality, beyond citing the authors’ previous work. Additionally, while the identification of 15 key compounds is valuable, the criteria for their selection should be more explicitly defined. The biological relevance of newly detected or tentatively identified compounds is only briefly mentioned and could be elaborated further to enhance the impact of the study.

In terms of presentation, the manuscript is generally well structured and clearly written. Figures are appropriate and informative, although some could benefit from improved readability, particularly PCA plots where labels and clustering interpretation could be more explicit. Minor typographical errors and inconsistencies are present and should be corrected during revision.

In conclusion, this manuscript presents novel and valuable data on the phytochemical variability of Buxus obtusifolia and is suitable for publication after minor to moderate revision. Addressing the points raised above, particularly regarding interpretation of seasonal effects, justification of methodological choices, and moderation of claims, will significantly strengthen the scientific rigor and clarity of the work.

Comments on the Quality of English Language

The manuscript is generally well written and demonstrates a good command of scientific English. The text is clear, coherent, and appropriate for publication. The authors successfully use discipline-specific terminology related to phytochemistry and metabolomics, and the overall structure facilitates comprehension of a technically complex dataset.

However, the language would benefit from careful proofreading to address minor grammatical, typographical, and stylistic issues. There are occasional spelling errors (e.g., “particulary,” “tepmereatures”), inconsistent spacing, and minor punctuation problems. Some sentences are overly long or complex, which slightly reduces readability; these could be improved by simplifying sentence structure or splitting into shorter statements.

In addition, there are instances of inconsistent terminology and formatting, particularly in the notation of ions, units, and abbreviations (e.g., spacing around symbols, variation in the presentation of m/z values). Ensuring uniformity throughout the manuscript would enhance professionalism and clarity.

A few passages would benefit from stylistic refinement to improve precision and avoid ambiguity, especially in the discussion section where interpretations are presented. These are not major issues but could be improved with light language editing.

Overall, the quality of English is acceptable for publication, but minor revisions are recommended to ensure clarity, consistency, and fluency

Author Response

Reviewer 1

Response: This formulation has been improved (also in accordance with reviewer 2).

The interpretation of abiotic factors influencing metabolite accumulation is plausible but remains correlative and should be framed accordingly to avoid overstatement.

Response: We have chosen the following formulations throughout the manuscript which are less likely to appear as overstatements:

Abstract: “…into possible association of some abiotic factors and the phytochemical profile of this plant. Studies including further populations of this species from different locations will have to show whether the present findings allow general conclusions with respect to the investigated compounds’ accumulation in response to external factors. Furthermore, the present results represent a basis to delineate the optimal harvest period for targeted isolation of larger quantities of bioactive aminosteroids for further development.”

Results and Discussion: Section 2.2: “the optimal harvest period for all B. obtusifolia aminosteroid alkaloids reported in our recent study [2] could be narrowed down…”

Section 2.5:” This relatively stable temperature pattern likely explains the absence of a clear and consistent seasonal separation or obvious sequential variation among the samples. However, subtle trends indicating an association to seasonal changes could be observed.”

Conclusions: “Direct comparison of the intensity of mass signals over the months for each of the bioactive aminosteroids identified in our previous study helped to narrow down the probably best time periods for their targeted isolation. “ and “Principal component analysis and volcano plot of the LC/MS dataset revealed clear organ-specific differences in alkaloid profiles, in addition to some less clear-cut trends in seasonal metabolite variation.”

Response: The thresholds were largely derived from our experience in previous similar studies (Szabó et al., Schäfer et al. 2025). We have added a short statement along with proper citations in section 3.3.

Additionally, the statistical treatment of replicate measurements could be described more rigorously, particularly regarding how technical versus biological variability is handled.

Response: Measurement of each sample was carried out three times as mentioned, i.e. three technical replicates. Due to the remote origin of the plant material, representing a rare species under protected conditions (with legal authorization, of course), unfortunately, precluded collection of a bigger number of samples and we also had no possibility to take into account biological variability in terms of different populations or geographic origin. So we hope the reviewer will accept that we worked with the material we could obtain and could not take into account biological variability in this study. To avoid misunderstandings, we added statements on this fact in the Abstract: “Studies including further populations of this species from different locations will have to show whether the present findings allow general conclusions with respect to the investigated compounds’ accumulation in response to external factors.” and Conclusions: “Future studies with different populations of B. obtusifolia will have to show whether the present findings on investigated compounds’ accumulation in response to external factors can be generalized. The present findings provide valuable insights into the phytochemical dynamics of the investigated population of B. obtusifolia and offer a basis for the targeted isolation of bioactive aminosteroids in future pharmacological studies.”

The use of Microsoft Excel for statistical testing (t-test) in volcano plot analysis is acceptable but may raise concerns regarding reproducibility and robustness; consideration of specialized statistical software would strengthen the methodological framework.

Response: Microsoft Excel was in fact only used for presentation purposes, i.e. to repeat the t-test and generate the (nicer) bubble volcano plot for the results. The t-test was also calculated (like the PCA) in the specialized statistical software Profile Analysis by the manufacturer of our mass spectrometer. The result was essentially identical.

Response: A statement on this aspect has been added to the Conclusions: “Direct comparison of the intensity of mass signals over the months for each of the bioactive aminosteroids identified in our previous study helped to narrow down the probably best time periods for their targeted isolation.“

Similarly, the linkage between environmental stress (temperature and drought) and alkaloid accumulation is discussed in a mechanistic manner that implies causality, whereas the presented data only demonstrate correlation.

Response: We are now using the term “subtle trend” and “some less clear-cut trends in seasonal metabolite variation” (see above).

The authors should revise such statements to reflect associative rather than causal relationships unless additional experimental evidence is provided.

Response: We have done our best to remove potentially over-interpretative formulations, see above.

Response: We are not aware of any previous systematic studies on metabolomic seasonality of Buxaceae alkaloids in the literature besides our own studies, although thoroughly sought for. If we overlooked such work, we would be grateful for a specific hint.

The biological relevance of newly detected or tentatively identified compounds is only briefly mentioned and could be elaborated further to enhance the impact of the study.

Response: Otherwise following this reviewer to be more cautious with our conclusions and interpretations, we chose not to speculate on the relevance of compounds that we only tentatively identified. We hope the reviewer can accept this.

Response: To improve readability of the PCA plots, Figure 3A and Figure 5, ellipses were modified so as to represent only analyses of samples which do not directly overlap with each other (but are nevertheless very close to each other). Furthermore, some arrows were added to assign samples more specifically.

Minor typographical errors and inconsistencies are present and should be corrected during revision.

Response: We have checked again for typographical errors and inconsistencies and corrected a few.

Comments on the Quality of English Language

Response: We did our best to eliminate all typos and minor grammatical errors.

Response: We did our best to be consistent in terminology and formatting throughout the manuscript.

Response: We have done our best to be as precise and unambiguous as possible.

Overall, the quality of English is acceptable for publication, but minor revisions are recommended to ensure clarity, consistency, and fluency

Response: We have done our best to ensure maximum clarity, consistency and fluency.

We cordially thank this reviewer for the meticulous work as well as the very constructive criticism to help us improve our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript title: Seasonal and Organ-Specific Variations of Alkaloids in Buxus obtusifolia (Mildbr.) Hutch: A Multivariate LC/MS Study

The manuscript regarding the topic and results presented is of interest to ……………. science community and revisions based on the comments below are recommended before considering for publication.

Clarity: The phrase "minimal seasonal trends" in the abstract is somewhat contradicted by later sections that highlight significant differences between dry and rainy seasons. Revising this for nuance (e.g., "minimal overall seasonal separation but distinct dry-season clusters") would improve clarity.

Major comments

Insufficient Abstract: In the abstract, the main aim and background of the manuscript are missing, the current version only highlights the result. In addition, it would be even better to have a sentence as a future perspective.
Abbreviations are typically defined the first time the term is used within the abstract and again in the main text, and then used exclusively throughout the remainder of the document. Please consider adhering to this convention.
In the introduction, the background, and a clear hypothesis are missing.
Lake of scientific literature to support the statements and findings throughout the manuscript…... I have made some suggestions for that and more need it….
More information is needed for ALL TABLE captions and define the abbreviations and units that are used. And adjust the significant figures for the table and manuscript.
Grammar and punctuation issues need to be addressed. I have selected/mentioned some as examples.
I have a major concern about the results and discussion section. The authors describe the results and compare the results with previous studies, however, insight mechanisms are still insufficient.
Use standalone keywords

Detailed comments:

Abstract

If the unit/abbreviation is not mentioned before, consider defining the abbreviation when mentioned for the first time.

Introduction:

Line 41: A reference is needed here, for example, you can use: https://doi.org/10.1007/978-3-030-38386-2_1

Line 42: A reference is needed here

Line 43-48: A complicated sentence, please revise and check the grammar

Line 47: A reference is needed here, for example, you can use:

https://doi.org/10.1016/j.scitotenv.2020.142822

line 52: you need to discuss other factors, drought…etc

R&D section

The authors note that temperature variation was minimal, except for January–March , which "likely explains the absence of a clear and consistent seasonal separation". However, the study does identify "optimal harvest periods" based on these variations. There is a slight contradiction between claiming a lack of "clear separation" while simultaneously recommending specific harvest windows.
The PC2 vs. PC1 scores plot captures 53.2% of the total variance. While significant, a large portion of the variance (nearly 47%) remains unexplained by the first two components. The authors could briefly discuss what other factors (e.g., specific minor metabolites or biological noise) might account for the remaining variance.

The manuscript mentions a "previously undescribed aminosteroid" (C23H39NO2) found in twigs. To strengthen the scientific impact, the authors should clarify if this is a "new" compound to science or simply one that hasn't been reported for this specific species.

In the "Elemental formula" column for compound 2, the formula is listed as C27H5N2O22+, is this chemically impossible for a steroidal alkaloid.
"alkloid" in the heading for section 2.6 should be "alkaloid".
"tepmereatures" in the environmental discussion should be "temperatures".
In Section 2.5, "twig samples collected in July appear distinctly separated in the upper left region..." should be checked against the figure to ensure "upper left" is the correct quadrant.

In MM section

Literature references are missing for all sub-sections. It would be better to cite the references that the procedure adopted.

Additional info is needed for the table caption, most importantly significant figures.

In the MM section, what is the quality control (QC) data? There is no mention of the QC.

What is the accuracy of the instruments, recovery, LOD, and LOQ ……. These parameters are needed to report the efficiency of any analytical system.

In general, how many times you’ve recorded the data,? duplicate? Triplicate?..... what you mentioned in the text is not clear, please elaborate more on this

The methodology states that the mean signal intensity was calculated from the "two most reproducible replicate measurements" out of three per month. Selecting only the "two most reproducible" replicates instead of averaging all three valid replicates can be seen as "cherry-picking" data. It is scientifically more rigorous to use all three replicates or provide a clear statistical justification for excluding a specific outlier.

Author Response

Reviewer 2

Clarity: The phrase "minimal seasonal trends" in the abstract is somewhat contradicted by later sections that highlight significant differences between dry and rainy seasons. Revising this for nuance (e.g., "minimal overall seasonal separation but distinct dry-season clusters") would improve clarity.

Response: We thank the reviewer for this suggestion. We have chosen the formulation “subtle overall seasonal changes with some distinct dry-season clustering”.

Major comments

Insufficient Abstract: In the abstract, the main aim and background of the manuscript are missing, the current version only highlights the result. In addition, it would be even better to have a sentence as a future perspective.

Response: To make background and aims even clearer, we have added some information: “…warranting more detailed studies on this interesting plant and its bioactive constituents.” and “….to gain insights into the dynamics of their formation and, potentially, the best times to harvest individual alkaloids.”

Regarding a clearer future perspective, we have modified and extended the last sentence of the abstract: “Studies including further populations of this species from different locations will have to show whether the present findings can be applied to draw general conclusions with respect to the investigated compounds’ accumulation in response to external factors. Furthermore, the present results represent a basis to delineate the optimal harvest period for targeted isolation of larger quantities of bioactive aminosteroids for further development.”

Abbreviations are typically defined the first time the term is used within the abstract and again in the main text, and then used exclusively throughout the remainder of the document. Please consider adhering to this convention.

Response: All abbreviations used in the abstract are now spelt out in full on first mentioning.

In the introduction, the background, and a clear hypothesis are missing.

Response: The background as well as clear aims and scope are cleary outlined in the first paragraph of the introduction. A few phrases were added to make it even clearer than before.

Lake of scientific literature to support the statements and findings throughout the manuscript…... I have made some suggestions for that and more need it….

Response: We did our best to support all statements and previous findings throughout the manuscript with relevant literature.

More information is needed for ALL TABLE captions and define the abbreviations and units that are used. And adjust the significant figures for the table and manuscript.

Response: We have added some explanation for abbreviations in the table caption. The abbreviations used in the table itself are tR(min) for retention time (minutes) and mass/charge ratio (m/z).

The number of significant figures for retention time (tR) and mass signals (m/z) is naturally different. While the former is expressed in minutes and decimal fractions of minutes (maximum 2 significant figures), a TOF mass spectrometer determines the mass/charge ratio (m/z) with a precision of about 5 significant digits but is usually rounded to 4 significant figures.

Grammar and punctuation issues need to be addressed. I have selected/mentioned some as examples.

Response: We have checked grammar and punctuation very carefully.

I have a major concern about the results and discussion section. The authors describe the results and compare the results with previous studies, however, insight mechanisms are still insufficient.

Response: We are not sure which insights or mechanisms the reviewer means, but we think that any further discussion of potential mechanisms underlying the observations would inevitably be speculative and mean to over-interpret the data (please compare statements of Reviewer 1). We have therefore not added any further mechanistic discussion.

Use standalone keywords

Response: We deleted the abbreviations in parentheses and think the keywords are appropriate. Please compare our previous paper: https://www.mdpi.com/2223-7747/14/19/3060). If further changes are required, we ask the editor to advise us.

Detailed comments:

Abstract

If the unit/abbreviation is not mentioned before, consider defining the abbreviation when mentioned for the first time.

Response: Abbreviations are now explained.

Introduction:

Line 41: A reference is needed here, for example, you can use: https://doi.org/10.1007/978-3-030-38386-2_1

Response: The suggested reference is not relevant in the context of line 41, where we just mentioned the fact that we have recently investigated the plant under study and briefly summarized our results. We also did not see any direct relationship between that article’s content and any other statements within our manuscript. We therefore did not include this reference.

Line 42: A reference is needed here

Response: Maybe the reviewer meant our reference [2]. We have added it once more.

Line 43-48: A complicated sentence, please revise and check the grammar

Response: We have checked the grammar and found no errors.

Line 47: A reference is needed here, for example, you can use:

https://doi.org/10.1016/j.scitotenv.2020.142822

Response: There are already two appropriate and relevant references cited [3,4]. This additional one would not add any further aspects (and in fact appears less relevant to the statement ending in line 47). We therefore did not include this reference.

line 52: you need to discuss other factors, drought…etc

Response: Discussion of these factors is already included: “It is widely established that abiotic factors such as fluctuating temperatures, light intensity, nutrient availability, and water stress influence growth as well as biosynthesis and accumulation of active principles in medicinal plants [3,4].” (Lines 45 -47 in the original version).

R&D section

The authors note that temperature variation was minimal, except for January–March , which "likely explains the absence of a clear and consistent seasonal separation". However, the study does identify "optimal harvest periods" based on these variations. There is a slight contradiction between claiming a lack of "clear separation" while simultaneously recommending specific harvest windows.

Response: This is not contradictory since we have a (more or less) continuous course of concentration for each compound (documented in Figure 1 and the many Supplementary figures for the individual time course of each compound) so that the best time interval for harvesting can be deduced individually for each compound. This is not directly related to the absence of a clear separation or an obvious time course in the PCA, which actually would be a function of all compounds’ time courses simultaneously. So we are quite sure that this is no contradiction.

The PC2 vs. PC1 scores plot captures 53.2% of the total variance. While significant, a large portion of the variance (nearly 47%) remains unexplained by the first two components. The authors could briefly discuss what other factors (e.g., specific minor metabolites or biological noise) might account for the remaining variance.

Response: The reviewer may not have noticed that we also investigated PC3 (section 2.5) whose information content explains another 7.0 % of variance. PCs 1-3, altogether, hence cover 60.2% (and thus the majority) of the total variance. All further PCs will individually have information content <7 %. It is quite normal that it is not easy (and not useful) to interpret them any further.

The manuscript mentions a "previously undescribed aminosteroid" (C23H39NO2) found in twigs. To strengthen the scientific impact, the authors should clarify if this is a "new" compound to science or simply one that hasn't been reported for this specific species.

Response: We have changed the sentence to: “Apart from an aminosteroid with the molecular formula C₂₃H₃₉NO₂ (7.68 min:m/z 362.3075) previously undescribed for B. obtusifolia and Buxaceae,…” and hope it is now clearer.

In the "Elemental formula" column for compound 2, the formula is listed as C27H5N2O22+, is this chemically impossible for a steroidal alkaloid.

Response: The formula C₂₇H₅₀N₂O₂²⁺is chemically possible for a Buxus alkaloid, of course. It is very common in the diamino series to have double protonation and obviously the N-oxide is also protonated at both sites (i.e. the amino group at C-3 and the N-oxide moiety at C-20). m/z is then [M+2H]²⁺.

"alkloid" in the heading for section 2.6 should be "alkaloid".

Response: This was corrected.

"tepmereatures" in the environmental discussion should be "temperatures".

Response: This was corrected.

In Section 2.5, "twig samples collected in July appear distinctly separated in the upper left region..." should be checked against the figure to ensure "upper left" is the correct quadrant.

Response: Yes, it appears that this is correct.

In MM section

Literature references are missing for all sub-sections. It would be better to cite the references that the procedure adopted.

Response: Descriptions of methods are accompanied by appropriate citations.

Additional info is needed for the table caption, most importantly significant figures.

Response: See above.

In the MM section, what is the quality control (QC) data? There is no mention of the QC.

Response: It is explained in section 3.2: “20 μL of each sample was pooled into a collective LC/MS vial (QCmix) and measured after every six samples”.

What is the accuracy of the instruments, recovery, LOD, and LOQ ……. These parameters are needed to report the efficiency of any analytical system.

Response: Detection limits and quantitation limits must be determined individually for analytes that are supposed to be quantified in terms of absolute concentration. This is not within the scope and not common practice in case a metabolic profiling study such as ours.

In general, how many times you’ve recorded the data,? duplicate? Triplicate?..... what you mentioned in the text is not clear, please elaborate more on this

Response: We have modified the respective statements and instead include all three replicates in the bar charts representing the bucket statistics. These now reflect n=3 ± sd.

We thank the reviewer for the very constructive criticism and for the time and effort spent to help us improve our work.

Reviewer 3 Report

Comments and Suggestions for Authors

the presentation of research background and scientific questions in the introduction still needs improvement, and we recommend rejection from the following aspects:

The authors only mentioned that their team has completed the first phytochemical investigation of this species, but did not summarize existing research advances on the alkaloid accumulation patterns of other Buxus species, nor clarified the complementary value of this study to the field. It is recommended to add relevant background to highlight the academic contribution of this work to the research on chemical variation in Buxus medicinal plants.
The current text states that "no previous studies have examined the seasonal and organ-specific variability of its phytochemical profile". However, it should clarify the core scientific question to be addressed, for example, "clarifying the accumulation pattern of active alkaloids in different seasons and organs to provide a scientific basis for determining the optimal harvest time and utilized plant part", which is very important to highlight the application value of the research.
the paper failed to explain the reason of UHPLC-QqTOF-MS combined with multivariate statistical analysis (PCA, volcano plot) was chosen for this study, and is is suggested to point out the applicability of this technical scheme to this research question (for example, untargeted metabolomics combined with statistical analysis can efficiently screen for signature differential components), so as to complete the research logic.
It is also recommended to briefly state the innovation of this study in conclusion compared with existing similar work, for example, this study is the first to clarify the accumulation dynamics of active alkaloids in the East African endemic medicinal plant B. obtusifolia, which provides fundamental data for the standardized development of this ethnomedicinal resource.

Author Response

the presentation of research background and scientific questions in the introduction still needs improvement, and we recommend rejection from the following aspects:

The authors only mentioned that their team has completed the first phytochemical investigation of this species, but did not summarize existing research advances on the alkaloid accumulation patterns of other Buxus species, nor clarified the complementary value of this study to the field. It is recommended to add relevant background to highlight the academic contribution of this work to the research on chemical variation in Buxus medicinal plants.
The current text states that "no previous studies have examined the seasonal and organ-specific variability of its phytochemical profile". However, it should clarify the core scientific question to be addressed, for example, "clarifying the accumulation pattern of active alkaloids in different seasons and organs to provide a scientific basis for determining the optimal harvest time and utilized plant part", which is very important to highlight the application value of the research.
the paper failed to explain the reason of UHPLC-QqTOF-MS combined with multivariate statistical analysis (PCA, volcano plot) was chosen for this study, and is is suggested to point out the applicability of this technical scheme to this research question (for example, untargeted metabolomics combined with statistical analysis can efficiently screen for signature differential components), so as to complete the research logic.
It is also recommended to briefly state the innovation of this study in conclusion compared with existing similar work, for example, this study is the first to clarify the accumulation dynamics of active alkaloids in the East African endemic medicinal plant B. obtusifolia, which provides fundamental data for the standardized development of this ethnomedicinal resource.

Response: We thank the reviewer for the suggested formulations. In fact, all these points were already covered -although maybe in other words- in the manuscript:

Previous work on other Buxus species and Buxaceae is mentioned and duly cited: [2, 10, 11, 19 – 26]. From this, the complementary value of the present study is implicitly clear.
The core scientific rationale and aim of the study is clearly mentioned in the introduction.
The relevance of our analytical method is evident and has been demonstrated many times before. References directly relevant in the present context are duly cited [10, 11, 20].
The innovation of this study becomes clear from the conclusions.

We therefore did not apply any further textual changes besides those following the comments of reviewers 1 and 2.

We cordially thank the reviewer for the time and effort spent on our manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive and well-designed metabolomic investigation of an endemic East African species, focusing on aminosteroid alkaloids. The study provides valuable and novel insights into the influence of abiotic factors on the phytochemical profile of Buxus obtusifolia, with particularly interesting findings regarding organ-specific differences and minimal seasonal variation, which are highly relevant for optimizing harvesting strategies of bioactive compounds. The application of advanced analytical techniques such as UHPLC/+ESI-QqTOF-MS/MS, combined with robust multivariate data analysis (PCA and volcano plot), is appropriate and executed at a very high level. The interpretation of the results is clear and scientifically sound. Moreover, the manuscript is technically excellent, with a strong experimental design, high-quality data processing, and clear, well-structured writing. I strongly recommend acceptance of this manuscript without any revisions and would like to congratulate the authors on an outstanding piece of work that offers interesting results and is prepared to an exceptionally high technical standard, representing a valuable contribution to the field.

Author Response

We thank the reviewer for the very appreciative and positive assessment!

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Seasonal and Organ-Specific Variations of Alkaloids in Buxus obtusifolia (Mildbr.) Hutch: A Multivariate LC/MS Study” presents a comprehensive metabolomic investigation of alkaloid variability using LC/MS combined with multivariate statistical analysis. The study is well structured, methodologically sound, and addresses a relevant topic in pharmacognosy and natural product research, particularly in the context of optimizing harvesting strategies for bioactive compounds.

The revised version shows clear improvement in clarity, data presentation, and methodological transparency. The integration of principal component analysis (PCA) and volcano plot analysis is appropriate and provides meaningful insights into both organ-specific and seasonal variation. The dataset appears robust, and the analytical workflow is reproducible.

However, several aspects of the manuscript would benefit from further refinement to strengthen its scientific impact, interpretative depth, and overall clarity prior to publication.

First, while the manuscript correctly states that this is the first study addressing seasonal and organ-specific alkaloid variation in Buxus obtusifolia, the novelty could be better contextualized within the broader framework of the Buxaceae family and phytochemical ecology. The Introduction would benefit from a clearer articulation of how this work advances current knowledge beyond descriptive profiling. In particular, the authors are encouraged to explicitly state the implications of their findings for pharmacological exploitation, metabolite optimization, or ecological adaptation mechanisms.

Second, the discussion of abiotic factors, especially temperature and precipitation, in relation to alkaloid accumulation is scientifically plausible but remains largely correlative. The manuscript occasionally adopts language that may imply causation. This should be moderated. The authors should clearly acknowledge that no controlled environmental experiments were conducted and that conclusions regarding abiotic stress effects are inferential. Including a short limitations paragraph would significantly strengthen the scientific rigor.

Third, the interpretation of the PCA results, while clear, remains primarily descriptive. The manuscript would benefit from a deeper biochemical or physiological interpretation of the observed clustering patterns. For example, the authors could elaborate on why certain aminosteroid alkaloids are more abundant in leaves compared to twigs, possibly discussing biosynthetic localization, transport mechanisms, or defensive roles of secondary metabolites.

Fourth, the volcano plot analysis is well executed statistically, but its biological interpretation is limited. The authors are encouraged to expand on the significance of low-abundance yet statistically significant metabolites. These may represent biosynthetic intermediates, degradation products, or novel compounds with potential pharmacological relevance. Highlighting this aspect would enhance the impact of the study.

Finally, the conclusion regarding the optimal harvesting period (December–March) should be more nuanced. It would be beneficial to specify whether this recommendation applies to total alkaloid yield or to specific high-value compounds. Additionally, linking this conclusion more explicitly to potential pharmaceutical applications would strengthen the practical relevance of the work.

In the Abstract, the content is comprehensive but somewhat dense. The authors may consider reducing methodological detail and placing greater emphasis on the key findings and their practical implications.

In the Introduction, the background is generally strong, but there is a minor editorial issue where an unfinished sentence appears (“or are there further influences…”). This should be corrected. Additionally, the discussion of influencing factors could be expanded to briefly include biotic stressors such as herbivory or microbial interactions.

The Materials and Methods section is detailed and largely reproducible. However, the rationale for specific methodological choices should be clarified. In particular, the authors should briefly justify the use of dichloromethane as extraction solvent and explain the selection of papaverine as an internal standard. A short explanation for choosing Pareto scaling over other normalization approaches would also be useful.

The Results section is logically organized and clearly presented. However, some repetition occurs in the description of PCA outputs and figures. Streamlining these sections would improve readability. Additionally, stronger cross-referencing between figures and tables (e.g., linking Figure 1 more explicitly to Table 1) would help guide the reader.

The manuscript would significantly benefit from a more clearly defined Discussion section. At present, interpretative elements are partially embedded within the Results. A dedicated Discussion would allow for deeper comparison with previous studies on Buxus species or other alkaloid-producing plants, as well as a more thorough exploration of ecological and pharmacological implications.

Regarding the figures, they are informative and appropriate. Minor improvements in readability (e.g., larger axis labels and clearer legends) are recommended. The PCA plots, in particular, would benefit from slightly enhanced visual clarity.

There are a few typographical and consistency issues that should be addressed. For example, “Mataboscape” should be corrected to “MetaboScape.” The manuscript alternates between “LC/MS” and “LC-MS,” and one format should be used consistently. Units should also be standardized in terms of spacing and formatting (e.g., μmol/L, °C).

Some sentences are overly long and could be simplified for clarity. Reducing repetition of phrases such as “previous study” and “characteristic compounds” would improve the flow of the text.

The inclusion of a graphical abstract summarizing the seasonal variation, organ specificity, and key alkaloids would greatly enhance the accessibility and visual appeal of the manuscript. Furthermore, a brief section outlining future research directions—such as multi-location sampling, controlled stress experiments, or isolation of newly detected metabolites—would add value.

The manuscript is of good quality and presents relevant and novel findings. It is suitable for publication after minor to moderate revisions addressing the points outlined above. With improved interpretative depth, clearer positioning of novelty, and minor editorial corrections, the work will make a valuable contribution to the field of natural product chemistry and phytochemical research.

Comments on the Quality of English Language

Overall, the quality of English is acceptable for publication, but minor revisions are recommended to ensure clarity, consistency, and fluency

Author Response

Reviewer 1

However, several aspects of the manuscript would benefit from further refinement to strengthen its scientific impact, interpretative depth, and overall clarity prior to publication.

Reply: It is quite explicitly stated in the manuscript how the present work advances current knowledge. We are also reluctant to add any further implications with respect to the points mentioned by the reviewer, since this would inevitably mean to imply causal relationships, and also an element of speculation, which the reviewer asked us not to do.

Reply: We have added a sentence to the Conclusions acknowledging that such controlled experiments will certainly be desirable in the future.

Reply: This would inevitably mean an interpretation the observations in terms of causal implications. This is exactly what the reviewer asked us to avoid in his/her round 1 report. We do not find ourselves able to follow this request.

Reply: A sentence taking this into account was added in the first paragraph of section 2.6.

Reply: It is clearly stated that this applies to the “targeted isolation of bioactive aminosteroids”. Targeted means that a particular compound can be isolated in a target-directed manner. “Bioactive” is defined in the introduction (but may certainly also include other bioactivities that may yet be discovered in the future).

Additionally, linking this conclusion more explicitly to potential pharmaceutical applications would strengthen the practical relevance of the work.

Reply: The pharmacological relevance of the compounds under study is clearly mentioned in the introduction: “Notably, some of these compounds exhibited promising in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense (Tbr), the causative agent of East African Human Trypanosomiasis and Plasmodium falciparum (Pf), the primary parasite responsible for tropical Malaria [2]. These findings prompted us to investigate the accumulation of the bioactive compounds in more detail.” We do not find it necessary to repeat this explicitly. We find it sufficient to mention again that they are “bioactive aminosteroids”.

In the Introduction, the background is generally strong, but there is a minor editorial issue where an unfinished sentence appears (“or are there further influences…”).

Reply: The unfinished sentence was corrected.

This should be corrected. Additionally, the discussion of influencing factors could be expanded to briefly include biotic stressors such as herbivory or microbial interactions.

Reply: Since we did not include such factors explicitly, adding them would also add an element of uncertainty which we would prefer to avoid.

Reply: The reason for using DCM (same solvent as used in our previous preparative study [2]) was added.

Regarding the other parameters of our analysis, we clearly mentioned that we used a methodology previously established (and obviously successfully used) in our group [10, 11]. This also includes the internal standard papaverine.

About the scaling method we describe specifically in section 2.3.: “The Pareto-scaled model was selected for further analysis, as it provides the most balanced representation of variance between high and low abundance metabolites, as previously described by both Szabó and Schmidt and Schäfer et al. of our group [10,11].” A similar statement is also to be found in section 3.4. We think this explanation should be sufficient.

Reply: We think that all of this is already presented very clearly and further streamlining in our opinion would not make it any clearer.

Reply: It is a matter of taste whether separate Discussion section (which would then necessarily be somewhat repetitive) is useful or not. We chose a combined Results and Discussion and would leave it at that.

Reply: We find the figures quite clear and leave it to the Editor whether any further improvements are mandatory.

Reply: We have done our very best to unify all expressions of this kind throughout the manuscript (except the references section, where different formats may be due to the cited articles’ titles).

Some sentences are overly long and could be simplified for clarity. Reducing repetition of phrases such as “previous study” and “characteristic compounds” would improve the flow of the text.

Reply: This is a stylistic issue and a matter of taste rather than of scientific relevance.

The inclusion of a graphical abstract summarizing the seasonal variation, organ specificity, and key alkaloids would greatly enhance the accessibility and visual appeal of the manuscript.

Reply: We recently submitted a very well-structured Graphical Abstract with a publication in an MDPI journal. It was quite laborious to produce but it never appeared. We would rather not waste time and effort again. Furthermore, a graphical abstract is not mandatory in this journal and for the present work would inevitably be very complex so we would prefer to omit it.

Furthermore, a brief section outlining future research directions—such as multi-location sampling, controlled stress experiments, or isolation of newly detected metabolites—would add value.

Reply: Future research directions as far as we see them are already outlined in the conclusions section. Adding anything else in this direction might appear an exaggeration which we would rather avoid.

Reply: We thank the reviewer for this positive assessment.

Comments on the Quality of English Language

Reply: We have done our best to correct all typographical and formal errors. In addition, each manuscript will also be checked again at the proof stage so that none of these should be left when the manuscript is published.

Overall, the quality of English is acceptable for publication, but minor revisions are recommended to ensure clarity, consistency, and fluency

Reply: We have done our very best to improve our manuscript as best we can. In addition, we are sure that any remaining minor issues will be fixed by the excellent editorial office staff at proof stage.