Bioactive Compounds Concentrations and Stability in Leaves of Ilex paraguariensis Genotypes
, Carlos André Stuepp 2, Cristiane Vieira Helm 3, Marcus Vinicius de Liz 4, Amanda Coelho de Miranda 4, Rafaela Imoski 4, Osmir José Lavoranti 3 and Ivar Wendling 3,*Round 1
Reviewer 1 Report (New Reviewer)
Comments and Suggestions for AuthorsThe manuscript «Bioactive concentrations and stability in Ilex paraguariensis genotypes» is devoted to evaluation of caffeine, theobromine, total phenolic compounds and protein on leaves of yerba mate genotypes for four consecutive years.
Some general and particular comments are given below to consider to improve the quality of the manuscript for readers.
1. The title of the manuscript should include «leaves».
2. I did not understand the relevance of this work. Based on the Introduction, it is unclear why this work was performed. Authors said that «Despite its variety of compounds, most research has focused on the caffeine, theobromine and theophylline contents in commercial products of yerba mate [3,4,11]». And in the end, the authors chose the same compounds for their study. Why? In addition, the authors did not mention a study on yerba mate genotypes («Vegetative propagation, chemical composition and antioxidant activity of yerba mate genotypes», DOI: https://doi.org/10.1017/S1479262121000150), which is very similar to their work.
3. Lines 63-64. «our hypothesis is that this variation is not influenced by the harvest year». It is unclear what the authors’ hypothesis is based on.
4. The references were listed out of order. The last reference in the Introduction was [16], and in the Materials and Methods there were already [42] and [25] references one after another. And so on in the text.
5. Line 92. HPLC is not High Efficiency Liquid Chromatography.
6. The method for selecting genotypes is unclear. When selecting research objects, the authors referred to reference [5], where I did not find descriptions of genotypes. The division of genotypes by year (2015-2018) is also unclear. How should I understand the year of the sample?
7. «We studied 54 genotypes for caffeine and 55 genotypes for theobromine». Change this sentence. Also, why was caffeine content determined in only 54 samples?
8. The authors determined the content of caffeine and theobromine using HPLC. Since the authors did not refer here to a previously developed HPLC method, it is necessary to provide a chromatogram for at least one genotype so that the reader can evaluate the separation of compounds.
9. Insert missing references in Materials and Methods: «Singleton and Rossi, 1965», «AOAC 2016», «AMMI».
10. Figures 1, 3 and 5 are low resolution and therefore uninformative. Therefore, I cannot evaluate the Results and Discussions sections on PCA.
11. There are no footnotes for tables 2, 4, 6, 8.
12. In the Discussion, paragraphs 303-307 and 330-335 are more like an Introduction.
13. The references are provided according to the rules of another journal, not Forests.
Author Response
We sincerely appreciate the suggestions made by the reviewers We are sure that they were very important in improving its quality. All suggestions and their respective comments and actions taken are described.
Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsL 26: it should be shown istead of showed
L. 62-66: the hypothesis: it's not clear what's the hypothesis is. The authors mention "our hypothesis is that this variation is not influenced
by the harvest year. " But what's about about the hypothesis - what else do they suggest instead of harvest year?
Material and Methods: as the stand and especially the soil conditions are important for the composition and concentration of bioactive components they should be mentioned in the text, e.g. which soil type, how large is the area, percipitation within each of the 4 years. The authors describe this aspect only in very general terms, but since they conclude that the variations are not influenced by the harvest year, the authors need to go into more detail about the conditions during the specific years
Author Response
We sincerely appreciate the suggestions made by the reviewers We are sure that they were very important in improving its quality. All suggestions and their respective comments and actions taken are described.
Author Response File: Author Response.pdf
Reviewer 3 Report (Previous Reviewer 5)
Comments and Suggestions for Authors1. Some information regarding the plant and its genetics (whether natural self or cross pollination) leading to within population genetic diversity etc. should be included in the introduction.
22. Most of the biochemical properties depend on climate. Need to explain why it is not so in the current study over the period of 4 years.
3 3. What exactly are the progenies: Are they breeding lines from a plant breeding experiment?
4 4. 175-176: It was observed that, even with only four evaluation years, it was possible to detect differences among them, indicating rather varied conditions for genotype evaluation. Need to be more explanative
5 5. Temporal variations of the same genotypes in biochemical properties can be discussed briefly in the discussion. Also included in the conclusion as well.
Comments on the Quality of English LanguageMinor English language editing is recommended.
Author Response
We sincerely appreciate the suggestions made by the reviewers We are sure that they were very important in improving its quality. All suggestions and their respective comments and actions taken are described.
Author Response File: Author Response.pdf
Reviewer 4 Report (New Reviewer)
Comments and Suggestions for AuthorsThe manuscript “Bioactive compounds concentrations and stability in Ilex para-guariensis genotypes” is interesting but not well prepared.
I have some major queries that should be taken into account in the revised manuscript.
Abstract should be improved in terms of Results HPLC analysis of Caffeine and theobromine, also include stability results.
Author did not used any guidelines for method validation ICH Q2 (R2) for method validation must be include the reference. Standards compounds and their preparation not available in the manuscript.
Method and assessment of stability of caffeine and theobromine and phenolic compounds not available only estimation and their results?
Author should provide HPLC chromatogram of caffeine and theobromine and how they prepared 55 yerba mate leaf samples?
How they check stability of these compounds in the samples?
References are not aligned and not properly sequenced throughout the manuscript.
% RSD of precision little high?
Author should include calibration curve for both the caffeine and theobromine.
Author should compare their results with previously published method? Also describe how this method is better than the previously reported methods?
Conclusion should be improved.
Also highlight the importance of this study?
Why author choose only caffeine and theobromine? Why not include theophylline as well?
Theophylline is one of the important constituents and present in the almost same concentration.
Author Response
We sincerely appreciate the suggestions made by the reviewers We are sure that they were very important in improving its quality. All suggestions and their respective comments and actions taken are described.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report (New Reviewer)
Comments and Suggestions for AuthorsThe authors responded to most comments. However, I still have some suggestions:
- Authors should move chromatograms to the Results section, as this does not apply to Materials and Methods.
- Figures 1, 3 and 5 are still low resolution and therefore uninformative and make the results difficult to interpret.
Author Response
We appreciate your suggestions made on the manuscript; they were very important in improving its quality. All suggestions and their respective comments and actions taken are described on the attached file
Author Response File: Author Response.pdf
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsM+M is now well described, but the problem is the research design. The authors took seedlings from one mother tree and demonstrated a high genetic variability. The conclusion that the variability of specific traits has a genetical background cann't be aproved by such an approach, because the mother trees were not investigated (an inheritance analysis is missing). There are no informations about the genetic makeup for the specific traits concerning the mother trees. I would suggest to include this point into the discussion part.
Author Response
We appreciate your suggestions made on the manuscript; they were very important in improving its quality. All suggestions and their respective comments and actions taken are described on the attached file
Author Response File: Author Response.pdf
Reviewer 4 Report (New Reviewer)
Comments and Suggestions for AuthorsAuthor significantly revised manuscript most of the point but still not answer satisfactorily for some points.
Also they answered in cover later but not included in the manuscript.
Query 2 responded in the author response but not available in the manuscript.
Query 8. calibration curve not available even in the author response and revised manuscript.
Query 9. not satisfactory response. they mention reference 19 (Duarte et al., 2020) but available in plant material but not discussed in discussion also my query was comparison of this study results with the previously reported method but no response.
even not much changed the abstract and conclusion.
These query must be responded
Author Response
We appreciate your suggestions made on the manuscript; they were very important in improving its quality. All suggestions and their respective comments and actions taken are described on the attached file
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsYerba mate is an important native forest species in Brazil. The MS revealed the variations of caffeine, theobromine, total phenolic compounds and proteins concentrations in yerba mate. However, the MS need present good background literature research. The section Materials and methods should present clear to enable other scientists to follow the results and continue recognition of other genetic doubts, especially the materials were not clearly described and the data analysis was too simple. It also requires a very through linguistic corrections to make it more understandable, having the entire manuscript checked by a native English speaker.
Reviewer 2 Report
Comments and Suggestions for AuthorsI recommend the rejection owing to too many flaws in the ms concerning the novelty and significance (e.g., experiment design, metabolite quantification, image quality, and the related results and discussion)
Reviewer 3 Report
Comments and Suggestions for AuthorsL72: the authors investigated different genotypes from a provenance and progeny trial...when I interpret this correct, it means that they 1.used no clonal plant material and 2. they used seedlings/ young trees from 2 different origins (provenances and progenies from a breeding trial (that's not clear)). That means the plant material per se is genetically inhomogenous and therefor to me the approach seems to be not suitable for answering the question the authors adress. The term "genotype" is not used correctly, because the authors use genotype in the sense of "Provenance" but not to describe the individual plant. In this study or in studies, which should be the basis for breeding programmes, a clear definition is essential.
As the authors correctly stated in the discussion part (L241-244) that "Caffeine (and cleary other components as well) content in teas varies greatly depending on the species, edaphoclimatic conditions of its cultivation and phenological stage of leaves [24,25]), which can influence the product quality, making it bitter and altering flavor of the final product [3,26]" , it's essentially important to pay attention on the study design. In the present study it would have been better to focus on specific plants (genotypes with a specific pheotype), to harvest always the same plants (year 1...4), leaves of the same age, position within the plant and same season. These aspects are not considered in the design resp. not mentioned in the M+M part.
The legend to the figures is not informative enough, there is no information about the abbreviations (EC35 etc., from the discussion part I learnt that these are the different genotypes) and it's not clear how the different years fit into the graphs.
Discussion: to me the link to the titel of the paper is missing, because the authors do not refer to the changes/ stability (over years) of the investigated components only in rudimentary form, but not in sufficient detail.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe caffeine, theobromine, total phenolic compounds and proteins of selected yerba mate genotypes of four years were determined. However, the novelty and quality of presentation seems not qualified for this journal.
1. PCA of the first component for these four bioactive compounds concentration (Figure 1, 3, 5, 7) and PCA of genotype-environment ratio according to PC1 and PC2 (Figure 2, 4, 6, 8) were not clearly showed in these figures. Normally, the concentration for four years of those bioactive compounds should be together by clearly showing the data of EC35-1 (Year 1), EC35-2 (Year 2), EC35-3 (Year 3) and EC35-4 (Year 4). However, these figures can not result in the conclusion that “The genetic materials are individually stable for concentration of those bioactive compounds”.
2. Though the analyses of moisture and total proteins were mentioned in Line 90, the methods for determination of total proteins were not adequately described. Moreover, no results of moisture data were showed anywhere else.
3. There’s two “1.” in the References section by mistake.
Reviewer 5 Report
Comments and Suggestions for Authors1. Methods of data analysis and the corresponding results should be presented more clearly. For example, the parameters used for each PCA should be indicated and the parameters identified to be contributing to variation should be identified. The principal components are better named as PC1 and PC2 etc. rather than using the terms, vertical axis and horizontal axis etc. Results of the AMMI analysis (genotypic stability) should be improved for better clarity.
2. Conclusions can be expanded a little more.
3. Minor language errors should be corrected.
