Characterization of Tocochromanols in Wild Hypericum perforatum Populations in Latvia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Paweł Górnaś and colleagues characterized lipophilic biomolecules in the medicinal plants St. John's wort (Hypericum perforatum L.). I have following suggestions:

1, For the title, I suggested to employ “Characterization of Tocochromanols in wild Hypericum perforatum populations in Latvia”.

2, For the Abstract, some values should be provided. For instance, The content of other tocotrienols was as follows α-T3 > γ-T3 > β-T3. H. perofratum location, harvest year, aerial part, and their interaction statistically impact the tocochromanols content. Please provide some values.

3, For the key words, Vitamin E should be deleted.

4, For the introduction, impact of study to the production of the medicinal plants St. John's wort (Hypericum perforatum L.) should be introduced.

5, For the materials and methods, genetic backgrounds of the medicinal plants St. John's wort (Hypericum perforatum L.) should be examined in the study. Growth conditions of medicinal plants St. John's wort (Hypericum perforatum L.), like temperature, light cycle, and life intensity should be described.

6, For the results, data in Tables 1, 2, 3 and 4 should be presented by Figures.

7, For the discussion, the section of results and discussion should be divided into separate result sections and discussion section.

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For literature we used references manager software therefore changes are not highlighted.

 

Reviewer 1

In this manuscript, Paweł Górnaś and colleagues characterized lipophilic biomolecules in the medicinal plants St. John's wort (Hypericum perforatum L.). I have following suggestions:

Comment 1: For the title, I suggested to employ “Characterization of Tocochromanols in wild Hypericum perforatum populations in Latvia”.

Response 1: Thank you for the suggestion. Title was updated according to suggestion.

Comment 2: For the Abstract, some values should be provided. For instance, The content of other tocotrienols was as follows α-T3 > γ-T3 > β-T3. H. perofratum location, harvest year, aerial part, and their interaction statistically impact the tocochromanols content. Please provide some values.

Response 2: Thank you for your comment. We have updated the abstract with some values. However, for the content of tocotrienols, it is not feasible to provide specific numbers as they vary significantly depending on each factor. Additionally, due also to the abstract limitation (200 words) providing more details is quite challenging.

Comment 3: For the key words, Vitamin E should be deleted.

Response 3: Thank you for your comment. “Vitamin E” key word was replaced with “Antioxidants”.

Comment 4: For the introduction, impact of study to the production of the medicinal plants St. John's wort (Hypericum perforatum L.) should be introduced.

Response 4: Thank you for your comment. The introduction part was updated according suggestion. (Page 3)

Comment 5: For the materials and methods, genetic backgrounds of the medicinal plants St. John's wort (Hypericum perforatum L.) should be examined in the study. Growth conditions of medicinal plants St. John's wort (Hypericum perforatum L.), like temperature, light cycle, and life intensity should be described.

Response 5: Thank you for your comment. The current research focused on understanding the accumulation of tocotrienols in various parts of St. John's wort and their variability depending on location and year of collection. To comprehend the underlying genetic basis, additional studies are necessary. The present research pertains to wild St. John's wort (as indicated in the title), not cultivated varieties. Agronomic factors were not the focus of this study. Meteorological data, such as air temperature (°C), precipitation (mm), and solar radiation (W/m²), were obtained from the nearest possible measurement stations and are provided in the Supplementary Materials. (Page 4, middle part)

Comment 6: For the results, data in Tables 1, 2, 3 and 4 should be presented by Figures.

Response 6: Thank you for your comment. The changes have been made according to suggestion. (Page 9-11)

Comment 7: For the discussion, the section of results and discussion should be divided into separate result sections and discussion section.

Response 7: Thank you for your comment. As far as we know, it is optional to combine the results and discussion sections. We prefer this approach, as it allows for an immediate comparison of the current study with previous research. However, several changes were made.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Research focused on the search, extraction and quantification of secondary metabolites that may have applications in different areas of science has been of great interest in recent decades. The manuscript “Tocochromanols in wild populations of Hypericum perforatum in Latvia: impact of location, year of harvest and aerial parts” is an example of this interest in the use of different agricultural resources to obtain different molecules of commercial interest. The evaluation of some factors that may influence the concentration of tocochromanols in St. John´s wort, in which part of this plant they are accumulated in higher concentration and what type of tocochromanols prevail in each aerial part constitutes an important advance focused on their best use.

The results presented in the manuscript are interesting and clearly described. However, the following suggestions derived from the review are intended to improve some minor details of the form of this document. To this end, the line referred to in the manuscript will be indicated by the letter “L” from now on. In the PDF file the lines have been highlighted for visualization.

 - In all the manuscript, standardize the spelling of St. John's wort (“W” in word wort, lowercase or uppercase).

-L121: Were three aerial parts of the plant separated? In the abstract, the authors also refer to the stems (L26), the same in L149.

It is suggested that the separated parts be described consistently in the manuscript. Although most of the results and discussions focused only on three aerial parts of the plant if the stem was initially considered, the reason for not following up on it is justified in the Results and Discussion section.

-Between lines 135-138, please indicate whether the study material collection was carried out on similar dates each year. That is, it should be made clear whether seasonality could be another factor of variation in the contents of tocochromanols detected.

-L142: Please review the grade format to indicate the GPS location.

-L160: Please refer more generally to the corresponding aerial part sample since all four or three parts of the plant studied were treated similarly, not just the leaves.

-L201: Please indicate the concentration range used in the calibration curves for tocopherols and tocotrienols.

-The quantification of tocochromanols is described by both RP-HPLC-FLD and LC-MS. Although quantification limits are described in section 2.5, it is unclear which system the quantitative data reported in the manuscript Tables and Table S1 were obtained from. Although it is obvious which system is more sensitive, it is suggested that this aspect be clarified in section 2.4 or 2.5, where the authors consider it best.

-L276-L277: The sentence appears to be incomplete. Please review.

-L305-L306: Please review the authors' assertion in this sentence. According to Table 1, the α-T3 content in the flower buds was five times HIGHER, not lower.

-L323-L324: Please review the concentration order of the tocochromanols.

-It is suggested to indicate the Table containing the results being compared at the beginning of the paragraph rather than at the end, e.g., paragraphs L362-L370, L377-L382, L389-L395 and L404-L413.

 

L417-L418: Please rephrase.

Comments for author File: Comments.pdf

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For literature we used references manager software therefore changes are not highlighted.

 

Reviewer 2

Comment 1: Research focused on the search, extraction and quantification of secondary metabolites that may have applications in different areas of science has been of great interest in recent decades. The manuscript “Tocochromanols in wild populations of Hypericum perforatum in Latvia: impact of location, year of harvest and aerial parts” is an example of this interest in the use of different agricultural resources to obtain different molecules of commercial interest. The evaluation of some factors that may influence the concentration of tocochromanols in St. John´s wort, in which part of this plant they are accumulated in higher concentration and what type of tocochromanols prevail in each aerial part constitutes an important advance focused on their best use.

The results presented in the manuscript are interesting and clearly described. However, the following suggestions derived from the review are intended to improve some minor details of the form of this document. To this end, the line referred to in the manuscript will be indicated by the letter “L” from now on. In the PDF file the lines have been highlighted for visualization.

Response 1: Thank you for the positive overview.

Comment 2: - In all the manuscript, standardize the spelling of St. John's wort (“W” in word wort, lowercase or uppercase).

Response 2: Thank you for noticing. We standardized the spelling of St. John`s wort in lowercase.

Comment 3: -L121: Were three aerial parts of the plant separated? In the abstract, the authors also refer to the stems (L26), the same in L149.

Response 3: Thank you for noticing. Indeed, we focused on three parts, since stems were poor in tocochromanols, and therefore were excluded from deeper studies. However, the plant was separated into four parts. To describe consistently text was updated to four parts.

Comment 4: It is suggested that the separated parts be described consistently in the manuscript. Although most of the results and discussions focused only on three aerial parts of the plant if the stem was initially considered, the reason for not following up on it is justified in the Results and Discussion section.

Response 4: Thank you for your comment. At the beginning of the Results and Discussion section, an explanation associated with the topic of stems was provided: “For the first tocochromanols screening, all four aerial parts (stems, leaves, flower buds, and flowers) of H. perforatum were analyzed (Figure S2, Supplementary Materials). The stems of St. John's exhibited the lowest content of tocochromanols, with an average of 6 mg/100 g dw of tocopherols, mainly α-T, and only 2 mg/100 g dw of tocotrienols, mainly δ-T3 (data not shown). Due to the low content of both tocochromanols, they were excluded from further comparative analyses. Eight tocochromanols, at least in trace amount, in leaves, flower buds, and flowers of wild St. John's wort, were detected (Table S1, Supplementary Materials).” We believe that this description will be clear for potential readers. (Page 6, middle part)

Comment 5: -Between lines 135-138, please indicate whether the study material collection was carried out on similar dates each year. That is, it should be made clear whether seasonality could be another factor of variation in the contents of tocochromanols detected.

Response 5: Thank you for your comment. Detailed data were provided. (Page 4, top and middle part)

Comment 6: -L142: Please review the grade format to indicate the GPS location.

Response 6: Thank you for your comment. The information about GPS locations were updated. (Page 6, top part)

Comment 7: -L160: Please refer more generally to the corresponding aerial part sample since all four or three parts of the plant studied were treated similarly, not just the leaves.

Response 7: Thank you for your comment. Additional description was provided. (Page 4, middle part)

Comment 8: -L201: Please indicate the concentration range used in the calibration curves for tocopherols and tocotrienols.

Response 8: Thank you for your comment. The missing data were updated. (Page 5, middle part)

Comment 9: -The quantification of tocochromanols is described by both RP-HPLC-FLD and LC-MS. Although quantification limits are described in section 2.5, it is unclear which system the quantitative data reported in the manuscript Tables and Table S1 were obtained from. Although it is obvious which system is more sensitive, it is suggested that this aspect be clarified in section 2.4 or 2.5, where the authors consider it best.

Response 9: Thank you for your comment. The missing information was updated: “The obtained results of the RP-HPLC-FLD analyses were quantitatively presented in the form of tables and figures.” and “Mass spectrometry (MS) was employed only to confirm the identification of tocopherols and tocotrienols in stems, leaves, flowers, and flower buds of St. John's wort obtained by RP-HPLC-FLD analysis (section 2.4.), according to the previously developed method [21].” (Page 5, middle part)

Comment 10: -L276-L277: The sentence appears to be incomplete. Please review.

Response 10: Thank you for your comment. The sentence was re-written. (Page 7, middle part)

Comment 11: -L305-L306: Please review the authors' assertion in this sentence. According to Table 1, the α-T3 content in the flower buds was five times HIGHER, not lower.

Response 11: Thank you for noticing our mistake. The sentence was re-written. (Page 8, bottom part)

Comment 12: -L323-L324: Please review the concentration order of the tocochromanols.

Response 12: Thank you for your comment. The order was corrected. (Page 9, middle part)

Comment 13: -It is suggested to indicate the Table containing the results being compared at the beginning of the paragraph rather than at the end, e.g., paragraphs L362-L370, L377-L382, L389-L395 and L404-L413.

Response 13: Thank you for your comment. The order was improved. Tables 1-4 were exchanged to figures.

Comment 14: L417-L418: Please rephrase.

Response 14: Thank you for noticing our mistake. The sentence was re-written. (Page 12, bottom part)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This work analyzes the accumulation and distribution of tocochromanols in the above-ground parts of St. John's wort. The analysis was conducted on plants collected from three geographical points over a period of 3 years. The data obtained may be useful for the medical and pharmacological industry and will help to use wild and cultivated plant materials more rationally. There are a few questions and comments:

1.      St. John's wort is a perennial plant, it was collected over a period of three years and different numerical values ​​of the content of individual metabolites were identified, therefore it is necessary to clarify whether the age of the harvested plant was taken into account? Were the climatic conditions different during the period of the work? It would be useful to provide data from official sources. What were the differences in the places where the plants were collected (soil, illumination, moisture, surrounding plant communities)?

2    Line 114. "Due to the observed remarkable variability of the hydrophilic phytochemicals in cultivated H. perforatum [6,19], it would be justified to perform the study focused on agronomic aspects affecting the profile of lipophilic secondary metabolites such as tocotrienols in St. John's Wort. For this purpose, in the current study, to better understand the accumulation of lipophilic secondary metabolites (tocopherols and tocotrienols) in wild H. perforatum, the plant was harvested for three years (2022–2024), at three different locations" - the authors' line of reasoning is not clear, in the case of agrotechnological cultivation of St. John's wort, almost all parameters affecting plant growth and accumulation of metabolites are controllable, how can the study of wild plant populations help to understand which agrotechnological approaches affect the accumulation of target components?

3.      Line 197. The ambient temperature does not play a role in this case, since the column is thermostatted, it is not necessary to indicate this data.

4.      According to the data in Tables 2 and 3, namely the value of the standard deviation, it follows that the indicators are extremely unstable among the analyzed populations, a strong impact of environmental factors within each population and by year is likely, how fair are the conclusions you made based on the data obtained?

 

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For literature we used references manager software therefore changes are not highlighted.

 

Reviewer 3

Comment 1: This work analyzes the accumulation and distribution of tocochromanols in the above-ground parts of St. John's wort. The analysis was conducted on plants collected from three geographical points over a period of 3 years. The data obtained may be useful for the medical and pharmacological industry and will help to use wild and cultivated plant materials more rationally. There are a few questions and comments:

Response 1: Thank you for the positive overview.

Comment 2: St. John's wort is a perennial plant, it was collected over a period of three years and different numerical values ​​of the content of individual metabolites were identified, therefore it is necessary to clarify whether the age of the harvested plant was taken into account? Were the climatic conditions different during the period of the work? It would be useful to provide data from official sources. What were the differences in the places where the plants were collected (soil, illumination, moisture, surrounding plant communities)?

Response 2: Thank you for your comment. We completely agree, which is why we have added to the manuscript data from meteo stations in the form of Supplementary Material and a discussion of the above-mentioned factors and their possible influence on the concentration of tocochromanols in different parts of H. perforatum. The following discussion was added: “The meteo conditions; average air temperature (°C), precipitation (rain) (mm), and the average solar radiation (W/m2), during the season of 2022-2024 for three main locations of wild H. perforatum collection are provided in Supplementary Materials.” and “Additionally, factors such as plant age, soil composition, and surrounding plant communities, in the current study, were not investigated. The climatic conditions, including precipitation, temperature, and solar radiation, relatively varied across each location of St. John's wort collection. All these factors may affect the observed variability in tocopherol and tocotrienol content in different parts of H. perforatum, over the three years of research. To better understand the accumulation process of tocochromanols in St. John's wort and the impact of environmental factors, further studies under controlled conditions are required to optimize the biosynthesis of those bioactive phytochemicals.” (Page 4, top and middle part; Page 12, bottom part)

Comment 3: Line 114. "Due to the observed remarkable variability of the hydrophilic phytochemicals in cultivated H. perforatum [6,19], it would be justified to perform the study focused on agronomic aspects affecting the profile of lipophilic secondary metabolites such as tocotrienols in St. John's Wort. For this purpose, in the current study, to better understand the accumulation of lipophilic secondary metabolites (tocopherols and tocotrienols) in wild H. perforatum, the plant was harvested for three years (2022–2024), at three different locations" - the authors' line of reasoning is not clear, in the case of agrotechnological cultivation of St. John's wort, almost all parameters affecting plant growth and accumulation of metabolites are controllable, how can the study of wild plant populations help to understand which agrotechnological approaches affect the accumulation of target components?

Response 3: Thank you for your comment. The text was improved to provide clarity of the present research. .” (Page 3, middle part)

Comment 4: Line 197. The ambient temperature does not play a role in this case, since the column is thermostatted, it is not necessary to indicate this data.

Response 4: Thank you for your comment. The information about ambient temperature was removed.

Comment 5: According to the data in Tables 2 and 3, namely the value of the standard deviation, it follows that the indicators are extremely unstable among the analyzed populations, a strong impact of environmental factors within each population and by year is likely, how fair are the conclusions you made based on the data obtained?

Response 5: Thank you for your comment. It should be taken into account that the obtained standard deviation in Tables 2 and 3 is based on 54 samples (n = 54) and they include leaves, flower buds, and flowers, e.g. for Year ANOVA (3 plant parts × 3 locations × 3 sub-locations × 2 independent experiments = 54). Statistical analysis indicates significant differences, although looking at the concentration values we may have doubts. We followed the results of the statistical analysis in the final interpretation.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

I suggest the following minor corrections that could improve the quality of the manuscript:

Lines 110-112 – “In cultivated H. perforatum has been observed that biomass as well as the yield of the secondary metabolite contents is by cultivation factors such as environment, agrotechnological techniques and the genotype.” – The sentence needs to be edited. For example: In cultivated H. perforatum, it has been observed that biomass, as well as secondary metabolite yield, are determined by/depend on cultivation factors such as environment, agrotechnical techniques, and genotype.

Line 160 – “0.1 g powdered leave sample” – Are all samples (stems, flower buds and buds) processed this way? Please clarify!

Table S1 – How is the Ts/T3s ratio calculated? For example, the table shows that the average values ​​for total Ts and T3s in the leaves are 80.4 and 13.7, respectively. Their ratio is 5.9, but the table indicates a value of 7.1. Please check and correct; “trace amount” (tr) is mentioned in the legend to this table, but “not detected” (nd) is used in the table. Although both are correct, please unify for more clarity!

Line 277 – “lower content of total tocotrienols was found in leaves and flowers” – “In leaves and flowers” should be added, because otherwise the sentence is incomplete and unclear.

Line 306 – “five times lower content of α-T3 in flower buds” – It should be “in flowers” not “in flower buds” or “five times higher content of α-T3 in flower buds” – Please check and correct!

Table 2 - The high standard deviation values ​​against the background of small differences in the means raise the question of how statistically significant differences in the content of metabolites, especially α-T3, were obtained. Please check and correct if needed!

Lines 369, 370 – “Content δ-T3 was statistically insignificant between the years” – It should be “Content of δ-T3 in the leaves was statistically insignificant between the years”, because the table shows that δ-T3 content in flowers, as well as in flower buds is statistically significant between the years.

Lines 392, 393 – “Opposite to Rēzekne was the concentration of those tocopherols, in the same parts, was the lowest.” – this sentence is unclear and needs to be edited. For example: In Rezekne, the concentration of these tocopherols, in the same parts, was the lowest.

I recommend adding more discussion to the interpretation of the results. Despite the scarce data available in the literature on the subject, an analogy can be sought with other secondary metabolites (in other plant species), the accumulation of which is influenced by factors such as habitat, plant age (in perennial species) and plant organs.

Thank You!

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For literature we used references manager software therefore changes are not highlighted.

 

Reviewer 4

Dear Authors, I suggest the following minor corrections that could improve the quality of the manuscript:

Comment 1: Lines 110-112 – “In cultivated H. perforatum has been observed that biomass as well as the yield of the secondary metabolite contents is by cultivation factors such as environment, agrotechnological techniques and the genotype.” – The sentence needs to be edited. For example: In cultivated H. perforatum, it has been observed that biomass, as well as secondary metabolite yield, are determined by/depend on cultivation factors such as environment, agrotechnical techniques, and genotype.

Response 1: Thank you for your comment. The sentence`s syntax was improved. (Page 3, top part)

Comment 2: Line 172 – “0.1 g powdered leave sample” – Are all samples (stems, flower buds and buds) processed this way? Please clarify!

Response 2: Thank you for your comment. The missing parts in the description were added. (Page 4, middle part)

Comment 3: Table S1 – How is the Ts/T3s ratio calculated? For example, the table shows that the average values for total Ts and T3s in the leaves are 80.4 and 13.7, respectively. Their ratio is 5.9, but the table indicates a value of 7.1. Please check and correct; “trace amount” (tr) is mentioned in the legend to this table, but “not detected” (nd) is used in the table. Although both are correct, please unify for more clarity!

Response 3: Thank you for your comment. The average ratio Ts/T3s represents an average of the fifty-four (n = 54) individual samples ratio, not a simple average of tocopherols divided average of tocotrienols content in specific plant parts, obtained in the left columns of the table. Below the table, we add an explanation to be clear. Calculation of the below listed Ts/T3s ratio for 54 individual samples of leaves gives 7.124782625. (ratio Ts/T3s of 54 individual samples of leaves: 6.02676189+6.191730759+6.086559652+6.200484838+7.193499891+6.998426306+18.02857114+19.30507113+5.750068736+5.818385903+6.10828723+6.383509292+6.838362402+6.147140654+5.53468876+5.284509184+9.576045013+9.744155304+7.608454543+7.51370751+5.948724217+5.694090591+3.496597919+3.387658675+6.011704274+5.73290405+6.476051299+6.412965637+15.36457397+16.21261611+4.224603845+4.648900006+8.470271215+9.113624005+10.51599795+9.816285477+4.919446801+5.069799082+4.024663179+3.726482821+2.083914257+1.8319462+2.297072613+2.342973964+12.0454823+12.67702915+3.6139654+3.660772763+2.108392387+2.014027494+4.908230175+4.857415226+15.80930584+16.88135271=7.124782625)

Comment 4: Line 277 – “lower content of total tocotrienols was found in leaves and flowers” – “In leaves and flowers” should be added, because otherwise the sentence is incomplete and unclear.

Response 4: Thank you for noticing. The missing words were added. (Page 8, middle part)

Comment 5: Line 306 – “five times lower content of α-T3 in flower buds” – It should be “in flowers” not “in flower buds” or “five times higher content of α-T3 in flower buds” – Please check and correct!

Response 5: Thank you for noticing. The correction was made. (Page 8, bottom part)

Comment 6: Table 2 - The high standard deviation values against the background of small differences in the means raise the question of how statistically significant differences in the content of metabolites, especially α-T3, were obtained. Please check and correct if needed!

Response 6: Thank you for your comment. It is correct. It should be taken into account that the obtained standard deviation in Tables 2 and 3 is based on 54 samples (n = 54) and they include leaves, flower buds, and flowers, e.g. for Year ANOVA (3 plant parts × 3 locations × 3 sub-locations × 2 independent experiments = 54). Statistical analysis indicates significant differences, although looking at the concentration values we may have doubts. We followed the results of the statistical analysis in the final interpretation.

Comment 7: Lines 369, 370 – “Content δ-T3 was statistically insignificant between the years” – It should be “Content of δ-T3 in the leaves was statistically insignificant between the years”, because the table shows that δ-T3 content in flowers, as well as in flower buds is statistically significant between the years.

Response 7: Thank you for your comment. The correction was made. (Page 10, bottom part)

Comment 8: Lines 392, 393 – “Opposite to Rēzekne was the concentration of those tocopherols, in the same parts, was the lowest.” – this sentence is unclear and needs to be edited. For example: In Rezekne, the concentration of these tocopherols, in the same parts, was the lowest.

Response 8: Thank you for your comment. The correction was made. (Page 11, bottom part)

Comment 9: I recommend adding more discussion to the interpretation of the results. Despite the scarce data available in the literature on the subject, an analogy can be sought with other secondary metabolites (in other plant species), the accumulation of which is influenced by factors such as habitat, plant age (in perennial species) and plant organs. Thank You!

Response 9: Thank you for your comment. Appropriate discusion was added. (Page 12, bottom part; Page 13, top part)

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

 

I appreciate your trust in listening to me about this study.

The study presented is interesting. It seeks to obtain more information about secondary compounds produced by the species H. perforatum, still in the wild, especially tocotrienols.

The introduction is very well written. It presents information about the current status of tocotrienols and the plant. It presented an adequate justification for advancing the studies. Therefore, I consider the introduction complete and the references presented faithfully support the study (hypotheses and objective).

The Material and Methods Item contains detailed information about where and how the study was conducted. Details of the methods used were presented.

The results are very well presented, including an important supplementary file.

The discussion sought to explain how the environment (year and location) affected the concentrations of metabolites in the plant. The discussion is not very in-depth, but it is most likely justified because it is a wild species, with little knowledge of how production factors impact the development and metabolism of the species. Therefore, although the discussion is timid, I consider it adequate for advancing knowledge.

As for the conclusions of the study, these are duly supported by the results. However, I believe it would be interesting to add a short text to highlight the relevance of interactions observed in the concentrations of metabolites, as these interactions should be studied further.

A few considerations were noted in the manuscript (attached)

Comments for author File: Comments.pdf

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font. For literature we used references manager software therefore changes are not highlighted.

 

Reviewer 5

Comment 1: I appreciate your trust in listening to me about this study. The study presented is interesting. It seeks to obtain more information about secondary compounds produced by the species H. perforatum, still in the wild, especially tocotrienols. The introduction is very well written. It presents information about the current status of tocotrienols and the plant. It presented an adequate justification for advancing the studies. Therefore, I consider the introduction complete and the references presented faithfully support the study (hypotheses and objective). The Material and Methods Item contains detailed information about where and how the study was conducted. Details of the methods used were presented. The results are very well presented, including an important supplementary file. The discussion sought to explain how the environment (year and location) affected the concentrations of metabolites in the plant. The discussion is not very in-depth, but it is most likely justified because it is a wild species, with little knowledge of how production factors impact the development and metabolism of the species. Therefore, although the discussion is timid, I consider it adequate for advancing knowledge.

Response 1: Thank you for the positive overview.

Comment 2: As for the conclusions of the study, these are duly supported by the results. However, I believe it would be interesting to add a short text to highlight the relevance of interactions observed in the concentrations of metabolites, as these interactions should be studied further.

Response 2: Thank you for your comment. The conclusion part was improved. (Page 14, top part)

Comment 3: A few considerations were noted in the manuscript (attached)

Response 3: Thank you. All comments and suggestions were taken into consideration and improved in the manuscript.

Comment 4: "... worldwide, which is rich in diverse secondary metabolites."

Response 4: Thank you for your comment. The correction was made. (Page 1)

Comment 5: Error???

Response 5: Thank you for your comment. The correction was made. (Page 1)

Comment 6: Please make sure to keep this information in mind. You said "near-global distribution", but you left out regions like tropical and subtropical, and furthermore, you mentioned that this genus is absent from extremely cold and arid environments. So I ask, does this genus really have a near-global distribution?

Response 6: Thank you for your comment. The correction was made. (Page 1, bottom part)

Comment 7: Are there references to this information?

Response 7: Thank you for your comment. The reference is the same as in the next sentence. (Page 2, top part)

Comment 8: Before that, it is not mandatory, but what do the authors think about including a short text about the characteristics of the plant (annual or perennial, size, start and duration of flowering, if possible in what situation they flower?

Response 8: Thank you for your comment. According to other recommendations, a bit different paragraph was added to the introduction part. (Page 3, middle part)

Comment 9: Please indicate for each year the times (at least the month, if possible the dates) of plant collection. Was there variation in these collection times? Could this have had an effect on the metabolite concentrations (Table 2 - supplementary)???

Response 9: Thank you for your comment. Detailed descriptions of plant material and potential impact were added to the material part and before the conclusion. (Page 4, top part; Page 12, bottom part, Page 13, top part)

Comment 10: I believe it would be interesting to add a short text to highlight the relevance of interactions observed in the concentrations of metabolites, as these interactions should be studied further

Response 10: Thank you for your comment. The conclusion part was improved. (Page 14, top part)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been revised in a satisfactory way.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made the necessary amendments, I believe that the materials can be published