Impact of Soil on Biomass Yield and Accumulation of Lipophilic Secondary Metabolites in Four Hypericum Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

You describe a straightforward protocol for the extraction and analysis of tocopherols and tocotrienols from four Hypericum species applying liquid-liquid extraction and HPLC equipped with a fluorescence detector. The manuscript is very well written and you do provide almost all the experimental details (see below for the few missing ones). While the introduction is on the long side, it does manage to explain the motivation for your study very well and will also allow non-expert readers to understand all the arguments made later in the article.
Your main emphasis is on the statistical evaluation of the concentrations you measured and this is the part of your manuscript that may profit from shortening. The Tables 1 to 7 are labelled as “ANOVA” instead of showing primary results (which they still do, but the label may confuse some readers). I recommend to use the tables in the main manuscript primarily to show your concentrations, and move the less important ANOVA results to the SI, where you already show most of the values resulting from the multiple ANOVAs you performed. I personally consider the chromatograms shown in Figure S3 to be more deserving of a spot in the main manuscript than presenting there all the different classification possibilities enumerated in Tables 1-7. The chromatograms show that you did optimize the chromatographic method to achieve baseline separation of all eight analytes and that your detection setup is indeed highly selective for tocochromanols as indicated by how little of the total signal area is not at the position of one of the eight standards. 
The analytical methodology you apply seems solid and you did ascertain the identity of all your analytes with authentic standard materials. You do not, however, present any method validation data.
- How high is the recovery for the eight tocochromanols you studied?
- What are the detection and quantification limits of your analytical method?
- What are the linear ranges for the eight tocochromanols?
- What are the reproducibility (if data is available) and repeatability of your analyses?
The answers to the second, third and fourth point are already available from your calibration curves (reproducibility only if you did repeat them on a different day with different operators and optimally with a different device), for the first one (which is also the most important one) you may need to spike some plant parts with standards before submitting them to the lyophilization, extraction and chromatographic separation steps.

Comments to specific lines in your manuscript:

51: The Chinese may frown on TCM being subsumed under "Western herbalism".

149, 96.2 % ethanol: Are these %(v/v)? The azeotrope available by distillation has 95.57 %(m/m) ≈ 96.5 %(v/v) ethanol.

181: How did you separate the plant parts?

184: How did you freeze the plant material before putting it into the lyophilisator?

189, dry mass: Please add a table to the SI with the dry mass content [ (g dw) / (g FW) ] of your plant samples. This will allow the estimation of the contents in fresh plant material from your dry weight-based values.

214: At which pressure did you evaporate the extraction liquids?

226: What was the guard column filled with? There are a large number of stationary phases sold under the Phenomenex brand.

232: What were the linear ranges and the detection/quantification limits of your method? Please provide this data (in a table in the SI).
Did you check the extraction method by spiking plant samples with tocopherols and tocotrienols? Were there any matrix effects on the obtained calibration factors? The latter may be checked by preparing calibration lines not in neat solvent but in a sample solution instead.

259: How did you determine the fresh weight of your plants? - Time from harvesting, method of washing and dry-blotting, ...

277, Figure 1: Please provide a table with the 36 FW values in the SI.

293, 3.3: Chapter 3.2 is presented after two chapters labelled 3.3 (line 429). Please check your numbering and/or arrangement of chapters.

297, (stems, leaves, flower buds, and flowers): Please provide an estimation of the mass fraction that each of these organs contribute to the total (aerial) plant (dry) mass.

487: In this line there is a sudden change from reporting the results to their interpretation. A chapter change may be in order.

505-507: Please provide a reference for that statement.

517-519: This sentence reads like a conclusion.

SI, Table S2: The planting dates would be of equal importance.

SI, Figure S2: Please add a legend for the four plant parts. I know it should be obvious but please do also consider readers struggling with English.

SI, Figure S3: What is the unit ("uV") of the ordinate on your chromatograms? Does the “wild” designation mean the sample whose chromatograms are shown here was not taken from the beds shown in figure S1?

SI: I would recommend to add a figure showing the structures of the eight tocochromanols you studied.

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 manuscript and supplementary materials have been improved accordingly. Provided changes are marked in red font. For the literature we used references manager software, therefore the changes are not highlighted.

Reviewer 1

Comment 1: You describe a straightforward protocol for the extraction and analysis of tocopherols and tocotrienols from four Hypericum species applying liquid-liquid extraction and HPLC equipped with a fluorescence detector. The manuscript is very well written and you do provide almost all the experimental details (see below for the few missing ones). While the introduction is on the long side, it does manage to explain the motivation for your study very well and will also allow non-expert readers to understand all the arguments made later in the article.

Response 1: Thank you for the positive overview.

 

Comment 2: Your main emphasis is on the statistical evaluation of the concentrations you measured and this is the part of your manuscript that may profit from shortening. The Tables 1 to 7 are labelled as “ANOVA” instead of showing primary results (which they still do, but the label may confuse some readers). I recommend to use the tables in the main manuscript primarily to show your concentrations, and move the less important ANOVA results to the SI, where you already show most of the values resulting from the multiple ANOVAs you performed.

Response 2: Thank you for your comment. We conducted a series of statistical analyses to demonstrate the presence or absence of statistical significance depending on the variables. Among all the tables, we selected only the most important ones for the manuscript. Including additional tables without statistical analysis could be confusing and potentially misleading for the reader. For these reasons, we recommend leaving the tables as they are.

 

Comment 3: I personally consider the chromatograms shown in Figure S3 to be more deserving of a spot in the main manuscript than presenting there all the different classification possibilities enumerated in Tables 1-7. The chromatograms show that you did optimize the chromatographic method to achieve baseline separation of all eight analytes and that your detection setup is indeed highly selective for tocochromanols as indicated by how little of the total signal area is not at the position of one of the eight standards. 
The analytical methodology you apply seems solid and you did ascertain the identity of all your analytes with authentic standard materials. You do not, however, present any method validation data.

- How high is the recovery for the eight tocochromanols you studied?
- What are the detection and quantification limits of your analytical method?
- What are the linear ranges for the eight tocochromanols?
- What are the reproducibility (if data is available) and repeatability of your analyses?
The answers to the second, third and fourth point are already available from your calibration curves (reproducibility only if you did repeat them on a different day with different operators and optimally with a different device), for the first one (which is also the most important one) you may need to spike some plant parts with standards before submitting them to the lyophilization, extraction and chromatographic separation steps.

Response 3: Thank you for your comment and positive overview of used chromatographic method. We would like to kindly emphasize that the current manuscript has been submitted to the journal Agriculture, which underscores the primary importance of agronomic techniques over chromatographic methods. Therefore, we do not recommend transferring the chromatogram to the main manuscript. Regarding the information related to method validation, we have included the appropriate details. All samples were prepared during the one day to avoid limiting external factors (human, instrument, day) potentially affecting the final results. (Page 5, middle part; Page 6, middle part)

Comment 4: Comments to specific lines in your manuscript: 51: The Chinese may frown on TCM being subsumed under "Western herbalism".

Response 4: Thank you for your comment. We appreciate your perspective on the classification of Traditional Chinese Medicine and have replaced the term - Western medicine for ‘global herbal traditions’, as well stating the use of H.perforatum in traditional Chinese, Western, Ayurvedic, ancient Greek and Islamic cultures. (Page 2, top part)

Comment 5: 149, 96.2 % ethanol: Are these %(v/v)? The azeotrope available by distillation has 95.57 %(m/m) ≈ 96.5 %(v/v) ethanol.

Response 5: Thank you for your comment. We used 96.2% (v/v) ethanol. The missing data was supplemented. (Page 4, middle part)

Comment 6: 181: How did you separate the plant parts?

Response 6: Thank you for your comment. Plant material paragraph was supplemented with more detailed description. (Page 5, top part)

Comment 7: 184: How did you freeze the plant material before putting it into the lyophilisator?

Response 7: Thank you for your comment. Plant material paragraph was supplemented with more detailed description to avoid any inconveniences. (Page 5, top part)

Comment 8: 189, dry mass: Please add a table to the SI with the dry mass content [ (g dw) / (g FW) ] of your plant samples. This will allow the estimation of the contents in fresh plant material from your dry weight-based values.

Response 8: Thank you for your comment. We appreciate your suggestion and would like to do this but only the whole sample was weighted. We did not weigh fresh or dry plant parts. The following information was added: „After freeze-drying, the moisture content of the samples was reduced to 3-5%.” (Page 5, top part)

Comment 9: 214: At which pressure did you evaporate the extraction liquids?

Response 9: Thank you for your comment. The information was added. (Page 5, bottom part)

Comment 10: 226: What was the guard column filled with? There are a large number of stationary phases sold under the Phenomenex brand.

Response 10: Thank you for your comment. “PFP” the information was added. (Page 6, top part)

Comment 11: 232: What were the linear ranges and the detection/quantification limits of your method? Please provide this data (in a table in the SI).
Did you check the extraction method by spiking plant samples with tocopherols and tocotrienols? Were there any matrix effects on the obtained calibration factors? The latter may be checked by preparing calibration lines not in neat solvent but in a sample solution instead.

Response 11: Thank you for your comment. The maximum information has been provided in the section 2.3. and 2.4. We did not observe the matrix effect on the fluorescence detector. “For tocochromanols extraction from all H. perforatum parts was applied the most frequent used protocol of saponification due to its highest recovery of those prenyllipids”. (Page 5, middle part)

Comment 12: 259: How did you determine the fresh weight of your plants? - Time from harvesting, method of washing and dry-blotting, ...

Response 12: Thank you for your comment. We incorporated this information into the manuscript to provide a comprehensive understanding of our methods. The missing information was provided in the material part. (Page 5, top part)

Comment 13: 277, Figure 1: Please provide a table with the 36 FW values in the SI.

Response 13: Thank you for your comment. Table S13 was added in the Supplementary Materials with mean values for biomass weight (g FW) of each species and soil.

Comment 14: 293, 3.3: Chapter 3.2 is presented after two chapters labelled 3.3 (line 429). Please check your numbering and/or arrangement of chapters.

Response 14: Thank you for noticing the error, we have corrected the numbering. (Page 8, bottom part)

Comment 15: 297, (stems, leaves, flower buds, and flowers): Please provide an estimation of the mass fraction that each of these organs contribute to the total (aerial) plant (dry) mass.

Response 15: Thank you for your comment. Thank you for the suggestion, but we did not look at the individual fractions of plant parts and their weight. We will certainly consider incorporating this aspect into our future studies.

Comment 16: 487: In this line there is a sudden change from reporting the results to their interpretation. A chapter change may be in order.

Response 16: Thank you for your comment. Thank you for suggestion, a new subsection titled “3.5. Agronomic, environmental, genetic, and other factors affecting the secondary metabolite concentrations” was implemented for better structure of the manuscript. (Page 15, middle part)

Comment 17: 505-507: Please provide a reference for that statement.

Response 17: Thank you for your comment. Reference has been added. (Page 16, top part)

Comment 18: 517-519: This sentence reads like a conclusion.

Response 18: Thank you for your comment. The syntax of the sentence has been improved to better fit the context of the discussion. „It is reported that ...” (Page 16, top part)

Comment 19: SI, Table S2: The planting dates would be of equal importance.

Response 19: Thank you for your comment. The planting dates are stated in the section 2.2. Plant material and we also added them in the Supplementary Materials.

Comment 20: SI, Figure S2: Please add a legend for the four plant parts. I know it should be obvious but please do also consider readers struggling with English.

Response 20: Thank you for your comment. The figure caption was added for clarity in the Supplementary Materials.

Comment 21: SI, Figure S3: What is the unit ("uV") of the ordinate on your chromatograms? Does the “wild” designation mean the sample whose chromatograms are shown here was not taken from the beds shown in figure S1?

Response 21: Thank you for your comment. Copy-Paste mistake. The “wild” was removed. "uV" is the unit on the chromatogram obtained from the fluorescence detector.

Comment 22: SI: I would recommend to add a figure showing the structures of the eight tocochromanols you studied.

Response 22: Thank you for your comment. The „Figure S4. Chemical structures of most common tocopherol and tocotrienol homologues” was added to Supplementary Materials.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper contains a study of the effect of different soil against lipophilic secondary metabolites in four Hypericum species, specifically the study compounds were tocotrienols and tocopherol. Although the secondary metabolites identification of Hypericum species, as well as the potential applications have already been reported, I find interesting the study of  less known hypericum species, as well as the related effect of different soil and the quantification of tocotrienol and tocopherol from different plant parts.

However, I have some recommendations.

Introduction

Lines 48-49. Authors mentioned that Hypericum species have a potential antitumor and antidepressant effects, I suggest that authors mentioned the bioactive compounds related to these effects and adding the references.

Line 53. H. inodorum needs to be writing on italics.

Lines 54-55. Authors mentioned that tocochromanols are lipophilic compounds with bioactive properties that could be found on Hypericum species, and in the next paragraph (lines 56-66) mentioned the tocotrienols group. To avoid doubts, I suggest that authors mentioned that on the tocochromanols group there are some compounds such as tocotrienols and others, which are mentioned or study on the paper.

Line 74. Which compounds have been identified on H. annalatum specie? I suggest that the authors mention this information.

Lines 87-88. Why is it important to study tocotrienols from Hypericum species? (human health?) I suggest that this information needs to be explained before or after these lines.

Lines 136-140. Why is different the lipophilic secondary metabolites on the different parts of the plant? How affect the type of soil on the lipophilic secondary metabolites accumulation?

Lines 140-142. On the title, the word biomass is present, and authors mentioned the word biomass on line 141. I suggest that authors describe if Hypericum species only are used as biomass of why authors study the biomass of the different plant species.

Results and discussion

Lines 286-292. Could only the nitrogen nutrition be the reason for the different biomass yield of the Hypericum species? Organic matter or another mineral content not affect this parameter?

Lines 293-294. I suggest that authors revised all the number of subtopics in the section of result and discussion, because need to be on ascend order (3.1, 3.2, etc)

Table 2. I suggest separating the information of the tocochromanol content for stems and another table for leaves.

Table 3. I suggest separating the information of the tocochromanol content for stems and another table for leaves.

 

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 manuscript and supplementary materials have been improved accordingly. Provided changes are marked in red font. For the literature we used references manager software, therefore the changes are not highlighted.

Reviewer 2

Comment 1: This paper contains a study of the effect of different soil against lipophilic secondary metabolites in four Hypericum species, specifically the study compounds were tocotrienols and tocopherol. Although the secondary metabolites identification of Hypericum species, as well as the potential applications have already been reported, I find interesting the study of  less known hypericum species, as well as the related effect of different soil and the quantification of tocotrienol and tocopherol from different plant parts.

Response 1: Thank you for the positive overview.

However, I have some recommendations.

Comment 2: Introduction. Lines 48-49. Authors mentioned that Hypericum species have a potential antitumor and antidepressant effects, I suggest that authors mentioned the bioactive compounds related to these effects and adding the references.

Response 2: Thank you for your comment. The missing information was supplemented. (Page 2, top part)

Comment 3: Line 53. H. inodorum needs to be writing on italics.

Response 3: Thank you for your comment. Mistake corrected. (Page 2, top part)

Comment 4: Lines 54-55. Authors mentioned that tocochromanols are lipophilic compounds with bioactive properties that could be found on Hypericum species, and in the next paragraph (lines 56-66) mentioned the tocotrienols group. To avoid doubts, I suggest that authors mentioned that on the tocochromanols group there are some compounds such as tocotrienols and others, which are mentioned or study on the paper.

Response 4: Thank you for your comment. We have revised the sentence to clarify that tocochromanol group includes tocopherols and tocotrienols, which are the most well-known. (Page 2, top part)

Comment 5: Line 74. Which compounds have been identified on H. annalatum specie? I suggest that the authors mention this information.

Response 5: Thank you for your comment. The missing information has been added. (Page 2, bottom part)

Comment 6: Lines 87-88. Why is it important to study tocotrienols from Hypericum species? (human health?) I suggest that this information needs to be explained before or after these lines.

Response 6: Thank you for your comment. Tocotrienols may have a significant contribution to human health. The missing information has been added. (Page 2, top part)

Comment 7: Lines 136-140. Why is different the lipophilic secondary metabolites on the different parts of the plant? How affect the type of soil on the lipophilic secondary metabolites accumulation?

Response 7: Thank you for your questions. Well, we do not know, therefore we come to the conclusion to investigate each part separately to better understand the distribution of those metabolites in different plant parts.

Comment 8: Lines 140-142. On the title, the word biomass is present, and authors mentioned the word biomass on line 141. I suggest that authors describe if Hypericum species only are used as biomass of why authors study the biomass of the different plant species.

Response 8: Thank you for your comment. The aim of the present study was rewritten to be more clear. (Page 4, top part)

Comment 9: Results and discussion. Lines 286-292. Could only the nitrogen nutrition be the reason for the different biomass yield of the Hypericum species? Organic matter or another mineral content not affect this parameter?

Response 9: Thank you for your comment. Yes, could. Therefore we provide the following explanation: „The influence of organic matter and other mineral content on the biomass yield of Hypericum species should not be overlooked. However, current studies, as well as previous reports, are insufficient to draw unambiguous conclusions.” (Page 8, middle part)

Comment 10: Lines 293-294. I suggest that authors revised all the number of subtopics in the section of result and discussion, because need to be on ascend order (3.1, 3.2, etc)

Response 10: Thank you for your comment. The mistake with wrong numbering has been corrected. (Page 8, bottom part)

Comment 11: Table 2. I suggest separating the information of the tocochromanol content for stems and another table for leaves.

Response 11: Thank you for your comment. We are afraid that due to applied statistical analysis, it cannot be done.

Comment 12: Table 3. I suggest separating the information of the tocochromanol content for stems and another table for leaves.

Response 12: Thank you for your comment. We are afraid that due to applied statistical analysis, it cannot be done.

Author Response File: Author Response.pdf