Could Supercritical Extracts from the Aerial Parts of Helianthus salicifolius A. Dietr. and Helianthus tuberosus L. Be Regarded as Potential Raw Materials for Biocidal Purposes?

Round 1

Reviewer 1 Report

The paper deals with the supercritical extracts from the aerial parts of Helianthus salicifolius and Helianthus tuberosus. In my opinion, the article is well written and understandable. Overall, this is an average experiment, and the authors have shown good writing skills, however, changes and perhaps some reconsiderations are advice. Therefore, the present manuscript needs to perform some changes before consideration to be published in Agriculture. My suggestions and questions are as follows:

 

In line 82, replace “Mg” for “mg”

 

In section 2.3, the spectrophotometric technique of Folin-Ciocaltau could be replaced in further experiments for more robust techniques that avoid false-positive amounts of TPC. It is now known that FC reaction does not only bound to polyphenolic compounds, but it does to a broad range of compounds like sugars, which overestimate the final amounts of TPC, therefore, correlation performed afterward might underestimate the power of the same compounds.

 

In section 2.5, also, the spectrophotometric technique DPPH change is advised for further experiments.

 

In line 152, 154, and table 1, replace “Mg” for “mg”

 

In table 3 and table 4, removal of the number within parenthesis has to be performed in order to simplify data. If “mode” is used instead of mean and standard deviation, this choice has to be explained in deep detail, therefore, subsection 2.6 statistical treatment has to be added. 

 

Table 3 and 4 could be summarized in one table and control responses is also advice in order to visualize and compare easily all data.

 

In figure 2 sub-figures a) and c) if the color does not change but the only tone, usage of different geometrical figures instead of just circles is advised. Sub-figure b) presented more than 1 empty space, these empty spaces should be removed or another type of representation had to be used in order to optimize graphical representation.

 

In lines 199-207, a description of figure 3 is made but not a discussion on the data is analyzed, for instance, how DPPH values could be explained due to the TPC? How do you describe the anti-correlation with that specific strain? Why are you only focusing on that strain? Where is the comparison explanation against controls? Why there is no comparison with other results of the same species or at least with related species?

 

In Lines 212-219, I have the impression that this is part of the introduction and not a discussion of your data and should be removed

 

Section 5, conclusions are very vague and robustness of data obtained should be concluded.

 

Finally, although the results of the microbiological assays are useful, the adoption of Folin and DPPH methods does not provide enough robustness to the final document, and therefore, more advanced analysis and better presentation and discussion of results it is mandatory to fulfill the minimum requirements of Agriculture journal. Supercritical extraction is a great method of extraction and the antimicrobial test is a great asset. 

Author Response

Dear Reviewer 1

 

We are kindly thankful for Your all valuable and constructive suggestions and the time spent on the review of our manuscript. We tried our best to improve the manuscript and hope that the correction will meet with approval. Here below we present a point-by-point list of the responses to the comments (in red). Moreover, our paper was also revised by a native speaker. The changes to the manuscript are highlighted by using yellow colored text.

 

Manuscript ID: agriculture-1003172

Title: Whether supercritical extracts from the aerial parts of Helianthus salicifolius and Helianthus tuberosus may be regarded as a potential raw materials for non-bioenergy purposes?

 

Comments and Suggestions for Authors

The paper deals with the supercritical extracts from the aerial parts of Helianthus salicifolius and Helianthus tuberosus. In my opinion, the article is well written and understandable. Overall, this is an average experiment, and the authors have shown good writing skills, however, changes and perhaps some reconsiderations are advice. Therefore, the present manuscript needs to perform some changes before consideration to be published in Agriculture. My suggestions and questions are as follows:

 

In line 82, replace “Mg” for “mg”

Response: The unit is correct, there must be Mg in this place, which means 1000 kg.

 

In section 2.3, the spectrophotometric technique of Folin-Ciocaltau could be replaced in further experiments for more robust techniques that avoid false-positive amounts of TPC. It is now known that FC reaction does not only bound to polyphenolic compounds, but it does to a broad range of compounds like sugars, which overestimate the final amounts of TPC, therefore, correlation performed afterward might underestimate the power of the same compounds.

Response: In further experiments we are going to perform a detailed studies needed to identify and to quantify constituents present in both extracts, for example by using GC-MS, LC-MS/MS and LC-MS QTOF MS/MS. TPC in the extracts using the Folin-Ciocalteu assay may be overestimated due to several constituents present in plant matrix. Therefore, some authors apply the correction of TPC, e.g. for ascorbic acid. In this paper we wanted to obtain preliminary data on TPC in general for indicating some relationship and trends between TPC and antimicrobial/antioxidant activity of both extracts, so we used the uncorrected TPC. We could not perform statistical analysis, concerning the correlation tests due to a limited amount of data. In further, more deatailed studies we are going to do statistical analysis (correlation tests), verifying our observations.

 

In section 2.5, also, the spectrophotometric technique DPPH change is advised for further experiments.

Response: The DPPH reagent method is one of the commonly used method to determine antioxidant activity. This method is very popular and easy to compare the activity with other data available in the literature. Thank you for this suggestion, we will consider extending the spectrum of antioxidant testing using other single electron transfer (SET) and hydrogen atom transfer (HAT) methods.

 

In line 152, 154, and table 1, replace “Mg” for “mg”

Response: Mg is the correct unit. There must be Mg in these places, which means 1000 kg.

 

In table 3 and table 4, removal of the number within parenthesis has to be performed in order to simplify data. If “mode” is used instead of mean and standard deviation, this choice has to be explained in deep detail, therefore, subsection 2.6 statistical treatment has to be added. 

Response: In Table 3 (previously Table 3 and Table 4), the numbers within parenthesis were removed. Of the three MIC, MBC and MFC values, the most common representative value, i.e. “mode” was showed. We did not include subsection statistical analysis but in subsection on determination of antimicrobial activity we added the sentence describing the choice of “mode”.

 

Table 3 and 4 could be summarized in one table and control responses is also advice in order to visualize and compare easily all data.

Response: The results presented in Table 3 and 4 were summarized in one table – Table 3 in order to compare both extracts. We suggest to leave data on MIC of the standard antimicrobial drugs into the text, if possible.

 

In figure 2 sub-figures a) and c) if the color does not change but the only tone, usage of different geometrical figures instead of just circles is advised. Sub-figure b) presented more than 1 empty space, these empty spaces should be removed or another type of representation had to be used in order to optimize graphical representation.

Response: The sub-figures a), b) and c) in Figure 2 were corrected and we hope that they are more clear.

 

In lines 199-207, a description of figure 3 is made but not a discussion on the data is analyzed, for instance, how DPPH values could be explained due to the TPC? How do you describe the anti-correlation with that specific strain? Why are you only focusing on that strain? Where is the comparison explanation against controls? Why there is no comparison with other results of the same species or at least with related species?

Response: We are focusing on S. aureus for two reasons: its higher sensitivity to both extracts compared to other strains and its involvement in human pathology and potential contamination of food or cosmetics. In Discussion we suggest that the observed anti-correlation may be due to differences in chemical composition of both extracts, e.g. sesquiterpens. We are going to perform further detailed studies using more advanced chromatographic techniques to verify our suggestion.

 

In Lines 212-219, I have the impression that this is part of the introduction and not a discussion of your data and should be removed

Response: This text was modified.

 

Section 5, conclusions are very vague and robustness of data obtained should be concluded.

Response: We hope, that the revised conclusions are more clear.

 

Finally, although the results of the microbiological assays are useful, the adoption of Folin and DPPH methods does not provide enough robustness to the final document, and therefore, more advanced analysis and better presentation and discussion of results it is mandatory to fulfill the minimum requirements of Agriculture journal. Supercritical extraction is a great method of extraction and the antimicrobial test is a great asset. 

Response: In this paper we wanted to present a possibility to use the supercritical extracts as antimicrobials (biocidals) and the relation of this activity to the most common phytochemical parameter, i.e. TPC. Now, we included data on ATR-FTIR spectra as preliminary analysis of both extracts. We know that further, detailed studies are needed to identify and to quantify constituents present in both extracts, for example by using GC-MS, LC-MS/MS and LC-MS QTOF MS/MS as well as verification of the extract activity against clinical S. aureus strains (MSSA, MRSA) due to high sensitivity of microorganism to both extracts.

 

On behalf of the Authors,

Anna Biernasiuk

Author Response File: Author Response.docx

Reviewer 2 Report

1. Table 1: State the number of replicates (n) for the data reported.

2. Discussion section should include a comparison with the reported activity (antioxidant, antibacterial) of the extracts from conventional solvents, such as methanol, water etc.

3. The discussion and conclusion is not linked to the title of the manuscript. Improve it so that the main aim of the title is being addressed, failing which the authors may consider changing the title. 

Author Response

Dear Reviewer 2

 

We are kindly thankful for Your all valuable and constructive suggestions and the time spent on the review of our manuscript. We tried our best to improve the manuscript and hope that the correction will meet with approval. Here below we present a point-by-point list of the responses to the comments (in red). Moreover, our paper was also revised by a native speaker. The changes to the manuscript are highlighted by using yellow colored text.

 

Comments and Suggestions for Authors

1. Table 1: State the number of replicates (n) for the data reported.

Response: As suggested by the Reviewer, information about number of replicates (n) were added to Table 1.

 

2. Discussion section should include a comparison with the reported activity (antioxidant, antibacterial) of the extracts from conventional solvents, such as methanol, water etc.

Response: Some literature data concerning the above problems were included in Discussion section. However, there are only some data on antimicrobial activity of Helianthus species using various methods and various parameters. In this paper we used the recommended microdilution method, allowing MIC determination.

 

3. The discussion and conclusion is not linked to the title of the manuscript. Improve it so that the main aim of the title is being addressed, failing which the authors may consider changing the title.

Response: The discussion and conclusion were modified and corrected. The title was also modified.

 

On behalf of the Authors,

Anna Biernasiuk

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for your answers and consideration.

Author Response

Response to Reviewer 1 Comments

 

Dear Reviewer,

We are kindly thankful for Your very valuable suggestions and reviewing our revised manuscript ID: agriculture-1003172 titled “Could supercritical extracts from the aerial parts of Helianthus salicifolius and Helianthus tuberosus be regarded as potential raw materials for biocidal purposes?”

 

In the re-submitted manuscript, we made appropriate modifications (minor corrections) according to Your comments. The changes to our article were highlighted (in red) by using the "Track Changes" function in Microsoft Word, so that they are easily visible to Reviewer and Editors. We tried our best to improve the manuscript and hope that the correction will meet with approval.

 

Review Report Form

• Is the research design appropriate? – Can be improved

 

Response:

We improved our research design and we hope it is appropriate.

 

Abstract:

We added text:

Lines 36-38: “The ATR-FTIR spectra of both extracts showed similar main vibrations of the functional groups typical for phytoconstituents possessing bioactivity.”

 

Line 39: “antioxidants and”

 

Lines 40-42: “The ATR-FTIR spectra of both extracts were characterized by similar main vibrations of the functional groups typical for phytoconstituents possessing bioactivity”

 

Introduction

We added text:

Line 71: “The biomass for the extraction purposes was collected on end of June.”

 

Lines 72-73: “together with the mode of action (bactericidal/fungicidal vs bacteriostatic/fungistatic)”

 

Line 73: “(Staphylococcus aureus)”

 

Line 74: “(Escherichia coli)”

 

Line 74: “from Candida spp.) –”

 

Lines 78-80: “in order to obtain preliminary data on the presence of functional groups characteristic for bioactive phytoconstituents.”

 

• Are the conclusions supported by the results? – Can be improved

 

Response:

We modified our conclusions and we hope they are appropriate and more clear.

 

Conclusions

We added text:

Lines 308 – 320:

“The presented data suggest that supercritical extracts with water as a co-solvent obtained from the aerial parts of H. salicifolius and H. tuberosus collected in summer period appeared to be a promising source of natural compounds with biocidal effect. They possessed antibacterial activity against Gram-positive (S. aureus) and Gram-negative (E. coli) species (MIC = 0.62-5 mg mL^-1), as well as antifungal activity against yeasts from Candida genus (MIC = 5-10 mg mL^-1). It is worth notifying their antistaphylococcal activity (MIC = 0.62-2.5 mg mL^-1). These extracts may be also regarded as natural potential antioxidants (EC₅₀ = 0.332-0.609 mg mL^-1). The ATR-FTIR spectra of both extracts showed similar main vibrations of the functional groups typical for phytoconstituents possessing bioactivity. The obtained data together with those from literature suggest that these extracts and their isolated bioactive compounds may be used as conservants in cosmetics and/or natural preservatives in food. However, further studies are needed to confirm the obtained results, to define and to quantify constituents present in both extracts, as well as to identify specific applications of supercritical extracts and their phytoconstituents from biomass of these two plant species.

 

We deleted text:

Lines 321-328:

“The supercritical extracts from H. salicifolius and H. tuberosus aerial parts with water as a co-solvent appeared to be a promising source of natural compounds with biocidal effect showing antibacterial activity against Gram-positive bacteria (especially S. aureus) and Gram-negative bacteria, as well as antifungal activity against fungi (represented by yeasts from Candida genus). Moreover, these extracts may be regarded as natural potential antioxidants. However, further studies are needed to confirm the obtained results and to identify specific applications of supercritical extracts from biomass of these two plant species, as well as to define and to quantify constituents present in both extracts.”

 

On behalf of the Authors,

Anna Biernasiuk

Author Response File: Author Response.docx