Pulsed Electric Field-Based Extraction of Total Polyphenols from Sideritis raiseri Using Hydroethanolic Mixtures

Round 1

Reviewer 1 Report

the current version is complete clear and can be accepted. Respect to the reported revisions highlighted in the pdf file, this reviewer has no other requests

Author Response

the current version is complete clear and can be accepted. Respect to the reported revisions highlighted in the pdf file, this reviewer has no other requests

Thank you very much.

Reviewer 2 Report

The manuscript was improved. Thank you to the Authors for the thorough revision.

I do have some minor comments.

I would still suggest making some corrections to the Figures. Please point out the statistical differences with the controls.
In section 2.4. Dry weight determination indicate the explanation of the authors
"In all PEF-based extractions, it is of paramount importance that the extractable tissue contains humidity, in order for electroporation to take place. Therefore, dry tissues cannot be used for PEF extractions. On top of that, in the case that dry tissue needs to be used, it is a common practice to re-hydrate the plant tissue [Carpentieri, S., Režek Jambrak, A., Ferrari, G., & Pataro, G. (2022). Pulsed Electric Field-Assisted Extraction of Aroma and Bioactive Compounds from Aromatic Plants and Food By-Products. Frontiers in nutrition, 8, 792203].

Additionally, practical recommendations are also welcome.

I would like to wish the Authors success in their further scientific work.

Author Response

The manuscript was improved. Thank you to the Authors for the thorough revision.

Thank you very much.

I do have some minor comments.

I would still suggest making some corrections to the Figures. Please point out the statistical differences with the controls.

Statistically significant differences between samples and control were added to the Figures.

In section 2.4. Dry weight determination indicate the explanation of the authors

"In all PEF-based extractions, it is of paramount importance that the extractable tissue contains humidity, in order for electroporation to take place. Therefore, dry tissues cannot be used for PEF extractions. On top of that, in the case that dry tissue needs to be used, it is a common practice to re-hydrate the plant tissue [Carpentieri, S., Režek Jambrak, A., Ferrari, G., & Pataro, G. (2022). Pulsed Electric Field-Assisted Extraction of Aroma and Bioactive Compounds from Aromatic Plants and Food By-Products. Frontiers in nutrition, 8, 792203].

The explanation was added in the text, according to the reviewer’s suggestion.

Additionally, practical recommendations are also welcome.

Thank you for your suggestion. In our paper, we tried our hardest to address and explain all of the issues.

I would like to wish the Authors success in their further scientific work.

Thank you very much.

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 and Authors' Reply

The authors analyze the extraction of polyphenols by means of the pulsed electric field technique using different hydroethanolic mixtures.
In general lines it can be said that:
The introduction must detail with some example any of the applications of this PEF extraction method that puts this technique in value

Examples of polyphenols extracted from plants with PEF were added in the introduction
section.

The materials and methods section is confusing. The extraction and determination of polyphenols from plant material is explained as independent processes. The authors should clarify the intermediate steps by which the samples go from the PEF extraction to the Folin-Ciocalteu assay or HPLC-based determination of total phenolic content.

 There are no steps between extraction and analysis. This is stated in Line 129 “Then the
supernatant (comprised of the extraction solvent and the polyphenols) was used immediately
subjected to further analyses, as stated in sections 2.6 and 2.7.”

Why determine the water content of plant material? Why was dry plant material not used
directly?
In all PEF-based extractions, it is of paramount importance that the extractable tissue contains
humidity, in order for electroporation to take place. Therefore, dry tissues cannot be used for
PEF extractions. On top of that, in the case that dry tissue needs to be used, it is a common
practice to re-hydrate the plant tissue [Carpentieri, S., Režek Jambrak, A., Ferrari, G., &
Pataro, G. (2022). Pulsed Electric Field-Assisted Extraction of Aroma and Bioactive
Compounds from Aromatic Plants and Food By-Products. Frontiers in nutrition, 8, 792203].
However, since the water content of the plant may vary due to environmental reasons, we
expressed our results to dry weight, so that there can be a benchmark for future reference. The
text was modified appropriately.

 The authors must take into account that if the supernatant analyzed by HPLC contains
different percentages of water and ethanol, the spectra, absorbances and retention times
may vary.
When a solution is injected into an HPLC system, then the organic compounds are retained by
the stationary phase while the solvent in which the compounds were dissolved and also injected,
cannot be retained and is eluted first. This is observed by the peak shape at the beginning of the
chromatogram. However, the compounds retained by the stationary phase are eluted in a
repeatable way, given the use of a specific mobile phase. Therefore, when they reach the
detector, they are dissolved in the mobile phase of the same consistency. As such, no such
problems, such as spectral differences, absorbance maxima and retention time arise, and this is
the reason why the HPLC-based separation has high reproducibility.

It is unlikely that the data conform to normality. Were the appropriate analyzes made to
verify the normality of the data? Indicate the type of analysis if this was so
The statistical analysis was carried out again using the non-parametric Kruskal Wallis test

The results and their statistical analysis are not shown in the most suitable way and the discussion is scarce

The results section and Figures were modified accordingly so that statistical analysis and
differences are discussed better.

The Analysis of polyphenols is always interesting due to their bioactivities, but the authors focus on total polyphenols without going into detail in indicating how each of these compounds is extracted depending on the mixtures used. Polyphenols are many and very diverse. For this reason, the results are not relevant and in the conclusions, it is not possible to specify which mixture would be the ideal one for which type of compound.

Our aim was not to examine the effect of the extraction parameters on specific polyphenols but
on the total polyphenolic content of the extracts. As stated by the reviewer, polyphenols are
many and when preparing extracts, it is impossible to extract only one phenolic compound,
without obtaining others as well. As such, we have not opted for individual differences, but for
the total content which may be more informative to readers and comparable to other studies

For all these reasons I think this work is not suitable to get accepted in this Journal.
In the attached file, different comments are made that can help the authors to improve the work.

Comments for author File: Comments.pdf

Reviewer 2 Report and Authors' Reply

The work is very interesting and seems to be complete. Form the analytical point of view i strongly encourage to use the quantitative analyses not by total peak area (very variable) but after a full method validation using chemical standards.

In this form the paper is not suitable for publication

 Our aim was not to examine the effect of the extraction parameters on specific polyphenols but
on the total polyphenolic content of the extracts. As stated by the reviewer, polyphenols are
many and when preparing extracts, it is impossible to extract only one phenolic compound,
without obtaining others as well. As such, we have not opted for individual differences, but for
the total content which may be more informative to readers and comparable to other studies