Red Clover and the Importance of Extraction Processes—Ways in Which Extraction Techniques and Parameters Affect Trifolium pratense L. Extracts’ Phytochemical Profile and Biological Activities

Round 1

Reviewer 1 Report

The present study titled “Red clover and the importance of extraction processes – ways in which extraction techniques and parameters affect Trifolium pratense L. extracts’ phytochemical profile and biological activities” is presenting, in detail, well-elaborated research to evaluate the profile of active phytochemicals obtained in different extraction methods.

Firstly, the authors reported on the obtaining and characterization (HPLC-MS, spectrophotometric methods: Folin-Ciocâlteu assay for TPC, TFC, DPPH assay, ABTS assay, FRAP assay ) of the extracts.

Thereafter, the authors determined the antimicrobial activity and the effect of the treatment with SE in the inflammation experimental model Wistar rats.

In general, the data are strong, with suitable controls, and convincingly show that the new formulation has great potential in further treatment against inflammation. The appropriate analyses are performed.

Overall, this is a well-performed study that I consider that is important and represent a good strategy to conveniently obtain new protocols to obtain biological active extracts.

 

The authors need to address the below comments to strengthen the quality of the manuscript:

The manuscript, including the Abstract part, should be checked for the English language.

E.g line 70-72“The main objective of this study was to evaluate the influence of the extraction method implied and the respective extraction parameters on phytochemical profile and on several biological activities of Trifolium pratense L. extracts, and to eventually offer a comparison of such aspects.” “

Line 20 “Several red clover extracts were performed,….”

Lines 333-335 “A prime example appeared for the DPPH assay, where the highest value was obtained by TBE, at the shorter extraction time used, i.e., 2 cycles of 5 min and a medium speed value, i.e., 6.000 rpm.”

The abbreviation should be kept constant. e.g. S or SE, as it is confusing for the reader (check the test, figures, and tables).

In M&M: 2.3.2. It is stated: “For this extraction method, the temperature of the heating plate was set to 210 °C in order  to permit the solvent to reach its boiling point and the duration of the extraction process was varied from 20 min to 40 min, and to 60 min, respectively. “ The phrase could be rewritten as the boiling point of the solvent mixture is about 79 ºC.

Please rewrite part 2.3.3. Turboextraction (TBE). It is not clear the steps were followed to obtain the extracts.

Please write all the names of organisms (e.g. Staphylococcus aureus) in italic. Also, “in vitro” and “in vivo” should be written in italic.

Please correct the phrase from line 256:” Once the phytochemical profile of the extracts was determined, the 60 min SE was selected for further determination of in vitro biological activities.”

Lines 345-346 should be rewritten: “This selection was based on the highest values found for the maximum number of the experiments presented above.”

Please add more discussion in the Discussion part on the obtained data, including some values and comparisons with the data from the literature. Please add more about the limitation of this study.

Please rewrite the Conclusion part: “As expected, generally, yields tended to increase proportionately along with extraction parameters values.”

Author Response

Reviewer #1 – Comments and replies

Line	Reviewer comments	Authors comments
	The manuscript, including the Abstract part, should be checked for the English language.	The manuscript, as well as the Abstract, has been grammatically checked by a native English speaker.
70-72	E.g line 70-72“The main objective of this study was to evaluate the influence of the extraction method implied and the respective extraction parameters on phytochemical profile and on several biological activities of Trifolium pratense L. extracts, and to eventually offer a comparison of such aspects.” “	The phrase has been reformulated.
20	Several red clover extracts were performed…	The phrase has been rewritten.
333-335	A prime example appeared for the DPPH assay, where the highest value was obtained by TBE, at the shorter extraction time used, i.e., 2 cycles of 5 min and a medium speed value, i.e., 6.000 rpm. The abbreviation should be kept constant. e.g. S or SE, as it is confusing for the reader (check the test, figures, and tables).	The authors would like to emphasize that abbreviations such as SE or TBE, etc. represent the extraction techniques. These abbreviations are provided in each of their respective descriptions, in Section 2. Materials and Methods. Furthermore, abbreviations such as S, T or U, etc. represent the sample/extract obtained through one of the studied extraction methods. Explanations of such aspects can be found at the beginning of Section 3. Results, more exactly, lines 333 – 339. However, we propose adding a new table, the current Table 1, that might provide the reader with an easier reading of the text.
	In M&M: 2.3.2. It is stated: “For this extraction method, the temperature of the heating plate was set to 210 °C in order  to permit the solvent to reach its boiling point and the duration of the extraction process was varied from 20 min to 40 min, and to 60 min, respectively. “ The phrase could be rewritten as the boiling point of the solvent mixture is about 79 ºC.	The phrase has been reformulated.
	Please rewrite part 2.3.3. Turboextraction (TBE). It is not clear the steps were followed to obtain the extracts.	The section has been rewritten.
	Please write all the names of organisms (e.g. Staphylococcus aureus) in italic. Also, “in vitro” and “in vivo” should be written in italic.	The requested modifications have been made.
256	Please correct the phrase from line 256:” Once the phytochemical profile of the extracts was determined, the 60 min SE was selected for further determination of in vitro biological activities.”	The phrase has been corrected.
345-346	Lines 345-346 should be rewritten: “This selection was based on the highest values found for the maximum number of the experiments presented above.”	The phrase has been rewritten.
	Please add more discussion in the Discussion part on the obtained data, including some values and comparisons with the data from the literature. Please add more about the limitation of this study.	The Discussion section was rewritten in the hope that it will be considered more satisfactory further on. References were modified accordingly.
	Please rewrite the Conclusion part: “As expected, generally, yields tended to increase proportionately along with extraction parameters values.”	The phrase has been reformulated.

Reviewer 2 Report

The paper focuses on the comparison of different extraction techniques on the effect of biological activity of Triforium extracts. The experiments were designed well. However, there are some needed to be revised before acceptance.

1. Titles of Tables should be described in more details. What does S T U in samples mean?

2. Discussion needs to be improved. What's the new findings of this paper? There are some results are contrary to previous reference. The explanations for your results and discussion between the difference with other reports are not enough.

Author Response

Reviewer #2 – Comments and replies

Line	Reviewer comments	Authors comments
	1. Titles of Tables should be described in more details. What does S T U in samples mean?	The authors propose a new table, the current Table 1, in hopes of facilitating the understanding of the sample nomenclature by readers.
	2. Discussion needs to be improved. What's the new findings of this paper? There are some results are contrary to previous reference. The explanations for your results and discussion between the difference with other reports are not enough.	The Discussion section was rewritten in the hope that it will be considered more satisfactory further on. References were modified accordingly.

Round 2

Reviewer 1 Report

The revision of the manuscript has solved all my questions. The authors rewrote the text with unclear phrases and adopted all my suggestions. I am satisfied with it. So, I suggest accepting.