HPLC-DAD Polyphenolic Profiling and Antioxidant Activities of Sorghum bicolor during Germination

Round 1

Reviewer 1 Report

I have carefully read manuscript agronomy-1105386 entitled “Polyphenol HPLC-DAD profiles and antioxidant activities of Sorghum bicolor during germination?” I have completed my review and submitted my recommendation for major revision of the manuscript.

After completing the reading of the manuscript, I find it interesting and think that it has the potential especially since it is about functional food. The possibility of applying the results of this research also highlights the potential of future publication.

All my suggestions regarding the major and minor changes, doubts and questions are given in the form of comments in the text itself. At the moment, before the further and more detailed consideration of the manuscript, I think that it is necessary to present the suggestions to the authors. I think the topic of the research itself is interesting and can attract readers, but the manuscript some refinement, both technical and substantive nature.

Comments for author File: Comments.pdf

Author Response

Reviewer 1 comments

 Point 1: Title change suggestion: HPLC-DAD polyphenolic profiling and ....

Response 1: We are grateful for the reviewer’s suggestions, which we have considered very useful and we modified the title as suggested.

Point 2: Albanus should be a hybrid of Sorghum bicolor. You need to emphasize this in the abstract. Whoever reads this will see only in the title, and the other in the abstract.

Response 2: We thank the reviewer for the observation. Indeed, the Albanus is a hibrid of Sorghum bicolor. We made the demanded modification in Abstract (line 25), as well as inside the article (Material and methods, Discussion, Conclusions)  

Point 3:  Indicate which procedures

Response 3: Thank you for the observation! We found your comments extremely helpful and have revised accordingly.

The manuscript has now been modified by rephrasing the Line 27 from Abstract („Ultrasound-assisted extraction in different solvents…”) and also the 2.3 subsection from Materials and Methods, lines 112-126 („The extraction procedure were carried out as follows: 20 g of each Sorghum bicolor samples were first subjected to ultrasound extraction with 200 mL of 3:1 hexane/isopropanol in order to remove the lipid content. The delipidized samples were then mixed with 200 mL of alcoholic solvents (methanol or ethanol). The extractions were performed in vessels fitted with an ascending refrigerant, in an ultrasonic bath, for 2 h.

The resulted alcoholic extracts were filtered through a filter paper, and then concentrated using a rotavapor until the dry extract. The crude obtained extracts were stored at 4 ºC until further use.”)

Point 4: Write the full name here, then specify the abbreviation (HPLC-DAD)

Response 4: Thank you for the comment! We modified the abstract as demanded. Please see lines 30-31: „High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD)…”

Point 5: I think total phenolic content (TPC) is more correct. Use TPC below.

Response 5: We thank the reviewer for the correction. We made the modification throughout the manuscript.

Point 6: I suggest that you avoid abbreviations like ABTS and DPPH here, but only explain the importance and conclusions you have obtained with these methods in relation to the subject of this paper.

Response 6: Thank you for the comment. The abstract has been modified accordingly. Please see lines 35-38: “The findings of this study showed that the TPC of sorghum extracts is strongly correlated with their antioxidant activity and overall, that the studied extracts presented a good radical scavenger activity, which supports the benefits of alimentary uses of Sorghum bicolor grains.”

Point 7: Why did you write flavones, when you analyze some other compounds from the group of flavonoids? Maybe you wanted to write flavonoids? There are no special conclusions regarding flavones only, in the paper.

Response 7: We thank the reviewer for the correction and we apologies for this error. We made correction throughout the manuscript and replace flavones with flavonoids. Please see lines 136, 186, 203, 253, etc.

Point 8: Is this relevant to the subject of this paper? It would be an analysis of stable isotope ratios.

Response 8: Thank you for the comments. Since the C4 carbon fixation or the Hatch-Slack pathway which is the photosynthetic mechanism characteristic for Sorghum bicolor, allows it to have a good photosynthetic yield, which gives it a better efficiency in conditions of heat and drought, we considered that this information could be included in our manuscript, part of plant description. We kindly ask the reviewer to accept our point of view. 

Point 9: Use molarity not normality

Response 9: Thank you for observation! We made the suggested correction (line 128).

Point 10: This should be total flavonoid content. You must correct this throughout the paper and state it in the abstract.

Response 10: We thank the reviewer for the correction and we apologies for this error. We made correction throughout the manuscript and introduced “total flavonoid content” in the abstract (Line 29)

Point 11: “no superscript”

Response 11: We appologies for the error. We made the correction (line 156)

Point 12: “0.1% trifluoracetic acid”

Response 12: We appologies for the error. We made the correction (lines 163, 164)

Point 13: “Are you sure? This is a very high flow. (1000mL/min)”

Response 13: We thank the reviewer for the observation. Indeed, the used elution flow was of 1mL/min. This was the condition for the utilized HPLC-DAD system. We replaced 1000mL/min to 1mL/min (lines 165), which is the usual mode to express this flow.

Point 14: Pay attention to the spaces. Also, there are repetitions of some compounds here. It should be stated exactly which wavelengths were used in the integration of certain peaks and quantification of the compounds

Response 14: We thank the reviewer for the comment and observations. We made the corrections regarding the spaces and also regarding the compounds and the wavelength used for their identification and quantification. Please see lines 167-170.

Point 15: What about LOD, LOQ and R2

Response 15: We thank the reviewer for signaling us the lack of those information, necessary to support the validity of the used method.

The used analytical method was performed according to a previous published article, as mentioned in the manuscript at section materials and methods. Based on the calibration curves of the used standards and on the performances of the HPLC-DAD system we have indicate in the revised manuscript the values for R2, LOD and LOQ. Please see lines 173-175.

Point 16: An introductory story should be given here in the results

Response 16: Thank you for the suggestion which can improve the quality of our manuscript.

We added the following paragraph to the results: “The aim of our research was to quantify phenolic and flavonoid compounds of a Romanian Sorghum bicolor hybrid, grains and germs in various stages of germination as well as to evaluate their antioxidant activity. The diversity of the composition in phenolic compounds and flavonoids has been highlighted for methanolic and ethanolic extracts by developing an HPLC-DAD method.” (lines 182-187).

Point 17: Where is the text related to this figure? First the text, then the figures

Response 17: Thank you for the suggestion which can improve the quality of our manuscript

Some corrections regarding the numbering and the titles of subsections from Results section. Also, each subsection has been supplemented with introductory comments for the presented results and figures. Please see lines 190-192, 204-208, 219-223 and 251-256.

Point 18: Fig 1 and 2 should be merged

Response 18: We thank the reviewer for the suggestion, but since the figures 1 and 2 present results which are discussed in separate section, we hope that the reviewer will accept to keep those figures separated.

Point 19: There are too many decimal places, two is enough

Response 19: We thank the reviewer for the suggestion, and we agree that the use of only two decimal facilitates the understanding of the results. Consequently, we made the suggested changes in Table 2.

Point 19: Very little discussion. For a serious discussion, the story needs to be expanded.

Response 19: We are grateful for the reviewer’s comment and suggestion, which we have considered very useful for the improvement of our manuscript. Consequently, we have supplemented the manuscript with consistent discussion regarding the performed research and the comparation of the obtained results with those obtained by other authors from studies regarding the subject approached in this article. 

Point 20: Neither the goal nor the scientific contribution of this paper is clear. I think that it should first be better defined, and accordingly, write a relevant discussion and conclusions

Response 20: We regret that the initial formulation of the purpose of our research was not clear enough, and consequently we made efforts to better define the scientific goal of this paper:

Lines 74-77: “The purpose of this study was to analyse the phenolic profile of sorghum grains and the antioxidant activity before and during germination, in order to sustain the beneficial effects of the use of this cereal in food products.”

Lines 280-287: “The aim of this study was to investigate the presence of antioxidant compounds, such as polyphenols and flavonoids, in the grains of a Sorghum bicolor hybrid cultivated in Romania, as well as the antioxidant activity in order to sustain the nutritional value of this cereal and the benefits for its use in human alimentation. The study analyzed the variation of the phenolic and flavonoid profile, as well as of the antioxidant activity during the germination process of sorghum grains, from the perspective of using this cereal in functional foods based on germinated sorghum.”

We think that the discussion and conclusion sections have now been improved in the revised manuscript, according to the Reviewer’s suggestion

Reviewer 2 Report

Dear Authors:

The topic of the manuscript is interesting and the study well conducted. My major suggestion is discussing the results in details, besides comparing the present findings with previously carried out similar investigation. Please find my comments below:

Abstract

L18: please use the approved plan's name instead of "Albanus sorghum" through whole text

L22-24: please add the evaluated values

L27: I would suggest to briefly mention a perspective for further similar investigations

Introduction

overall, this part must be completed with the similar references formerly performed on this species.

L48: add "and" before "cardiovascular disease"

L49: remove "while" please

L57-59: please re-write "The extraction solvent best suited for extracting total phenolics as well as specific polyphenol classes is variable for each plant type and part"

Materials and Methods

L74: please correct "10 015 kg/ha"

L77: "minutes" to "min" through whole the manuscript, please; moreover, unify please "hours" to "h"

L117: Any positive control has been used in DPPH assay?

L134-135: two solvent systems have been applied in HPLC-DAD analysis? please make it clear

Results

In Figure 1, what is the differences of the four analysis, on the other hand the horizontal axis has no definitions, as same as the Figure 2.

L154: the results of total phenolic and flavonoid contents are not described well in the "Discussion" part. I must be discussed the reason for being different between methanolic and ethanolic extracts. Only mentioning L210,211: "Methanol was more effective than ethanol for total phenolic content extraction from the samples" is not enough. Please use the literature, as well as in case of the differences in germination stages; for instance please discuss the reason in L163-166: "The highest concentration of flavonoids for the methanolic extracts was obtained after 36h of germination (7.8 mgEqR/g sample), while for the ethanolic extracts it was after 24h of germination (3.34 mg EqQ/g"

In Table 1, please define samples' names as footnote; as well as in Table 2 in terms of compounds' names

Discussion

- this sections needs to be supplied by comparing previously performed studies similar to the present work

- as above mentioned the rationale for being difference of phytochemical contents and antioxidant potencies have to be discussed, that's the reason of the study

Reference

This part must be in accordance with the "Author's guideline". Please revise it

Good luck!

Author Response

Reviewer 2 comments

Point 1: The topic of the manuscript is interesting and the study well conducted. My major suggestion is discussing the results in details, besides comparing the present findings with previously carried out similar investigation. Please find my comments below

Response 1: We are grateful for the Reviewer’s comments and suggestions, which were very useful for the significant improvement of our manuscript. We believe that after considering all the changes introduced in the revised manuscript, suggested by the reviewer, the quality of the manuscript was significantly improved.

Point 2: L18: please use the approved plan's name instead of "Albanus sorghum" through whole text

Response 2: We thank the reviewer for the suggestion. Since the Albanus is a hibrid of Sorghum bicolor, we made the demanded modification in Abstract (line 25), as well as throughout the manuscript (Material and methods, Discussion, Conclusions), referring to the used plant as “Albanus hybrid of Sorghum Bicolor”, “Sorghum bicolor hybrid” or “Albanus variety of Sorghum bicolor”

Point 3: 2 L22-24: please add the evaluated values

Response 3: The authors would like to thank the reviewer for this comment, which is very useful for the quality improvement of our manuscript. We have introduced the suggested addition, in the Abstract, lines 33-34: “…1853mg GAE/100 g for the methanolic extract and 1726 mg GAE/100 g for the ethanolic extract”

Point 4: L27: I would suggest to briefly mention a perspective for further similar investigations

Response 4: We appreciated the reviewer’s observation, which we also believe it is important.

We have added supplementary details regarding the scope of our study from the perspective of further use of this plant material. Please see:

Lines 35-38: “The findings of this study showed that the TPC of sorghum extracts is strongly correlated with their antioxidant activity and overall, that the studied extracts presented a good radical scavenger activity, which supports the benefits of alimentary uses of Sorghum bicolor grains”

Lines 74-77: “The purpose of this study was to analyse the phenolic profile of sorghum grains and the antioxidant activity before and during germination, in order to sustain the beneficial effects of the use of this cereal in food products.”

Lines 280-287: “The aim of this study was to investigate the presence of antioxidant compounds, such as polyphenols and flavonoids, in the grains of a Sorghum bicolor hybrid cultivated in Romania, as well as the antioxidant activity in order to sustain the nutritional value of this cereal and the benefits for its use in human alimentation. The study analyzed the variation of the phenolic and flavonoid profile, as well as of the antioxidant activity during the germination process of sorghum grains, from the perspective of using this cereal in functional foods based on germinated sorghum.”

Lines 362-363: “Specific phenolic compounds exhibited diverse variation patterns, and further research is required for the clarification of the biochemical mechanisms involved”.

Point 5: overall, this part must be completed with the similar references formerly performed on this species

Response 5: The authors would like to thank the reviewer for this comment. We think that the introduction has now been improved in the revised manuscript, as well as the discussion section according to the Reviewer’s suggestion. References regarding similar studies performed on this species has been used for this purpose. Please see lines 49-51, lines 251-256, lines 278-280, lines 300-312, table 3 and line 337-347

Point 6: L48: add "and" before "cardiovascular disease"; L49: remove "while" please; L57-59: please re-write "The extraction solvent best suited for extracting total phenolics as well as specific polyphenol classes is variable for each plant type and part"; "

Response 6: The authors would like to thank the reviewer for those observation and we apologies for the errors.

We made the suggested corrections in the revised manuscript. Please see new lines 63, 64, 72-74

Point 7: L74: please correct "10 015 kg/ha

Response 7: Thank you for this comment. In the revised manuscript we expressed the production as T grains/ha. Please see new line 93: “The production obtained was approximately 10 T grains/ha”

Point 8: L77: "minutes" to "min" through whole the manuscript, please; moreover, unify please "hours" to "h

Response 8: We thank the reviewer for the observation. We made the suggested corrections throughout the manuscript.

Point 9: L74: L117: Any positive control has been used in DPPH assay?

Response 9: We thank the reviewer for signaling the lack of this important information and we apologies for this error.

The positive control used for both DPPH and ABTS assays was Trolox. We added this information in the revised manuscript, lines 148 and 156: “Trolox (15.63– 250µg/mL) was used as positive control.”

Point 10: L134-135: two solvent systems have been applied in HPLC-DAD analysis? please make it clear

Response 10: We thank the reviewer for the comment, which allow us to improve the description of the used method for HPLC-DAD analysis. We apologies for the initial ambiguity.

A mobile phase from two solvent-systems in gradient elution has been used in our method.

 Please see new lines 162-165: “The mobile phase consisted of a two-solvent system, used in gradient elution. The solvents used were (A) 0.1 % trifluoroacetic acid (TFA) in water and (B) 0.1 % TFA in acetonitrile. The gradient elution was 2 -100 % B at 30 °C for 60 minutes and the elution flow was set at 1 mL/min.”

Point 11: In Figure 1, what is the differences of the four analysis, on the other hand the horizontal axis has no definitions, as same as the Figure 2.

Response 11: We thank the reviewer for the observation and we apologies for the signaled problem in Figures 1 and 2, which was a problem generated by the software used to make the graphics. We replaced the Figures in the revised manuscript and we hope that now the information presented in these figures is clearer. The Figures present the TPC and TFC obtained for the alcoholic extracts from the sorghum samples.

We mention that, during the restoration of the graphs in Figures 1 and 2, an error was found in the way of expressing the concentration of polyphenols and flavonoids, the initial data being calculated for 10 g of the sample but expressed as being calculated for 1 g of the sample. In order to correct this error and to correlate the data obtained by us with others presented in the literature, the calculation and expression of the total phenolic and flavonoids content was modified per 100 g sample.

Point 12: L154: the results of total phenolic and flavonoid contents are not described well in the "Discussion" part. It must be discussed the reason for being different between methanolic and ethanolic extracts. Only mentioning L210,211: "Methanol was more effective than ethanol for total phenolic content extraction from the samples" is not enough. Please use the literature, as well as in case of the differences in germination stages; for instance please discuss the reason in L163-166: "The highest concentration of flavonoids for the methanolic extracts was obtained after 36h of germination (7.8 mgEqR/g sample), while for the ethanolic extracts it was after 24h of germination (3.34 mg EqQ/g"

Response 12: We thank the reviewer for the comment and suggestions which were very useful for the quality improvement of our manuscript.

In the revised manuscript Discussion part was significantly modified and we hope that we managed to respond to reviewer’s expectations. From the best of our knowledge, the higher polarity of methanol compared to ethanol could justify the differences observed between the composition of methanolic and ethanolic extracts.

Regarding the differences observed for the TPC and TFC during the germination process, further research is required for the clarification of the biochemical mechanisms involved.

Point 13: In Table 1, please define samples' names as footnote; as well as in Table 2 in terms of compounds' names

Response 13: We thank the reviewer for the suggestion. In the revised manuscript we have made the requested changes (please see Tables 1 and 2)

Point 14: - this sections needs to be supplied by comparing previously performed studies similar to the present work

- as above mentioned the rationale for being difference of phytochemical contents and antioxidant potencies have to be discussed, that's the reason of the study

Response 14: We are grateful to the reviewer for those comments and sugestions, which we considered very useful for the quality improvement of our manuscript. We think that the discussion section has now been improved in the revised manuscript, according to the reviewer’s suggestion. References regarding similar studies performed on this species has been used for this purpose. Please 251-256, lines 278-280, lines 300-312, table 3 and line 337-347

Point 15: - This part must be in accordance with the "Author's guideline". Please revise it

Response 15: Thank you for this observation and we apologies for not following the "Author's guideline" in the original manuscript.

The Reference section has now been revised according to "Author's guideline".

Reviewer 3 Report

Sorghum bicolor has wide-range of economical uses such as biofuel and fiber production, animal feed and silage. It also has an increasing importance in human nutrition.

In my opinion this article can be accepted after minor revision.

Please follow the amendments below:

1. Lines 72-73: it is worth indicating whether, and if so, how much the temperature and precipitation deviated from the norm during the growing season of Sorghum bicolor.
2. Line 74: please change the yield for 10 Mg/ha.
3. Line 77: please specify the wavelength of radiation generated by this lamp.
4. Lines 87-89: please enter the name of the chemical compound for the first time, followed by the chemical formula in brackets, and then use it to shorten the description. Please apply the same principle to all chemical compounds in the article. 
5. All the figures need to be corrected. There is no scale on them, which makes them unreadable: it is difficult to read the results and the values. For example it is impossible to verify the results described in 157, 165, 166th lines. The percentage scales on the side of the Figures 3 and 4 are also necessary to improve readability.
   
6. Line 207: I suggest you do not use 'study suggest' in disscusion. 'Showed' will be better.
7. Line 234: The findings showed...
8. Please add some articles to the References. 26 is not so much. Please also include more studies on bioactive compounds in sorghum grain from Europe, e.g. articles written by Mrs. Przybylska-Balcerek.

I think it would be better if the authors indicated one corresponding author instead of three. For future readers it will make easier to contact the authors about the content of the publication. Please consider it.

Author Response

Reviewer 3 comments

Sorghum bicolor has wide-range of economical uses such as biofuel and fiber production, animal feed and silage. It also has an increasing importance in human nutrition.

In my opinion this article can be accepted after minor revision.

Response: We gratefully acknowledge this Reviewer’s comments, which were very useful to encourage us thorough revision and the significant improvement of our manuscript. Thank you!

Point 1: Lines 72-73: it is worth indicating whether, and if so, how much the temperature and precipitation deviated from the norm during the growing season of Sorghum bicolor

Response 1: We thank the reviewer for the suggestion which can which can help increase the value of the manuscript. As suggested, additional information on climate conditions during the cultivation of the analyzed species was added both in Materials and methods subsection 2.1 (please see new lines 91-92) as well as in the Discussion section (please see new lines 288-300).

Point 2: Line 74: please change the yield for 10 Mg/ha.

Response 2: Thank you for this comment. In the revised manuscript we expressed the production as T grains/ha. Please see new line 93: “The production obtained was approximately 10 T grains/ha”. We hope that you will agree with this change.

Point 3: Line 77: please specify the wavelength of radiation generated by this lamp.

Response 3: We thank the reviewer for the suggestion which corrects our omission. The emission wavelength of UV lamp was 254 nm. We made the necessary correction, as suggested, in the revised manuscript (please see line 96).

Point 4: Lines 87-89: please enter the name of the chemical compound for the first time, followed by the chemical formula in brackets, and then use it to shorten the description. Please apply the same principle to all chemical compounds in the article.

Response 4: Thank you for the suggestion. We have revised section 2.3 and 2.2 materials and methods description, including sample description and we hope that now anusless repetition are avoided. Please see new lines 95-111

Point 5: All the figures need to be corrected. There is no scale on them, which makes them unreadable: it is difficult to read the results and the values. For example, it is impossible to verify the results described in 157, 165, 166th lines. The percentage scales on the side of the Figures 3 and 4 are also necessary to improve readability

Response 5: We thank the reviewer for the observation and we apologies for the signaled problem in Figures 1 and 2, which was a problem generated by the software used to make the graphics. We replaced the Figures in the revised manuscript and we hope that now the information presented in these figures is clearer. We also modified figures 3 and 4 as suggested.

We mention that, during the restoration of the graphs in Figures 1 and 2, an error was found in the way of expressing the concentration of polyphenols and flavonoids, the initial data being calculated for 10 g of the sample but expressed as being calculated for 1 g of the sample. In order to correct this error and to correlate the data obtained by us with others presented in the literature, the calculation and expression of the total phenolic and flavonoids content was modified per 100 g sample.

Point 6 and 7: Line 207: I suggest you do not use 'study suggest' in discussion. 'Showed' will be better

Line 234: The findings showed...

Response 6: We thank the reviewer for the observation. In the revised manuscript, discussion section was substantially modified and we considered reviewer suggestion to replace “study suggest” with “showed”. Line 234 was also modified, please see new line 370

Point 8: Please add some articles to the References. 26 is not so much. Please also include more studies on bioactive compounds in sorghum grain from Europe, e.g. articles written by Mrs. Przybylska-Balcerek

Response 8: We are grateful to the reviewer for those comments and suggestions, which we considered very useful for the quality improvement of our manuscript. We think that the discussion section has now been improved in the revised manuscript, according to the reviewer’s suggestion. References regarding similar studies performed on this species has been used for this purpose, including recent articles written by Mrs Przybylska-Balcerek Please see lines 252-253, lines 300-312, lines 322-326, lines 330-331, table 3, lines 337-345 and References 23, 24, 25, 26, 27, 32, 33

Additional comment: I think it would be better if the authors indicated one corresponding author instead of three. For future readers it will make easier to contact the authors about the content of the publication. Please consider it.

Response: Considering the similar implication of those authors to this article, and their complementary competencies, we would prefer to keep all three authors as corresponding authors. We hope that the reviewer will understand and accept our decision

Round 2

Reviewer 1 Report

Dear,

The manuscript has been significantly improved and now warrants publication in Agronomy.

All the best

Reviewer 2 Report

Dear Authors,

Thank you for the precise revision. The present form can be considered for further publication process, in my opinion.

Good luck!