Effect of Spirulina (Formerly Arthrospira) Maxima against Ethanol-Induced Damage in Rat Liver

Round 1

Reviewer 1 Report

The study is interesting, the draft was well-prepared, but the statistic analysis has a major problem and must be improved.

Line 106. How many rats are in each group?

Line 197. 'multiple comparison tests'  should be marked with the style of 'a,b,c,d,e,f,g' instead of only comparison with the control. It would be more interesting to compare PH-SP vs. PH, and PH-EtOH vs.PH-EtOH-SP. Please revise the significant differences in all tables. And revise the entire draft accordingly.

Line 434. The conclusion should be improved, and it is too short now.

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors

The study is interesting, the draft was well-prepared, but the statistic analysis has a major problem and must be improved.

Thank you very much for the suggestion, the statistical analysis was improved

Line 106. How many rats are in each group?

Response.

Thank you very much in advance for the suggestion, the correction was made in the writing.

Line 197. 'multiple comparison tests'  should be marked with the style of 'a,b,c,d,e,f,g' instead of only comparison with the control. It would be more interesting to compare PH-SP vs. PH, and PH-EtOH vs.PH-EtOH-SP. Please revise the significant differences in all tables. And revise the entire draft accordingly.

 Response.

Thanks for the recommendation, the statistical analysis and the interpretation of the results were modified.

Line 434. The conclusion should be improved, and it is too short now.

Response.

Thank you very much for the observation the conclusion has been modified.

Reviewer 2 Report

In the manuscript “Effect of Spirulina (formerly Arthrospira) maxima against ethanol-induced damage in rat liver” authors evaluated the protective effect of spirulina (SP) against ethanol (EtOH)-induced cytotoxicity, in a context of liver regeneration after 70% partial hepatectomy (PH) in Wistar rats. They investigated multiple liver and blood parameters 7 days after PH through five experimental groups: Control, PH, PH-EtOH, PH-SP and PH-SP-EtOH. Results are overall convincing and show a beneficial effect of SP in this context of EtOH consumption during hepatic regeneration after PH, and thus on several biological parameters. Nevertheless, I will have a few comments:

Major comments:

1.       Authors wrote in the conclusion, “our study results indicate that SP exerts a protective effect against the liver damage induced by EtOH administration”. They also wrote similar conclusions throughout the manuscript. I suggest authors to be more specific, and to mention the experimental context of regeneration after PH. It is not directly shown in this publication that SP protect against ethanol-induced cytotoxicity, as SP, EtOH and SP-EtOH have been administered only to animals after PH, and not on heathy animals.

2.       Better quality images in figure 1 would highly improve the figure.

3.       Throughout the manuscript, authors preformed statistical analysis by comparing PH, PH-SP, PH-EtOH and PH-SP-EtOH only to the control. I would also compare every condition to the PH experimental group. Also, the most appropriate comparison to conclude whether SP can protect from EtOH-induced toxicity would be PH-EtOH vs. PH-EtOH-SP.

Minor comments:

1.       Please add scale bar on images in figure 1.

2.       Please mention PH-SP group instead of “SP” group line 391

3.       Please correct the typo line 370.

Author Response

Response to Reviewer 2 Comments

In the manuscript “Effect of Spirulina (formerly Arthrospira) maxima against ethanol-induced damage in rat liver” authors evaluated the protective effect of spirulina (SP) against ethanol (EtOH)-induced cytotoxicity, in a context of liver regeneration after 70% partial hepatectomy (PH) in Wistar rats. They investigated multiple liver and blood parameters 7 days after PH through five experimental groups: Control, PH, PH-EtOH, PH-SP and PH-SP-EtOH. Results are overall convincing and show a beneficial effect of SP in this context of EtOH consumption during hepatic regeneration after PH, and thus on several biological parameters. Nevertheless, I will have a few comments:

Major comments:

1. Authors wrote in the conclusion, “our study results indicate that SP exerts a protective effect against the liver damage induced by EtOH administration”. They also wrote similar conclusions throughout the manuscript. I suggest authors to be more specific, and to mention the experimental context of regeneration after PH. It is not directly shown in this publication that SP protect against ethanol-induced cytotoxicity, as SP, EtOH and SP-EtOH have been administered only to animals after PH, and not on heathy animals.

 Response.

Thank you very much for the observation the conclusion has been modified.

2. Better quality images in figure 1 would highly improve the figure.

Response.

Thanks for the observation, figure 1 has been improved.

3. Throughout the manuscript, authors preformed statistical analysis by comparing PH, PH-SP, PH-EtOH and PH-SP-EtOH only to the control. I would also compare every condition to the PH experimental group. Also, the most appropriate comparison to conclude whether SP can protect from EtOH-induced toxicity would be PH-EtOH vs. PH-EtOH-SP.

Response.

Thanks for the recommendation, the statistical analysis and the interpretation of the results were modified.

Minor comments:

1. Please add scale bar on images in figure 1.

Response. 

Thanks for the suggestion, scale was added

2. Please mention PH-SP group instead of “SP” group line 391

 Response.

Thank you very much in advance for the suggestion, the correction was made in the writing.

3. Please correct the typo line 370.

Response.

Thank you very much in advance for the suggestion, the correction was made in the writing.

Reviewer 3 Report

Ms. Ref. No.: applsci-1849377

Title:

 Effect of Spirulina (formerly Arthrospira) maxima against eth-2 anol-induced damage in rat liver

Dear editor

This study aimed to study the protective properties of SP on ethanol (EtOH)-induced toxicity by examining the effects on antioxidant enzyme activity in partial post-hepatectomy liver regeneration in male Wistar rats. Besides, the effects were analyzed on serum parameters and histopathological changes in the liver of rats.  

The experiment has been set up well. The writing is relatively good. But some errors exist in manuscript as mentioned below.

Comments to Author

Abstract:

The Keywords should be providing in alphabetic order and each word should be capitalized.

Introduction

Line 47 move “studies” after “in vitro”

Line 60 delete” among others”

Lines 67-70 the authors stated some enzymes, among them glutathione (GSH) which is not enzyme. Delete “glutathione” from the list.

Line 76 change “and these are the markers that are most specific for ”  to  “and these are the most specific markers for ”

Line 78 is unclear for me. Revise please.

Line 80 delete “,etc”

Material and methods

Line 108 what is “PH-EtOH”?

Line 125 what is “CP”?

Line 147 which spectrum?

Lin e158 what is “U”?

Line 163 which spectrum?

Line 168 which spectrum?

Line 173 which spectrum?

Results

Lines 205-206 delete “The values are expressed as the mean ± SEM in each experimental group. These are 205 illustrated in “

In  table 1: the SEM values for body weight gain in PH-EtOH and PH-SP are very high. re-check please.

In  table 4: the SEM values for CAT and GSH in PH-EtOH are very high. re-check please.

The sub titles are not complete revise them

“Influence on body and liver weights” influence of what?

 “Liver histology” incomplete???

 “Effect of Spirulina on Biochemical Parameters” just spirulina?

“Influence on Antioxidant Biomarkers“ influence of what?

Discussion

In this section we do not refer to the figure nor table, revise please.

Line 301 delete “to the that”

Lines 370-372 “These were is reflected in the PH-EtOH group, where the level of albumin was lowest compared to the control group, while in the groups treated with SP, the levels were very similar to normal values” is unclear for me. Revise please.

Line 378 delete “to this”

Line 379 add a “,” after “liver”

Line 379-382 revise please

Lines 399- 402 revise please.

Line 403 delete “to this”

Conclusion

This section is very short. There is no information about PH-EtOH-SP

Author Response

Response to Reviewer 3 Comments

 Effect of Spirulina (formerly Arthrospira) maxima against eth-2 anol-induced damage in rat liver

Dear editor

This study aimed to study the protective properties of SP on ethanol (EtOH)-induced toxicity by examining the effects on antioxidant enzyme activity in partial post-hepatectomy liver regeneration in male Wistar rats. Besides, the effects were analyzed on serum parameters and histopathological changes in the liver of rats.  

The experiment has been set up well. The writing is relatively good. But some errors exist in manuscript as mentioned below.

Comments to Author

Abstract:

The Keywords should be providing in alphabetic order and each word should be capitalized.

Response.

Thank you very much in advance for the suggestion, the correction was made in the writing.

Introduction

Line 47 move “studies” after “in vitro”

Response.

Thank you very much for the observation, the modification was made in the file

Line 60 delete” among others”

Response.

Thank you very much for the suggestion, that word was removed from the file

Lines 67-70 the authors stated some enzymes, among them glutathione (GSH) which is not enzyme. Delete “glutathione” from the list.

Response.

Thank you very much for the suggestion, glutathione was removed from the list.

Line 76 change “and these are the markers that are most specific for ”  to  “and these are the most specific markers for ”

Response.

Thank you very much for the recommendation, the phrase in the file was changed.

Line 78 is unclear for me. Revise please.

Response.

Thanks for the observation, the wording was improved

Line 80 delete “,etc”

Response.

Thank you very much for the suggestion, that word was removed from the file.

Material and methods

Line 108 what is “PH-EtOH”?

Response.

Thank you very much for the observation, it improved in writing

Line 125 what is “CP”?

Response.

Thank you very much for the suggestion, that word was removed from the file.

Line 147 which spectrum?

Response.

Thank you very much for your observation, the spectrum was added.

Line 158 what is “U”?

Response.

Thank you very much for the observation, following the instructions provided by the manufacturer U: SOD units, final activity in U/min/mg/protein

Line 163 which spectrum?

Response.

Thank you very much for your observation, the spectrum was added

Line 168 which spectrum?

Response.

Thank you very much for your observation, the spectrum was added

Line 173 which spectrum?

Response.

thank you very much for the suggestion, the spectrum was added.

Results

Lines 205-206 delete “The values are expressed as the mean ± SEM in each experimental group. These are 205 illustrated in “

Response.

Thank you very much for the suggestion, that phrase was removed from the file.

In  table 1: the SEM values for body weight gain in PH-EtOH and PH-SP are very high. re-check please.

Response.

Thanks for the suggestion, the observed values were corrected

In  table 4: the SEM values for CAT and GSH in PH-EtOH are very high. re-check please.

Response.

Thanks for the suggestion, the values were corrected

The sub titles are not complete revise them

“Influence on body and liver weights” influence of what?

Response.

Thank you very much for your recommendation, the subtitle was modified

 “Liver histology” incomplete???

Response.

Thank you very much for your suggestion, the subtitle was modified

 “Effect of Spirulina on Biochemical Parameters” just spirulina?

Response.

Thank you very much for the recommendation, the subtitle was modified

“Influence on Antioxidant Biomarkers“ influence of what?

Response.

Thanks for the observation, the subtitle was improved

Discussion

In this section we do not refer to the figure nor table, revise please.

Response.

Thanks for the observation, the tables and figures have been referred to in the discussion.

Line 301 delete “to the that”

Response.

Thank you very much for the suggestion, that phrase was removed from the file.

Lines 370-372 “These were is reflected in the PH-EtOH group, where the level of albumin was lowest compared to the control group, while in the groups treated with SP, the levels were very similar to normal values” is unclear for me. Revise please.

Response.

Thanks for the observation the text has been modified

Line 378 delete “to this”

Response.

Thank you very much for your observation, that phrase was removed from the file.

Line 379 add a “,” after “liver”

Response.

Thank you for your input, it has been modified.

Line 379-382 revise please

Response.

Thank you very much for your observation, it has been modified.

Lines 399- 402 revise please.

Response.

Thanks for the suggestion, the wording was improved

Line 403 delete “to this”

Response.

Thank you very much for your observation, that phrase was removed from the file.

Conclusion

This section is very short. There is no information about PH-EtOH-SP

Response.

Thank you very much for the observation the conclusion has been modified.

Round 2

Reviewer 1 Report

The authors have tried to improve the manuscript, but have not fully compared the significant differences in the tables. Although the author may not necessarily present all comparisons in data, it would be of great value to readers for analyzing the data by themselves via the significant differences. I would recommend the authors learn enough knowledge about multi-comparison analysis from the internet (i.e. the Youtube video is good) or consult a statistician, then carefully revise the significant differences data.

Correcting for multiple comparisons in R (STAT 320 lab_multiple_testing video 1 or 1), is a nice 9 mins statistic video:

https://www.youtube.com/watch?v=pevqgRKjlk4

Please compare significant differences by multi-comparison analysis for the following tables:

Table 1, each column

Table 3, each column

Table 4, each column

Please mark the significant differences systematically by 'abce...' style, and normally mark the highest value from a.

Several examples (please do NOT cite):

Effect and mechanism of psyllium husk (Plantago ovata) on myofibrillar protein gelation: Table 1

https://www.sciencedirect.com/science/article/abs/pii/S002364382031639X

Instrumental measurement of cooked rice texture by dynamic rheological testing and its relation to the fine structure of rice starch:  Table 1 and 2.

https://www.sciencedirect.com/science/article/abs/pii/S0144861716302740

Author Response

Thanks for the suggestion. Statistics corrected as suggested by reviewer in tables. Thank you

Reviewer 2 Report

The authors of the manuscript Named "Effect of Spirulina (formerly Arthrospira) maxima against eth-anol-induced damage in rat liver" improved the manuscript by performing a more robust statistical comparison between every groups. Also, the the conclusion is now in line with the overall message of the manuscript. 

A scale bar was added onto the images in figure 1, nevertheless, I am not sure if the actual size of the latest is actually 1micrometer as indicated in the legend. The authors should verify this information before publication of the manuscript. 

Author Response

Thanks for the comment. The correct is 15 micrometer. Thank you

Round 3

Reviewer 1 Report

It is interesting that the 259.52+-2.2a and 245.98+-3.0a in Table1 do not have significant differences. Also, 59.81+-1.0a and 44.30+-2.2ab do not have significant differences. Obvieosuly, those values have no overlaps between standard deviations and huge differences of average. The author used a t-test, which is a generally more rigorous test than others. I would recommend the authors double-check the statistic program before final submission.

This would not be accepted in the top 25% (Q1) of food science journals, but I can accept it now for this journal. However, it would be appreciated that the authors carefully check again before publication.