The Anti-Inflammatory Effects of Broccoli (Brassica oleracea L. var. italica) Sprout Extract in RAW 264.7 Macrophages and a Lipopolysaccharide-Induced Liver Injury Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. This study was performed using powdered broccoli sprouts, did the authors compared the quantity of sulforaphane obtained from fresh broccoli sprouts?

2. Quality and resolution of Figure 1 should be improved. The axis and labels can be made bigger.

3. Improve the resolution and font size of the axis and labels for Figure 2. The scale bar although present for the cell images, the size is not indicated in the figure legend.

4. The concentrations of BSE should be indicated in ug/mL instead of % throughout all the results. This makes interpretation of the results clearer for the readers. 

5. The ladder and the MW size is missing for the western blot images. Figure 3 and 5.

6. For all figures, increase the font size for the axis and labels. It is difficult to read. Quality and resolution of the images should also be improved. 

7. Figure legend for Figure 5 is incorrectly labeled. The scale bar size is missing in the descriptions.

8. Apart from retention time, did the authors use other methods to support the identification of sulforaphane from their water and ethanolic extracts?

 

 

Comments on the Quality of English Language

Authors should edit the writing of their manuscript to improve its clarity and scientific soundness.

Author Response

1) Comments and Suggestions for Authors

1. This study was performed using powdered broccoli sprouts, did the authors compared the quantity of sulforaphane obtained from fresh broccoli sprouts?

A: Thank you for your comment`s Powdered broccoli sprouts is pure freeze-dried broccoli sprouts turned into powder. We used that one for experimental consistency.

2. Quality and resolution of Figure 1 should be improved. The axis and labels can be made bigger.

A: Resolution of Figure 1 was improved, and replaced

3. Improve the resolution and font size of the axis and labels for Figure 2. The scale bar although present for the cell images, the size is not indicated in the figure legend.

A: Resolution of Figure 1 was improved and replaced. We provided scale bar in Figure 2 legend

4. The concentrations of BSE should be indicated in ug/mL instead of % throughout all the results. This makes interpretation of the results clearer for the readers. 

A: Yes. We agreed that. We replaced according to reviewer`s comments.

5. The ladder and the MW size is missing for the western blot images. Figure 3 and 5.

A: Yes. We added.

6. For all figures, increase the font size for the axis and labels. It is difficult to read. Quality and resolution of the images should also be improved. 

A: Yes. We added and changed all image with high resolution.

7. Figure legend for Figure 5 is incorrectly labeled. The scale bar size is missing in the descriptions.

A: Thank you for your comment. we edited and corrected. Line: 385, 388

8. Apart from retention time, did the authors use other methods to support the identification of sulforaphane from their water and ethanolic extracts?

A: Thank you for your comment, For the identification of sulforaphane, we used retention time values from HPLC analysis and compared them to the standard compound. As is well known, HPLC can identify a compound by qualitative analysis, which compares the retention time of the peak in the extract with the standard compound in the presence of the standard compound. For more accurate identification, we used a photodiode array detector and confirmed that the retention time of the peak in the extract and the peak of the standard compound matched.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study examined the effects of sulforaphane-rich broccoli sprout extract (BSE) on inflammation, both in controlled laboratory settings (in vitro) and in living organisms (in vivo). Among the different BSE extracts tested, the one containing 70% ethanol was found to have the highest sulforaphane content. The results demonstrated that BSE significantly reduced the expression of pro-inflammatory cytokines and mediators in stimulated cells, leading to improved survival rates and reduced liver damage in mice with endotoxemia. Additionally, BSE was shown to decrease the infiltration of hepatic macrophages. These findings propose BSE as a promising nutraceutical for mitigating excessive immune responses in inflammatory diseases. However, there are several suggestions for improving the manuscript:

1. It is recommended to include a figure illustrating the impact of ethanolic extract with sulforaphane on pro-inflammatory cytokine production, with a focus on the NF-kB pathway. You can refer to Figure 9 in the paper with DOI: 10.3390/antiox10121941 as an example.

2. Please specify the number of replicates and trials conducted in the LPS assay to enhance experimental transparency.

3. It was observed that 0.1% BSE exhibited the highest anti-inflammatory effect. Have you had the chance to quantify the sulforaphane content in this extract?

4. In Figure 3, consider showing immunostaining data that illustrates the difference in fluorescence intensity between the cytoplasm and the nucleus as a result of inhibiting the nuclear translocation of NF-kB transcription factors.

5. To further elucidate the effects of sulforaphane, it is suggested to perform molecular docking experiments to demonstrate the binding affinity of this compound to crucial enzymes involved in the NF-kB pathway. Reference these studies for the protocol: DOI: 10.1016/j.bmc.2012.10.051 and DOI: 10.3390/nu15040883.

6. Enrich the discussion by citing relevant studies that explore the inflammatory-modulating potential of native plants like Uvaria alba, which has shown inhibition of the NF-kB pathway and PDE enzyme while upregulating the cytoprotective NRF2 cascade. Please reference the following studies: DOI: 10.1021/acsomega.2c06451 and DOI: 10.1021/acsomega.1c00137.

7. Discuss the limitations of the current study and propose future experiments that could address these limitations, providing a more comprehensive view of the research's scope and potential areas for further investigation.

 

Comments on the Quality of English Language

Minor english editing is required.

Author Response

2) Comments and Suggestions for Authors

This study examined the effects of sulforaphane-rich broccoli sprout extract (BSE) on inflammation, both in controlled laboratory settings (in vitro) and in living organisms (in vivo). Among the different BSE extracts tested, the one containing 70% ethanol was found to have the highest sulforaphane content. The results demonstrated that BSE significantly reduced the expression of pro-inflammatory cytokines and mediators in stimulated cells, leading to improved survival rates and reduced liver damage in mice with endotoxemia. Additionally, BSE was shown to decrease the infiltration of hepatic macrophages. These findings propose BSE as a promising nutraceutical for mitigating excessive immune responses in inflammatory diseases. However, there are several suggestions for improving the manuscript:

1. It is recommended to include a figure illustrating the impact of ethanolic extract with sulforaphane on pro-inflammatory cytokine production, with a focus on the NF-kB pathway. You can refer to Figure 9 in the paper with DOI: 10.3390/antiox10121941 as an example.

A: Thank you for your comment`s. we reflected to our graphic abstract.

1. Please specify the number of replicates and trials conducted in the LPS assay to enhance experimental transparency.

A: We performed that minimum over the three biological and three experimental (Technical) replications. We described in material and method section.  Line: 228-229

1. It was observed that 0.1% BSE exhibited the highest anti-inflammatory effect. Have you had the chance to quantify the sulforaphane content in this extract?

A: We did not measure but we can calculate based on HPLC result. Probably. 50-60 mg of Sulforaphane per 10g BSE which we extracted and dried.

1. In Figure 3, consider showing immunostaining data that illustrates the difference in fluorescence intensity between the cytoplasm and the nucleus as a result of inhibiting the nuclear translocation of NF-kB transcription factors.

A: Thank you for your comment`s. we finished this project almost two years ago, and currently we don`t have this extract anymore for additional experiment. Please, considered this situation.

5. To further elucidate the effects of sulforaphane, it is suggested to perform molecular docking experiments to demonstrate the binding affinity of this compound to crucial enzymes involved in the NF-kB pathway. Reference these studies for the protocol: DOI: 10.1016/j.bmc.2012.10.051 and DOI: 10.3390/nu15040883.

A: According to our manuscripts, we used BSE for experiment. Of course, this extract included sulforaphane (Figure 1) but also maybe other functional materials are also in there. In the other hands, BSE is no single compound. So, it is impossible to perform molecular docking using BSE

1. Enrich the discussion by citing relevant studies that explore the inflammatory-modulating potential of native plants like Uvaria alba, which has shown inhibition of the NF-kB pathway and PDE enzyme while upregulating the cytoprotective NRF2 cascade. Please reference the following studies: DOI: 10.1021/acsomega.2c06451 and DOI: 10.1021/acsomega.1c00137.

A: Thank you for your comment`s and suggestion. We additionally described in discussion part according to your comment and references which reviewer provided. Line: 456-461

1. Discuss the limitations of the current study and propose future experiments that could address these limitations, providing a more comprehensive view of the research's scope and potential areas for further investigation.

 A: Thank you for your comment. we agreed that and added in discussion part. Line: 479-486

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The paper has been revised and can now be accepted for publication.