Effect of Bergamot Leaves (Citrus bergamia) in the Crosstalk between Adipose Tissue and Liver of Diet-Induced Obese Rats

Round 1

Reviewer 1 Report

This article is interesting because insulin resistance is a serious problem for many patients in the world. Unfortunately, the authors should clarify which component of the bergamot extract definitely affects this health problem. The extract contains flavonoids, vitamin C, melittin and brutieridin and we do not know what works. Maybe it's worth doing research with individual compounds and comparing with the effect of the entire extract.

Author Response

Dear reviewer, thank you for your feedback on our study. We appreciate your comment regarding specific compounds identification responsible for the extract beneficial action. However, we would like to clarify that the primary aim of our study is not to identify the individual compounds responsible for the observed effect, but rather to observe the synergism of all the compounds. Our focus is to investigate the overall extract effect and its potential as a therapeutic agent, rather than on identifying specific compounds. We believe that our approach provides a more comprehensive understanding of the extract's beneficial properties and its potential as a nutraceutical and potential as natural therapeutic. For sure, the identification and bioavailability of the compounds is essential for understanding the extract, so this will be our next step in further studies.

Reviewer 2 Report

The research content is not complex, and the experimental technology is also very traditional, but the application of matrix diagrams to display correlation analysis is relatively novel.

I have several questions:

1. Does visceral fat include perirenal fat and mesenteric fat? Why use epididymal fat instead of visceral fat when studying adipocytokines and oxidative stress?

2. Please show the liver HE.

3. Why only three groups of data are compared, while the experimental method describes four groups.

4. What are the effective components of bergamot leaves extract?

Author Response

1. Does visceral fat include perirenal fat and mesenteric fat? Why use epididymal fat instead of visceral fat when studying adipocytokines and oxidative stress?

R: Dear Reviewer, we considered visceral fat as mesenteric fat and retroperitoneal as perirenal fat. Epididymal fat is often used in scientific analysis and research for a few specific reasons:

The use of epididymal fat from Wistar rats is a preferred choice for analyses in scientific research due to its easy accessibility and availability in relatively pure amounts, ensuring consistency and reliability of analysis results. The unique lipid composition of epididymal fat, which includes long-chain fatty acids and high concentration of cholesterol, is relevant for certain types of analyses such as lipid metabolism, lipid transport and the inflammatory response. Epididymal fat is commonly used in research related to obesity, diabetes, and inflammation. It is easily accessible and available in relatively pure amounts, important to ensure consistency and reliability of analysis results.

Oliveira, C. A., Lopes, L. F., Biancardi, M. F., de Paula, F. J., & de Carvalho, M. H. (2019). Comparative study of the epididymal adipose tissue of Wistar rats fed with different high-fat diets. Brazilian journal of medical and biological research, 52(2), e7735. https://doi.org/10.1590/1414-431X20187735

Heilbronn, L. K., & Ravussin, E. (2003). Adipose tissue metabolism and inflammation in obesity. Inflammatory Research, 52(2), 199-205. https://doi.org/10.1007/s00011-003-1186-7

2. Please show the liver HE.

R: Dear reviewer, we have included the liver HE figures in the manuscript (Figure 1, page 6).

3. Why only three groups of data are compared, while the experimental method describes four groups.

R: Dear reviewer, previous studies conducted by our research group using the same experimental model of diet-induced obesity with four groups did not show any toxicity in the control group supplemented with bergamot extract at the same time that was not observed any other beneficial effect. Therefore, we opted to use only 3 groups. We apologize for the typing error in the methodology, which has already been corrected and updated. The changes are highlighted in yellow (Page 3, line 105).

4. What are the effective components of bergamot leaves extract?

R: Dear reviewer, the qualitative and semi-quantitative profile of the bergamot leaf extract was already carried out and published by one of the authors of the present manuscript. However, the authors believe that the introduction was a bit too general. For this reason, we have included a brief description of the extract's composition and its beneficial health implications in the introduction (Page 2, line 16) and discussion (Page 11, line 330; Page 11, line 359).

Baron, G.; Altomare, A.; Mol, M.; Garcia, J.L.; Correa, C.; Raucci, A.; Mancinelli, L.; Mazzotta, S.; Fumagalli, L.; Trunfio, G.; et al. Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave. Antioxidants 2021, 10, 141, doi:10.3390/antiox10020141. 

Reviewer 3 Report

Dear author, 

i have highlighted some minor changes but not limited to these, you can recheck you article for mistakes like these.

Comments for author File: Comments.pdf

Author Response

1. I have highlighted some minor changes but not limited to these, you can recheck you article for mistakes like these.

R: Dear reviewer, thank you for your considerations on our manuscript. The grammar of the article has been revised and updated.

Reviewer 4 Report

 

After reading the article Effect of Bergamot Leaves (Citrus bergamia) in the crosstalk between adipose tissue/liver of diet-induced obese rats, I found the results they obtained interesting. However, I have some doubts:

1. Why didn't you add the results of the control+BLE group?

2. Why did they not include a reference drug?

3. Did they carry out experiments to see the chemical composition?

 

4. Why didn't you try more doses?

Author Response

1. Why didn't you add the results of the control+BLE group?

R: Dear reviewer, previous studies conducted by our research group using the same experimental model of diet-induced obesity with four groups did not show any toxicity in the control group supplemented with bergamot extract at the same time that was not observed any other beneficial effect. Therefore, we opted to use only 3 groups.

2. Why did they not include a reference drug?

R: Our research aims to investigate the potential benefits and therapeutic properties of the bergamot extract, particularly those derived from the leaf. As such, we chose to focus on evaluating the efficacy of the extract rather than comparing it to a reference drug. Furthermore, including a reference drug in the study would require additional experimental groups and animals, which would raise ethical concerns regarding animal welfare.

3. Did they carry out experiments to see the chemical composition?

R: Dear reviewer, the qualitative and semi-quantitative profile of the bergamot leaf extract was already carried out and published by one of the authors of the present manuscript.

Baron, G.; Altomare, A.; Mol, M.; Garcia, J.L.; Correa, C.; Raucci, A.; Mancinelli, L.; Mazzotta, S.; Fumagalli, L.; Trunfio, G.; et al. Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave. Antioxidants 2021, 10, 141, doi:10.3390/antiox10020141. 

4. Why didn't you try more doses?

R: The dose was established according to literature data that used this concentration from fruit juice in an experimental model of non-alcoholic fatty liver disease and based on previously studies already published by our research group. Futhermore, the dose of 50mg/kg of animal body weight can be easily extrapolated to an adult of 70kg, resulting in a total tose of 3.5g, which can be easily consumed and encapsulated, making it a viable option for future researches involving supplementation in adults.

Musolino, V., Gliozzi, M., Scarano, F. et al. Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease.Sci Rep 10, 2565 (2020). https://doi.org/10.1038/s41598-020-59485-3

Lu, K.; Yip, Y.M. Therapeutic Potential of Bioactive Flavonoids from Citrus Fruit Peels toward Obesity and Diabetes Mellitus. Futur. Pharmacol. 2023, 3, 14–37, doi:10.3390/futurepharmacol3010002.

Palacio, T.L.N.; Siqueira, J.S.; de Paula, B.H.; Rego, R.M.P.; Vieira, T.A.; Baron, G.; Altomare, A.; Ferron, A.J.T.; Aldini, G.; Kano, H.T.; et al. Bergamot ( Citrus Bergamia ) Leaf Extract Improves Metabolic, Antioxidant and Anti-Inflammatory Activity in Skeletal Muscles in a Metabolic Syndrome Experimental Model. Int. J. Food Sci. Nutr. 2022, 1–8, doi:10.1080/09637486.2022.2154328.

Siqueira, J.S.; Vieira, T.A.; Nakandakare-Maia, E.T.; Palacio, T.L.N.; Sarzi, F.; Garcia, J.L.; de Paula, B.H.; Bazan, S.G.Z.; Baron, G.; Tucci, L.; et al. Bergamot Leaf Extract Treats Cardiorenal Metabolic Syndrome and Associated Pathophysiological Factors in Rats Fed with a High Sugar Fat Diet. Mol. Cell. Endocrinol. 2022, 556, 111721, doi:10.1016/j.mce.2022.111721.

Nakandakare-Maia, E.T.; Siqueira, J.S.; Ferron, A.J.T.; Vieira, T.A.; Palacio, T.L.N.; Grandini, N.A.; Garcia, J.L.; Belin, M.A.; Altomare, A.; Baron, G.; et al. Treatment with Bergamot (Citrus Bergamia) Leaves Extract Attenuates Leptin Resistance in Obese Rats. Mol. Cell. Endocrinol. 2023, 111908, doi:10.1016/j.mce.2023.111908.

 

Round 2

Reviewer 2 Report

The research content of this article is not complex, but starting from the dialogue between adipose tissue and liver, the entry point is very novel, and the application of matrix diagrams to display relevant analysis in the study is also very enlightening. The author induced obesity in rats through a high sugar diet, demonstrating that Bergamot Leaves extract can act on the inflammation and oxidative stress in the adipose tissue/liver axis in obese rates. At the same time, the author carefully answered the reviewer's questions. I think it is a worthwhile article to publish. But there is still a question: why choose a high sugar diet instead of a high fat diet to induce obesity models?

Author Response

Dear Reviewer,
Thank you for your feedback. We appreciate your comments regarding the choice of the "high sugar-fat" diet in our study. We chose to include sugar in the diet to mimic the Western diet, which is known for its high consumption of both sugar and fat. By including sugar in the diet, we aimed to replicate the effects of excessive consumption of these two components.Furthermore, the inclusion of sugar in the diet also allowed us to investigate the synergistic effects of these two nutrients when consumed together, as the combination of sugar and fat may lead to different metabolic and physiological effects compared to the isolated consumption of fat. We believe that this choice of diet allowed us to investigate the potential impact of excessive sugar and fat intake on the outcomes of our study.

Reviewer 4 Report

the authors responded to all comments

Author Response

Thank you for your feedback.