Antioxidant, Anti-Obesity, and Hypolipidemic Effects of Polyphenol Rich Star Anise (Illicium verum) Tea in High-Fat-Sugar Diet-Induced Obesity Rat Model

Round 1

Reviewer 1 Report

In the present study the authors investigate the effects of star anise tea (SAT) on oxidative stress, obesity and related biochemical parameters in high-fat-sugar-diet rat model; the authors also compared the effects of SAT with the effect of orlistat the rat model. They conclude that the treatment with SAT has beneficial effects by decreasing Body Mass Index, has a hipolypidemic effect, diminish oxidative stress marker by increase SOD activity, total antioxidant capacity and GSH levels. Also, SAT decrease the transaminases levels and kidney parameters.

The use of natural dietary to prevent metabolic diseases such as obesity, is currently eliciting considerable interest, but there are some details that must be reviewed by the authors:

1. Did the authors evaluate the effect of the treatments in control rats? I suggest they must include: NC+ SAT250; NC+SAT500 and NC+orlistat to show the effect in healthy rats (as a negative controls); it would be important to show the data in each table and discuss the results.

2. What is the rationale for the dose selected for SAT?

3. Why did the authors choose orlistat to compare the effect of SAT and did not use a natural compound? It would desirable that the authors evaluate the SAT effect on the inhibition of gastric and pancreatic lipases. Moreover, there are many reports regarding the pleiotropic effect of orlistat. Please include a paragraph in discussion section for each result about the effect of orlistat.

3. The authors must report the amount of visceral adipose tissue of each experimental group and not only the BMI index.

4. Did the authors evaluate the fatty acid contents in their SAT? Despite the extract has a high content of polyphenols, other significant components such as fatty acids (linoleic, oleic and palmitic) could contribute to the beneficial effect that the authors describe in their work. Therefore, this reviewer recommend do not attribute the entire effect to polyphenols due that the authors did not administrated only the polyphenols of SAT. Even, the title of their manuscript could be SAT extract instead polyphenols.

5. It would be desirable that the authors performed some histological stains to show the fatty acid accumulation in liver.

6. Please substantiate the hypothesis to explain the SAT effect on the decrease the levels of LDL cholesterol.

7. Please improve the figures, specially figure 2.

7. The discussion section must be improved.

8. There are many typing errors in text and tables that should be corrected.

9. Please is very important that the authors check and unify the abbreviations throughout the text. Please define abbreviations only the first time that they are used.

Author Response

The manuscript has been carefully revised in all aspects. A number of additional improvements have been made, regarding grammar, clarity and language. All the new changes/amendments have been marked by blue color.

 

Reviewer 1

General Comment: In the present study the authors investigate the effects of star anise tea (SAT) on oxidative stress, obesity and related biochemical parameters in high-fat-sugar-diet rat model; the authors also compared the effects of SAT with the effect of orlistat the rat model. They conclude that the treatment with SAT has beneficial effects by decreasing Body Mass Index, has a hipolypidemic effect, diminish oxidative stress marker by increase SOD activity, total antioxidant capacity and GSH levels. Also, SAT decrease the transaminases levels and kidney parameters.

The use of natural dietary to prevent metabolic diseases such as obesity, is currently eliciting considerable interest, but there are some details that must be reviewed by the authors:

Comment: Did the authors evaluate the effect of the treatments in control rats? I suggest they must include: NC+ SAT250; NC+SAT500 and NC+orlistat to show the effect in healthy rats (as a negative controls); it would be important to show the data in each table and discuss the results.

Response: The aim of the present research work was to investigate and validate the potential of polyphenol rich star anise tea (SAT) on oxidative stress, obesity and related biochemical parameters in high-fat-sugar-diet (HFSD) induced obesity model in rats. Therefore following groups were created; Normal Control (NC) group [Received normal feed (approx. 20 g/rat/day)], High fat diet Control (HFDC) group [Received HFD (approx. 20g /rat/day)], Positive Control (PC) group [Received HFD (approx. 20g /rat/day) plus orlistat 250 mg/kg Body Weight/day for 28 days], SA-250 group [Received HFD (approx. 20g/rat/day) supplemented with star anise extract (250 mg/kg BW/day for 28 days)] and SA-500 group [Received HFD (approx. 20g/rat/day) supplemented with star anise extract (500 mg/kg BW/day for 28 days)]. The study showed the protective effects of treatment groups against obesity and normal control group was taken to investigate the effectiveness of the obesity model (high fat and sugar diet). This is the reason that effect of treatments on the normal rats was not added in the manuscript.  

Comment: What is the rationale for the dose selected for SAT?

Response: The toxicity study was performed and the effective doze was calculated and added in the manuscript.

Comment: Why did the authors choose orlistat to compare the effect of SAT and did not use a natural compound? It would desirable that the authors evaluate the SAT effect on the inhibition of gastric and pancreatic lipases. Moreover, there are many reports regarding the pleiotropic effect of orlistat. Please include a paragraph in discussion section for each result about the effect of orlistat.

Response: Different practices are in use to control obesity related issues, including utilization of synthetic drugs like orlistat. Currently, the only medication authorized by European Medicines Agency (EMA) for the treatment of chronic obesity is orlisat has also been associated with side effects such as gastrointestinal reactions, oily spotting, abdominal cramps and liquid stools (Rufino et al., 2020; Shang et al., 2020). Moreover, its pharmacological effects rely on the patients' compliance to certain dietary restrictions, limiting its efficacy (Rufino et al., 2020). More data on the effect of orlistat is added in the introduction and also in the discussion section.  

[Rufino A.T., Costa, V.M., Carvalho, F., Fernandes, E. (2020). Flavonoids as antiobesity agents: A review. Medicinal Research Reviews, 41(1), 556-585.]

[Shang, A.; Gan, R.Y.; Xu, X.Y.; Mao, Q.Q.; Zhang, P.Z.;  Li, H.B. Effects and mechanisms of edible and medicinal plants on obesity: an updated review. Crit. Rev. Food Sci. Nutr. 2020, 61(12), 2061-2077.]

Comment: The authors must report the amount of visceral adipose tissue of each experimental group and not only the BMI index.

Response: Percentage of body weight gain and body mass index (BMI) was measured as indicators of obesity as reported previously (Rodriguez-Peres et al., 2019; Iftikhar et al., 2022). Moreover the kidney index (KI) and liver index (LI) were calculated and reported. The biochemical biomarkers including total cholesterol (TC) and triglyceride (TGL), High-density Lipoprotein (HDL), Low-density Lipoprotein (LDL) and Very Low-Density Lipoprotein (VLDL) cholesterols were estimated. Furthermore, both non-enzymatic and enzymatic defense against oxidative stress were studied by measuring reduced glutathione (GSH) and superoxide dismutase (SOD) levels and the total antioxidant activity was determined by estimating total antioxidant capacity (TAC). Serum creatinine and alkalinephosphate levels were checked to access the kidney function. Serum Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), bilirubin total (BT) levels were measured to evaluate the liver function along with the histology. The visceral adipose tissue was not determined in this study.

[Rodríguez-Pérez C, Segura-Carretero A, del Mar Contreras M. Phenolic compounds as natural and multifunctional anti-obesity agents: A review. Critical reviews in food science and nutrition 2019;59(8):1212-1229.]

[Iftikhar N, Hussain AI, Chatha SAS, Sultana N, Rathore HA. Effects of polyphenol‐rich traditional herbal teas on obesity and oxidative stress in rats fed a high‐fat–sugar diet. Food Science & Nutrition 2022;10(3):698-711.]

Comment:  Did the authors evaluate the fatty acid contents in their SAT? Despite the extract has a high content of polyphenols, other significant components such as fatty acids (linoleic, oleic and palmitic) could contribute to the beneficial effect that the authors describe in their work. Therefore, this reviewer recommend do not attribute the entire effect to polyphenols due that the authors did not administrated only the polyphenols of SAT. Even, the title of their manuscript could be SAT extract instead polyphenols.

Response: Fatty acid composition was determined on GC-FID but not included in the study due to the minor concentration (due to the hydrophobic nature of the oil). Authors agreed with the suggestion of the reviewer and revised the title and the concerned part of the manuscript. The star anise polyphenol is now replaced with the polyphenol rich star anise tea in the title and the manuscript. Due to the polar nature of polyphenols, the water extract/tea has rich concentration of polyphenols.

Comment:  It would be desirable that the authors performed some histological stains to show the fatty acid accumulation in liver.

Response: Primary objective of the study was to appraise and validate the potential of polyphenol rich star anise tea (SAT) on oxidative stress, obesity and related biochemical parameters in high-fat-sugar-diet (HFSD) induced obesity model in rats. We added histopathological findings in the study as preliminary findings to see impact of high fat diet on kidney and liver. We observe insignificant changes of HFD on liver and kidney and could not proceed with histopathology. However, we liked the idea of the reviewer and will include histopathological findings of liver and kidney in HFD model of metabolic syndrome.

 

Comment: Please substantiate the hypothesis to explain the SAT effect on the decrease the levels of LDL cholesterol.

Response: The discussion section is improved in the manuscript and elaborated. More discussion is added in the section 3.3.2.

Comment:  Please improve the figures, specially figure 2.

Response: Corrections have been made.

Comment: The discussion section must be improved.

Response: Discussion section is improved and more detail is added along with more literature;

[18] Sarikahya NB, Varol E, Okkali GS, Yucel B, Margaoan R, Nalbantsoy A. Phenolic profile and chemical content of propolis samples in different colors from Turkiye.  Antioxiants 2022; 10, 2075.

[33] Haque RA, Hasanudin N, Iqbal MA, Ahmad A, Hashim S, Abdul Majid A, Ahamed MBK. Synthesis, crystal structures, in vitro anticancer, and in vivo acute oral toxicity studies of bis-imidazolium/benzimidazolium salts and respective dinuclear Ag(I)-N-heterocyclic carbene complexes. Journal of Coordination Chemistry 2013; 66(18): 3211-3228.

[42] Vamanu E. Polyphenolic nutraceuticals to combat oxidative stress through microbiota modulation. Frontiers in Pharmacology 2019; 10: 492.

 

Comment:  There are many typing errors in text and tables that should be corrected.

Response: Corrections have been made.

Comment:  It is very important that the authors check and unify the abbreviations throughout the text. Please define abbreviations only the first time that they are used.

Response: Corrections have been made.

Reviewer 2 Report

Dear authors,

After the review process, I have several comments: in the introduction (line 50), you should include more findings related to the link between obesity and microbiota dysbiosis; also, you are strongly encouraged to present details that refer to the polyphenolic nutraceuticals to combat oxidative stress through microbiota modulation; the lend of the figures should be placed after the image; in the case of figure 3, you should mention how was realized, in the legend; also, in the discussion, because you tested products from the plant, you should add comment their results realized correlations between microbiota bioactivity and bioavailability of functional compounds.

Best regards!

Author Response

The manuscript has been carefully revised in all aspects. A number of additional improvements have been made, regarding grammar, clarity and language. All the new changes/amendments have been marked by blue color.

Reviewer 2

After the review process, I have several comments: in the introduction (line 50), you should include more findings related to the link between obesity and microbiota dysbiosis; also, you are strongly encouraged to present details that refer to the polyphenolic nutraceuticals to combat oxidative stress through microbiota modulation; the lend of the figures should be placed after the image; in the case of figure 3, you should mention how was realized, in the legend; also, in the discussion, because you tested products from the plant, you should add comment their results realized correlations between microbiota bioactivity and bioavailability of functional compounds.

Response: The introduction section in improved and more finding was added from literature to link the obesity, polyphenmols and the microbiota and the role of polyphenols against oxidative stress. Discussion section is also improved with following updated literature as well;

Rufino A.T., Costa, V.M., Carvalho, F., Fernandes, E. (2020). Flavonoids as antiobesity agents: A review. Medicinal Research Reviews, 41(1), 556-585.

Shang, A.; Gan, R.Y.; Xu, X.Y.; Mao, Q.Q.; Zhang, P.Z.;  Li, H.B. Effects and mechanisms of edible and medicinal plants on obesity: an updated review. Crit. Rev. Food Sci. Nutr. 2020, 61(12), 2061-2077.

 Sarikahya NB, Varol E, Okkali GS, Yucel B, Margaoan R, Nalbantsoy A. Phenolic profile and chemical content of propolis samples in different colors from Turkiye.  Antioxiants 2022; 10, 2075.

Haque RA, Hasanudin N, Iqbal MA, Ahmad A, Hashim S, Abdul Majid A, Ahamed MBK. Synthesis, crystal structures, in vitro anticancer, and in vivo acute oral toxicity studies of bis-imidazolium/benzimidazolium salts and respective dinuclear Ag(I)-N-heterocyclic carbene complexes. Journal of Coordination Chemistry 2013; 66(18): 3211-3228.

Vamanu E. Polyphenolic nutraceuticals to combat oxidative stress through microbiota modulation. Frontiers in Pharmacology 2019; 10: 492.

 

The lend of the figures are placed at correct position. The discussion section is now improved with updated literature.   

 

Round 2

Reviewer 1 Report

I evaluated the revised version of the manuscript entitled: “Antioxidant, Anti-obesity, and Hypolipidemic Effects of Polyphenol Rich Star Anise (Illicium verum) Tea in High-Fat-Sugar Diet-induced Obesity Rat Model”.

The authors have satisfactorily addressed the concerns of this Reviewer and made the necessary changes to the manuscript.

Reviewer 2 Report

No other comments