Antidiabetic and Antihyperlipidemic Activities and Molecular Mechanisms of Phyllanthus emblica L. Extract in Mice on a High-Fat Diet

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To understand if the Phyllanthus emblica L. extract has beneficial effects on diabetes and high lipidic associated diseases, the authors performed quite interesting assays not only to search biochemical changes when this extract is administrated to an animal model of these conditions, but also tried to understand the mechanisms of action of the extract that possibly explain the biochemical results. Here are some doubts and suggestions that will help to improve the work:

-           The title of the article is too long. If the authors want the article to be more appealing, I advise you to shorten it. The length of the abstract should also be reduced.

-           In the abstract it is stated that “Our present results showed that HFD-induced mice exhibited hyperglycemia, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia, higher blood levels of glycosylated HbA1C, and higher blood urea nitrogen (BUN) and plasma creatinine concentrations than those of vehicle-treated HFD mice”. In this context, does the statement “…than those of vehicle-treated HFD mice” is correct or did you want to compare with EPE treated mice?

-           Regarding the first sentence of the introduction section, the appropriate reference must be added.

-           In the introduction section, the meaning of the abbreviation AMPK appears two times with two different meanings. The meaning of the abbreviation NOD in paragraph 11 is also not provided. The meaning of HFD also appears two times. Please correct it.

-           Along all introduction section, a quite extensive description of receptors, proteins/enzymes and possible molecules that play an important role in type 2 diabetes and insulin resistance is performed. However, I’ve noted a lack of rational interconnection between all the presented information making it difficult for readers to make a logical and interrelated reasoning of all the ideas. Thus, I advise you to revise the introduction section and present the information in a more summarized and interconnected way.

-           Were fenofibrate and metformin oral solutions prepared in water? Please also explain the choice of the in vivo tested doses.

-           What was the time between the last administration and animals’ sacrifice? Have urine samples been collected right after the last administration? And what was the blood volume collected for further analysis? And does that volume have been enough for all the reported measurements?

-           How do the authors explain that HF mice gained more body weight but consumed less food than control group?

-           Figure 3B shows that body weight gain of HFD treated mice was lower than the of control (not treated and subjected to low fat diet) mice. Does it mean that the extract has weight loss properties like those already observed for fenofibrate and metformin?

-           The title of the section 3.2.2 should be changes since in this section results of the urine analysis is also presented

-           - Figure 6D also shows a high concentration of a compound in EA-4 with a retention time of 22.267 min. Which compound is it? Is it possible that this unknown compound could also be responsible for the best activity of EA-4 in phospo-AMPK7/total-AMPK?

-           It would be interesting to also study the effects of the extract in GLUT4 expression in adipose tissue and compare with the expression in muscle tissue. Is it possible for the authors to perform that test?

 

Comments on the Quality of English Language

The grammar and language can be improved in some cases.There is also a lack of connection between some sentences that in some cases makes it difficult to understand the pretended message

 

Author Response

Outline the changes made:

 

Reviewer: 1

1. As Reviewer suggests, “The title of the article is too long. If the authors want the article to be more appealing, I advise you to shorten it. The length of the abstract should also be reduced.”

And revised into

----> Title:

Antidiabetic and Antihyperlipidemic Activities and Molecular Mechanisms of Phyllanthus emblica L. Extract in Mice on a High-Fat Diet

 

Abstract: We planned to explore the protective activities of extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders including hyperlipidemia, visceral obesity, and renal dysfunction in high-fat diet (HFD)-progressed T2DM mice. Mice treatments included 7 weeks of HFD induction followed by EPE, fenofibrate (Feno), or metformin (Metf) treatment daily for another 4-week HFD in HFD-fed mice. Finally, we harvested blood to analysis some tests on circulating glycemia and blood lipid levels. Western blotting analysis was performed on target genes expressions in peripheral tissues. The present findings indicated that EPE treatment reversed the HFD-induced increases in blood glucose, glycosylated HbA1_C, and insulin levels. Our findings proved that treatment with EPE in HFD mice effectively controls hyperglycemia and hyperinsulinemia. Our results showed that EPE reduced blood lipid levels, including a reduction in blood TG, TC, and FFA; moreover, EPE reduced blood leptin levels and enhanced adiponectin concentrations. EPE treatment in HFD mice reduced BUN and creatinine in both blood and urine, and lowered albumin levels in urine; moreover, EPE decreased circulating concentrations of inflammatory NLRP3 and KIM-1. These results indicated that EPE displayed antihyperglycemic, antihyperlipidemic activities but alleviated renal dysfunction in HFD mice. The histology examinations indicated that EPE treatment decreased adipose hypertrophy and hepatic ballooning, thus contributing to amelioration of lipid accumulation. EPE treatment decreased visceral fat amounts and led to improve systemic insulin resistance. For target gene expression levels, EPE enhanced AMPK phosphorylation expressions both in livers and skeletal muscles, and elevated the muscular membrane GLUT4 expressions. Treatment with EPE reduced hepatic G6Pase and PEPCK expressions to suppress glucose production in the livers and decreased phosphorylation of GSK3β expressions to affect hepatic glycogen synthesis, thus convergently contributing to an antidiabetic effect and improving insulin resistance. The mechanism of the antihyperlipidemic activity of EPE involved a decrease in the hepatic phosphorylation of mTORC1 and p70S6K1 expressions to improve insulin resistance but also a reduction in hepatic SREBP-1c expressions, and suppression of ACC activity, thus resulting in the decreased fatty acid synthesis but elevated hepatic PPARα and SREBP-2 expressions, resulting in lowering TG and TC concentrations. Our results demonstrated that EPE improves insulin resistance and ameliorates hyperlipidemia in HFD mice.

 

 

2. As Reviewer suggests, “In the abstract it is stated that “Our present results showed that HFD-induced mice exhibited hyperglycemia, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia, higher blood levels of glycosylated HbA1C, and higher blood urea nitrogen (BUN) and plasma creatinine concentrations than those of vehicle-treated HFD mice”. In this context, does the statement “…than those of vehicle-treated HFD mice” is correct or did you want to compare with EPE treated mice?”

And revised into

----> The present findings indicated that HFD-induced animals exhibited higher blood glucose, TC, TG, glycosylated HbA1_C, and insulin levels, and higher blood urea nitrogen (BUN) and plasma creatinine concentrations than those of CON mice (the control mice).

 

 

3. As Reviewer suggests, “Regarding the first sentence of the introduction section, the appropriate reference must be added. “

And revised into

----> The 10^th revised statistical epidemiological analysis of International Diabetes Federation prophesied that incidence of diabetes mellitus may estimate 783 million by 2045 [1] ….

[1]  Diabetes now affects one in 10 adults worldwide. International Diabetes Federation 2021.

 

 

4. As Reviewer suggests, “In the introduction section, the meaning of the abbreviation AMPK appears two times with two different meanings. The meaning of the abbreviation NOD in paragraph 11 is also not provided. The meaning of HFD also appears two times. Please correct it. “

----> (1) AMPK: activation of AMP-activated protein kinase (AMPK) [3,8]. AMPK plays a target in the remedy of T2DM [3].

     (2) NOD: in two non-obese diabetes (NOD)-induced

     (3) HFD: C57BL/6J mice fed a high-fat diet (HFD)

And others have been corrected

 

 

5. As Reviewer suggests, “Along all introduction section, a quite extensive description of receptors, proteins/enzymes and possible molecules that play an important role in type 2 diabetes and insulin resistance is performed. However, I’ve noted a lack of rational interconnection between all the presented information making it difficult for readers to make a logical and interrelated reasoning of all the ideas. Thus, I advise you to revise the introduction section and present the information in a more summarized and interconnected way.”

And revised into

----> Done. According to Reviewer 3, Introduction have been revised shorten and some mentioned in the discussion. Please see the manuscript.

 

 

6. As Reviewer suggests, “Were fenofibrate and metformin oral solutions prepared in water? Please also explain the choice of the in vivotested doses.”

And revised into

---->

For example, the dosage of Feno is 250 mg/kg bw, and preparation of Feno solution, calculation, and determination of choice concentrations in Feno-treated group was described as the following. To make the solution of dosage 250 mg/ body weight of Feno, for 100 g body weight mice, 25 mg of Feno and vehicle (ex: carboxymethylcellulose; CMC) were added to make the total volume of the solution up to 1 mL, that is, 2.5 g Feno were dissolved in 0.5% CMC to make the 100 mL total volume of the solution. Whenever administration of Feno according to the body weight of the mice at that day (if body weight is 30 g), we administered 0.3 mL Feno to the mice.

 

Calculation and preparation of the solution of dosage Feno (250 mg/g body weight):

25 mg/100 g body weight −> dissolved in 1 mL solvent, that is,

25: 1=X mg: 100 mL,

X = 25 x 100 (mg),

X= 2500/1000 (g),

X=2.5 (g), that is,

2.5g Feno −> dissolved in solvent (ex: CMC) to make up the 100 mL total volume of the solution.

Feno and Metf was dissolved in CMC solvent.

 

The dose of 10 mg/kg body weight/day for a mouse can be converted to a daily dose of about 52.64 mg for a 70 kg human subject, which is equivalent to about 0.752 mg/kg body weight/day, based on the guideline on estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers issued by U.S. Food and Drug Administration (FDA) in 2005.

To Convert Animal Dose in mg/kg to HED in mg/kg

HED= animal dose in mg/kg x (animal weight in kg/human weight in kg)^0.33

If mice = 27.52g bw

Human dose= 10 mg/kg x (27.52/1000/70)^0.33

Human dose (mg/kg) =10 x 0.0752 = 0.752

70kg human dose = 0.752 x70 =52.64

 

Fenofibrate (Feno; 250 mg/kg/day body wt, from Sigma Chemical Co, St. Louis, MI, USA)

The adapted dosage in the paper is according to Rai Ajit K. Srivastava, 2009. Fenofibrate ameliorates diabetic and dyslipidemic profiles in KKAy mice partly via down-regulation of 11β-HSD1, PEPCK and DGAT2. Comparison of PPARα, PPARγ, and liver x receptor agonists. European Journal of Pharmacology 2009, 258-263.

Therefore, we adapted dosage of Feno is 250/10  x 52.64 = 1316 mg/70kg above usual Feno ‘s daily dose =300mg; nevertheless, the tolerance of mice is better than human.

 

 

7. As Reviewer suggests, “What was the time between the last administration and animals’ sacrifice? Have urine samples been collected right after the last administration? And what was the blood volume collected for further analysis? And does that volume have been enough for all the reported measurements?”

And revised into

----> (1) The time of daily treatment drug is about AM 8:30~9:30. After treatment with EPE, Feno, or Metf for 28 days, the mice were sacrificed. The night (about PM 8:30~9:30) before sacrifice, food was removed and mice was fasting for 12-h. During the procedure, 100 μl blood sample was collected from the retro-orbital sinus of mice and for blood glucose levels analysis. At the day of sacrifice, 0.6-0.8 mL blood sample was collected from the posterior vena cava of mice, and then put it in the heparin tube, and followed by mixture, centrifugation at 3300 rpm for 10 minutes, and then the adapted 0.3-0.4 mL plasma for later analysis.

(2) After treatment with drugs, a single mouse was put in metabolic cab, and a 24-h urine sample was collected for analyses of renal function.

 

 

8. As Reviewer suggests, “How do the authors explain that HF mice gained more body weight but consumed less food than control group?”

And revised into

----> The HF mice consumed HFD, which is a high caloric diet and contains more fat, and therefore HFD mice gained more body weight. The Control mice consumed more food since CON mice hypothalamus did not recognize they already ate food (because the hypothalamus only recognize fat as they already ate food), and therefore hypothalamus constantly stimulated more adipocytokine such as ghrelin and CCK to eat more food.

 

 

9. As Reviewer suggests, “Figure 3B shows that body weight gain of HFD treated mice was lower than the of control (not treated and subjected to low fat diet) mice. Does it mean that the extract has weight loss properties like those already observed for fenofibrate and metformin?”

And revised into

----> Yes. Figure 3B shows that body weight gain of EPE-treated HFD mice was lower than the of control (not treated and subjected to low fat diet) mice. It means that the extract has weight loss properties like those already observed for fenofibrate and metformin.

 

 

10. As Reviewer suggests, “The title of the section 3.2.2 should be changes since in this section results of the urine analysis is also presented”

And revised into

----> 3.2.2. Blood Glucose, Biochemical Parameters, and Urine Analysis

 

11. As Reviewer suggests, “Figure 6D also shows a high concentration of a compound in EA-4 with a retention time of 22.267 min. Which compound is it? Is it possible that this unknown compound could also be responsible for the best activity of EA-4 in phospo-AMPK7/total-AMPK?”

And revised into

----> We did not analysis this compound (a retention time of 22.267 min), and could not make sure what is this compound.

Nevertheless, it is sure that the rich main component chebulagic acid in EPE is responsible for antihyperglycemic and antihyperlipidemic activities based on the results of HPLC analysis.

Since the high peak we can say this compound (a retention time of 22.267 min) could absorb UV, the peak is high but it doesn’t mean that its concentration is high.

 

 

12. As Reviewer suggests, “It would be interesting to also study the effects of the extract in GLUT4 expression in adipose tissue and compare with the expression in muscle tissue. Is it possible for the authors to perform that test?”

And revised into

----> Since the skeletal muscle plays the majority (75%) of glucose uptake [44], therefore we did not assess the GLUT4 expressions in adipose tissue and we merely analysis the membrane GLUT4 expressions in skeletal muscle. And we have no idea analysis “adipose” “membrane” GLUT4 expressions in mice.

 

 

13. As Reviewer suggests, “Comments on the Quality of English Language The grammar and language can be improved in some cases. There is also a lack of connection between some sentences that in some cases makes it difficult to understand the pretended message”

And the authors response---->

The manuscript had already improved by American Journal of Editing (AJE) Order ID: CLPMHL93) INVOICE # SCLPMHL93 (Services: - Standard Editing and – Standard Editing) in June 21 2023) We could improve on the Quality of English Language by CIMB Journal assistants and I will pay, and nevertheless at the present because other Reviewer’s comments are too many points to improve this point in time, please give me additional time, thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper titled “Antidiabetic and Antihyperlipidemic Activities of Phyllanthus emblica L. Extract by the Promotion of AMPK Activation and the Inhibition of Hepatic Gluconeogenesis and Fatty Acid Synthesis in Mice on a High-Fat Diet” investigates the protective effects and mechanisms of the ethyl acetate extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders in high-fat diet (HFD)-induced diabetic mice. The study demonstrates that EPE treatment effectively reduced blood glucose, insulin, glycosylated HbA1c, triglycerides, total cholesterol, and leptin levels, while increasing adiponectin levels. Additionally, EPE improved renal function and reduced markers of inflammation. Furthermore, EPE decreased visceral fat, adipose tissue hypertrophy, and hepatic lipid accumulation, enhancing insulin sensitivity. Overall, the study found that EPE exhibited significant antihyperglycemic, antihyperlipidemic, and antidiabetic effects, thereby improving the metabolic health of HFD-fed mice.

1.     In Figure 5, it appears that the stripe for the ladder has a background, raising concerns about whether the marker was run each time with other samples.

2.     Did the study adequately explore the dose-response relationship of EPE by including a sufficient range of dosages? How were the specific dosages of EPE selected, and are these doses relevant to potential human applications?

3.     Were appropriate control groups included to clearly distinguish the effects of EPE from those of the high-fat diet and standard treatments like metformin and fenofibrate? Specifically, were there vehicle-treated groups that received no EPE but were otherwise treated identically to the experimental groups?

 

4.      Is the 4-week treatment period sufficient to observe the long-term effects of EPE on insulin resistance, hyperlipidemia, and renal dysfunction? Should the study have included a longer follow-up period to assess the sustainability of the observed effects after the cessation of treatment?

Comments on the Quality of English Language

The quality of the paper is good, and no further questions are necessary

Author Response

Outline the changes made:

 

Reviewer: 2

1. As Reviewer suggests, “In Figure 5, it appears that the stripe for the ladder has a background, raising concerns about whether the marker was run each time with other samples.”

And revised into

----> Sample and marker ladder run together, because marker ladder did not have Chemiluminescent effects. In the present experiments, all samples react with antibodies, and then react with Chemiluminescent to form images, and then the images were taken with white light, and therefore the backgrounds have color differences.

 

 

2. As Reviewer suggests, “Did the study adequately explore the dose-response relationship of EPE by including a sufficient range of dosages? How were the specific dosages of EPE selected, and are these doses relevant to potential human applications? ”

And revised into

----> (1) Yes. This study contains three dosages of EPE.

(2) No specific dosages of EPE selected.

(3) The dose of 10 mg/kg body weight/day for a mouse can be converted to a daily dose of about 52.64 mg for a 70 kg human subject, which is equivalent to about 0.752 mg/kg body weight/day, based on the guideline on estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers issued by U.S. Food and Drug Administration (FDA) in 2005.

To Convert Animal Dose in mg/kg to HED in mg/kg

HED= animal dose in mg/kg x (animal weight in kg/human weight in kg)^0.33

If mice = 27.52g bw

Human dose= 10 mg/kg x (27.52/1000/70)^0.33

Human dose (mg/kg) =10 x 0.0752 = 0.752

70kg human dose = 0.752 x70 =52.64

 

Fenofibrate (Feno; 250 mg/kg/day body wt, from Sigma Chemical Co, St. Louis, MI, USA)

The adapted dosage in the paper is according to Rai Ajit K. Srivastava, 2009. Fenofibrate ameliorates diabetic and dyslipidemic profiles in KKAy mice partly via down-regulation of 11β-HSD1, PEPCK and DGAT2. Comparison of PPARα, PPARγ, and liver x receptor agonists. European Journal of Pharmacology 2009, 258-263.

Therefore, we adapted dosage of Feno is 250/10  x 52.64 = 1316 mg/70kg above usual Feno ‘s daily dose =300mg.

Therefore, 400 mg EPE is 400/250 x 1316 = 2105.6 mg/70 kg human subject.

 

 

3. As Reviewer suggests, “Were appropriate control groups included to clearly distinguish the effects of EPE from those of the high-fat diet and standard treatments like metformin and fenofibrate? Specifically, were there vehicle-treated groups that received no EPE but were otherwise treated identically to the experimental groups?”

And revised into

----> In the present study, the control group is merely treated with vehicle and no EPE-treated because previous study showed that Phyllanthus emblica display the antihyperglycemic and antihyperlipidemic activities in various animal models, and the aim of the present study focus on T2DM and hyperlipidemia in HFD mice, and Institutional Animal Care and Use Committee emphasizes 3R rules (especially Reduce), and therefore no vehicle-treated groups that received no EPE.

 

 

4. As Reviewer suggests, “Is the 4-week treatment period sufficient to observe the long-term effects of EPE on insulin resistance, hyperlipidemia, and renal dysfunction? Should the study have included a longer follow-up period to assess the sustainability of the observed effects after the cessation of treatment?”

And revised into

----> Yes. This animal study is induced by 7-week HFD or LD and then treated with EPE for 4-week period, and the total period procedure is 11-week, and blood glucose level and plasma triglyceride is above the hyperglycemic and hyper triglyceridemic levels. According the previous others animal studies, the perfect period is 12-16-week. Our experience is that if the experiment procedure is too long, whenever mice body weight is over 40 g and quickly die (due to metabolic disorders) and will bring the disease to the other mice.

 

 

5. As Reviewer suggests, “Comments on the Quality of English Language

The quality of the paper is good, and no further questions are necessary”

And revised into

----> Thank you

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The aim of this study is to demonstrate the protective activity of ethyl acetate extract from the fruit of the plant species Phyllanthus emblica L. (EPE) against insulin resistance and metabolic disorders (hyperglycemia, hypertriglyceridemia, hypercholesterolemia, visceral obesity and renal dysfunction) in a mouse model of type 2 diabetes mellitus (T2DM) induced by a high-fat (hypercaloric) diet. The study is very interesting, it is designed and carried out appropriately, which allows reaching a conclusion clearly supported by the results obtained. Based on the above, it can be considered that this original research article could become of interest to the scientific community if the necessary modifications are made. The authors do not exceed the number of self-citations, however, it would be convenient to add a greater number of updated references in order to better justify the findings found. Although the figures included are appropriate and easy to interpret, specifically in Figure 3, 3A to 3H do not appear, so the text is not understandable. The conclusion is consistent with the central theme and is supported by the results obtained, which could be sufficient to affirm that EPE can have beneficial effects for patients with T2DM in terms of improving insulin resistance and reducing hyperlipidemia. However, it is recommended that authors work on the observations and suggestions made below:

1.   Abstract: Follow the author's guidelines regarding the length of the abstract, as it is very long, so it is suggested that it be more specific.

2.     Introduction: Like the abstract, this section is very long and some of the information would be more appropriate to mention in the discussion. It is suggested to include more information regarding the studies carried out with the evaluated plant specie (Phyllanthus emblica L.).

3.    Materials and Methods: Although some methodologies have already been reported in previous studies, it would be convenient to describe them in more detail in this work.

4.      Results: It is necessary to improve the description of the results in terms of wording. Specifically, in sections 3.2.1. (Metabolic Parameters) and 3.2.2. Blood Glucose, Biochemical Parameters and Monitoring of Renal Function, Figures 3A-3H are mentioned in the text, but they do not appear in Figure 3. In section 3.2.3. it is necessary to better explain the morphological findings found in both the liver and adipose tissue. In addition, it is convenient to point out in the micrographs the changes found in each of the treatment groups.

5.   Discussion: This section is adequately described, however, it would be convenient to add more updated references that support the findings found and, as previously mentioned, some of the paragraphs of the Introduction should be moved to this section. In addition, it is suggested to expand the information regarding the mechanisms of action associated with chebulagic acid.

6.      Conclusion: It is consistent with the central theme and is supported by the results obtained, which may be sufficient to demonstrate that EPE is capable of improving insulin resistance and attenuating the hyperlipidemia that occurs in mice with T2DM induced by HFD.

7.      References: It is necessary to add a greater number of updated references in order to better justify the findings found.

Comments on the Quality of English Language

The manuscript must be reviewed by a native English speaker, since there are spelling and punctuation errors, which cause the ideas that are intended to be communicated to be unclear.

Author Response

Outline the changes made:

 

Reviewer: 1

1. As Reviewer suggests, “Abstract: Follow the author's guidelines regarding the length of the abstract, as it is very long, so it is suggested that it be more specific.”

And revised into

----> Abstract: We planned to explore the protective activities of extract of Phyllanthus emblica L. (EPE) on insulin resistance and metabolic disorders including hyperlipidemia, visceral obesity, and renal dysfunction in high-fat diet (HFD)-progressed T2DM mice. Mice treatments included 7 weeks of HFD induction followed by EPE, fenofibrate (Feno), or metformin (Metf) treatment daily for another 4-week HFD in HFD-fed mice. Finally, we harvested blood to analysis some tests on circulating glycemia and blood lipid levels. Western blotting analysis was performed on target genes expressions in peripheral tissues. The present findings indicated that EPE treatment reversed the HFD-induced increases in blood glucose, glycosylated HbA1_C, and insulin levels. Our findings proved that treatment with EPE in HFD mice effectively controls hyperglycemia and hyperinsulinemia. Our results showed that EPE reduced blood lipid levels, including a reduction in blood TG, TC, and FFA; moreover, EPE reduced blood leptin levels and enhanced adiponectin concentrations. EPE treatment in HFD mice reduced BUN and creatinine in both blood and urine, and lowered albumin levels in urine; moreover, EPE decreased circulating concentrations of inflammatory NLRP3 and KIM-1. These results indicated that EPE displayed antihyperglycemic, antihyperlipidemic activities but alleviated renal dysfunction in HFD mice. The histology examinations indicated that EPE treatment decreased adipose hypertrophy and hepatic ballooning, thus contributing to amelioration of lipid accumulation. EPE treatment decreased visceral fat amounts and led to improve systemic insulin resistance. For target gene expression levels, EPE enhanced AMPK phosphorylation expressions both in livers and skeletal muscles, and elevated the muscular membrane GLUT4 expressions. Treatment with EPE reduced hepatic G6Pase and PEPCK expressions to suppress glucose production in the livers and decreased phosphorylation of GSK3β expressions to affect hepatic glycogen synthesis, thus convergently contributing to an antidiabetic effect and improving insulin resistance. The mechanism of the antihyperlipidemic activity of EPE involved a decrease in the hepatic phosphorylation of mTORC1 and p70S6K1 expressions to improve insulin resistance but also a reduction in hepatic SREBP-1c expressions, and suppression of ACC activity, thus resulting in the decreased fatty acid synthesis but elevated hepatic PPARα and SREBP-2 expressions, resulting in lowering TG and TC concentrations. Our results demonstrated that EPE improves insulin resistance and ameliorates hyperlipidemia in HFD mice.

 

 

2. As Reviewer suggests, “Introduction: Like the abstract, this section is very long and some of the information would be more appropriate to mention in the discussion. It is suggested to include more information regarding the studies carried out with the evaluated plant specie (Phyllanthus emblica L.).”

And revised into

----> Introduction have been revised shorten and some mentioned in the discussion. Please see the manuscript.

 

Phyllanthus emblica L. (Figure 1), known as the Indian gooseberry, belongs to the family Euphorbiaceae and is the most important medicinal agent in the traditional Indian system of medicine.……………

1. emblica contains rich in polyphenols those exhibits an antioxidant activity. Evidences have shown that P. emblica displays an antioxidant activity [28,29,31,32]. The fruits are used to treat ailments including diabetes, antitussive [28], liver complaints, inflammation, cardiac disorders, jaundice, and cataractogenesis [35]. Some of the recent in vitro studies revealed anti-inflammatory [36], nephrotoxicity modulation [37], and hepatoprotective [38] properties.

 

 

3. As Reviewer suggests, “Materials and Methods: Although some methodologies have already been reported in previous studies, it would be convenient to describe them in more detail in this work.”

And revised into

----> The time of daily treatment drug is about AM 8:30~9:30. After treatment with EPE, Feno, or Metf for 28 days, the mice were sacrificed. The night (about PM 8:30~9:30) before sacrifice, food was removed and mice was fasting for 12-h. During the procedure, 100 μl blood sample was collected from the retro-orbital sinus of mice and for blood glucose levels analysis. At the day of sacrifice, 0.6-0.8 mL blood sample was collected from the posterior vena cava of mice, and then put it in the heparin tube, and followed by mixture, centrifugation at 3300 rpm for 10 minutes, and then the adapted 0.3-0.4 mL plasma for later analysis.

After treatment with drugs, a single mouse was put in metabolic cab, and a 24-h urine sample was collected for analyses of renal function.

 

4. As Reviewer suggests, “Results: It is necessary to improve the description of the results in terms of wording. Specifically, in sections 3.2.1. (Metabolic Parameters) and 3.2.2. Blood Glucose, Biochemical Parameters and Monitoring of Renal Function, Figures 3A-3H are mentioned in the text, but they do not appear in Figure 3. In section 3.2.3. it is necessary to better explain the morphological findings found in both the liver and adipose tissue. In addition, it is convenient to point out in the micrographs the changes found in each of the treatment groups.“

And revised into

----> 3.2.1. Body Weight, Weight Gain, Liver Weights, and White Adipose Weights

---->3.2.2. Blood Glucose, Biochemical Parameters, and Urine Analysis

 

Histopathology Examination

HFD-feeding progressed a hepatic ballooning degeneration in HF mice in comparison to those of CON mice. Ballooning degeneration (as the arrow indicated) is a form death of hepatic parenchymal cell and its nucleolus is squeezed into the other side, and it was owing to glycogen accumulation within the nucleus. HFD developed obesity and insulin resistance. Insulin levels influenced the storage of hepatic glycogen. EPE1-, EPE2-, EPE3-, Feno-, or Meft- treated mice significantly decreased the degree of ballooning degeneration (Figure 4A).

 

The appearance of adipocyte showed a string-like cytosol surrounding a vacuole, and it is owing to it is embedded in paraffin resemble immersed in lipid solvents and could remove all the fats. The unobvious nucleus (N) could be carefully observed in the other side of cells. HFD-feeding developed hypertrophy of the adipocytes in comparison to CON mice in epididymal WAT. The average area of adipocytes in HF mice is larger than those of CON mice. Treatment with EPE2, EPE3, or Feno reduced adipocyte hypertrophy in comparison to those of HF mice (Figure 4B).

 

 

5. As Reviewer suggests, “Discussion: This section is adequately described, however, it would be convenient to add more updated references that support the findings found and, as previously mentioned, some of the paragraphs of the Introduction should be moved to this section. In addition, it is suggested to expand the information regarding the mechanisms of action associated with chebulagic acid.“

----> Chebulagic acid has an antiangiogenic effect associated with VEGFR2 signaling pathway inhibition, and this is important in the context of inflammation as a risk factor for tumor development, co-dependence of inflammation and angiogenesis, and the anti-inflammatory actions of chebulagic acid [79]. Chebulagic acid and chebulinic acid are demonstrated to inhibit TGF-β1 induced fibrotic changes in the chorio-retinal endothelial cells and as potential adjuvants in the management of diabetic retinopathy [80]. EPE is demonstrated to protect from diabetic nephropathy by its maker component chebulagic acid with an antiangiogenic effect involved in VEGF inhibition and anti-inflammatory in db/db mice [55].

[79]  Xu, Y.; Tang, G.; Zhang, C.; Wang, N.; Feng, Y. Gallic acid and diabetes mellitus, its association with oxidative stress. Molecules 2021, 26(23), 7115.

 

6. As Reviewer suggests, “Conclusion: It is consistent with the central theme and is supported by the results obtained, which may be sufficient to demonstrate that EPE is capable of improving insulin resistance and attenuating the hyperlipidemia that occurs in mice with T2DM induced by HFD.”

----> Thank you

 

7. As Reviewer suggests, “References: It is necessary to add a greater number of updated references in order to better justify the findings found.”

And revised into

----> [35]  Sowmya, V.; Baliga, M.S. Food and dietary supplements in the prevention and treatment of disease in older adults. 2015, 63-67. Edited by Ronald Ross Watson 2015 Elsevier Inc.

[36]  Golechha, M.; Sarangal, V.; Ojha, S.; Bhatia, J.; Arya, D.S. Anti-inflammatory effect of Emblica officinalis in rodent models of acute and chronic inflammation: involvement of possible mechanisms. Int. J. Inflamm. 2014, 1, 178408.

[37]  Sinha, R., Jindal, R., Faggio, C. Nephroprotective effect of Emblica officinalis fruit extract against malachite green toxicity in piscine model: Ultrastructure and oxidative stress study. Micro. Res. Tech. 2021, 84(8), 1911−1919.

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8. As Reviewer suggests, “Comments on the Quality of English Language The manuscript must be reviewed by a native English speaker, since there are spelling and punctuation errors, which cause the ideas that are intended to be communicated to be unclear.”

And the authors response---->

The manuscript had already improved by American Journal of Editing (AJE) Order ID: CLPMHL93) INVOICE # SCLPMHL93 (Services: - Standard Editing and – Standard Editing) in June 21 2023) We could improve on the Quality of English Language by CIMB Journal assistants and I will pay, and nevertheless at the present because other Reviewer’s comments are too many points to improve this point in time, please give me additional time, thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, all the questions and revisions were properly answered by the authors and I think the manuscript can be published. However, I think the abstract continue to be too long and without any reference to  any background information (abstracts at the begining should contain a little introduction to the studied topics). In my prespective, the abstract needs to be improved and shortened.

Comments on the Quality of English Language

The english language seems to be better (of course some details could be improved with another review) but i will not ask to the authors to pay for another review to the english. If you can, please do another revision only to corret some aspects

Reviewer 2 Report

Comments and Suggestions for Authors

The updated version looks pretty good. I don’t have any other questions.