The Protective Effect of Myristica fragrans Houtt. Extracts Against Obesity and Inflammation by Regulating Free Fatty Acids Metabolism in Nonalcoholic Fatty Liver Disease

Round 1

Reviewer 1 Report

In this study authors examined the effects of alcohol extract of nutmeg on the inhibition of lipid synthesis and inflammation in vitro and in vivo in a mouse model of NAFLD (High Fat Diet). Alcohol extract of nutmeg inhibited the aggravation of obesity and chronic low-grade inflammation by downregulating lipid-gene  expression in the liver

The quality of the article is fair. Data obtained aim to provide alternative and complementary medical therapies with the regulatory property of hepatic lipid metabolism to current pharmaceuticals for the treatment of NAFLD.

MAJOR REVISION: The background of the study is well described even if results are not clearly and logically presented. The authors must clarify the results sections to avoid confusion. The paper would benefit from stylistic changes to the way it has been written for a stronger, clearer, and more compelling argument

MAJOR REVISION

Introduction

This section is too long. I suggest the author reduces this section to keep only the most important elements strictly necessary to frame the issue

-line 100. AIM of the study. Please reformulated the sentence. The aim of a study describes what you hope to achieve, not what you have already achieved

Material and Methods

The research method is appropriate to the objectives of the study but Authors should improve the flow and readability of the section, to present these elements as clearly and as logically as possibly..

-line 140. The number of animals used for each group of experiments is missing. Please add it

-line 147. Please specify which reference standard was used for the establishment of the mouse model for NAFLD

-line 149.The dosage indicated is not complete and exhaustive. The daily dose per kg of animal body weight should be reported. What do you mean by oral administration? bolus, tablet, oral powder, oral paste, oral solution and premix or gavage? Please specify

Results

Results are not clearly and logically presented. Figures are not correctly labelled. Authors should review the exposure sequence in this section and make improvements to the figures reported. These add clarity to the data presented.

-line 239. The results reported in the literature must be compared with the results of one's work in the Conclusions section not in the Results. In addition, reference 38 is not appropriate for the sentence. It reports the historical method of lipid extraction and not the role of nutmeg in lipid metabolism.

-line 243-244. Please remove the sentence. Comments on the results are to be found in the conclusions.

-line 262-264. Authors reported no significant change of TNF-α and IL-6  in  AEN group versus control group. Have the authors proved to stimulate macrophages with LPS?

Perhaps the authors would have obtained different results if they stimulated  macrophages with LPS ( Jin H.; 2012 Phytother. Res. 26: 1320–1326)  Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. It has been conventionally used to study inflammation, due to the abundance of inflammatory effects that it generates through TLR4 signaling (Tucureanu MM.; Int J Nanomedicine. 2018; 13: 63–76)

-FIG.2 E-F Please remove letters and insert the horizontal square bracket with the respective significance values

-FIG.3 Please maintain the same sequence of groups in all graphs (e.g. CON; HF; HF+AEN)

-FIG 3 D. Please remove letters and insert the horizontal square bracket with the respective significance values

-FIG 4. Authors should indicate the respective number above or below the figures. Please add correct labels to figure 4A and 4B and use the same sequence in the text in according to what is shown in in Figure 4

-FIG 4C-4-D Please remove letters and insert the horizontal square bracket with the respective significance values

FIG 5E-5H Please remove letters and insert the horizontal square bracket with the respective significance values

FIG 6B. Band analysis. Please remove letters and insert the horizontal square bracket with the respective significance values

Discussion

Authors reported that in their HFD-induced NAFLD animal model the body weight and blood glucose of the HF mice increased confirming the association between the progression of NAFLD and the occurrence of obesity and hyperglycemia. In  addition authors observed a significant increase in pro-inflammatory cytokines (TNF-407 α, IL-6 and IL-1β) in the high fat diet-induced mice. This association as well as the increase in proinflammatory factors are now widely consolidated. The authors should mainly focus their attention on discussing the results obtained regarding the ability of the AEN to intervene in the metabolic signaling pathway of fatty acids.

Please remove the references to the figures.  Data have already been reported in the Results section

References

Authors don’t use references in the correct style and some are incomplete. I suggest to carefully revise some references (e.g. 17; 47; 50;54;55;56).

MINOR REVISION

Introduction

-line 48-50. Please reformulated the sentence for clarity.

-line 77-78. Please reformulated the sentence for clarity

-line 83-84. Please check the font used

-line 96. Please also add information about nutmeg toxicity and abuse

Material and Methods

-line 116-127. Please check the time used in the sentences and standardize the format. Simple past and simple present were used at the same time

-line 148. Please reformulated the sentence “Through screening and dose determination by lavage in the pre-experiment” for clarity. If necessary, add further description of the method used in the pre-experiment or bibliographic citation

-line 163-171. Please reformulated the section 2.5 describing the paraffin-embedded method separately from the cryostat method

-line 174. Please reformulated the sentence “of liver tissue, adipose tissue, and high fat feed” What do you mean by high-fat feed among the tissues analyzed?

-line 186. Please check the use of ANE instead of AEN

-line 188. Please check the use of ANE instead of AEN

-line 204. Please specify which type of tissues were lysed

Please check punctuation and uppercases

Statistical Analysis

Please expand this section. For example, were your variables all normally distributed? If not, describe which nonparametric tests were used.

Results

-line 261. Please check the use of ANE instead of AEN

-line 264. Please rewrite the sentence more clearly and appropriately

-Line 280-285. Please remove sentence. They are redundant and already described in the methods. Instead it might be helpful to add a flowchart of experimental design.

-Line 287-301 The sentences appear to be solid, but the exposure is unclear, which makes it difficult to follow it. Please rewrite more clearly and linearly. The comments to the figures should follow the  same order shown in the figures.

-line 356. Please rewrite the sentence more appropriately

-line 363. Please rewrite the sentence more appropriately

-line 364-366 Please rewrite the sentence more clearly and appropriately. In the way it was written, the significance of AEN treatment in reducing proinflammatory factors levels has been lost

-line 366.  Please remove the sentence. Comments on the results are to be found in the conclusions.

Author Response

Please see the attachment.

Response to Reviewer 1 Comments

Comments and Suggestions for Authors

In this study authors examined the effects of alcohol extract of nutmeg on the inhibition of lipid synthesis and inflammation in vitro and in vivo in a mouse model of NAFLD (High Fat Diet). Alcohol extract of nutmeg inhibited the aggravation of obesity and chronic low-grade inflammation by downregulating lipid-gene expression in the liver

The quality of the article is fair. Data obtained aim to provide alternative and complementary medical therapies with the regulatory property of hepatic lipid metabolism to current pharmaceuticals for the treatment of NAFLD.

MAJOR REVISION: The background of the study is well described even if results are not clearly and logically presented. The authors must clarify the results sections to avoid confusion. The paper would benefit from stylistic changes to the way it has been written for a stronger, clearer, and more compelling argument.

MAJOR REVISION

Introduction

Point 1: This section is too long. I suggest the author reduces this section to keep only the most important elements strictly necessary to frame the issue

-line 100. AIM of the study. Please reformulated the sentence. The aim of a study describes what you hope to achieve, not what you have already achieved

Response to 1: Thank you very much for your professional comments. In this section, we had described some mechanisms involved in lipid metabolism and NAFLD to introduce the function of AEN better and let readers understand more clearly. However, this part is not refined enough. According to your kindly suggestion, we reduced redundant parts of this section to highlight the most important elements. Then we adjusted the structure of Introduction to make it more coherent. The first and third paragraphs were combined, focusing on the pathogenesis of NAFLD from lipid metabolism and inflammation respectively and the importance of fatty acids in regulating NAFLD was proposed. The second paragraph is the background of the molecular mechanisms of fatty acid synthesis, and the third paragraph is the related information of Myristica fragrans. Please see the highlighted parts in Introduction. (-line 35-36, 39, 40, 44-47, 51, 53-67, 68-70, 72-75, 77-79)

–line 100.We revised the aim of this study according to your professional suggestion. (-line 89-91).

Material and Methods

Point 2: The research method is appropriate to the objectives of the study but Authors should improve the flow and readability of the section, to present these elements as clearly and as logically as possibly.

-line 140. The number of animals used for each group of experiments is missing. Please add it

-line 147. Please specify which reference standard was used for the establishment of the mouse model for NAFLD

-line 149.The dosage indicated is not complete and exhaustive. The daily dose per kg of animal body weight should be reported. What do you mean by oral administration? bolus, tablet, oral powder, oral paste, oral solution and premix or gavage? Please specify

Response to 2: Thank you for your professional advice.

-line 140. We are sorry for our mistake. The number of animals was added according to you advice (-line129, 134-135).

-line 147. M Van Heek et al studied on establishing a model of high fat diet obesity in mice by using 45% high-fat diet in 1997[1]. Then researchers modified fat calories (40%~71%) to form Van Heek series of high-fat obesity model feed. Among them, 60% high-fat diet, represented by Research Diets Company D12492, was widely used in the researches of obesity, diabetes, NAFLD and so on[2,3]. Reference standard for the establishment of the mouse model for NAFLD was added in 2.3 section according to your kind advice (-line136-138).

-line 149. We are sorry for the confusion caused by our unclear description. The “oral administration” means gavage of AEN (250 mg/kg bw, resolved in 0.5% CMC). Daily dose of gavage was 250 mg/kg bw. The above information was specified in Section 2.3 (-line139-145).

Results

Point 3: Results are not clearly and logically presented. Figures are not correctly labelled. Authors should review the exposure sequence in this section and make improvements to the figures reported. These add clarity to the data presented.

-line 239. The results reported in the literature must be compared with the results of one's work in the Conclusions section not in the Results. In addition, reference 38 is not appropriate for the sentence. It reports the historical method of lipid extraction and not the role of nutmeg in lipid metabolism.

-line 243-244. Please remove the sentence. Comments on the results are to be found in the conclusions.

Response to 3: Thank you for your professional advice. We adjust the exposure sequence of Fig. 3A-H according to your kind suggestion.

-line 239. According to your suggestion, the sentence “It has also been reported by others that nutmeg is potentially valuable as an edible plant for its role in lipid metabolism” was removed. The corresponding comparative discussion of nutmeg function was given in Discussion section (-line -line420-422). We are sorry for our mistake presenting the wrong reference in this part. Reference [38] were replaced with the right one and transfer to Discussion Section. Please see line -line420-422.

-line 243-244.We removed this sentence “We believe that these findings will enrich follow-up investigations” according to your suggestion.

Point 4:-line 262-264. Authors reported no significant change of TNF-α and IL-6 in AEN group versus control group. Have the authors proved to stimulate macrophages with LPS?

Perhaps the authors would have obtained different results if they stimulated macrophages with LPS (Jin H.; 2012 Phytother. Res. 26: 1320–1326) Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. It has been conventionally used to study inflammation, due to the abundance of inflammatory effects that it generates through TLR4 signaling (Tucureanu MM.; Int J Nanomedicine. 2018; 13: 63–76)

Response to 4: Thank you very much for your professional comments. As we all know, lipopolysaccharide is a component of cell wall in gram negative bacterium. It is an endotoxin that can stimulated macrophages producing inflammatory cytokines and chemokines. As you mentioned above, it has been conventionally used to study inflammation [4,5]. Besides, reports in recent years have shown that NAFLD and gut microbiome are inextricably linked. The interactions between the gut and liver, called the gut–liver axis, play an essential role in NAFLD development and evolution. Studies in animal whose gut microbiota are manipulated, and observational studies in patients with NAFLD, have provided considerable evidence that gut microbiota dysbiosis contributes to the pathogenesis of NAFLD. Gut microbiota dysbiosis increases gut permeability to bacterial products and increases hepatic exposure to injurious substances that increase hepatic inflammation and fibrosis. A large portion of the liver blood that comes from the intestine exposes the liver to the metabolic products form the gut microbiome, including LPS[6,7]. Therefore, according to your professional suggestion, in subsequent studies of NAFLD and gut microbiome, we will consider LPS as macrophage-derived mediator of inflammation.

However, in this study, we focus on the stimulation by free fatty acids (FFAs) on macrophages. Because FFA is the key to lipid metabolism and inflammation, we want to identify whether AEN down-regulate the inflammation by inhibiting fatty acids synthesis in vitro. It was also confirmed by subsequent animal experiments. FFAs, especially saturated FFAs, have been studied for a long time to explain the relationship between lipid metabolism and inflammation. Toll like receptors 2 and 4 are activated by free fatty acids to generate abundance of inflammatory effects [8,9]. It has been used in study on inflammation as an in vitro model[10]. Therefore, in this study, FFAs were chose as a mediator of inflammation on macrophage.

Point 5:-FIG.2 E-F Please remove letters and insert the horizontal square bracket with the respective significance values

-FIG.3 please maintain the same sequence of groups in all graphs (e.g. CON; HF; HF+AEN)

-FIG 3 D. Please remove letters and insert the horizontal square bracket with the respective significance values

-FIG 4. Authors should indicate the respective number above or below the figures. Please add correct labels to figure 4A and 4B and use the same sequence in the text in according to what is shown in Figure 4

-FIG 4C-4-D Please remove letters and insert the horizontal square bracket with the respective significance values

FIG 5E-5H Please remove letters and insert the horizontal square bracket with the respective significance values

FIG 6B. Band analysis. Please remove letters and insert the horizontal square bracket with the respective significance values

Response to 5: Thank you for your professional suggestions. We adjusted the format of all the figures according to your kind advices.

Discussion

Point 6: Authors reported that in their HFD-induced NAFLD animal model the body weight and blood glucose of the HF mice increased confirming the association between the progression of NAFLD and the occurrence of obesity and hyperglycemia. In addition authors observed a significant increase in pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the high fat diet-induced mice. This association as well as the increase in proinflammatory factors are now widely consolidated. The authors should mainly focus their attention on discussing the results obtained regarding the ability of the AEN to intervene in the metabolic signaling pathway of fatty acids.

Please remove the references to the figures.  Data have already been reported in the Results section.

Response to 6: Thank you for your professional advice. According to your suggestion, some descriptions of common sense that has been widely consolidated were removed and the discussion on regarding the ability of the AEN to intervene in the metabolic signaling pathway of fatty acids were strengthened in Discussion section.(-line397-401.-line412-415) The references to the figures and the data in Discussion section were also removed according to your kind advice.

References

Point 7: Authors don’t use references in the correct style and some are incomplete. I suggest to carefully revise some references (e.g. 17; 47; 50; 54; 55; 56).

Response to 7: Thank you for your professional advice. We carefully revised the references. All the references are provided by google academic and edited in “nutrients” style by EndNoteX9.

MINOR REVISION

Point 1: Introduction

-line 48-50. Please reformulated the sentence for clarity.

-line 77-78. Please reformulated the sentence for clarity

-line 83-84. Please check the font used

-line 96. Please also add information about nutmeg toxicity and abuse

Response to 1: Thank you for your professional advice.

-line 48-50. -line 77-78.We revised the sentences for clarity and combined some paragraphs (line40-51).

-line 83-84.We revised the font used (line50-51).

-line 96.We added some information about nutmeg toxicity and abuse (line77-79). “Although nutmeg has a long history of abuse[11,12], even higher doses (20–80 g of powder) had been ingested (adult), a life-threatening situation was never observed[13]”.

Point 2: Material and Methods

-line 116-127. Please check the time used in the sentences and standardize the format. Simple past and simple present were used at the same time

-line 148. Please reformulated the sentence “Through screening and dose determination by lavage in the pre-experiment” for clarity. If necessary, add further description of the method used in the pre-experiment or bibliographic citation

-line 163-171. Please reformulated the section 2.5 describing the paraffin-embedded method separately from the cryostat method

-line 174. Please reformulated the sentence “of liver tissue, adipose tissue, and high fat feed” What do you mean by high-fat feed among the tissues analyzed?

-line 186. Please check the use of ANE instead of AEN

-line 188. Please check the use of ANE instead of AEN

-line 204. Please specify which type of tissues were lysed

Please check punctuation and uppercases

Response to 2: Thank you for your professional advice.

-line 116-127.We adjusted the tense of the sentence according to your kind advice (line 105-116).

-line 148. We added the description of the method used in the pre-experiment or bibliographic citation according to your kind advice. Please see line136-145.

-line 163-171.We separated the method for paraffin-embedded and cryostat to make it clearer in section 2.5 according to your kind advice. Please see line158-166.

-line 174.We analyzed the proportion of fatty acids in liver, epididymal fat, blood and high fat feed by GC-MS. The analysis of fatty acids in high-fat diet is necessary because the fatty acids obtained from food greatly affect the proportion of fatty acids in the body. The content of saturated fatty acids (C14:0, C16:0, C18:0) and monounsaturated fatty acids (C16:1, C18:1) in high fat feed is high. The proportion of fatty acids in high-fat diet group showed the same trend in Fig 5A and B. This gives us a more intuitive understanding that the high-fat diet changes the proportion of fatty acids in NAFLD mice.

-line 186. -line 188. We are sorry for our mistakes. All the ANE were substituted with AEN.

-line 204. The liver tissue was specified according to your kind advice. (-line 199).

This section was revised carefully for punctuation and uppercases.

Point 3: Statistical Analysis

Please expand this section. For example, were your variables all normally distributed? If not, describe which nonparametric tests were used.

Response to 3: Thank you for your professional advice. Since the sample size was small (<100), we chose Shapiro-Wilk test to test the normal distribution by SPSS19.0. All data basically conform to normal distribution.

For example, we input the body weight data of mice HF group into SPSS19.0 to test normality in Shapiro-Wilk test (n=10). We got a significant difference 0.037 (less than 0.05), which is accord with normally distribution.

PS: If the sample size is greater than 100, we will use Kolmogorov-Smirnov test.

We expanded this section according to your kind advices. Please see line 223-225

Point 4: Results

-line 261. Please check the use of ANE instead of AEN

-line 264. Please rewrite the sentence more clearly and appropriately

-Line 280-285. Please remove sentence. They are redundant and already described in the methods. Instead it might be helpful to add a flowchart of experimental design.

-Line 287-301 The sentences appear to be solid, but the exposure is unclear, which makes it difficult to follow it. Please rewrite more clearly and linearly. The comments to the figures should follow the same order shown in the figures.

-line 356. Please rewrite the sentence more appropriately

-line 363. Please rewrite the sentence more appropriately

-line 364-366 Please rewrite the sentence more clearly and appropriately. In the way it was written, the significance of AEN treatment in reducing proinflammatory factors levels has been lost

-line 366.  Please remove the sentence. Comments on the results are to be found in the conclusions.

Response to 4: Thank you for your professional advice.

-line 261. We are sorry for our mistakes. The ANE were substituted with AEN. (-line255-256.)

-line 264. We rewrote the sentence more clearly according to your advice. (-line258-259)

-line 280-285. We removed the sentence according to your advice.

-line 287-301. According to your advice, we reordered the statements and made comments to the figures following the same order shown in the figures (-line 272-288).

-line 356. We rewrote the sentence more clearly according to your advice. (-line345-346).

-line 363. We rewrote the sentence more clearly according to your advice. (-line353).

-line 364-366.We rewrote the sentence more clearly and appropriately according to your advice. (-line354-355).

-line 366. We removed the sentence according to your advice.

Reference

1. Van Heek, M.; Compton, D.S.; France, C.F.; Tedesco, R.P.; Fawzi, A.B.; Graziano, M.P.; Sybertz, E.J.; Strader, C.D.; Davis, H.R., Diet-induced obese mice develop peripheral, but not central, resistance to leptin. The Journal of clinical investigation 1997, 99, 385-390.
2. Jiang, Y.; Gao, Y.Q.; Zhu, M.Q.; Man, L.I.; Zhang, X.; Hepatology, D.O., Establishment of the nonalcoholic fatty liver disease model in c57bl/6 mice. Journal of Bengbu Medical College 2018.
3. Leng, L.; Jiang, Z.Q.; Ji, G.Y., [effects of soybean isoflavone on liver lipid metabolism in nonalcoholic fatty liver rats]. Zhonghua Yu Fang Yi Xue Za Zhi Chinese Journal of Preventive Medicine 2011, 45, 335-339.
4. Jin, H.; Zhu, Z.G.; Yu, P.J.; Wang, G.F.; Zhang, J.Y.; Li, J.R.; Ai, R.T.; Li, Z.H.; Tian, Y.X.; Zhang, W.X., Jia‐Jie, Myrislignan attenuates lipopolysaccharide‐induced inflammation reaction in murine macrophage cells through inhibition of nf‐κb signalling pathway activation. Phytotherapy Research 2012, 26, 1320-1326.
5. Tucureanu, M.M.; Rebleanu, D.; Constantinescu, C.A.; Deleanu, M.; Voicu, G.; Butoi, E.; Calin, M.; Manduteanu, I., Lipopolysaccharide-induced inflammation in monocytes/macrophages is blocked by liposomal delivery of gi-protein inhibitor. International journal of nanomedicine 2018, 13, 63.
6. Leung, C.; Rivera, L.; Furness, J.B.; Angus, P.W., The role of the gut microbiota in nafld. Nature Reviews Gastroenterology & Hepatology 2016, 13, 412-425.
7. Safari, Z.; Gérard, P., The links between the gut microbiome and non-alcoholic fatty liver disease (nafld). Cellular and Molecular Life Sciences 2019, 76, 1541-1558.
8. Nguyen, M.T.A.; Favelyukis, S.; Nguyen, A.K.; Reichart, D.; Scott, P.A.; Jenn, A.; Liu-Bryan, R.; Glass, C.K.; Neels, J.G.; Olefsky, J.M., A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via toll-like receptors 2 and 4 and jnk-dependent pathways. Journal of Biological Chemistry 2007, 282, 35279-35292.
9. Shi, H.; Kokoeva, M.V.; Inouye, K.; Tzameli, I.; Yin, H.; Flier, J.S., Tlr4 links innate immunity and fatty acid–induced insulin resistance.
10. Gao, M.; Ma, Y.; Liu, D., Rutin suppresses palmitic acids-triggered inflammation in macrophages and blocks high fat diet-induced obesity and fatty liver in mice. Pharmaceutical Research 2013, 30, 2940-2950.
11. Forrester, M.B., Nutmeg intoxication in texas, 1998–2004. Human & experimental toxicology 2005, 24, 563-566.
12. Sangalli, B.C.; Sangalli, B.; Chiang, W., Toxicology of nutmeg abuse. Journal of Toxicology: Clinical Toxicology 2000, 38, 671-678.
13. Stein, U.; Greyer, H.; Hentschel, H., Nutmeg (myristicin) poisoning—report on a fatal case and a series of cases recorded by a poison information centre. Forensic science international 2001, 118, 87-90.

Reviewer 2 Report

The manuscript (ID nutrients-880460) is focused on the biochemical effects of nutmeg in LO2, RAW264.7 and in vivo models of mice. The scope of the journal  concerns novel insights into the impacts of nutrition on human health and the outcomes of animal studies that have relevance to human health. In general, reading the manuscript I had an impression of too much details of molecular science. In my opinion, the Authors should force the nutrition aspect in their manuscript, in the Introduction and Discussion sections. The introduction should be shortened. The Authors tested extract of Myristica fragrans in different in vitro and in vivo models. However, the usage of the term „low-grade inflammation” based on these studies is far-fetched. There is no background in this manuscript for this title. Therefore, the title should be changed. The Authors did not discussed with previous reports on extracts of M. fragrans seeds in the quite similar field of studies, e.g.  Biomedical and Pharmacology Journal, 2019, 12(2), pp. 669-676, Molecules, 2011, 16(8), pp. 7132-7142, Journal of Medicinal Food, 2016, 19(10), pp. 952-960.

More correlation with nutrition are required in the manuscript.

The full name of Myristica fragrans Houtt. should be included in the title and in the introduction in addition to family Myristicaceae.

The series numer is required for purchased nutmeg.

1. 272-274 Please remove „After the polymerase …trend in vitro”
2. 419 Please check the assignement of figure „Fig. 1E-F”, it seems it is not well-referred.

The units of concentrations should be changed in the entire manuscript. Please express all in „µg/mL” instead of „mg/L”.

The Authors declared the concentration range used in the in vitro studies 25, 50, 100, and 200 mg/L but in Figure 2 it is not clear why only cell viability was studied in this concentration range. Moreover, the cell viablity decreased gradually. Therefore, testing of cytotoxic extract in cell lines is not justified. What was the concentration of DMSO in wells? The declaration is that extracts were dissolve din DMSO. „The control cells received only the solvent (DMSO).”? What about any positive controls, in cytokines secretion tests.

It is required to provide the dose of AEN in mg/kg b.w. Please recalculate 50 mg/ml p.o.

The quality of structures in Figure 1 should be improved.

Figure 3. Please use the same order of samples CON, HF, HF+AEN.

Figure 4. Please improve dimensions

1. 461 Please remove the statement „AEN effectively alleviates chronić low-grade inflammation in mice.” It is not adequate.

Author Response

Please see the attachment.

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

The manuscript (ID nutrients-880460) is focused on the biochemical effects of nutmeg in LO2, RAW264.7 and in vivo models of mice. The scope of the journal concerns novel insights into the impacts of nutrition on human health and the outcomes of animal studies that have relevance to human health. In general, reading the manuscript I had an impression of too much details of molecular science. In my opinion, the Authors should force the nutrition aspect in their manuscript, in the Introduction and Discussion sections.

Point1: The introduction should be shortened. The Authors tested extract of Myristica fragrans in different in vitro and in vivo models. However, the usage of the term, low-grade inflammation” based on these studies is far-fetched. There is no background in this manuscript for this title. Therefore, the title should be changed.

Response to 1: Thank you very much for your professional comments. According to your kind suggestion, we reduced redundant parts in Introduction section to highlight the important elements (-line36-91). Also, we changed the title “The protective effective of nutmeg extracts against obesity and chronic low-grade inflammation by regulating free fatty acids metabolism in nonalcoholic fatty liver disease” to “The protective effective of Myristica fragrans Houtt. extracts against obesity and inflammation by regulating free fatty acids metabolism in nonalcoholic fatty liver disease”(-line2-5). Besides, all the “chronic low-grade inflammation” were changed to “inflammation/ inflammatory response” in the manuscript to make it more appropriate according to your advice (-line 22,-line25,-line 33,-line446).

Point 2: The Authors did not discussed with previous reports on extracts of M. fragrans seeds in the quite similar field of studies, e.g.  Biomedical and Pharmacology Journal, 2019, 12(2), pp. 669-676, Molecules, 2011, 16(8), pp. 7132-7142, Journal of Medicinal Food, 2016, 19(10), pp. 952-960.

Response to 2: Thank you very much for your professional comments and the references on nutmeg extracts you provided. Myristicin has anti-inflammatory properties related with its activities in inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-stimulated macrophages via the calcium pathway[1]. A standardized blend comprising three extracts from Myristica fragrans, Astragalus membranaceus and Poria cocos can protect acute liver toxicity model mice induced by acetaminophen (APAP) and carbon tetrachloride (CCl4) [2]. According to your suggestion, the last two references were cited in this article (-line82-85). But for the first reference “Biofilm Forming Abilities of Microorganisms Associated with Diabetic Wound Infection: A Study from a Tertiary Care Hospital”[3], which aimed at isolating bacteria from chronic diabetic foot ulcers to check for its antibiotic susceptibility and biofilm forming ability, no information about Myristica fragrans Houtt could be found in this article. Besides, we added additional references to introduce the research background of Myristica fragrans Houtt.and its extracts[4]. (-line81-82)

Point 3: More correlation with nutrition are required in the manuscript. In my opinion, the Authors should force the nutrition aspect in their manuscript, in the Introduction and Discussion sections.

Response to 3: Thank you very much for your professional comments. We added some content about diet, energy and nutrition about Myristica fragrans Houtt. in Introduction section and Discussion section. For example, we have added some information about the main fatty acids and amino acids in nutmeg of Myristica fragrans Houtt.[5,6] to analyze the nutrients (-line72-75). We have found that AEN contained 8.57% tetradecanoic acid (myristic acid).Therefore we have discussed the nutrition and function aspect in dietary myristic acid[7,8](-line 439-441).

Point 4: The full name of Myristica fragrans Houtt.should be included in the title and in the introduction in addition to family Myristicaceae. The series number is required for purchased nutmeg.

Response to 4: Thank you very much for your professional comments. We added the full name of Myristica fragrans Houtt.in the title and the Introduction Section (-line2.-line22.-line74) according to your kind suggestion.

The series number of purchased nutmeg is 270028428 (Tong Ren Tang, Beijing) was also added in Section 2.1 according to your advice (-line 95).

Point 5:1.272-274 please remove, after the polymerase …trend in vitro”

1.419 Please check the assignement of figure „Fig. 1E-F”, it seems it is not well-referred.

The units of concentrations should be changed in the entire manuscript. Please express all in „µg/mL” instead of „mg/L”.

Response to 5: Thank you very much for your professional comments.

-line272-274 According to your advice, we removed this sentence.

-line 419. We are very sorry for our mistake that given the wrong figure references in this part. Considering the Reviewer 1’s suggestion that references to the figures which have already been reported in the Results section should not appear in the Discussion Section, we deleted all references to figures in Discussion section. Also, we replaced all “mg/L” with “µg/mL” according to your kind advice.

Point 6: The Authors declared the concentration range used in the in vitro studies 25, 50, 100, and 200 mg/L but in Figure 2 it is not clear why only cell viability was studied in this concentration range. Moreover, the cell viablity decreased gradually. Therefore, testing of cytotoxic extract in cell lines is not justified. What was the concentration of DMSO in wells? The declaration is that extracts were dissolve din DMSO. „The control cells received only the solvent (DMSO).”? What about any positive controls, in cytokines secretion tests.

Response to 6: Thank you very much for your professional comments. We found that the alcohol extract of nutmeg could inhibit lipid production in Mortierella alpina ATCC32222(MA) [9] (Fig. 1A), then we try to verify its ability in fatty acid synthesis inhibition in hepatocytes. For the experiment of cell viability, we hope to use this experiments with a concentration gradient of AEN to find the optimal concentration of AEN for cell model treatment. With the treatment by optimal concentration of AEN, we aimed to observe significant inhibition on the synthesis of fatty acids by AEN with acceptable low cell toxicity, to prove that AEN can inhibit the synthesis of fatty acids in vitro. Because 200 mg/L of AEN was used to treat Mortierella alpina ATCC32222 (MA) to identify the inhibition activity on lipid synthesis, and the tolerance of mold is higher than cells, the concentration below 200 mg/L were set to do a gradient experiment.

We are sorry for the confusion caused by our unclear descriptions for AEN solvents.  We added same information about the two AEN solvents in manuscript (-line 98-100). DMSO in AEN was pure (100%) as solvent. When we stimulated cells with difference concentration of AEN, it was diluted 1,000 times per well. That means the ratio of the DMSO to the culture medium was 1:1000. Taking the 6-well plate as an example, we usually added 2-3 μL AEN/DMSO in 2-3 mL medium per well. In the blank control group, we added the same amount of pure DMSO to prove that no side effects could be caused by this concentration of DMSO.

For cytokines secretion tests, we did not use a positive control because our purpose was to prove that the inflammatory responses in macrophages induced by FFAs could be inhibited by FFAs. Therefore, no positive control was used to quantify the ability of AEN to inhibit the inflammatory response in macrophages caused by FFA. Thank you for your professional suggestion. We will add a positive control in future studies to quantify the ability of AEN to inhibit inflammatory responses.

Point 7: It is required to provide the dose of AEN in mg/kg b.w. Please recalculate 50 mg/ml p.o.

The quality of structures in Figure 1 should be improved.

Response to 7: Thank you very much for your professional comments. According to your advice, we substituted “50 mg/ml p.o.” with “250 mg/kg b.w” (-line 143-144). Besides, we splatted Figure 1 into two parts to improve the quality and make the content clearer.

Point 8: Figure 3. Please use the same order of samples CON, HF, HF+AEN.

Figure 4. Please improve dimensions

461 Please remove the statement „AEN effectively alleviates chronić low-grade inflammation in mice.” It is not adequate.

Response to 8: Thank you very much for your professional comments.

Figure 3. We uniformly adjusted the order of samples in Figure 3 to “CON, HF, HF+AEN”.

Figure 4. According to your advice, we splatted Figure 4 into two parts to improve the quality and make the content clearer.

–line 461 According to your advice, we substituted the sentence “AEN effectively alleviates chronić low-grade inflammation in mice.” with “AEN down-regulate proinflammatory level in liver of mice” (-line445-446).

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9. Ding, Y.; Gu, Z.; Wang, Y.; Wang, S.; Chen, H.; Zhang, H.; Chen, W.; Chen, Y.Q. Clove extract functions as a natural fatty acid synthesis inhibitor and prevents obesity in a mouse model. Food & Function 2017, 10.1039.C1037FO00096K.

Round 2

Reviewer 1 Report

Authors well revised sentence in according to suggestions. They adjusted the exposure and format of figures according to advices, gaining in quality of presentation. I suggest its pubblication in this revised form

Reviewer 2 Report

The Authors improved the manuscript to a large extent. In experiments there is a lack of positive control. The Autors provided the response for this point.

L. 241 „Mortierella alpina” in italics.

L. 262 shoud be „cell viability” instead of „activity”

The editorial mistakes should be carefully revised and improved. 

Author Response

Thank you for your kind advice. We have completed the adjustment accordingly