Exploring the Potential Lipid-Lowering and Weight-Reducing Mechanisms of FH06 Fermented Beverages Based on Non-Targeted Metabolomics and Network Pharmacology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In their manuscript, the authors have identified the possible lipid-lowering and weight loss-promoting active components in a herbal beverage and the targets and related pathways using non-targeted metabolomics and network pharmacology methods. On the whole, the lack of the experimental validation for some key components or targets reduces the scientific soundness of this study that is predictive so far.

Major Comments

It is premature to tell about any action mechanism as in the title which does not match the reported data.  Indeed, the action mechanisms have not been unraveled, and identification of potentially active compounds and targets with pathways is not enough to claim about any mechanism. So, the title must be revised, as well as any mentioning of the action mechanism in the text.

The authors have described the production of FH06 supernatant (subsection 2.1) but the protocol for UPLC-MS/MS has been omitted in M&M.

The authors have mentioned that “a total 445 compounds were obtained from FH06 by UPLC-MS/MS (Table S1)” but at the time of reviewing Table S1 is unavailable via the link indicated (L344-5)

The authors often use abbreviations without spelling, especially for bioinformatics tools. It is necessary to spell out all abbreviations, except for the commonly accepted in biology.

Overall, the description of results is too technically written.

What are OB and DL values in Table 1?

Much more references are needed to describe the biological activities possessed by the key compounds  (LL 130 – 177) than in the current ms.

L249: This is not a chapter.

The conclusion” the research results can provide a …. basis for further research and development on the medicinal and edible value of  FH06” obviously reflects a too intermediate significance of this work.

Comments on the Quality of English Language

English should be checked carefully in view of some errors and roughly written phrases.. 

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

 

In your comments and suggestions for authors, you mentioned:

In their manuscript, the authors have identified the possible lipid-lowering and weight loss-promoting active components in a herbal beverage and the targets and related pathways using non-targeted metabolomics and network pharmacology methods. On the whole, the lack of the experimental validation for some key components or targets reduces the scientific soundness of this study that is predictive so far.

Major Comments

It is premature to tell about any action mechanism as in the title which does not match the reported data.  Indeed, the action mechanisms have not been unraveled, and identification of potentially active compounds and targets with pathways is not enough to claim about any mechanism. So, the title must be revised, as well as any mentioning of the action mechanism in the text.

Your comments are very good. Indeed, our presentation needs to be more rigorous. Our topic has been revised to :Exploring the potential lipid-lowering and weight-reducing mechanisms of FH06 fermented beverages based on non-targeted metabolomics and network pharmacology.

Now I will respond to the comments one by one.

 

（1）The authors have described the production of FH06 supernatant (subsection 2.1) but the protocol for UPLC-MS/MS has been omitted in M&M.

Response（1）: The relevant method (L97-122) has been added after section 2.1, and the modified version is shown in the following figure.

 

（2）The authors have mentioned that “a total 445 compounds were obtained from FH06 by UPLC-MS/MS (Table S1)” but at the time of reviewing Table S1 is unavailable via the link indicated (L344-5).

Response（2）: Due to the careless use of synonyms during translation, some inappropriate names appeared. They have been checked and modified according to Table S1.

（3）The authors often use abbreviations without spelling, especially for bioinformatics tools. It is necessary to spell out all abbreviations, except for the commonly accepted in biology.

Response（3）: The complete information has been supplemented using abbreviated language. (L135-136), modified as shown in the following figure.

（4）Overall, the description of results is too technically written.

Response（4）: I'm very sorry, dear reviewer. We still don't quite understand how to make revisions based on your feedback. Due to the tight deadline for modification, this part has not been modified yet. If possible, please provide further guidance on the modifications.

（5）What are OB and DL values in Table 1?

Response（5）: In section 2.3 The FH06 compound screening and target prediction section indicates the meanings of OB and DL. (L116-118), modified as shown in the following figure.

（6）Much more references are needed to describe the biological activities possessed by the key compounds  (LL 130 – 177) than in the current ms.

Response（6）: Partial references have been added to L130-177 to illustrate the bioactive functions of this key compound. (L165-214), modified as shown in the following figure.

（7）L249: This is not a chapter.

Response（7）: The L249 format has been modified. The modified version is shown in the following figure.

（8）The conclusion” the research results can provide a …. basis for further research and development on the medicinal and edible value of  FH06” obviously reflects a too intermediate significance of this work.

Response（8）: As you mentioned, the significance of the research conclusion is somewhat broad, so we have changed it to "The research results can provide theoretical and scientific basis for further research and development of FH06 associated lipid-lowering products." (L377-394), as shown in the revised figure.

Finally, we asked our colleagues who are proficient in English to help revise the language expression and grammar of the article.

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1. Introduction is like a narration. There is no proper references given. Without perticular references not allowed discussion at introduction. Try to add references more and more.

2. Abstact need revision its lack of aim and what achieved.

3. Table 1. There was no parameters of OD and DL. Just numbers mentioned.

4. Line 130-137  in results and discussion. Its like introduction part

5. Results and discussion. Try to explain the research results and highlight the key results.

6. Conclusion also need amendments, like key result mention is necessary.

Comments on the Quality of English Language

Minor editing needed 

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

 

Now I will respond to the comments one by one.

 

（1）Introduction is like a narration. There is no proper references given. Without perticular references not allowed discussion at introduction. Try to add references more and more.

Response（1）: We have added some references in the introduction section to introduce the research background and methods used in the article. (L46-86), modified as shown in the following figure.

 

（2）Abstact need revision its lack of aim and what achieved.

Response（2）: The abstract section has been supplemented with the missing purpose and results as required.

 

（3）Table 1. There was no parameters of OD and DL. Just numbers mentioned.

Response（3）: In section 2.3 The FH06 compound screening and target prediction section indicates the meanings of OB and DL. (L116-118), modified as shown in the following figure.

（4）Line 130-137  in results and discussion. Its like introduction part

Response（4）: The effective compound Genistein of Line130-137 has been re discussed and analyzed. (L165-175), modified as shown in the following figure.

（5）Results and discussion. Try to explain the research results and highlight the key results.

Response（5）: The results and discussion of sections 3.2, 3.3, and 3.4 were explained to highlight the key findings. (L280-288, 306-308, 319-322), modified as shown in the following figure.

（6）Conclusion also need amendments, like key result mention is necessary.

Response（6）: Revised some of the results in the conclusion and provided key findings and research implications. (L377-394), modified as shown in the following figure.

 

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comments and Suggestions for Authors

1. Introduction is like a narration. There is no proper references given. Without perticular references not allowed discussion at introduction. Try to add references more and more.
2. Abstact need revision its lack of aim and what achieved.
3. Table 1. There was no parameters of OD and DL. Just numbers mentioned.
4. Line 130-137  in results and discussion. Its like introduction part
5. Results and discussion. Try to explain the research results and highlight the key results.
6. Conclusion also need amendments, like key result mention is necessary.

 

Comments on the Quality of English Language

Minor editing needed 

Submission Date

21 April 2024

Date of this review

01 May 2024 06:29:08

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The study investigates the lipid-lowering mechanism and weight loss effects of FH06 fermentation broth using nontargeted metabolomics and network pharmacology. With some improvements, the paper could make a significant contribution to the understanding of traditional Chinese medicine in obesity treatment.

-The abstract effectively summarizes the study's objectives, methods, and key findings. However, the opening sentence, "To explore the mechanism by which FH06 fermentation broth reduces lipid levels and weight loss via network pharmacology and untargeted metabolomics," should be modified. For example, a brief statement about the importance of understanding the mechanisms behind FH06's effects on lipid reduction and weight loss could provide a stronger opening for the abstract.

-In the Introduction, the section introducing FH06 could be expanded more to provide details about its composition, traditional use, and previous research findings. This would help readers unfamiliar with FH06 understand its significance.

-In the M&M, the preparation method for FH06 fermentation follows a previously established protocol. It would be beneficial to include a comment on the stability of the prepared FH06 extract, considering its potential impact on reproducibility and practical application.

-Moreover, it would be useful to provide a brief explanation of ADME filtering for readers who may not be aware with the term.

-The methods for visualization using R language are appropriate, but it might be helpful to include a sentence explaining why the top 10 genes or pathways were selected.

-In the Results, the description of individual compounds like genistein, quercetin, etc is informative and enriches the understanding of FH06's potential mechanisms. However, it would be positive to specify how these compounds were selected for further analysis.

-The construction of the PPI network is described clearly, and the analysis provides valuable insights into potential mechanisms of action. However, the authors could discuss the biological significance of the interactions and how they relate to FH06's effects on fat metabolism in more detail.

-The discussion of key targets like TP53, SRC, AKT1, and STAT3 is informative, highlighting their roles in fat metabolism and potential implications for FH06's effects. However, it would be helpful to include some analysis or speculation on how FH06 might modulate these targets specifically.

-In general, it is important to include some discussion on key findings or patterns observed in all the networks described, both in the text, as well as in the figure legends.

In the Conclusion, mentioning that the research results can provide a basis for further research and development on FH06's medicinal and edible value is appropriate. Please provide specific suggestions for future research directions or potential applications of FH06 in clinical practice.

Comments on the Quality of English Language

moderate editing

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

 

In your comments and suggestions for authors, you mentioned:

The study investigates the lipid-lowering mechanism and weight loss effects of FH06 fermentation broth using nontargeted metabolomics and network pharmacology. With some improvements, the paper could make a significant contribution to the understanding of traditional Chinese medicine in obesity treatment.

Now I will respond to the comments one by one.

 

（1）The abstract effectively summarizes the study's objectives, methods, and key findings. However, the opening sentence, "To explore the mechanism by which FH06 fermentation broth reduces lipid levels and weight loss via network pharmacology and untargeted metabolomics," should be modified. For example, a brief statement about the importance of understanding the mechanisms behind FH06's effects on lipid reduction and weight loss could provide a stronger opening for the abstract.

Response（1）: Indeed, as you mentioned in the opening statement, making a brief statement on the importance of FH06's mechanism behind its lipid-lowering and weight loss effects can provide a more powerful starting point for the abstract. Therefore, we briefly explained its importance in the opening remarks. (L20-21), modified as shown in the following figure.

 

（2）In the Introduction, the section introducing FH06 could be expanded more to provide details about its composition, traditional use, and previous research findings. This would help readers unfamiliar with FH06 understand its significance.

Response（2）: We have conducted extensive research and discussion during the literature analysis of the question you raised. Currently, most of the research on FH06 has focused on its beneficial antioxidant properties, flavor, and potential as a functional food. In this context, we have introduced the components and types of compounds contained in FH06, as well as the potential effects it has. On this basis, we conducted relevant research on its mechanism of inhibiting obesity. (L46-81), modified as shown in the following figure.

 

（3）In the M&M, the preparation method for FH06 fermentation follows a previously established protocol. It would be beneficial to include a comment on the stability of the prepared FH06 extract, considering its potential impact on reproducibility and practical application.

Response（3）: When preparing samples, we divide them into three batches. Fermented and unfermented samples are made in one batch per week, and then the samples are frozen at -80 ℃. Finally, one sample is randomly selected from each batch of the three batches for testing. The results indicate that the stability of samples prepared from different batches is still relatively good.

 

（4）Moreover, it would be useful to provide a brief explanation of ADME filtering for readers who may not be aware with the term.

Response（4）: As you said, I have explained ADME in the article. ADME refers to the metabolic dynamics of drugs, mainly studying the dynamic changes in the body's disposal of drugs. This includes the absorption, distribution, biochemical conversion (or metabolism), and excretion processes of drugs in the body, especially the temporal variation of blood drug concentration. The metabolism of drugs is related to human age, gender, individual differences, and genetic factors. (L119-123), modified as shown in the following figure.

 

（5）The methods for visualization using R language are appropriate, but it might be helpful to include a sentence explaining why the top 10 genes or pathways were selected.

Response（5）: Additional explanation has been provided on why the top 10 genes were selected for visualization. (L148-150), modified as shown in the following figure.

（6）In the Results, the description of individual compounds like genistein, quercetin, etc is informative and enriches the understanding of FH06's potential mechanisms. However, it would be positive to specify how these compounds were selected for further analysis.

Response（6）: As you mentioned, we have described and analyzed some key compounds in FH06, and we selected these compounds based on oral bioavailability (OB) and drug like properties (DL).

（7）The construction of the PPI network is described clearly, and the analysis provides valuable insights into potential mechanisms of action. However, the authors could discuss the biological significance of the interactions and how they relate to FH06's effects on fat metabolism in more detail.

Response（7）: . As you suggested, we have added a discussion on the biological significance of key targets and FH06 in section 3.2. (L280-289), modified as shown in the following figure.

（8）The discussion of key targets like TP53, SRC, AKT1, and STAT3 is informative, highlighting their roles in fat metabolism and potential implications for FH06's effects. However, it would be helpful to include some analysis or speculation on how FH06 might modulate these targets specifically.

Response（8）: We analyzed the KEGG enrichment pathway of these key targets, and found that these targets in the 40 pathways of FH06 treating obesity are mainly related to chemical carcinogenesis, receptor activation, lipid and atherosclerosis, neuroactive ligand receptor interaction, prostate cancer, endocrine resistance, AGE-RAGE signaling pathway in diabetes complications, HIF-1 signaling pathway, inflammatory mediators regulation of TRP channel, regulation of lipolysis of adipocytes, FoxO signaling pathway and sphingolipid signaling pathway. This means that these targets are specifically regulated through these signaling pathways.

（9）In general, it is important to include some discussion on key findings or patterns observed in all the networks described, both in the text, as well as in the figure legends.

Response（9）: For this issue, we considered it this way. We first detected the compounds in FH06 using network pharmacology methods and screened them in TTD and DisGeNET databases. Then, we constructed a PPI network to analyze these effective proteins, enriched their effective targets with GO and analyzed them with KEGG. Finally, we analyzed the network of metabolite target pathway. Through this process, we found that all results are presented in the form of key compounds, target proteins, and pathways. Therefore, we focused on discussing key compounds and targets when constructing relevant networks.

（10）In the Conclusion, mentioning that the research results can provide a basis for further research and development on FH06's medicinal and edible value is appropriate. Please provide specific suggestions for future research directions or potential applications of FH06 in clinical practice.

Response（10）: As you mentioned, we have added the value of FH06 in terms of medicinal and usage in the conclusion section, and explained its positive significance for the treatment of obesity in clinical practice. (L390-395), modified as shown in the following figure.

 

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

I recommend that the manuscript be accepted after minor revisions have been made. With that, below are my comments and suggestions for the authors.

 Abstract. Please, add more results found in your study.

The UPLC/MS/MS method was used to determine compounds in the FH06 fermentation broth, is mentioned in the Abstract and Introduction, but is not described in Materials and Methods.

Line 53, 85, References: “Lactobacillus fermentum” should be written in italics.

Line 89: “The above experimental test results“- what test results are at stake? The preparation and fermentation of the FH06 extract is described above.

TCMSP database - please provide a link to the website and add that TCMSP is a pharmacology database of Traditional Chinese Medicine.

The top 20 compounds were selected (Table 2) and only six were described, including three listed only in the Table 1 (demethylwedelolactone, sinensetin and isosinensetin). Why only these 6 were focused on? What is the significance of the remaining compounds listed in Table 2?

Line 83: Is reference number [1] correct?

Line 187: The abbreviation PPI is explained in line 183.

Line 212: Add reference number.

The diagram (Figure 1) shows that TP53, SRC, AKT1, STAT3, ESR1 may be key targets of FH06 in the regulation of fat metabolism . TP53, SRC, AKT1, STAT are describe, ESR1 is not. Why?

The conclusion from the manuscript does not correspond to the Results and Analysis. The conclusion lists the compounds that are listed in Table 1, but does not describe them in the results.

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

 

In your comments and suggestions for authors, you mentioned:

I recommend that the manuscript be accepted after minor revisions have been made. With that, below are my comments and suggestions for the authors.

 

Now I will respond to the comments one by one.

 

（1）Abstract. Please, add more results found in your study.

Response（1）: We have added relevant research findings in the abstract section. (L35-39), modified as shown in the following figure.

（2）The UPLC/MS/MS method was used to determine compounds in the FH06 fermentation broth, is mentioned in the Abstract and Introduction, but is not described in Materials and Methods.

Response（2）: The relevant methods (L97-112) have been added after section 2.1, as shown in the modified figure below.

（3）Line 53, 85, References: “Lactobacillus fermentum” should be written in italics.

Response（3）: Latin names such as "Lactobacillus fermentum" have been adjusted to italics. (L60, L92), modified as shown in the following figure.

（4）Line 89: “The above experimental test results“- what test results are at stake? The preparation and fermentation of the FH06 extract is described above.

Response（4）: The UHPLC-QE-MS conditions and methods have been added in section 2.2, and the above test results refer to the effective compound components of FH06 obtained using this method. (L97-112), modified as shown in the following figure.

（5）TCMSP database - please provide a link to the website and add that TCMSP is a pharmacology database of Traditional Chinese Medicine.

Response（5）: We have explained TCMSP in the relevant section. (L114-116), modified as shown in the following figure.

（6）Line 83: Is reference number [1] correct?

Response（6）: . Both Line 83 and Line 43 are sourced from literature: “[1]Zhang, X.; Ha, S.; Lau, H.C.; Yu, J. Excess body weight: Novel insights into its roles in obesity comorbidities. Semin Cancer Biol 2023, 92, 16-27, doi:10.1016/j.semcancer.2023.03.008.”

（7）Line 187: The abbreviation PPI is explained in line 183.

Response（7）: The PPI has been adjusted to be explained in the methodology section. (L135-137), modified as shown in the following figure.

（8）Line 212: Add reference number.

Response（8）: The reference for Line 212 can be found in [15,16]. (L448-454), modified as shown in the following figure.

（9）The diagram (Figure 1) shows that TP53, SRC, AKT1, STAT3, ESR1 may be key targets of FH06 in the regulation of fat metabolism . TP53, SRC, AKT1, STAT are describe, ESR1 is not. Why?

Response（9）: Thank you for pointing out the problem. Although ESR1 is a key target in the regulation of fat metabolism, ESR1 mediates the corresponding biological effects of estrogen and regulates the expression of different genes in different tissues. ESR1 plays an important role in maintaining blood glucose homeostasis, bone and cardiovascular health, reproductive capacity and neurological function. We have supplemented this part of the content. ( L280-283 ), see figure below for modifications.

（10）The conclusion from the manuscript does not correspond to the Results and Analysis. The conclusion lists the compounds that are listed in Table 1, but does not describe them in the results.

Response（10）: Thank you for pointing this out. In our study, we found that we obtained a total of 85 effective compounds, and we explained the conditions of compound screening in section 2.3 ( oral bioavailability ( OB ) ≥ 30 %, drug similarity ( DL ) ≥ 0.18, and finally obtained effective compounds of FH06 ). We introduced some effective compounds in section 3.1, and did not introduce all compounds due to space reasons.

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authrs have imroved the ms but some points should be checked and considered. 

Abastract: the first and second sentences are incomplete.

The authors should check dashes in words after formatting.

Comments on the Quality of English Language

Some typing problems should be solved.

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

 

In your comments and suggestions for authors, you mentioned:

The authrs have imroved the ms but some points should be checked and considered. Major Comments

Now I will respond to the comments one by one.

 

（1）Abastract: the first and second sentences are incomplete.

Response（1）: The first sentence and second round have been rewritten (Line20-22), as shown in the revised figure below.

（2）The authors should check dashes in words after formatting.

Response（2）: We have checked the dashes in the article.

 

Finally, we asked our colleagues who are proficient in English to help revise the language expression and grammar of the article.

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have indeed covered nearly all the points raised.

However, I will emphasize two aspects:

• The beginning of the abstract.

Rephrasing to incorporate the suggested sentence:

"Investigating the intricate pathways through which FH06 fermentation broth exerts its lipid-lowering and weight loss effects is pivotal for advancing our comprehension of metabolic regulation and therapeutic interventions."

• In the conclusion, the authors should be more specific in the end and add something like:

"Our research opens a promising avenue for obesity through FH06-based interventions. By providing a robust theoretical foundation, we advocate for the development of FH06-assisted lipid-lowering products, potentially revolutionizing current approaches to weight management. The elucidation of FH06's pharmacological profile leads to diverse applications, spanning medicinal therapeutics to functional foods. Future research  may explore novel formulations and delivery systems, maximizing the therapeutic potential of FH06 and harnessing its medicinal and edible value to its fullest extent."

 

Comments on the Quality of English Language

minor editing

Author Response

A short cover letter for detailing the changes

 

Dear Professor,

First of all, thank you for seriously pointing out the shortcomings in our article, so that we can have the opportunity to improve.

In your comments and suggestions for authors, you mentioned:

The authors have indeed covered nearly all the points raised.

However, I will emphasize two aspects.

Now I will respond to the comments one by one.

（1）The beginning of the abstract.

Rephrasing to incorporate the suggested sentence:

"Investigating the intricate pathways through which FH06 fermentation broth exerts its lipid-lowering and weight loss effects is pivotal for advancing our comprehension of metabolic regulation and therapeutic interventions."

Response（1）:Your suggestion is very good. We have made modifications to the abstract, as shown in the figure below (Line20-22).

（2）In the conclusion, the authors should be more specific in the end and add something like:

"Our research opens a promising avenue for obesity through FH06-based interventions. By providing a robust theoretical foundation, we advocate for the development of FH06-assisted lipid-lowering products, potentially revolutionizing current approaches to weight management. The elucidation of FH06's pharmacological profile leads to diverse applications, spanning medicinal therapeutics to functional foods. Future research  may explore novel formulations and delivery systems, maximizing the therapeutic potential of FH06 and harnessing its medicinal and edible value to its fullest extent."

Response（2）: We have revised the conclusion section according to the suggestions, as shown in the figure below (Line391-398).

Finally, we asked our colleagues who are proficient in English to help revise the language expression and grammar of the article.

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

 

Kind regards,

Mr. Hao-Ming Wang

Author Response File: Author Response.docx