Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the current study the authors demonstrated that Padina minor ethanolic extract improves metabolic parameters and adipose tissue characteristics in HFD-induced obese rats, potentially through modulation of pathways related to adipogenesis and lipid metabolism, supporting its potential as a marine-derived nutraceutical candidate for obesity management.  Further studies are required to confirm its mechanisms and clinical relevance.

Some suggestions:

1. Page 3, add please more details related to “Species identity was confirmed morphologically by a phycologist using standard taxonomic keys, and cross-checked against Algae Base”
2. Point 2.2. Animal treatment:

-in what consists HDF and which is its origin?

-how did you decided to administer  25/50/100 mg P minor /kg BW. Did you performed acute toxicity studies?

- please add how was the food intake and Lee index recorded throughout the study. Please specify how were the rats caged, namely how many/cage

3. At biochemical analysis, molecular analysis and histological examination (points 2.4-2.7) you must add the devices and the methods used to perform the analysis. Take them please from the Suppl information and add them in the article.

4.Why you didn’t you determine also HbA1c? You determined only glucose.

5. Please add how were the mice euthanized.

6.Primer Sequences Used for Quantitative Real-Time PCR (qPCR) must be included in the article not at Suppl Material.

7.Point 2.7. and Figure 4 - What kind of microscope was used to performe the histological examination? Please add.

8. Add please the normal values for the biochemical parameters presented in Table 2.
9. Please underline what brings new your study.
10. Page 18 - In my opinion the number of references is too small
11. The article is not formatted according to the requirements of the Nutrients Journal and no template was used.

 

Author Response

Dear Reviewer,

We sincerely thank you for your careful evaluation of our manuscript entitled:

“Marine-Derived Padina minor Extract Improves Lipid and Glucose Metabolism in Obese Rats: Evidence for PPARγ and ADIPOR1 Modulation”

We greatly appreciate your constructive comments and valuable suggestions, which have significantly improved the quality, clarity, and scientific rigor of our manuscript.

In response to your comments, we have carefully revised the manuscript and addressed all concerns point-by-point. All modifications made in the revised manuscript have been highlighted in red text for ease of review.

The major revisions include:

• Expanded taxonomic identification details of Padina minor, including morphological characteristics, voucher specimen information, and AlgaeBase verification.
• Added detailed composition and preparation of the high-fat diet (HFD), including caloric values and origin.
• Clarified the rationale for selecting the extract doses (25, 50, and 100 mg/kg BW) based on previous anti-obesity studies and gallocatechin gallate equivalence.
• Added detailed descriptions of food intake recording, Lee index calculation, and housing conditions (1 rat per cage).
• Included instruments and analytical methods used for biochemical, molecular, and histological analyses.
• Added explanation regarding the absence of HbA1c measurement.
• Included euthanasia procedures and anesthetic doses.
• Moved qPCR primer sequences into the main manuscript as requested.
• Added details regarding the microscope used for histological examination.
• Added normal reference ranges for biochemical parameters.
• Clarified the novelty and scientific contribution of the study.
• Expanded and updated the references section with additional recent literature.
• Reformatted the manuscript according to the official Nutrients (MDPI) template requirements.

We believe that these revisions have substantially strengthened the manuscript and adequately addressed all reviewer concerns.

Thank you again for your valuable feedback and consideration.

Sincerely,

Anton Bahtiar, Ph.D.
Corresponding Author
Faculty of Pharmacy, Universitas Indonesia

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review Report

concerning the manuscript entitled „Marine-Derived Padina minor Extract Improves Lipid and Glucose Metabolism in Obese Rats: Evidence for PPARγ and ADIPOR1 Modulation”

by Anton Bahtiar and others

Manuscript ID: nutrients-4286952

 

General:

The article submitted for review by Anton Bahtiar and colleagues evaluates the potential antiobesity properties of an ethanol extract from the brown algae Padina minor in a rat model of obesity induced by a high-fat diet (HFD). The work is interesting, experimentally well-designed, and contributes to the search for natural, marine bioactive compounds with metabolic activity. The authors combine phytochemical analysis (GC-MS, LC-MS/MS) with the assessment of physiological, biochemical, and molecular parameters (qPCR, ELISA), which is a strength of the manuscript. The article is written with great care, containing all the essential elements of the original publication, and the results are presented clearly, although with minor editorial errors. The work contributes new knowledge about the previously little-studied species Padina minor in the context of obesity.

 

Major Comments:

(1) In Table 5 – for the PM-M group, the CNR1 column gives a value of -45.00 ± 35.355. The negative standard deviation and negative mean for protein levels (which cannot be negative) suggest an error in the calculations or presentation of the data. (2) In the text (e.g., pp. 9, 13) and below the tables, the authors use a significance level of p < 0.001 as the only threshold. Currently, it is recommended to report exact p values (e.g., p = 0.002) or at least ranges (e.g., p < 0.01, p < 0.05). Strict adherence to <0.001 for all differences may be insufficient. (3) Table 6 lacks data for the PM-L and PM-M groups on Adipor1 and Pparγ expression. The authors report only NC, HFD, ORL, and PM-H. Why were lower doses omitted? (4) in section 2.2.3 it is written: "Obesity was induced [...] for 90 days. The treatment phase continued once rats reached approximately 50% body-weight gain." There is no information about the average time of obesity induction – did all rats achieve 50% body-weight gain after 90 days? Or maybe earlier? (5) Section 2.7's description of the histology is very brief. Information is missing regarding the method and number of independent observers used to measure adipocyte diameter. Furthermore, Table 4 provides "Adipose cell perimeters" – is this circumference? Why not a more standard parameter like surface area or diameter? (6) In sections 3.7 and 3.8, the authors analyze PPARγ at both the protein (ELISA – Table 5) and mRNA (qPCR – Table 6) levels. However, the results are not fully consistent: in Table 5, PM-H results in a reduction of PPARγ (0.94 vs. 3.58 in HFD), and in Table 6, PPARγ mRNA expression in the PM-H group is 1.98 (an increase relative to NC, but a decrease relative to HFD? HFD=2.82). The value of 1.98 in PM-H is higher than in ORL (0.98) and is difficult to interpret as strong inhibition. A test of the significance of the difference between PM-H and ORL is missing. (7) Why were samples from only 6 animals per group used for gene expression analysis (qPCR), but Table 6 is missing data for two extract doses (PM-L and PM-M)? Were these samples not analyzed, or were the results statistically insignificant? (8) The "Discussion" section is extensive and well-grounded in the literature. However, it lacks a critical response to certain limitations, such as why the higher dose of the extract (100 mg/kg) did not always perform better than the average (50 mg/kg) – e.g., in Table 4 for the PM-M (5.05) vs. PM-H (5.50) adiposity index. Is there a plateau effect? (9) The authors rightly mention the multicomponent effects of the extract, but they do not attempt to assess which compounds (flavonoids, sterols, phenolic acids) may be most responsible for the observed effects. A correlation analysis between the content of individual compounds and potency could be suggested. (10) How did the authors ensure that the extract did not degrade during storage (conditions: -20°C, up to 30 days)? Was stability testing of the active compounds, especially polyphenols, performed? (11) Were any adverse effects observed following the administration of the extract (e.g., behavioral changes, diarrhea, changes in the weight of internal organs other than adipose tissue)? The article does not mention this. (12) I suggest proofreading the entire manuscript due to syntactic errors.

 

Minor Comments:

(1) Table 2 should be reorganized because it is unclear which results refer to "before HFD," "after HFD," and "after treatment." The current formatting (duplicate indicator names in the first column) is misleading. For example, in Table 2, the "Cholesterol" row appears multiple times, but the values refer to different measurement times. Similarly, Table 3 also requires reformatting. Perhaps adding colors would be a good idea? (2) The test contains numerous typos, e.g., missing spaces, unnecessary spaces, missing hyphens, and missing periods. Summary and Recommendation: This article presents valuable and innovative results indicating the potential of Padina minor to alleviate metabolic disorders associated with obesity. The study was conducted in accordance with ethical animal practices, using randomization and blinding, which enhances its credibility. The main concerns concern inconsistencies in the tables (data errors, missing groups, negative values) and the incomplete presentation of qPCR results. Major Review.

Comments on the Quality of English Language

I suggest proofreading the entire manuscript due to syntactic errors.

Author Response

Dear Reviewer,

We sincerely thank you for your careful evaluation of our manuscript and for your thoughtful and constructive comments. Your detailed feedback has greatly helped us improve the scientific rigor, clarity, and overall quality of the manuscript.

In response to your suggestions, we have carefully revised the manuscript and addressed all comments point-by-point. For ease of evaluation, all revisions and newly added text in the manuscript have been highlighted in blue text.

The major revisions made in response to your comments include:

• Re-evaluation and correction of the CNR1 ELISA data presentation, including clarification regarding values below the assay detection limit.
• Revision of statistical reporting throughout the manuscript using graded significance thresholds (p < 0.05, p < 0.01, and p < 0.001) instead of a single universal threshold.
• Clarification regarding the selection of the PM-H group for downstream qPCR analysis and explanation for exclusion of PM-L and PM-M groups from molecular analysis.
• Clarification of the obesity induction timeline and confirmation that all HFD-fed rats completed the standardized 90-day induction period.
• Expanded histological methodology, including adipocyte morphometric analysis, blinded observers, and clarification of adipocyte perimeter measurements.
• Revision of the interpretation of PPARγ protein and mRNA expression results, including discussion of post-transcriptional regulation and addition of statistical comparison between PM-H and ORL groups.
• Inclusion of a more critical discussion regarding the non-linear dose-response relationship and possible plateau/saturation effects.
• Addition of discussion regarding the multicomponent nature of the extract and future directions involving bioactivity-guided fractionation and compound-specific analysis.
• Clarification of extract storage conditions and acknowledgement of the absence of formal stability testing for polyphenolic compounds.
• Addition of statements regarding daily toxicity monitoring and the absence of observable adverse effects during treatment.
• Thorough proofreading and correction of grammatical, syntactic, typographical, and formatting inconsistencies throughout the manuscript.
• Reorganization and reformatting of Tables 2 and 3 to improve readability and interpretation of the different experimental time points.

We believe that these revisions have substantially strengthened the manuscript and adequately addressed all reviewer concerns.

We sincerely appreciate your valuable comments and thoughtful suggestions, which have significantly improved the quality of our work.

Sincerely,

Anton Bahtiar, Ph.D.
Corresponding Author
Faculty of Pharmacy, Universitas Indonesia

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Point 10. In my opinion the number of references is too small.

You answered: We have carefully revised the manuscript to increase the number of relevant references, particularly in the Introduction and Discussion sections. Additional recent and high-quality studies have been incorporated to better support the interpretation of our findings and to place our results in the context of current literature. We believe these additions have strengthened the scientific background and discussion of the study.

In the revised manuscript you have the same number of references. In my opinion the number of references is too small.

 

 

 

 

Author Response

Dear Reviewer,

Thank you for your valuable comment regarding the number of references included in the manuscript. We sincerely apologize that the previous revision did not sufficiently address this concern.

In response to your suggestion, we have carefully revised the manuscript and substantially expanded the reference list, particularly in the Introduction and Discussion sections, to provide a stronger scientific foundation and broader contextualization of our findings.

Specifically, we incorporated several recent and relevant references related to:

• obesity pathophysiology and current therapeutic strategies,
• marine-derived anti-obesity bioactive compounds,
• metabolic effects of flavonoids and brown algae constituents,
• molecular regulation involving PPARγ, ADIPOR1, ESR1, and CNR1,
• fucoxanthin and phenolic compounds in obesity management,
• adiponectin signaling and metabolic homeostasis.

The revised manuscript now contains substantially more up-to-date literature, including multiple studies published between 2020 and 2026. We believe these additions significantly strengthen the scientific background, mechanistic discussion, and interpretation of our results within the context of current research.

Thank you again for your constructive feedback, which has helped us improve the quality of the manuscript.

Sincerely,

Anton Bahtiar, Ph.D.
Corresponding Author

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

After carefully examining the authors’ responses and the updated manuscript, I am pleased to confirm that the authors have addressed all of my comments, suggestions, and questions in a thorough and satisfactory manner. They have implemented the requested revisions and provided the necessary clarifications and additions, which have significantly improved the overall quality and clarity of the work.

In my opinion, the manuscript now meets the standards required for publication and may be considered suitable for acceptance. However, I respectfully acknowledge that the final decision rests with the Editorial Board, and I am fully supportive of their judgment in this matter.

Comments on the Quality of English Language

no additional comments

Author Response

Dear Reviewer,

We sincerely thank you for your careful re-evaluation of our revised manuscript and for your positive and encouraging feedback.

We are truly grateful for your thoughtful comments, constructive suggestions, and detailed evaluation throughout the review process. Your insights have significantly contributed to improving the scientific quality, clarity, methodological rigor, and overall presentation of our manuscript.

We greatly appreciate your acknowledgment that the revised manuscript has satisfactorily addressed all comments and now meets the standards required for publication. Your supportive assessment is highly encouraging to us.

Thank you once again for your valuable time, expertise, and constructive guidance during the review process. We also fully respect and appreciate the final decision of the Editorial Board regarding the manuscript.

Sincerely,

Anton Bahtiar, Ph.D.
Corresponding Author
Faculty of Pharmacy, Universitas Indonesia

 

Author Response File: Author Response.pdf