Effects of Passiflora edulis Leaf Extract on Lipid Accumulation in HepG2 Cells: In Vitro Evidence and Molecular Docking Analysis Involving PPARα and SREBP-1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Effect of Ethanolic Extract of Passion Fruit Leaves on Transcriptional Factors (SREBP-1 and PPARa) and Their Lipid Response in HepG2 Cells" investigates the hypolipidemic effects of Passiflora edulis leaf extract. The authors provide a comprehensive study combining phytochemical characterization, in vitro assays (cell viability, lipid quantification), and in silico molecular docking. The topic is interesting and falls within the scope of the journal.

However, there are several issues regarding data presentation, abbreviations, and the interpretation of the mechanistic study that need to be addressed before publication. I recommend acceptance after Minor Revisions.

1. The title and conclusion emphasize the effect on transcriptional factors (SREBP-1 and PPAR-α). However, the study relies solely on molecular docking predictions and the phenotypic outcome, e.g. lipid reduction to support this. There is no direct experimental evidence showing that the extract actually alters the expression or phosphorylation levels of both transcriptional factors in HepG2 cells. Please rephrase sentences in the Abstract, Discussion, and Conclusion to reflect that the interaction with these transcription factors is predicted by in silico modeling rather than definitively proven as the sole mechanism.

 

2. In Figures 1, 2, and 3, the X-axis or legends contain the text "24 horas" and "48 horas", which is in Spanish. Please update all figures to English ("hours").

 

3. In Figure 2 and 3, is the "Control" group treated with the vehicle (solvent) used for the extract and OA? Please specify the vehicle used (e.g., DMSO, Ethanol) and confirm the final concentration was non-toxic.

 

Please check the manuscript for minor grammatical errors, e.g. the compound "espinosin" on line 217 and 514 should be spelled "spinosin".

Author Response

We thank the reviewer for their time and valuable comments, which significantly improve the quality of our article. Responses to each of the comments made are included in the attached document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript entitled: “Effect of ethanolic extract of passion fruit leaves on transcriptional factors (SREBP-1 and PPARα) and their lipid response in HepG2 cells”. The manuscript addresses an important and timely issue concerning the pathogenesis of NAFLD and the potential role of phytochemical interventions. Conceptually, the study design is appropriate and translationally relevant. However, in its current form, the work remains preliminary and largely screening-based, and it lacks direct mechanistic evidence to substantiate the conclusions regarding PPARα/SREBP-1 modulation. Several methodological and interpretational aspects require clarification, and some conclusions appear overstated.

• At present, there is no molecular validation of PPARα and SREBP-1 activity, which represents the most critical limitation of the study. The authors infer PPARα activation and SREBP-1 inhibition solely on the basis of molecular docking analyses. This is insufficient to support mechanistic claims. Western blotting and/or qPCR analyses of PPARα, SREBP-1c, FASN, ACC, SCD1, and CPT1A/ACOX1 (β-oxidation markers) are essential and should be considered the minimum requirement to confirm pathway modulation. Without these data, the conclusions are not adequately supported.
• The Introduction should end with a clearly defined aim of the study and, ideally, explicit research hypotheses. This would substantially improve clarity and help readers better understand the manuscript’s objectives and rationale.
• The concentrations used (up to 3000 µg/mL; 3 mg/mL) and IC₅₀ values in the mg/mL range are non-physiological and extremely high. The observed effects may result from nonspecific toxicity or membrane disruption rather than specific biological activity. This also limits the translational relevance of the findings, as the real bioavailability of such doses is questionable. The authors should justify the choice of concentrations, focus on lower and more physiologically relevant doses, report extraction yield and equivalent dry-leaf concentrations, and consider fractionation or identification of the active fraction. These issues should also be explicitly discussed as study limitations.
• The authors do not discuss important limitations of the HepG2 model, including low CYP450 activity, altered lipid metabolism, and the transformed (cancer) phenotype. These aspects should be addressed in the Discussion. Alternatively, additional hepatic models such as Huh7, HepaRG, or primary hepatocytes would strengthen the study.
• The calculation of IC₅₀ values for triglyceride and cholesterol reduction is unconventional and methodologically questionable. IC₅₀ is typically applied to cytotoxicity or enzyme inhibition assays. For lipid metabolism studies, results are more appropriately presented as percentage reduction or fold-change relative to controls.
• The study relies excessively on molecular docking. In addition to the suggested molecular assays (Western blot or qPCR), the authors should compare their findings with known modulators, such as fenofibrate (PPARα agonist) or fatostatin (SREBP inhibitor), to provide pharmacological context and validation.
• Chromatographic analysis or deeper chemical characterization of the extract is lacking. Without such data, it is unclear which compounds are responsible for the observed effects.
• Appropriate experimental controls are also missing, including solvent (DMSO/vehicle) controls, positive controls (e.g., fenofibrate), and oxidative stress/ROS measurements.
• Regarding statistics, the manuscript does not clearly specify the value of n or whether replicates are biological or technical. The simultaneous use of ANOVA, Duncan, and Tukey tests appears excessive. One-way ANOVA followed by Tukey’s post-hoc test would be sufficient. The authors should justify the chosen statistical methods and clarify whether normality assumptions were tested.

Author Response

We thank the reviewer for their time and valuable comments, which significantly improve the quality of our article. Responses to each of the comments made are included in the attached document.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,
You have addressed and improved most of the previously concerning aspects of the manuscript. Despite some remaining limitations, these have now been clearly acknowledged and discussed. Therefore, I believe that, in its current form, the manuscript can be accepted.