-->PONE-D-25-60521-->-->Biochemical and Antidiabetic Properties of Elaeocarpus angustifolius Blume: In vitro, In vivo, and In silico Insights-->-->PLOS One

Dear Dr. Adhikari,

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The abstract should be further improved, along with the conclusion, and the title did not describe the whole picture of the manuscript.

add more recent related reported work and highlight the gap of the current study in abstract part

Reviewer #2: Article review

Title: Biochemical and Antidiabetic Properties of Elaeocarpus angustifolius Blume: In vitro, In vivo, and In silico Insights.

The manuscript is skilfully written. The article evaluates the antidiabetic potential of Elaeocarpus angustifolius bark extract through phytochemical, in vitro, in vivo, and computational analyses, structured across standard scientific sections with varying strengths in clarity and depth. While the study provides valuable insights and a robust dataset, several weaknesses and limitations can be discerned in its different components. The results can be presented in a more suitable manner. In summary, certain remarks ought to be taken into account.

Title and abstract

The title adequately reflects the study content. Please add from Kirtipur, Kathmandu .

The abstract concisely outlines objectives, methods, key results (e.g., IC50 values for α-glucosidase 0.79 μg/mL, glucose reduction 21%, corilagin docking ]10.3 kcal/mol), and implications for drug development, effectively highlighting antioxidant/antidiabetic activities. Strengths include quantitative highlights and logical flow from in vitro to in silico data. The abstract provides a dense summary of methods and results but lacks precision in quantifying key outcomes, such as exact reductions in glucose levels or statistical significance beyond p<0.01 mentions. It overstates findings by claiming "potent" inhibition without contextualizing IC50 values against standards like acarbose, potentially misleading readers on comparative efficacy. Additionally, it prematurely highlights Corilagin as the "most active compound" without briefly noting limitations like in vivo dose translation.

1. Introduction:

The introduction provides diabetes epidemiology (e.g., projections to 552 million by 2030 M, complications, synthetic drug limitations, and plant-based rationale, justifying Elaeocarpus study via traditional uses and phytochemical gaps. It clearly states aims (antioxidant/antidiabetic assays plus in silico support). Shortcomings include outdated statistics (e.g., 2013 data), repetitive prevalence claims, and superficial traditional evidence without cultural specificity beyond Nepal.

The introduction compiles diabetes statistics from outdated sources (e.g., projections to 2030 from 2013 data) without updating to current global prevalence figures, undermining relevance given diabetes cases exceeded 500 million by 2021. It lists complications and synthetic drug side effects generically without specific evidence tying them to the plant's context, weakening the rationale for studying Elaeocarpus angustifolius. Traditional uses are asserted broadly (e.g., for diabetes in Nepal) but lack cited ethnographic studies, relying on vague references that do not justify the research gap convincingly

2. Materials and Methods

Detailed subsections cover chemicals (Sigma-Aldrich sources), plant collection (voucher KATHI 64207), and neonatal STZ rat model (n=6/group, 28 days), extractions (methanol maceration, 12.4% yield), assays (DPPH, enzyme inhibition with formulas), LC/MS (Thermo/SCIEX, gradient specified), and computations (AutoDock Vina, GROMACS MDM. Strengths: Ethical approval, statistical tools (ANOVA, t-test, p<0.05M, reproducible protocols with references. Weaknesses: Incomplete details (e.g., STZ dose, assay modifications, no blinding/power analysis), formula errors (e.g., incomplete LDL equation), and single-fraction LC/MS limitation.

Plant extraction uses basic maceration without yield optimization or validation against advanced techniques like ultrasound-assisted extraction, limiting reproducibility. Animal model employs neonatal STZ induction for type-2 diabetes in adult rats, but details on litter size, sex distribution, or long-term hyperglycemia confirmation via OGTT thresholds are absent, risking variability. In vivo dosing (1.25 g/kg methanol extract) vastly exceeds typical herbal doses without fractionation for safety, and ethical approval cites a mismatched institution (Bangladesh vs. Nepal collection), raising procedural concerns. Computational methods mix tools (AutoDock Vina, GROMACS) without full parameter disclosure, such as grid box validation beyond CASTp or force field justification for ligands

3. Results

Results report phytochemicals (flavonoids, tannins present), TPC/TFC (182.73 mg GAE/g, 48.78 mg QE/g), IC50s (e.g., ethyl acetate DPPH 1.47 μg/mL vs. quercetin 4.87), in vivo changes (glucose -21%, glycogen 14.06 mg/mL), 13 LC/MS compounds (e.g., corilagin), and docking (corilagin -10.3 kcal/mol). Tables/figures (e.g., Table 1 masses, Fig. 1 glucose) support data with means±SD and p-values. Issues: Combined results/discussion blurs separation; missing raw curves, baselines, or multiple comparisons adjustments; contradictory weight data unclarified.

Phytochemical screening is qualitative only, with no quantification beyond TPC/TFC, missing opportunities to correlate specific compounds to activity via HPLC standardization. In vitro IC50 values for fractions vary widely (e.g., DCM poor at 16.97 µg/mL for α-glucosidase), but no dose response curves or replicates beyond mean±SD are shown, questioning reliability. In vivo data reports body weight decreases in extract group (p<0.001 vs. controls) without exploring toxicity, and glycogen assay uses outdated anthrone method prone to interference. LC/MS identifies 13 compounds but provides inconsistent RTs/mz (e.g., rutin/geraniin at similar times) without purity confirmation or standards

4. Discussion

Discussion integrates findings (e.g., corilagin stability via MD RMSD 1.47 Å) with prior Elaeocarpus studies, proposing mechanisms (e.g., non-competitive inhibition). Discussion repeats results with superficial comparisons to prior Elaeocarpus studies, ignoring contradictions like weaker in vivo effects vs. standards. It extrapolates Corilagin's docking/MD stability to "promising lead" despite ADMET flaws (3 Lipinski violations, immunotoxicity), without dose-response or selectivity data

5. Conclusion

Conclusions emphasize bark as "promising natural source." Flaws: Over-relies on comparisons without effect sizes; ignores limitations (e.g., small n, no toxicity); speculates drug potential sans ADMET details or human relevance. Overall, the article advances plant antidiabetic research but requires methodological refinements for higher impact. Conclusion advocates clinical validation but overlooks scalability issues like bark sourcing sustainability and lacks nuance on side effects beyond synthetics

References

o The reference list is relevant but does not include recent works to provide balanced context.

o There is an absence of discussion relating results to the results to reference, recent, or even potentially conflicting literature or alternative hypotheses.

Reviewer #3: The manuscript presents a comprehensive in vitro, in vivo, and in silico investigation into the antidiabetic potential of Elaeocarpus angustifolius bark. The study is well-motivated, the experimental design is generally sound, and the findings are promising. However, several critical issues must be addressed to strengthen the manuscript's validity, clarity, and compliance with journal standards before it can be considered for publication.

Major Revisions Required:

1. The ethics statement on page 3 notes approval from the Bangladesh University of Health Sciences (BUHS) ERC. However, the in vivo study was conducted on rats in Nepal, while the approving body is in Bangladesh. Please clarify: Where exactly were the animals housed and the experiments performed? Provide the name and location of the animal facility.

2. Clarify the anesthesia/euthanasia method: The manuscript states anesthesia was performed using halothane. Please specify the method of euthanasia at the end of the experiment (e.g., overdose of anesthetic, cervical dislocation, etc.) as required by PLOS ONE's animal research guidelines (page 4 of the submission).

3. The Methods section (page 12) lists five groups but only names four (Group 1: NWC, Group 2: DWC, Group 3: GT, Group 5: Extract treated). There is a numbering error (Group 4 is missing). Please correct the group numbering and labeling throughout the manuscript and figures for consistency.

4. Justify the extract dosage: The dose of 1.25 g/kg is very high. Please provide a rationale for this specific dose (e.g., based on preliminary toxicity studies or literature). A brief discussion on the feasibility of translating such a high dose to human use should be added to the Discussion or Conclusion.

5. Clarify statistical tests: The legend for Figures 1 and 2 states a paired sample t-test was used for post-hoc comparison after ANOVA. This is unconventional. For comparing multiple groups, appropriate post-hoc tests (e.g., Tukey's, Bonferroni) should be used following ANOVA. Please specify the exact post-hoc test used and ensure it is appropriate for the experimental design.

6. For the in vitro enzyme inhibition assays (α-glucosidase and α-amylase), specify the positive control concentration range used alongside the samples. Currently, only the IC₅₀ of acarbose is given. This is necessary to validate the assay performance under the experimental conditions used for the extracts.

7. The Financial Disclosure section (page 2) states "The author(s) received no specific funding for this work." However, the Acknowledgements section (page 37) thanks the Asian Network of Research on Antidiabetic Plants (ANRAP) for "financial support." This is a direct contradiction. The authors must correct the Financial Disclosure statement to accurately reflect any and all sources of funding or material support received for this study, as per PLOS ONE policy.

8. Abstract: "significant progress in the discovery of antidiabetic drugs had been made" – change "had" to "has."

9. Page 23, Line 342: "The results (S4 Table) indicated..." – When referencing supporting information, briefly state the key finding in the main text (e.g., "The methanol extract exhibited the highest inhibition... (S4 Table)").

10. Discussion: The discussion could be strengthened by more directly comparing the potency (IC₅₀ values) of Corilagin's in silico binding with the observed in vitro activity of the ethyl acetate fraction. A sentence or two on this would better integrate the computational and experimental findings.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:Amal El Rherabi

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Biochemical and Antidiabetic Properties of Elaeocarpus angustifolius Blume: In vitro, In vivo, and In silico Insights

PONE-D-25-60521R1

Dear Dr. Adhikari,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Jorddy Neves Cruz

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

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-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The manuscript title "Biochemical and Antidiabetic Properties of Elaeocarpus angustifolius Blume: In vitro, In vivo, and In silico Insights" can be accepted in the present form.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

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