Insight into the Hypoglycemic Effects of Pinus nigra Arn. Bark Extracts Through In Silico and In Vivo Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The bark was collected in April 2018. When the extract was extracted, and when the silicon, in vivo experiment was done? If the bark or the extract kept long time, what’s influence will happen to them?

The chemicals in Pinus nigra extract were not analyzed, which should also be discussed in the limitation?

The conclusions should highlight your novel finding.

Comments on the Quality of English Language

No comments.

Author Response

The bark was collected in April 2018. When the extract was extracted, and when the silicon, in vivo experiment was done? If the bark or the extract kept long time, what’s influence will happen to them?

Response: We appreciate this comment. We are completely aware that achieving stable pine bark extracts involves careful control over extraction, using protective measures for bioactive ingredients stability. Hence, the bark was keep protected from light in a cool, dry place. Bark in a dark, cold place is relatively stable for preserving hydrophilic extractives and polyphenols. Hence, extracts were prepared within the two months of the sampling period and kept at room temperature, protected from light for maximum 48h before chemical characterization. After chemical characterization profiling immediately, the in vivo experiments were carried out. Fresh ethanol extracts were prepared for in vivo experiments following the same procedure in order to ensure the stability of phytoconstituents. In silico analysis was renewed and improved in 2025 using the latest version of software for molecular docking and visualization of obtained ligand-target complexes. Please see the revised version of section 2.1.

The chemicals in Pinus nigra extract were not analyzed, which should also be discussed in the limitation?

Response: Thank you for this valuable comment. We agree that the lack of comprehensive chemical profiling of the tested P. nigra bark extract should be explicitly acknowledged as a limitation. Accordingly, we have revised the Limitations (Discussion) to state that detailed qualitative/quantitative characterization of individual constituents (e.g., by HPLC–MS-based profiling) was not performed within the present work, which precludes direct attribution of the observed biological effects to specific compounds and further supports interpreting the in silico findings as hypothesis-generating.

The conclusions should highlight your novel finding.

Response: In addition, we have revised the Conclusions to clearly highlight the novelty of our study. Specifically, we now emphasize that, to our knowledge, this is the first in vivo evaluation of a Serbia-origin P. nigra bark extract demonstrating hypoglycaemic and hypolipidaemic effects in an alloxan-induced diabetic rat model, including additive efficacy when combined with metformin or gliclazide.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I attach a review of the article „Insight Into the Antidiabetic Effects of Pinus nigra Arn. Bark Extracts Through In Silico and In Vivo Analysis”.

Main notes:

• In title is Pinus nigra Arn. – the correct name is Pinus nigra
• Unacceptable form of Abstract.
• Unacceptable presentation and description of statistical analyses. The form of presenting the results of statistical analyses in Tables presented by the authors is outside the generally accepted standard. Very often, the consequence of this is a description containing too many results of statistical analyses, which makes the text unreadable. For example Line 291-294: …no statistically significant differences in baseline glucose values between groups at the beginning of the experiment (F 292 = 1.739; p = 0.115). However, on the final day of treatment, a statistically significant difference in glycaemia was recorded (F = 7.332; p < 0.0001).
• Tables – difference “in columns” should be presented in accordance with the rule (standard): values represented with different letters (e.g. small letters) are statistically different at p<0.05.
• Tables – difference “before-after” should be presented in accordance with the rule (standard): values represented with different letters (e.g. capital letters) or (*) are statistically different at p<0.05.
• Results of ANOVA (F-values; p-values) should be presented in additional Table. Constantly including analysis results in the text is unnecessary ballast. During description of statistically significant differences or their absence, it is enough to cite the adequate Tables.
• Text and Tables need improvement: see Table 1: data of Δ HOL and Δ TG should be in one line , see Line 332-352 (italics); There are more errors in the manuscript.
• Additional literature: Oshetkova, D.; Klimowicz, A. Antioxidative and Photoprotective Activity of Pinus nigra, Pinus strobus and Pinus mugo. Appl. Sci. 2025, 15, 209. Nisca, A.; Stefanescu, R.; Stegdrus, D.I.; Mare, A.D.; Farczadi, L.; Tanase, C. Comparative study regarding the chemical composition and biological activity of pine (Pinus nigra and sylvestris) bark extracts. Antioxidants 2021, 10, 327.

Author Response

• In title is Pinus nigra Arn. – the correct name is Pinus nigra 

Response: Thank you for noting this. We have corrected the title by removing the author citation “Arn.” and now use the scientific name as Pinus nigra, in accordance with the reviewer’s recommendation and journal style.

• Unacceptable form of Abstract.

Response: Thank you for this comment. We have revised the Abstract to fully comply with the journal instructions: it is now a single paragraph (~200 words), follows a structured-abstract style without headings (background–methods–results–conclusion), includes the animal species/strain and key treatments, and avoids introducing results not presented in the main text or overstating conclusions. 

• Unacceptable presentation and description of statistical analyses. The form of presenting the results of statistical analyses in Tables presented by the authors is outside the generally accepted standard. Very often, the consequence of this is a description containing too many results of statistical analyses, which makes the text unreadable. For example Line 291-294: …no statistically significant differences in baseline glucose values between groups at the beginning of the experiment (F 292 = 1.739; p = 0.115). However, on the final day of treatment, a statistically significant difference in glycaemia was recorded (F = 7.332; p < 0.0001).

Response: Thank you for this important remark. We agree that reporting extensive test outputs in the Results narrative reduced readability. We have therefore streamlined the Results section by removing most F- and p-values from the text and focusing the narrative on the direction and magnitude of effects with direct references to the corresponding Tables.

• Tables – difference “in columns” should be presented in accordance with the rule (standard): values represented with different letters (e.g. small letters) are statistically different at p<0.05.

Response: We agree and have revised all relevant Tables to follow the standard compact-letter display. Specifically, statistically significant between-group differences within the same column are now indicated by different lowercase letters (one-way ANOVA with Tukey’s HSD, p < 0.05), as stated in the updated table footnotes.

• Tables – difference “before-after” should be presented in accordance with the rule (standard): values represented with different letters (e.g. capital letters) or (*) are statistically different at p<0.05.

Response: We agree and have standardized the within-group comparisons accordingly. Baseline-to-post treatment differences within the same group are now marked with an asterisk (*) (paired-sample t-test, p < 0.05), as specified in the revised table notes. This replaces the previous non-standard and overly detailed footnote system.

• Results of ANOVA (F-values; p-values) should be presented in additional Table. Constantly including analysis results in the text is unnecessary ballast. During description of statistically significant differences or their absence, it is enough to cite the adequate Tables.

Response: We fully agree. All omnibus ANOVA outputs (F-values and p-values) for the primary outcomes are now consolidated in an additional supplementary table (Table S1). The Results text now primarily cites the relevant Tables (Tables 2–3) and no longer repeatedly includes ANOVA outputs, thereby reducing unnecessary statistical “ballast”.

• Text and Tables need improvement: see Table 1: data of Δ HOL and Δ TG should be in one line , see Line 332-352 (italics); There are more errors in the manuscript.

Response: Thank you for this comment. We have reformatted Table to improve readability and to ensure that ΔHOL and ΔTG values are presented on a single line by shortening column headers, adjusting column widths, and harmonizing table layout. In addition, we corrected unintended italic formatting in the indicated text segment (Lines 332–352), retaining italics only where required (e.g., scientific names). Finally, we performed a thorough proofreading of the manuscript to identify and correct additional formatting inconsistencies (tables/labels/italics and related typographical issues).

• Additional literature: Oshetkova, D.; Klimowicz, A. Antioxidative and Photoprotective Activity of Pinus nigra, Pinus strobus and Pinus mugo. Appl. Sci. 2025, 15, 209. Nisca, A.; Stefanescu, R.; Stegdrus, D.I.; Mare, A.D.; Farczadi, L.; Tanase, C. Comparative study regarding the chemical composition and biological activity of pine (Pinus nigra and sylvestris) bark extracts. Antioxidants 2021, 10, 327.

Response: Thank you for the suggested literature. We have added both references to the manuscript and cited them in the Introduction/Discussion to strengthen the context regarding (i) antioxidant/photoprotective activities of Pinus extracts and (ii) prior chemical-composition and bioactivity profiling of P. nigra bark extracts, which also supports our emphasis on the need for detailed chemical characterization and standardization in future work.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors report on their study of Pinus nigra bark extracts, whose antidiabetic potential they analysed in silico and in vivo.
They conducted this study in light of the growing number of studies on the biological effects of botanical extracts and the use of a standardised formulation such as Pycnogenol®.
The paper is well organised, starting with the abstract, which is comprehensive, informative, clear in its presentation and also in indicating what was observed, including the critical aspects of the results.
In the introduction, the state of the art in the field of research is well reported with references to many up-to-date bibliographical references.
The materials and methods section is also good, accurate, thorough and detailed. The methods are described in a comprehensive and informative manner for anyone wishing to carry out a similar study. The targets of the in silico analyses were appropriately chosen for their involvement in diabetic pathology. The choice of which extract to use for the in vivo analyses is also appropriate.
The results are reported in a fluent and informative manner, and the images of the in silico analysis and tables are adequate. The statistical analysis is carried out correctly.
The discussion is extensive, with each result being thoroughly examined on the basis of the relevant scientific literature in the field.
It is particularly commendable that both the critical issues of some results and the need for the study to continue with further investigations such as binding tests, enzymatic analyses and safety assessments of the extracts are highlighted.
A good paper.

Author Response

Dear Reviewer,

We sincerely thank Reviewer 3 for the exceptionally positive, thorough, and professional evaluation of our manuscript. We highly appreciate the time and effort devoted to the careful assessment of all aspects of the study, including the clarity of the language, the appropriateness of the research design, the completeness of the methodological description, the correctness of the statistical analysis, and the interpretation of the results.

We are pleased that the manuscript was recognized as providing a comprehensive and up-to-date overview of the current state of research in the field, and that both the in silico and in vivo approaches, as well as the selection of targets and extracts for further investigation, were considered appropriate. We are particularly grateful for the acknowledgment of our critical approach in the Discussion section, including the explicit recognition of the study’s limitations and the need for further mechanistic, binding, enzymatic, and safety investigations.

The comments of Reviewer 3 represent an important endorsement of the quality of our work and serve as a strong motivation for us to continue research in this area. We sincerely appreciate the constructive and encouraging nature of this review.

Kind regards,
The Authors

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I think “polyphenols” should be included in the title.

Why these compounds “catechin, epicatechin, taxifolin, caffeic, ferulic, p-coumaric, protocatechuic, and syringic acids” were selected? What’s the standard for this selection?

The diabetic Wistar rats was induced by alloxan. What’s the advantage of induction by alloxan compared with that by streptozotocin?

The format of Table 4 should be re-organized.

In Conclusions, the authors just said “extract”, not focus on the bioactivities chemical markers such as “polyphenols”.

The former question “The bark was collected in April 2018. When the extract was extracted, and when the silicon, in vivo experiment was done? If the bark or the extract kept long time, what’s influence will happen to them?” was not explained.

Comments on the Quality of English Language

No comments.

Author Response

Comment 1: I think “polyphenols” should be included in the title.

Response 1: Thank you for this suggestion. We have revised the manuscript title to improve clarity and readability by explicitly including “polyphenols”, in line with the focus of our chemical characterization and bioactivity assessment. The updated title now emphasizes the polyphenol-related component of the Pinus nigra bark extract evaluated through us in silico and in vivo analyses.

Comment 2: Why these compounds “catechin, epicatechin, taxifolin, caffeic, ferulic, p-coumaric, protocatechuic, and syringic acids” were selected? What’s the standard for this selection?

Response 2: Thank you for requesting clarification regarding the rationale for compound selection. We have revised the manuscript to explicitly state that the set of caffeic acid, catechin, epicatechin, ferulic acid, p-coumaric acid, protocatechuic acid, syringic acid, and taxifolin was chosen using a marker-based approach, guided by the well-established polyphenolic fingerprint of Pycnogenol® and our preliminary chemical characterization of the obtained P. nigra Arn. bark extracts [7,21]. Accordingly, these compounds were included as representative low-molecular-weight polyphenolic markers commonly reported in pine bark preparations, and the supporting references [7,21] were retained in the revised text.

Comment 3: The diabetic Wistar rats was induced by alloxan. What’s the advantage of induction by alloxan compared with that by streptozotocin?

Response 3: Thank you for this important question. We selected alloxan because it induces a reproducible insulin-deficient diabetic phenotype primarily via preferential pancreatic β-cell vulnerability to oxidative stress, providing a rapid and widely used rat model for first-line screening of hypoglycaemic effects of polyphenol-rich plant extracts. We have now clarified this rationale in the Methods section and added appropriate references comparing alloxan and streptozotocin models, as well as prior studies that used alloxan-induced diabetes in the context of pine bark preparations, to support comparability with the existing literature.

Comment 4: The format of Table 4 should be re-organized.

Response 4: Thank you for pointing this out. We apologize for the formatting issue in Table 4, which occurred due to an inadvertent error during document preparation/upload. The table has now been reformatted and reorganized to improve readability and to ensure consistent structure and presentation of the results.

Comment 5: In Conclusions, the authors just said “extract”, not focus on the bioactivities chemical markers such as “polyphenols”.

Response 5: Thank you for this helpful suggestion. We have revised the Conclusions to explicitly emphasize the bioactivities observed and to link them to the polyphenol-rich chemical profile of the P. nigra bark extract. Specifically, we now refer to the hypoglycaemic and lipid-related effects in the applied models and discuss them in the context of key polyphenolic markers (caffeic acid, catechin, epicatechin, ferulic acid, p-coumaric acid, protocatechuic acid, syringic acid, and taxifolin) rather than using the generic term “extract” alone.

Comment 6: The former question “The bark was collected in April 2018. When the extract was extracted, and when the silicon, in vivo experiment was done? If the bark or the extract kept long time, what’s influence will happen to them?” was not explained.

Response 6: We appreciate this comment and agree that maintaining extract stability requires controlled handling and storage. The bark was collected in April 2018 and stored in airtight containers, protected from light and moisture, in a cool, dry place °Cuntil extraction. All types of extracts intended for chemical characterization (ethanol, methanol and acetone) were prepared in May 2018 (i.e., within two months after sampling) and were kept at room temperature (≈20 °C), protected from light, for a maximum of 48 h prior to analysis. Hence, the chemical profiling was performed immediately thereafter, within 48h (May 2018), and the in vivo experiments were initiated shortly after (June–July 2018). Importantly, for the in vivo study, fresh ethanol extracts were prepared immediately before administration using the same procedure to avoid any potential changes associated with prolonged storage; thus, no long-term storage of the extract was involved for in vivo testing. We acknowledge that prolonged storage may affect the relative abundance of individual phenolics; therefore, storage time was minimized and light exposure was avoided. In silico analysis was renewed and improved in 2025 using updated docking and visualization software. Please see the revised Section 2.1.

Reviewer 2 Report

Comments and Suggestions for Authors

Minor revision

Line 32-34: ...eration.. Keywords: Pinus nigra bark; plant metabolites; antidiabetic potential; hypoglycaemic; hypolipidaemic; in silico; in vivo...

Rev: ???

Rev: in silico and in vivo should be in italics – the note applies to the entire manuscript.

Table 4 requires correction

Line 521-532: …

Rev: ??? requires correction.

Author Response

Comment 1: Line 32-34: ...eration.. Keywords: Pinus nigra bark; plant metabolites; antidiabetic potential; hypoglycaemic; hypolipidaemic; in silico; in vivo...

Rev: ???

Response 1: Thank you for this remark. We have corrected the typographical and formatting issues in the Keywords section (Lines 32–34) and revised the text accordingly.

Comment 2: Rev: in silico and in vivo should be in italics – the note applies to the entire manuscript.

Response 2: We thank the Reviewer for this remark. We have revised the manuscript formatting and ensured that the terms in silico and in vivo are consistently italicized throughout the entire manuscript, including the Keywords section.

Comment 3: Table 4 requires correction

Response 3: Thank you for pointing this out. We apologize for the formatting issue in Table 4, which occurred due to an inadvertent error during document preparation/upload. The table has now been reformatted and reorganized to improve readability and to ensure consistent structure and presentation of the results.

Comment 4: Line 521-532: …

Rev: ??? requires correction.

Response 4: Thank you for this comment. We carefully reviewed the text in Lines 521–532 and corrected the identified typographical/formatting issues to improve clarity and consistency.