Geopropolis from Melipona orbignyi and Melipona quadrifasciata anthidioides Enhances Oxidative Stress Resistance and Lifespan in Caenorhabditis elegans

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I reviewed the manuscript entitled Geopropolis from Melipona orbignyi and Melipona quadrifasciata anthidioides Enhances Oxidative Stress Resistance and  Lifespan in Caenorhabditis elegans.

 

I agree to accept this manuscript after major revision. 

 

 

1) Abstract, reactive oxygen species (ROS), hydroethanolic extracts of geopropolis from Melipona orbignyi (HGMO) and Melipona quadrifasciata anthidioides (HGMQ), these abbreviations appear no more than three times in the abstract, so there is no need to use them; the full terms should be used instead. Excessive abbreviations can confuse readers. Please review and revise similar issues throughout the text according to this principle.

2) In previous studies, we demonstrated the antimicrobial, anti-inflammatory, and antimutagenic properties, as well as a potent antioxidant action against free radicals and lipid peroxidation, of geopropolis of the species M. orbignyi and M. q. anthidioides [12, 19]. When "M. orbignyi" and "M. q. anthidioides" are mentioned for the first time, the full name should be written first, followed by the abbreviation in parentheses. The abbreviation can then be used directly thereafter.

3) Figure 1. All abbreviations in the figure should be listed with their full names in the footnotes; when statistics are involved, p should be italicized. Other figures also exhibit similar issues. Please inspect and revise them as well.

4) HGMO was able to maintain BSA integrity at 39.75 ± 0,51 and 97.18 ± 0,18% at concentrations of 12.5 and 250 μg/mL, respectively (Figure 1A). 0,51 should change to 0.51; 0,18 should change to 0.18.

5) after 24 and 48 hours, hours should change to h. To use international units instead of words, please check and correct similar issues throughout the text.

6) 2.3. In vivo tests using the Caenorhabditis elegans model, in second-level headings, the first letter of every major word should be capitalized. Third-level headings follow the same requirement. Please check and correct them accordingly.

7) Figure 5. * vs. CT20 °C; # vs. CT37 °C; *, A space should be inserted between a number and its unit, except for the degree Celsius symbol (°C) and the percentage sign (%). Please review and revise the entire text according to this principle.

8) Table 1. "Longevity" refers to different dosage groups, so it would be more appropriate to change it to "Group."

9) Regarding protection against oxidative stress, the extracts were able to increase the viability of C. elegans exposed to Juglone, a substance that promotes the generation of intracellular ROS H2O2 and O2• [45] and reduces nematode survival.  The specific substances following ROS should be clarified within parentheses: the original phrasing "intracellular ROS H2O2 and O2•" could lead readers to misinterpret ROS, H2O2, and O2• as three distinct substances listed in parallel. In reality, H2O2 and O2• are two specific types of ROS. Therefore, a more precise approach is to enclose the specific substances in parentheses to indicate that they are subtypes of ROS.  O2• should be corrected to O2•⁻: the standard chemical formula for the superoxide anion radical in reactive oxygen species is O2•⁻. The original notation O2• is incomplete, lacking the superscript indicating the negative charge, which is a technical detail that requires correction.  The comprehensive revision suggestion is as follows: Regarding protection against oxidative stress, the extracts were able to increase the viability of C. elegans exposed to Juglone, a substance that promotes the generation of intracellular ROS (such as H2O2 and O2•⁻) and reduces nematode survival.

10) In the section on "DNA-induced oxidative damage," were control group data (such as data using only oxidants) provided?

11) In Figure 4, does "JC: juglone control" correspond to "CJ"? The notations in the text appear inconsistent. Please check and correct them accordingly.

12) Was the rationale for selecting 37°C and 6 h as stress conditions explained in the thermal stress experiment?

13) In Section 4.3.1, are the electrophoresis conditions (voltage, duration) for SDS-PAGE applicable to all samples?

14) The rationale for selecting juglone as an oxidative stress inducer should be explained.

15) The formatting of the references is inconsistent. Some references are missing page numbers or volume information. Please review and revise them according to the journal's requirements.

16) The research background should thoroughly elaborate on the differences between geopropolis and traditional propolis, as well as their respective research value. Additional geographical and ecological context should also be supplemented.

17) The paper should clearly specify the differences in chemical composition between HGMO and HGMQ and attempt to explain their potential variations in bioactivity.

18) Should the conclusion section explicitly outline the specific limitations of this study and propose clear directions for future research (e.g., validation in mammalian models, isolation and identification of active components, etc.) to enhance the rigor and foresight of the study?

19) This study investigated the protective effects of hydroethanolic geopropolis extracts from Melipona orbignyi (HGMO) and Melipona quadrifasciata anthidioides (HGMQ) against oxidative stress, along with their toxicity and influence on lifespan. In vitro assessments revealed that both extracts reduced protein oxidation induced by AAPH and prevented DNA fragmentation caused by hydrogen peroxide and UV radiation. In vivo evaluation using Caenorhabditis elegans showed no toxicity and demonstrated enhanced resistance to juglone-induced oxidative stress and thermal stress. Additionally, all tested concentrations of HGMO and HGMQ consistently extended nematode lifespan. These findings indicate that geopropolis extracts protect biomolecules from oxidative damage, improve stress resilience, and promote longevity, supporting their potential development for preventing or treating oxidative stress-related diseases.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

 Geopropolis from Melipona orbignyi and Melipona quadrifasciata anthidioides Enhances Oxidative Stress Resistance and Lifespan in Caenorhabditis elegans

Formatting:

• There is an obvious problem with the indentation. Please fix.
• Formula should be formatted as per the MDPI format.
•  

Title:

• The study is exploratory and mechanistic and not definitively causal; therefore, I suggest that the authors use the term modulatory. The title, however, is informative and specific.

 

Authors and affiliations:

• Fix the indentation of the affiliations. Also, include each author's email address and middle initial as early as possible.

 

Abstract:

• In the aim, please include Caenorhabditis elegans after lifespan.
• Overall, the abstract is clear and concise, but could benefit more if specific, significant data are included, especially in the case of the reduction of protein oxidation and DNA fragmentation, toxicity, and enhanced lifespan (actual data or %)

 

Keywords:

• Consider including geopropolis, species of bees, and assays performed. Please maximize the number of specific keywords to improve visibility.

 

Introduction:

• Overall, the introduction is logically presented, statements are cited appropriately, the research gap is well defined and supported by literature, and the hypothesis is presented.
• The aims and objectives, though, should be clarified, and authors should explicitly mention that the toxicity was also analysed.
•  

 

Results:

• In the case of all figures, please explain why only one direction of the error bar is shown. Is it possible to show both directions?
• 1. Protein oxidation. Please guide the readers on what the assay is all about before presenting the results. Please include positive and negative controls and incorporate the statistical analysis into the results section.
• Figure 1. A and B images are not uniform in the sense that A does not have tailings and fragments as compared to B. Please explain the difference and provide context on how QA is performed.
• Figure 2. The figure is broken, and the labels on the x-axis of B are not thoroughly visible. Please define all the abbreviations in the figure description. Also, what are O and AG?
• 2. Please incorporate statistical analysis into your results description.
• 3.1. Please mention what the control/s are
• 3.2., 2.3.3., Please incorporate statistical analysis into your results description.

 

Discussion:

• The discussion can be improved by ensuring that each of the results in the assays performed is discussed in terms of patterns, trends, and possible explanations for the mechanism or observed results.
• Discussion should be improved
• Authors should also discuss the limitations and future directions of the study.
• Please maintain caution in relating the results of this study to human pathways since a different model or system was used. Perhaps include disclaimer.

Methodology:

• 2. Please add details on the nature of the geopropolis samples. For example, did it undergo drying, age/time prior to extraction?
• 2. Please specify the rotavap temperature, brand, and model, as well as the shaker's agitation brand and model.
• 2. Please specify the shaking speed.
• 2. Please revise the section title. Probably Geopropolis Sampling and Extraction?
• Overall, the rest of the methodologies are described in detail and, in my assessment, can be reproduced by other researchers.
•  

Author Response

Please see the attached file.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Geopropolis from Melipona orbignyi and Melipona quadrifasciata anthidioides Enhances Oxidative Stress Resistance and Lifespan in Caenorhabditis elegans” addresses an interesting and relevant topic, exploring the antioxidant and lifespan-extending effects of geopropolis extracts using in vitro assays and the C. elegans model. However, several conceptual, methodological, and interpretative weaknesses currently limit the suitability of the manuscript for publication. My observations are listed below:

 

Narrow the scope of the Introduction to oxidative stress biology and aging models, rather than disease prevention, to avoid expectation mismatch. The Introduction repeatedly links oxidative stress to chronic non-communicable diseases (CNCDs) and aging (lines 52–60), culminating in translational framing, but no disease-specific models or mammalian systems are used later in the manuscript.

The authors explicitly state that the chemical composition of HGMO and HGMQ was characterized in previous publications (lines 446–453).

The DNA fragmentation assay employs 30% H₂O₂ and UV irradiation. These conditions are highly non-physiological, yet results are interpreted as strong DNA protection – the authors should Temper conclusions regarding superiority.

 

Additionally, the claim that extracts are: “more effective than quercetin and gallic acid” is based on single concentrations, without dose–response comparisons, please clarify that this assay reflects chemical DNA protection, not cellular genomic protection.

 

In Table 1, only the highest concentration (250 µg/mL) shows a statistically significant increase in mean lifespan for both extracts, whereas lower concentrations do not; therefore, the statement in the Results section that lifespan was increased at all concentrations is not statistically supported and should be revised and the authors should revise this part

The Discussion extensively attributes the observed effects to DAF-16, SKN-1, IIS, JNK, and HSF-1 pathways, although no mutant strains, reporter assays, or gene expression analyses were performed; these statements are therefore speculative and should be clearly framed as such or substantially reduced.

Although several bioactive compounds are discussed, the manuscript does not establish any direct relationship between extract composition, differences between HGMO and HGMQ, and the observed biological effects, which should be acknowledged as a limitation or supported by comparative interpretation of Table S1.

The conclusion that these findings support the development of geopropolis-based products for preventing or treating oxidative stress-related diseases is not justified by the exclusive use of in vitro assays and a C. elegans model and should be reframed to reflect their preclinical, model-specific nature and the need for further validation.

Author Response

Please see the attached file

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The authors of the manuscript, "Geopropolis from Melipona orbignyi and Melipona quadrifasciata anthidioides Enhances Oxidative Stress Resistance and Lifespan in Caenorhabditis elegans," present an extensive and interesting study. The study demonstrates that hydroalcoholic extracts of geopropolis from Melipona orbignyi (HGMO) and Melipona quadrifasciata anthidioides (HGMQ) protect biomolecules (proteins and DNA) from oxidative stress and prolong the life of Caenorhabditis elegans without showing toxicity. This suggests the extracts have therapeutic potential against diseases related to oxidative stress.

While the manuscript is clearly written and the results are interesting, there are some flaws that need improvement.

 

Points to be addressed as “Minor Issues”:

Introduction.

Authors are requested to check for typos. For example, check line 41 for typos, as well as the full names of species mentioned for the first time in the body of the article, excluding the abstract (see line 73, for example).

Results.

1. In Figure 2, authors should pay closer attention to the figure's formatting. In the PDF, part of the X-axis is cut off at the end of the page.
2. In Section 2.3.1, the authors should justify their statement that the small differences observed in Figure 3 are not significant. To this end, please indicate any tests performed and any corrections applied, as there are many comparisons.

Discussion.

Some references must be anticipated in line 420. Strong statements are not supported by bibliographic citations. They are fine subsequently, but the reader remains somewhat perplexed.

Materials and Methods.

1. In lines 442 and 443, the names of the species may be abbreviated.
2. In lines 449–453, reference numbers are sufficient, and it is unnecessary to cite the titles of the articles.
3. In lines 495–496, the authors must specify whether the measurements are in triplicate and which ANOVA is used (one-way or two-way). Leaving it up to the reader's intuition can complicate matters for the authors.

 

Points that must be addressed as “Major Issues”:

Results.

1. Table 1. The authors are requested to highlight the word "Table 1" in bold and the name of the species in italics.

1a) The first column is not "longevity," but rather "treatments."

1b) After all this work, the hazard ratio (HR) derived from Cox regression is missing. This value could be useful for calculating estimated benefits at different doses. For example, if an HR is << 1 with a significant P value at 250 μg/mL, it is a strong confirmation of the adopted model. If it is not possible to include HR, or if the value is unavailable, the authors must justify its absence in the discussion.

Materials and Methods:

1. In lines 481 and 482, the sentence is unclear. Did the authors perform only one experiment? While it is acceptable to make four gels and take measurements in triplicate, at least two replications of the experiment are necessary. The authors are requested to correct or complete the final sentence.
2. Add the survival analysis part (Kaplan–Meier curves and subsequent log-rank and possibly Cox regression) to section 4.5. Additionally, the authors cite the Mantel-Cox test, which should be included in this section.

 

Author Response

Please see the attached file

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has made the modifications as requested and addressed my concerns, therefore I agree to accept it in its current form.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thanks for addressing my comments and suggestions. I do not have any issues in accepting this paper.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors seem to have taken the suggestions into account, and the manuscript shows clear improvements

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been improved according to the comments provided in the previous review. In my opinion, the manuscript can be published.