Chemical Composition, Antimicrobial, and Repellant Properties of Lavandula stoechas and Artemisia absinthium Essential Oils Against Ephestia kuehniella

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

Manuscript: "Chemical composition, Antimicrobial, and Repellant properties of Lavandula stoechas and Artemisia absinthium essential oils against Ephestia kuehniella"

This manuscript presents a broad investigation into the potential applications of two essential oils, covering their chemical analysis, antimicrobial properties, and insecticidal effects. The ambition of the study is commendable, and the data generated are of interest. However, the work is undermined by several significant issues in its structure, methodology, and interpretation that require substantial revision before it can be considered a robust scientific contribution.

1- The most significant structural flaw is the complete lack of a standalone Discussion section. The authors have chosen to intersperse interpretive comments within the Results section. This format conflates the presentation of data with its interpretation, preventing a cohesive synthesis of the study's overall findings. A dedicated discussion is necessary to:

2- Integrate the disparate results from the chemical, bacteriological, mycological, and entomological assays into a unified narrative. For instance, how does the significant difference in chemical composition (beyond just camphor content) between the two oils explain the observed variations in their biological activities? The current format does not allow for such a cross-cutting analysis.

3- Thoroughly and critically compare the findings with the existing literature, moving beyond simple statements of agreement or disagreement.

3- Frankly address the study's limitations—a critical component of scholarly work that is currently absent.

4- Provide a nuanced conclusion that is a direct and logical outcome of the interpreted results, rather than the generalized summary currently offered.

5- The use of bacterial strains identified only to the genus level (e.g., Staphylococcus spp., Salmonella spp.) is a critical flaw. Biological activity can be highly species- and even strain-specific. Without precise identification, the results are of limited value, as they are not reproducible and cannot be reliably compared to other studies. All test organisms must be identified to the species level.

6- The methodology for the repellent effect is inadequately described and appears contradictory. The methods state that filter paper was "impregnated with pure essential oil", which implies a non-quantified, saturated dose. However, the results are presented for various concentrations measured in "µl/ml", a unit which is ambiguous in this context. Is this a volume-for-volume dilution in a solvent, or a dose per unit area of paper? This lack of precision makes the experiment impossible to replicate.

7- The authors state that the oils were active against all tested strains "except against B. cereus and P. aeruginosa". However, Table 3 reports an inhibition zone of 6.0 mm for both. According to the study's own criteria, a diameter below 8 mm constitutes "weak" activity, not a total lack of it. This is an inaccurate interpretation of their own data.

8- The manuscript frequently draws conclusions that are not fully supported by the data presented.

9- The assertion that the oils' action is "independent of their resistance mechanisms" is a significant overstatement. This conclusion is based on testing only a single MRSA strain and a single ESBL-producing strain. To make such a broad claim would require a much larger panel of resistant isolates with well-characterized resistance mechanisms.

10- The discussions on the mechanisms of antibacterial and antifungal action ⁷ are entirely speculative. While citing potential mechanisms from literature is standard, the authors present these possibilities without any supporting experimental data from their own work (e.g., cell membrane integrity assays, mitochondrial function tests). This should be clearly framed as speculation that requires further investigation.

11- The paper misses an opportunity for a deeper analysis of the structure-activity relationship. For example, L. stoechas EO was substantially more toxic to larvae than A. absinthium EO, despite having a lower percentage of camphor. Why? The discussion is superficial and does not attempt to correlate this difference with other major components like fenchone ⁸ or the synergistic effects that are briefly mentioned but not explored.

12- The manuscript requires a thorough round of proofreading to correct several errors, such as "femelle fecundity" instead of "female fecundity" in Table 12 ⁹ and inconsistent statistical notations in figures. The graphical presentation, particularly in Figure 7, is confusing due to the inconsistent use of logarithmic and linear scales for the x-axis on graphs that are presented side-by-side.

13- The paper fails to explore its most interesting findings in sufficient depth. For instance, L. stoechas oil was found to be significantly more toxic to larvae than A. absinthium oil despite containing less camphor. This discrepancy is a key finding but is never properly interrogated. A meaningful discussion would have attempted to correlate this superior activity with other major compounds or potential synergistic effects. Furthermore, the report of "null" fecundity is presented without any supporting observational detail (e.g., were mating behaviors observed? was the effect on males or females?), reducing a potentially significant finding to a simple data point.

14- The authors state they corrected mortality using Abbott's formula but fail to report the mortality rates in the control groups. This is a critical omission, as it prevents the reviewer from assessing the baseline health of the test organisms and verifying the data treatment.

15- The identification of chemical constituents via GC-MS relies solely on library matching and retention time. The standard and expected practice for robust identification is the calculation and comparison of Retention Indices (e.g., Kováts indices). The absence of this validation step makes doubts on the accuracy of the reported chemical profiles.

16- The repellency assay methodology is particularly problematic. It is described with a lack of quantitative precision, stating the use of "pure essential oil" while presenting results in ambiguous units of "µl/ml". This prevents any other research group from being able to replicate the experiment accurately.

Finally, In its current form, the manuscript reads as a preliminary report of a collection of loosely connected experiments. To elevate this work to the level of a good scientific paper, the authors must: Restructure the paper to include a distinct and comprehensive Discussion section. Address the methodological shortcomings, particularly by identifying all microbial strains to the species level and by clarifying the experimental protocol for the repellency assay. Temper the conclusions to align strictly with the presented data, avoiding overstatement and clearly delineating between empirical findings and speculation. Conduct a thorough proofread to improve the overall quality of the presentation. Addressing these critical points would significantly strengthen the manuscript's contribution to the field.

Author Response

I would like to thank you for all your constructive comments and for the interest you have shown in our work. I am forwarding you the responses to all your comments, and all the requested changes are highlighted in yellow in the manuscript.

 

• The most significant structural flaw is the complete lack of a standalone Discussion section. The authors have chosen to intersperse interpretive comments within the Results section. This format conflates the presentation of data with its interpretation, preventing a cohesive synthesis of the study's overall findings. A dedicated discussion is necessary.

Response : You are absolutely right, a discussion section has been added to the manuscript.

 

• Integrate the disparate results from the chemical, bacteriological, mycological, and entomological assays into a unified narrative. For instance, how does the significant difference in chemical composition (beyond just camphor content) between the two oils explain the observed variations in their biological activities? The current format does not allow for such a cross-cutting analysis.

Response : This section has been completely revised according to your recommendations.

 

• Thoroughly and critically compare the findings with the existing literature, moving beyond simple statements of agreement or disagreement.

Response : The new discussion resumes the in-depth discussion appropriately, taking into account recent work.

 

• Frankly address the study's limitations—a critical component of scholarly work that is currently absent.

Response: This consideration has been addressed in the new version of the article.

 

• Provide a nuanced conclusion that is a direct and logical outcome of the interpreted results, rather than the generalized summary currently offered.

Response : Indeed, we have added a discussion section where we compared our findings with the existing literature, mentioned our study’s limitations and provided conclusions for our results, Page, 12.

 

• The use of bacterial strains identified only to the genus level (e.g., Staphylococcus spp., Salmonella spp.) is a critical flaw. Biological activity can be highly species- and even strain-specific. Without precise identification, the results are of limited value, as they are not reproducible and cannot be reliably compared to other studies. All test organisms must be identified to the species level.

Response: Indeed, we removed all test organisms that were not identified to the species level Table 2, page, 8.  

 

• The methodology for the repellent effect is inadequately described and appears contradictory. The methods state that filter paper was "impregnated with pure essential oil", which implies a non-quantified, saturated dose. However, the results are presented for various concentrations measured in "µl/ml", a unit which is ambiguous in this context. Is this a volume-for-volume dilution in a solvent, or a dose per unit area of paper? This lack of precision makes the experiment impossible to replicate.

Response: We have added more details to the experimental section, sub-section, 2.7.1. Repellent Effect of Essential Oils, page, 4.  

 

• The authors state that the oils were active against all tested strains "except against  cereusand P. aeruginosa". However, Table 3 reports an inhibition zone of 6.0 mm for both. According to the study's own criteria, a diameter below 8 mm constitutes "weak" activity, not a total lack of it. This is an inaccurate interpretation of their own data. 

Response: Indeed, we removed the sentence: "except against B. cereus and P. aeruginosa" and retained: “both EOs were remarkably more active against Gram-positive bacteria, with IZDs ranging from 8.1 to 25.3 mm, than Gram-negative bacteria, whose IZDs varied from 7.1 to 15.3”, section: Results, lines: 243-244, page, 8.

 

• The manuscript frequently draws conclusions that are not fully supported by the data presented.

Response: Indeed, we retained only the necessary conclusions and we added the most important perspectives.

 

• The assertion that the oils' action is "independent of their resistance mechanisms" is a significant overstatement. This conclusion is based on testing only a single MRSA strain and a single ESBL-producing strain. To make such a broad claim would require a much larger panel of resistant isolates with well-characterized resistance mechanisms.

Response: Indeed, we have added this sentence: “However, further studies including a broader range of well-characterized resistant isolates are required to confirm this observation”, in the discussion section, lines: 461-462, page, 14.

 

• The discussions on the mechanisms of antibacterial and antifungal action ⁷are entirely speculative. While citing potential mechanisms from literature is standard, the authors present these possibilities without any supporting experimental data from their own work (e.g., cell membrane integrity assays, mitochondrial function tests). This should be clearly framed as speculation that requires further investigation.

Response: Indeed, we have clarified this by adding:” It should be noted that the mechanisms proposed in this section are hypothetical and derived from published literature. The absence of mechanistic assays such as    membrane integrity or mitochondrial function tests constitutes a limitation of our study. Therefore, these mechanisms remain speculative and require further experimental validation”, to the discussion section, lines: 505-509, page, 15.   

 

• The paper misses an opportunity for a deeper analysis of the structure-activity relationship. For example,  stoechas EO was substantially more toxic to larvae than A. absinthium EO, despite having a lower percentage of camphor. Why? The discussion is superficial and does not attempt to correlate this difference with other major components like fenchone ⁸ or the synergistic effects that are briefly mentioned but not explored.

Response: Indeed, more explanations were added in the discussion part, lines: 567-596, page, 16.

 

• The manuscript requires a thorough round of proofreading to correct several errors, such as "femelle fecundity" instead of "female fecundity" in Table 12 ⁹and inconsistent statistical notations in figures. The graphical presentation, particularly in Figure 7, is confusing due to the inconsistent use of logarithmic and linear scales for the x-axis on graphs that are presented side-by-side.

Response: Indeed, we have corrected the errors, and we deleted the Figure 7, to avoid any confusion.  

 

• The paper fails to explore its most interesting findings in sufficient depth. For instance,  stoechasoil was found to be significantly more toxic to larvae than A. absinthium oil despite containing less camphor. This discrepancy is a key finding but is never properly interrogated. A meaningful discussion would have attempted to correlate this superior activity with other major compounds or potential synergistic effects. Furthermore, the report of "null" fecundity is presented without any supporting observational detail (e.g., were mating behaviors observed? was the effect on males or females?), reducing a potentially significant finding to a simple data point.

Response: Indeed, all these aspects were discussed in discussion section, lines: 556-625.

• The authors state they corrected mortality using Abbott's formula but fail to report the mortality rates in the control groups. This is a critical omission, as it prevents the reviewer from assessing the baseline health of the test organisms and verifying the data treatment.

Response: No mortality was detected in the control groups. this information has been added to the results

 

16- The identification of chemical constituents via GC-MS relies solely on library matching and retention time. The standard and expected practice for robust identification is the calculation and comparison of Retention Indices (e.g., Kováts indices). The absence of this validation step makes doubts on the accuracy of the reported chemical profiles.

Response: Indeed, all Kovats indices were calculated and showed in the table 1, page: 6.

 

17-The repellency assay methodology is particularly problematic. It is described with a lack of quantitative precision, stating the use of "pure essential oil" while presenting results in ambiguous units of "µl/ml". This prevents any other research group from being able to replicate the experiment accurately.

Response : 500μl of pure essential oil was placed on half of a disc with a diameter of 9 cm. This information has been added to the protocol page 4.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the chemical composition, antimicrobial, antifungal, and repellent properties of essential oils from Lavandula stoechas and Artemisia absinthium. The findings offer valuable insights into their potential as sustainable alternatives for food preservation.
My key remarks are as follows:
1. Please explain the significant difference in camphor retention times observed between Lavandula stoechas essential oil (Table 1) and Artemisia absinthium essential oil (Table 2).
2. The introduction, presented as a single paragraph, reduces readability. It is generally recommended to break down the introduction into several paragraphs, with each paragraph focusing on a specific aspect or idea to improve flow and comprehension for the reader.
3. The phrase "a very good activity" (line 24) is subjective. Please quantify this statement with specific data.
4. Line 142, it should be "anti-fungal activity".
5. Ensure consistent capitalization across all headings throughout the manuscript.
6. As Section 3 includes both results and extensive discussion, please retitle it "Results and Discussion."
7. In Section 3.5, the word "toxicity" may not be the most precise term, as it give the impression that it is poisonous or cause harm (to human). It is suggested to use the term "insecticidal activity".
8. Please add standard deviation bars to Figures 3, 4, 6, and 7 to represent data variability.

Author Response

Reviewer 2

I would like to thank you for all your constructive comments and for the interest you have shown in our work. I am forwarding you the responses to all your comments, and all the requested changes are highlighted in yellow in the manuscript.

1. Please explain the significant difference in camphor retention times observed between Lavandula stoechas essential oil (Table 1) and Artemisia absinthium essential oil (Table 2).

Response: Different overall composition: The essential oils of Lavandula stoechas and Artemisia absinthium have radically different chemical profiles.

o    L. stoechas: Rich in camphor and fenchone.

o    A. absinthium: Rich in thujone (α and β), sabinene and terpenyl acetates.

Competitive interactions: In A. absinthium essential oil, camphor is a minor component (2.69%) drowned out in a matrix dominated by other very abundant molecules such as thujones. These major compounds can ‘monopolise’ the interaction sites of the stationary phase, forcing camphor to pass more quickly into the mobile phase, which reduces its retention time (RT = 11.864 min).

Chemical environment: Conversely, in L. stoechas essential oil, camphor is a major component (28.31%) within a different chemical matrix (rich in fenchone, pinene, etc.). The overall interactions with the column are different, allowing camphor to interact longer with the stationary phase, which increases its retention time (RT = 13.769 min).

2. The introduction, presented as a single paragraph, reduces readability. It is generally recommended to break down the introduction into several paragraphs, with each paragraph focusing on a specific aspect or idea to improve flow and comprehension for the reader.

Response: The introduction has been amended in accordance with your recommendations.

3. The phrase "a very good activity" (line 24) is subjective. Please quantify this statement with specific data.

Response: Both essential oils (EOs) were more active against Gram-positive bacteria, with inhibition zone diameters (IZDs) exceeding 15 mm. In contrast, IZDs ranged from 8 to 14 mm against Gram-negative strains.

4. Line 142, it should be "anti-fungal activity".

Response: Indeed, it is an antifungal activity.

5. Ensure consistent capitalization across all headings throughout the manuscript.
   6. As Section 3 includes both results and extensive discussion, please retitle it "Results and Discussion."

Response: We have divided the section into two parts.

7. In Section 3.5, the word "toxicity" may not be the most precise term, as it give the impression that it is poisonous or cause harm (to human). It is suggested to use the term "insecticidal activity".

Response: ok it’s done: Determination of the Essential Oils' Insecticidal Activity.

8. Please add standard deviation bars to Figures 3, 4, 6, and 7 to represent data variability

Response : It’s done

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. Line 56-57. Family names (Asteraceae, Lamiaceae) must be written in italics.
2. Line 106. Here a source should be indicated in square brackets, not just a placeholder link.
3. The dominant volatile compounds you listed range from 31.83% to 3.93%. My suggestion is to indicate 4–5 dominant compounds whose content exceeds 5%.
4. Table 1. Please correct the compound name and write cis-vebemol in italics.
5. L-alpha-Terpineol. Please correct L- α-Terpineol.
6. Figure 1and 2. You show a GC-MS chromatogram; my suggestion is to label the main compounds in it.
7. Table 2. Please correct the compound names and write cis and trans in italics. Use the symbol α instead of writing “alfa”.
8. References 1–10 should be written without a leading zero.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

I would like to thank you for all your constructive comments and for the interest you have shown in our work. I am forwarding you the responses to all your comments, and all the requested changes are highlighted in yellow in the manuscript.

 

1. Line 56-57. Family names (Asteraceae, Lamiaceae) must be written in italics.

Response: ok it’s done

2. Line 106. Here a source should be indicated in square brackets, not just a placeholder link.Johnson, R. D., III (Ed.). (2021). NIST Computational Chemistry Comparison and Benchmark Database(NIST Standard Reference Database Number 101). National Institute of Standards and Technology. https://doi.org/10.18434/T47C7Z.

Response: Ok it’s done, reference number (10), line 101, page 3.  

3. The dominant volatile compounds you listed range from 31.83% to 3.93%. My suggestion is to indicate 4–5 dominant compounds whose content exceeds 5%.

Response: We thank you for your suggestion regarding the selection of dominant volatile compounds. However, we have chosen to present compounds with concentrations ranging from 31.83% to 3.93%, as this range includes all compounds that contribute significantly to the overall chemical profile, in line with common practice in scientific literature. Limiting the selection to only compounds exceeding 5% would risk excluding certain key compounds which, although present in lower concentrations, may have very interesting activities either alone or in combination with other molecules.

4. Table 1. Please correct the compound name and write cis-vebemol in italics.

Response: ok it’s done, Table 1, page, 6.

5. L-alpha-Terpineol. Please correct L- α-Terpineol.

Response: ok it’s done, Table 1, page, 6.

6. Figure 1and 2. You show a GC-MS chromatogram; my suggestion is to label the main compounds in it.

Response: We have removed the figures, and we grouped all compounds in the Table 1, page, 6.   

7. Table 2. Please correct the compound names and write cisand trans in italics. Use the symbol α instead of writing “alfa”.

Response: ok it’s done, Table 1, page, 6.

8. References 1–10 should be written without a leading zero.

Response: ok it’s done, Table 1, page, 6.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors present an interesting study on the antimicrobial properties and repellant effect against Ephestia kuehniella of L. stoechas and A. absinthium essential oils. The authors characterized the EO's composition, by GC/MS, and tested the antimicrobial activity on several bacteria strain, both Gram+ and Gram-. The methods used for this study were well described and the results are clear and well exposed, both in tables and graphs. The proposed discussion is consistent with the observed results and well argued. For these reasons, I believe that this work can be accepted for publication in this journal.

However, some minor revisions would be necessary in order to make the work more readable and better discussed. Specifically:

Introduction: the introduction is lacking in the section regarding the state of the art about the studies on the antimicrobial and antifungal properties of essential oils. References as recent as possible should be included. About the reference [1], the authors should be cite a work written in english. 

Materials and methods: Just a few suggestions to make the section more readable.
Paragraphs 2.1 and 2.2 are easier to read if reported together.
The division into subparagraphs of paragraph 2.4 is not necessary.
Line 134-136 should be moved in the results and discussion section.

Results

Table 1 and table 2 are not cited in the paragraphs 3.1, nor are figure 1 and figure 2. However, it might be better to report the contents of Tables 1 and 2 in a single table, so as to make the result of the analysis of the chemical composition of both essential oils more readable and easily comparable.

In Figure 3, 4, 5 and 6 the standard deviation is missing, or incomplete.

Pay attention to the significant figures in the remaining tables. Does it make sense to report two significant figures for those percentage values? Reporting just one is probably sufficient. The 0, as the first non-decimal digit, should not be reported.
Authors should also standardize the formatting of table borders.

Reference formatting should be standardized. DOI reported for the reference [31] give an error 404.

More generally, a careful review of text formatting and spacing is recommended. In some places, the text is in a different font than the previous one (e.g., line 458), and some subparagraphs have italicized text, while others do not.

Author Response

Reviewer 4

I would like to thank you for all your constructive comments and for the interest you have shown in our work. I am forwarding you the responses to all your comments, and all the requested changes are highlighted in yellow in the manuscript.

1- Introduction : the introduction is lacking in the section regarding the state of the art about the studies on the antimicrobial and antifungal properties of essential oils. References as recent as possible should be included. About the reference [1], the authors should be cite a work written in english. 

Response : It’s done

2- Materials and methods: Just a few suggestions to make the section more readable.
Paragraphs 2.1 and 2.2 are easier to read if reported together.

Response: Ok it’s done

3- The division into subparagraphs of paragraph 2.4 is not necessary.

Response: Ok it’s done

4- Line 134-136 should be moved in the results and discussion section. 

Response: The lines are moved to the results section, section: 3.2. Antibacterial activity, page, 7.

5- Table 1 and table 2 are not cited in the paragraphs 3.1, nor are figure 1 and figure 2. However, it might be better to report the contents of Tables 1 and 2 in a single table, so as to make the result of the analysis of the chemical composition of both essential oils more readable and easily comparable.

Response: Indeed, the tables 1 and 2 were grouped in a single Table 1, page 6.

6- In Figure 3, 4, 5 and 6 the standard deviation is missing, or incomplete.

Response : it’s done

7- Pay attention to the significant figures in the remaining tables. Does it make sense to report two significant figures for those percentage values? Reporting just one is probably sufficient. The 0, as the first non-decimal digit, should not be reported.

Response : It’s done

8- Authors should also standardize the formatting of table borders.   

Response: Ok it’s done.

 

9- Reference formatting should be standardized. DOI reported for the reference [31] give an error 404.  

Response: Ok it’s done.

10- More generally, a careful review of text formatting and spacing is recommended. In some places, the text is in a different font than the previous one (e.g., line 458), and some subparagraphs have italicized text, while others do not.

Response :  The manuscript has been completely revised and corrected according to your recommendations.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In my opinion the authors have made the necessary revisions. I believe the manuscript is acceptable for publication in its current form, after linguistic review.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments.