Unlocking the Value of Nine Olive Leaf Varieties: A Dual Assessment of Phenolic Composition and Antioxidant Properties
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript evaluated the phenolic composition and antioxidant potential of different olive leaves varieties and the influence of extraction methods and types of varieties on these parameters. The design of study is well conducted, and the aim of valorising this by-product is well presented, well-founded, and highlighted. However, I have some major concerns and minor comments/suggestions, which I mention as follows and that should be considered before acceptance for publication.
Major points:
1) First of all, with all due respect to the work done by the authors, I fear that the study itself does not bring enough novelty to what has already been described for olive leaves. While the authors do highlight the need to value this by-product, having identified which varieties could have the greatest potential for high value-added incorporation into nutraceuticals, for example, on the other hand, the assays performed to ascertain antioxidant activity are only in vitro spectrophotometric assays and do not guarantee the scientific robustness necessary to make health-promoting claims in nutraceutical products. Furthermore, the phenolic profile is not exactly known, based only, once again, on spectrophotometric assays. Therefore, I think the article could be enriched if more robust assays were performed, such as the determination of antioxidant activity in cell lines and a more in-depth determination of the composition of the extracts (qualitative and quantitative), for example by HPLC. However, if it is not possible to carry out the experiments, I suggest that the authors refer to this study as a preliminary study, since this data is already an important starting point for screening what may have the most potential to advance to future studies.
2) Furthermore, considering that the authors intend to add value to olive leaves and integrate them into a circular economy context, why did they only use classic extraction techniques? These techniques are usually very time-consuming and require large quantities of solvent, for example, which hinders their applicability in an industrial context. I think the article could benefit from the addition of at least one green extraction methodology to enhance the visibility and relevance of the study.
3) Figure 1: The results presented correspond to the extracts prepared by which extraction method?
4) Figure 2: In this case, the results obtained for each extraction method correspond to which variety? Considering that the authors analysed those parameters for all varieties prepared by both extraction methods (according to the information presented in Methodologies), I consider that they should present all results or, at least, indicate to which extraction method correspond the results present in Figure 1 and to which variety correspond the results present in Figure 2.
5) Table 2: Same question as Figures 1 and 2. In general, I think these results could be presented in a better way, as they seem a bit confusing to the reader.
6) Lines 392-394: I think it could be interesting to discuss these findings.
Minor points:
1) Line 111: “but also” is repeated.
2) Line 212: The authors mentioned 18 extracts. However, in lines 133-135 the authors mentioned that for each variety, the extracts were prepared in triplicate. Could you please clarify this information?
3) Lines 333-334: Please rephrase. The sentence implies that the values of Haouzia and Picual are statistically comparable to the standard, but in reality, Manzanilla is the one whose values are comparable and close.
Author Response
Responses to Reviewer 1
We would like to sincerely thank the reviewer for the careful and insightful evaluation of our manuscript. The thoughtful comments and constructive suggestions have greatly contributed to improving the clarity, coherence, and scientific rigor of the work. We appreciate the time and expertise dedicated to reviewing our study, and we have revised the manuscript accordingly. Our detailed responses to each comment are presented below in italics.
Major points:
Comment 1: First of all, with all due respect to the work done by the authors, I fear that the study itself does not bring enough novelty to what has already been described for olive leaves. While the authors do highlight the need to value this by-product, having identified which varieties could have the greatest potential for high value-added incorporation into nutraceuticals, for example, on the other hand, the assays performed to ascertain antioxidant activity are only in vitro spectrophotometric assays and do not guarantee the scientific robustness necessary to make health-promoting claims in nutraceutical products. Furthermore, the phenolic profile is not exactly known, based only, once again, on spectrophotometric assays. Therefore, I think the article could be enriched if more robust assays were performed, such as the determination of antioxidant activity in cell lines and a more in-depth determination of the composition of the extracts (qualitative and quantitative), for example by HPLC. However, if it is not possible to carry out the experiments, I suggest that the authors refer to this study as a preliminary study, since this data is already an important starting point for screening what may have the most potential to advance to future studies.
Response 1: We thank the reviewer for this valuable observation. We fully agree that the present study constitutes a preliminary screening aimed at identifying the olive leaf varieties with the highest phytochemical and antioxidant potential. Following the suggestion, we have now clarified this point in the Introduction (please see lines 110-120). This framing reinforces the role of the current dataset as a starting point for future detailed investigations on compound-specific profiling, green extraction optimization, and industrial valorization.
Comment 2: Furthermore, considering that the authors intend to add value to olive leaves and integrate them into a circular economy context, why did they only use classic extraction techniques? These techniques are usually very time-consuming and require large quantities of solvent, for example, which hinders their applicability in an industrial context. I think the article could benefit from the addition of at least one green extraction methodology to enhance the visibility and relevance of the study.
Response 2: We thank the reviewer for this insightful comment. We fully agree that green extraction technologies, such as ultrasound-assisted, microwave-assisted, or pressurized liquid extraction, offer important advantages in terms of time, energy efficiency, and solvent reduction, and are highly relevant for circular economy strategies. However, the present study was designed as a preliminary screening to establish baseline differences among nine olive cultivars under two well-established conventional extraction conditions. Classical methods such as maceration and Soxhlet were intentionally selected because they (i) remain reference techniques for comparative phytochemical studies, (ii) allow direct benchmarking of varietal differences without technological bias, and (iii) provide data that can be reliably compared with existing literature.
To address the reviewer’s suggestion, we have added a paragraph to the Discussion acknowledging the limitations of classical techniques and highlighting the need for integrating green extraction methodologies in future steps of this research (please see lines 679-685). This study therefore serves as a foundational evaluation upon which more advanced, sustainable extraction strategies will be developed.
Comment 3: Figure 1: The results presented correspond to the extracts prepared by which extraction method?
Response 3: We thank the reviewer for this observation. Figure 1 has now been modified to clearly present the mean values obtained from both extraction methods. The updated figure displays side-by-side bars for maceration and Soxhlet extracts for all measured parameters, ensuring accurate comparison and improved clarity.
Comment 4: Figure 2: In this case, the results obtained for each extraction method correspond to which variety? Considering that the authors analysed those parameters for all varieties prepared by both extraction methods (according to the information presented in Methodologies), I consider that they should present all results or, at least, indicate to which extraction method correspond the results present in Figure 1 and to which variety correspond the results present in Figure 2.
Response 4: We thank the reviewer for this helpful remark. In response, Figure 2 has been merged with Figure 1, and all results from both extraction methods (maceration and Soxhlet) and all varieties are now presented together in a single, comprehensive new Figure 1. This correction ensures full clarity regarding which extraction method and which variety each result corresponds to.
Comment 5: Table 2: Same question as Figures 1 and 2. In general, I think these results could be presented in a better way, as they seem a bit confusing to the reader.
Response 5: We thank the reviewer for this comment. To improve clarity, we have updated Table 2 by inserting, under each antioxidant assay (DPPH and ABTS), the results for every variety obtained from both extraction methods (maceration and Soxhlet). This restructuring ensures that the data are easier to follow and that the contribution of each extraction method is clearly identifiable for all varieties.
Comment 6: Lines 392-394: I think it could be interesting to discuss these findings.
Response 6: We appreciate the reviewer’s suggestion. However, these findings were already discussed in the submitted version of the manuscript (please see lines 579-589). This section provides an interpretation of the observed trend and contextualizes it within the relevant literature. Nevertheless, we have carefully rechecked the text to ensure that the discussion is clear and sufficiently detailed.
Minor points:
Comment 1: Line 111: “but also” is repeated.
Response 1: Thank you for pointing this out. We have deleted the repeated “but also” to improve clarity. Please see line 114.
Comment 2: Line 212: The authors mentioned 18 extracts. However, in lines 133-135 the authors mentioned that for each variety, the extracts were prepared in triplicate. Could you please clarify this information?
Response 2: We thank the reviewer for this helpful observation. We clarify that a total of 54 extracts were obtained in this study. For each of the nine olive varieties, three independent extraction replicates were prepared for each extraction method (maceration and Soxhlet), resulting in: 9 varieties × 2 extraction methods × 3 independent extracts = 54 extracts. We have corrected the total number of extracts in line 222.
Comment 3: Lines 333-334: Please rephrase. The sentence implies that the values of Haouzia and Picual are statistically comparable to the standard, but in reality, Manzanilla is the one whose values are comparable and close.
Response 3: Thank you for pointing this out. We have rephrased the sentence to accurately reflect the new results of Table 2 (please see lines 401-403).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
You have conducted very interesting research on different varieties of olive leaves originating from Morocco, evaluating their TPC, TFC, and antioxidant activity using conventional extraction methods – Soxhlet and maceration with absolute ethanol – and comparing the results.
I recommend this manuscript for acceptance in the journal Horticulturae, after minor revision.
My suggestions are as follows:
- Line 111: The phrase “but also” appears twice in the sentence.
- Line 165: (w/v) should be italicised (w/v). Is the solvent removed in the rotary evaporator to dryness, or is it just concentrated? In the next paragraph, you state, “the extraction yield was determined as the ratio of the dry mass of the recovered crude extract to the initial dry mass of olive leaves.” Please clarify.
- Line 212: Are there only 18 extracts? In lines 133–134, you state, “For each variety, one composite sample was prepared, and all extractions and analytical measurements were performed in triplicate to ensure precision and reproducibility.”
-Section 2.7.1: Is the reference antioxidant Trolox or ascorbic acid? In line 220, Trolox is mentioned, while in line 224, ascorbic acid is referenced.
- Table 2: There should be two values for each variety – for example, two DPPH values for ARB (obtained from Soxhlet and maceration) and two ABTS values (obtained from Soxhlet and maceration), and the same for the other varieties.
- Line 512: “Demonstrated” and “found” appear consecutively in the sentence.
- Lines 535–536: Is the flavonoid content higher in Soxhlet extraction? (“Soxhlet extracts were aligned with higher total phenolic and flavonoid contents as well as stronger antioxidant activities, ...”).
Author Response
Responses to Reviewer 2
We express our sincere appreciation to the reviewer for the attentive and insightful assessment of our manuscript. The constructive remarks and valuable suggestions provided have significantly enhanced the clarity, precision, and overall quality of the revised paper. We are grateful for the time and expertise devoted to this review. Our detailed responses to each comment are presented below in italics.
Comment 1: Line 111: The phrase “but also” appears twice in the sentence.
Response 1: Thank you for pointing this out. We have deleted the repeated “but also” to improve clarity. Please see line 114.
Comment 2: Line 165: (w/v) should be italicised (w/v). Is the solvent removed in the rotary evaporator to dryness, or is it just concentrated? In the next paragraph, you state, “the extraction yield was determined as the ratio of the dry mass of the recovered crude extract to the initial dry mass of olive leaves.” Please clarify.
Response 2: We thank the reviewer for this comment. In line 169 (w:v) was italicized. We confirm that the solvent was removed completely: the extracts were evaporated to dryness under reduced pressure at 40–50 °C using a rotary evaporator. We have clarified this point in the revised manuscript. Please see line 161 and line 170. We clarify also that the extraction yield was calculated by dividing the dry mass of the recovered extract obtained after complete solvent removal (i.e., after evaporation to dryness) by the initial dry mass of the olive leaves used for the extraction. We have revised the text accordingly for clarity (please see lines 176-177).
Comment 3: Line 212: Are there only 18 extracts? In lines 133–134, you state, “For each variety, one composite sample was prepared, and all extractions and analytical measurements were performed in triplicate to ensure precision and reproducibility.”
Response 3: We thank the reviewer for this helpful observation. We specify that a total of 54 extracts were obtained in this study. For each of the nine olive varieties, three independent extraction replicates were prepared for each extraction method (maceration and Soxhlet), resulting in: 9 varieties × 2 extraction methods × 3 independent extracts = 54 extracts. We have corrected the total number of extracts in line 222.
Comment 4: Section 2.7.1: Is the reference antioxidant Trolox or ascorbic acid? In line 220, Trolox is mentioned, while in line 224, ascorbic acid is referenced.
Response 4: Thank you for your valuable comment. You are correct that the original wording created confusion regarding the standard used in the DPPH assay. The mention of Trolox resulted from an inadvertent mix-up during the writing of the DPPH method description, as Trolox is used in our ABTS assay but not in the DPPH protocol. In the DPPH test, ascorbic acid was the only reference antioxidant employed. To correct this, we have revised the text to clearly indicate that only ascorbic acid was used as the standard, and that it was prepared and treated in the same manner as the extracts (please see lines 229-235).
Comment 5: Table 2: There should be two values for each variety – for example, two DPPH values for ARB (obtained from Soxhlet and maceration) and two ABTS values (obtained from Soxhlet and maceration), and the same for the other varieties.
Response 5: Thank you for this observation. We have revised Table 2 to include separate values for each variety corresponding to the two extraction methods (Soxhlet and maceration) for both DPPH and ABTS assays, as suggested. This restructuring ensures that the data are easier to follow and that the contribution of each extraction method is clearly identifiable for all varieties.
Comment 6: Line 512: “Demonstrated” and “found” appear consecutively in the sentence.
Response 6: Thank you for your careful reading. We apologize for the oversight in the manuscript; the word “found” was inadvertently left in during revision. The sentence has now been corrected for clarity (please see line 627.).
Comment 7: Lines 535–536: Is the flavonoid content higher in Soxhlet extraction? (“Soxhlet extracts were aligned with higher total phenolic and flavonoid contents as well as stronger antioxidant activities, ...”).
Response 7: Thank you for your comment. We clarify that while Soxhlet extraction resulted in higher yield, total phenolic content, and stronger antioxidant activities, maceration favored the retention of higher levels of flavonoids. To make this distinction clear, we have corrected and revised the sentence (please see lines 642-643).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
I have carefully read your manuscript titled: „Unlocking the Value of Nine Olive Leaf Varieties: A Dual Assessment of Phenolic Composition and Antioxidant Properties“, ID - horticulturae-3998079.
In my opinion, the work needs to expand on the results section, as the introduction outlines the intention of a "dual comparative approach that simultaneously examines the influence of inter-varietal diversity and extraction methodology on the phytochemical evaluation and bioactivity of olive species in Moroccan conditions." After considering mentioned recommendations, it is necessary to review and adequately supplement other parts of the manuscript, such as the abstract, methodology, and discussion. For now, I do not recommend publishing the manuscript. You can find my reasoning in the attached file.
Comments for author File: Comments.pdf
Author Response
Responses to Reviewer 3
We sincerely thank the reviewer for the thorough and insightful evaluation of our manuscript. The constructive comments and thoughtful suggestions have been extremely helpful in enhancing the clarity, accuracy, and overall quality of the revised paper. We truly appreciate the time and expertise devoted to reviewing our work. Our detailed responses to each comment are presented below in italics.
Justification:
Comment 1: The results (Figure 1) show the average values of the parameters „Y“; „TPC“; „TFC“; „TCT“ in extracts of varieties and do not indicate the values of the parameters for both selected extraction methods. Displaying them would clearly show the influence of the extraction method on the quality of the extract obtained from a specific variety using a specific method.
Response 1: Thank you for this insightful comment. We have revised Figure 1 to present the values of each parameter (Y, TPC, TFC, TCT) separately for the two extraction methods (Soxhlet and maceration) across all varieties. This modification allows a clearer visualization of the influence of the extraction method on the phytochemical composition and quality of the extracts from each specific variety.
Comment 2: Comparison of average values of selected parameters according to extraction methods (Figure 2), which compares selected parameters depending on the extraction method, does not characterize the situation in a specific variety.
Response 2: Thank you for this comment. Figure 2 has been merged into the revised Figure 1, which now presents the values of all selected parameters for each variety according to the extraction method. This provides a clearer representation of the influence of extraction methods on the phytochemical composition of each specific variety.
Comment 3: A similar method of evaluation is also offered in the case of antioxidant activity (Table 2), where it is desirable to clarify which extract and which method is responsible for a specific antioxidant activity value.
Reponse 3: Thank you for this suggestion. We have revised Table 2 to clearly indicate the antioxidant activity values for each variety according to the extraction method (Soxhlet or maceration). This clarification ensures that the contribution of each extraction method to the observed antioxidant activity is clearly identifiable.
Comment 4: Similar studies present the above-mentioned metabolic analyses of „TPC“; „TFC“; „TCT“ as indicative and are often supplemented by additional analyses that better specify the phytochemical composition of extracts (e.g., HPLC combined with DAD/MS using external commercial standards, etc.). In the literature used, the authors refer to works that define the phytochemical content of the leaves of certain olive varieties. Therefore, based on the information already available, the authors could have supplemented at least the analyses of the phytochemical composition of extracts and varieties that have not yet been analyzed in this way, or analyzed all the alcohol extracts obtained and evaluated. This would have provided a much more valuable picture of their composition and biological potential, which the authors strongly emphasize.
Reponse 4: We thank the reviewer for this valuable and well-taken comment. We agree that complementary phytochemical analyses would provide a more detailed characterization of the extracts and further strengthen the assessment of their biological potential. However, we would like to emphasize that the present work represents a preliminary study aimed at comparing extraction methods and establishing baseline data on the phenolic content and antioxidant activity of different olive varieties. A comprehensive profiling of the phytochemical composition of the extracts, particularly for varieties that have not yet been investigated, was beyond the scope of the current study. Nevertheless, we fully acknowledge its importance, and future work will focus on performing detailed phytochemical analyses as well as exploring greener and more selective extraction techniques to better characterize and valorize the bioactive compounds in olive leaves. We have added a statement in the manuscript to clarify the scope of this study (please see lines 110-120) and outline these future research directions (please see lines 679-685).
Additional comments:
Comment 1: In sections 2.4. – 2.7. concerning the analysis of total phenol content, flavonol content, condensed tannins content, and ABTS radicalcation scavenging activity, information on the concentrations of the analyzed leaf extracts and standards used (such as gallic acid, quercetin, catechin, and trolox) is missing.
Response 1: Thank you for highlighting this. We have updated sections 2.4.–2.7. to include the specific concentrations of the leaf extracts and the standards (gallic acid, quercetin, catechin, and trolox) used in each assay. This additional information ensures that the methodology is fully transparent and reproducible. Please see lines 184, 190, 197, 198, 212.
Comment 2: Line 273 refers to the „variety factor“ and line 277 to the „varietal factor“. Are these different parameters or is this an error in the wording?
Response 2: We thank the reviewer for noticing this inconsistency. The terms “variety factor” and “varietal factor” refer to the same parameter. This was an error in wording. For consistency, we have revised the manuscript to use a single term “variety factor” throughout the text (please see line 288).
Comment 3: In the results section of Table 1, what exactly does the parameter "Df" mean?
Response 3: Thank you for pointing this out. The parameter “Df” in Table 1 refers to the “degree of freedom”, which represents the number of independent values that can vary in the statistical analysis. It is used to determine the significance of differences among groups. We have clarified this in the table caption to ensure the parameter is clearly understood by readers (please see line 296).
Comment 4: In the discussion section, lines 441–443, the sentence "The present study evaluated the influence of varietal diversity and extraction method on the phytochemical composition and antioxidant potential of olive leaves cultivated under Moroccan conditions" is inaccurate; the extraction method does not affect the phytochemical content of the leaves, but rather the phytochemical content of the leaf extracts.
Response 4: Thank you for this comment. We have corrected the sentence to more accurately reflect the focus of the study: “The present study evaluated the influence of varietal diversity and extraction method on the phytochemical composition and antioxidant potential of leaf extracts from nine olive varieties cultivated under Moroccan conditions.” This revision clarifies that the varietal diversity and extraction method affect the composition of the extracts, not the leaves themselves (please see lines 526-527).
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript was carefully revised and improved according to suggestions and is now suitable for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsAccepting the comments point by point and subsequently incorporating them into the original version of the manuscript resulted in a significant improvement. The results section became more comprehensible and significantly reflects the original intention of the work to comparatively assess the influence of inter-varietal diversity and extraction methodology on the phytochemical quality of plant extracts from selected varieties of olive leaves as possible sources of bioactive substances. The chosen methodological procedure and phytochemical analyses are adequate for the study's aim of conducting a preliminary analysis of plant material in order to outline its potential as a source of biologically active phytochemicals. The significant contribution of the work is the determination and verification of the extraction process in the targeted extraction of substances from biological material and the preliminary phytochemical-biological characterization of leaf ethanol extracts. The work meets the parameters of a preliminary study, which can be followed up with further study, as the authors emphasized in the text.
