Synthesis and Application of Natural Deep Eutectic Solvents (NADESs) for Upcycling Horticulture Residues

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the issue that is discussed on the present study is very interesting and yet, a comprehensive study regarding a wide variety of NADES on experimental extractions of plant materials does not exist. So, it is very good that this gap is filled with that study. But, because myself as a researcher, I have occupied with DES, and NADES, I observed some issues that need to be clarified and corrected to accept in present form. Below I present each one of them

Comment #1: Line 37: The use of the word "benign" is not appropriate the way and the field you are using it. Please replace it with "friendly" or something else more fit.

Comment #2: This comment refers to the whole introduction: The research regarding DES and NADES, is not a new field and at least a decade ago from now, researchers globally highlighted the use of these extraction media. So, I am very curious and I need this to be clarified and corrected, why you did not insert citations regarding other researches in the last two paragraphs (Lines 42-59) where you expand your objectives. Please add more literature content.

Comment #3: (Lines 42-59) Apart from the addition of new literatures regarding NADES in the introduction, clearly the last two paragraphs are totally blank of citations. So, where did you find these mechanisms you are talking about? Are these not published in other researches? The addition of citations for what you are referring to is crucial for integrity of the research. Please add.

Comment #4: Section 2.1.2: It is crucial to enlighten us with the particle size (mm) of the plant residue.

Comment #5: Results section: You are using a very interesting and correct screening statistical technique, which enables you to present your results in a variety of forms and figure types. Instead, you chose to present tables with data and some simple bar figures where the x-axis has so many data (NADES treatments) and any reader and researcher could not make any comparisons between different treatments. The presentation of data needs totally different formation. The correct and efficient way for the readers is to present more adequate figures such as contour plots or surface plots or other types and insert your raw data tables (except of the model data) as supplementary material.

Author Response

Comments 1: Line 37: The use of the word "benign" is not appropriate the way and the field you are using it. Please replace it with "friendly" or something else more fit.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have replaced the word “benign” with the word “pleasant”. The sentence reads in the revised manuscript as: “NADESs, a class of environmentally pleasant solvents, are composed of natural, biodegradable, and non-toxic components such as sugars, amino acids, organic acids, and salts”.

Comments 2: This comment refers to the whole introduction: The research regarding DES and NADES, is not a new field and at least a decade ago from now, researchers globally highlighted the use of these extraction media. So, I am very curious and I need this to be clarified and corrected, why you did not insert citations regarding other researches in the last two paragraphs (Lines 42-59) where you expand your objectives. Please add more literature content.

Response 2: We appreciate the honorable reviewer's observation. In our most recent publication, the honorable reviewers suggested that we move most of the citations to the discussion section of the manuscript. We followed that suggestion in the current manuscript. However, we have added fifteen citations to the introduction. We still prefer a short but impactful introduction to a worded lengthy one.

Comments 3: (Lines 42-59) Apart from the addition of new literatures regarding NADES in the introduction, clearly the last two paragraphs are totally blank of citations. So, where did you find these mechanisms you are talking about? Are these not published in other researches? The addition of citations for what you are referring to is crucial for integrity of the research. Please add.

Response 3: We have added 15 more citations to the introduction (lines 42-59)

Comments 4: Section 2.1.2: It is crucial to enlighten us with the particle size (mm) of the plant residue.

Response 4: We did not consider the particle size of the plant residue as a factor in our design because, from our preliminary studies, it had no effect on the extraction process.

Comments 5: Results section: You are using a very interesting and correct screening statistical technique, which enables you to present your results in a variety of forms and figure types. Instead, you chose to present tables with data and some simple bar figures where the x-axis has so many data (NADES treatments) and any reader and researcher could not make any comparisons between different treatments. The presentation of data needs totally different formation. The correct and efficient way for the readers is to present more adequate figures such as contour plots or surface plots or other types and insert your raw data tables (except of the model data) as supplementary material.

Response 5: Our decision to present our results in simple figures and tables stems from suggestions we got from reviewers in our last publication. We also observed that simple figures and tables help nonscientific and nontechnical readers comprehend the materials easily leading to a broader readership. However, we present the contour plots of the factors’ effects on TPC extraction as supplementary plots (SP 1 to SP 14) in the supplementary information document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the submitted Manuscript ("horticulturae-3555753...") , the authors investigated a horticulture-related application of green extraction (e.g., valorizing horticultural byproducts like fruit peels, seeds, or floral waste using NADES). The authors synthesized 14 NADESs, optimized extraction conditions via RSM, and evaluated antioxidant/antiradical activities (TPC, TFC, CUPRAC, FRAP, DPPH). The results explore broader applications of NADESs for upcycling horticultural residues (e.g., African nutmeg peels, other plant waste).

Overall, the submitted manuscript directly addresses horticultural waste valorization, a key focus area for Horticulturae, which is alignment with journal scope. Meanwhile, the manuscript introduces 14 novel NADES formulations (e.g., citric acid-glycine, citric acid-xylitol) with potential for diverse horticultural applications, which also indicates some novelty at a certain degree. However, before the manuscript can be accepted for publication in the journal of Horticulturae, the following minor or major issues should be carefully addressed.

1. The authors have already published similar paper in their recent paper “Okeke, U.J.; Micucci, M.; Mihaylova, D.; Cappiello, A. The Effects of Experimental Conditions on Extraction of Polyphenols from African Nutmeg Peels Using NADESs-UAE: A Multifactorial Modelling Technique. Sci Rep 2025, 15, 4890, doi:10.1038/s41598-025-88233-8.”, while in the submitted manuscript, the authors expanded on the published paper’s scope, clarified how the broader horticultural focus (beyond African nutmeg peels) and novel NADES formulations provided unique contributions. If the authors told more differentiations from their published work, it will make the manuscript more readable.
2. For data interpretation, if the authors gave more discussions on mechanistic insights (e.g., why fructose-based NADES outperformed others in TPC extraction), and compared results with traditional solvents (e.g., ethanol, methanol) to emphasize NADES advantages, this will be more particularly helpful to the readers.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comments 1: The authors have already published similar paper in their recent paper “Okeke, U.J.; Micucci, M.; Mihaylova, D.; Cappiello, A. The Effects of Experimental Conditions on Extraction of Polyphenols from African Nutmeg Peels Using NADESs-UAE: A Multifactorial Modelling Technique. Sci Rep 2025, 15, 4890, doi:10.1038/s41598-025-88233-8.”, while in the submitted manuscript, the authors expanded on the published paper’s scope, clarified how the broader horticultural focus (beyond African nutmeg peels) and novel NADES formulations provided unique contributions. If the authors told more differentiations from their published work, it will make the manuscript more readable.

Response 1: We thank the honorable reviewer and have provided a summarized differentiation in the discussion section of the revised manuscript as follows:

“In our recent work [3], we focused on a single NADES-CaFr11, providing in-depth exploration, analysis, and discussion of its efficient phenolic extraction capability, as well as anti-radical scavenging efficacy. In contrast, in the present work, we broaden the scope significantly, expanding the discussion to include thirteen additional NADESs synthesized with similar and different HBDs at different molar ratios. We aim to expand the NADES candidates, providing diversification which enhances the breadth of the research, offering a more comprehensive exploration of the application of NADESs in extraction studies and potentially introducing new perspectives, comparisons, and relationships between the synthesized NADESs”.

Comments 2: For data interpretation, if the authors gave more discussions on mechanistic insights (e.g., why fructose-based NADES outperformed others in TPC extraction), and compared results with traditional solvents (e.g., ethanol, methanol) to emphasize NADES advantages, this will be more particularly helpful to the readers.

Response 2: We thank the honorable reviewer for the insight, hence our submission in the revised manuscript that:

“Among the synthesized NADESs, fructose-based NADESs at 1:1 molar ratio provided the most excellent extraction media for polyphenol extraction. However, at a 2:1 molar ratio, it provided the weakest extraction efficiency. More studies are required to establish the reason for such a phenomenon. We cannot accurately speculate that the differences in physical properties between the two fructose-based NADES formulations were responsible for such a substantial performance gap”. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Udodinma Jude Okeke et al have submitted the manuscript entitled 'Synthesis and application of Natural Deep Eutectic Solvents (NADESs) for Upcycling Horticulture Residues '. After close evaluation of the manuscript I recommend revision according to the next comments:

1. In Introduction I suggested to underline some additional benefits of NADESes. To be polar NADES are able for co-extraction of some elements from plant materials. It was shown recently, that NADES have a limited ability to coextract elements from the plant materials. NADES extracts from plants and seaweeds possess no health risk for both ingestion and topical application.
2. The stabilizing effect of NADES on active metabolites in extracts was reported. The stability of total phlorotannins content and dynamic of antioxidant activity of F. vesiculosus NADES extracts was recently studied during 360 days of storage.
3. In Sect. 2.1.2: please indicate who have identified Monodora myristica and provide voucher of specimen
4. In Sect. 2.2.1 please clarify which method was used ultrasound-assisted or microwave technique?
5. All Tables/ Figures must be self-explanatory. Please explain all abbreviations after each Table/ Figure.
6. Table 8: please indicate statistically significant difference between Physical Properties  for different NADES.
7. Table 6 is not clear. Please provide FTIR spectra for clarification.
8. Figures 1-5: no statistics was provided. It looks as results of single experiments were provided. Statistically significant differences must be indicated.
9. For comparison, DPPH scavenging activity must be calculated as IC50
10. Positive control for all antioxidant methods must be used.
11. The discussion is weak. Authors must compare own results with the results published in literature.  For example, physicochemical and antimicrobial properties of lactic acid-based natural deep eutectic solvents as a function of water content were were published recently..
12. Conclusions must be based on statistics. Please update conclusion accordingly.
13. 13, The limitation of the study must be indicated.

Author Response

Comments 1: In Introduction I suggested to underline some additional benefits of NADESes. To be polar NADES are able for co-extraction of some elements from plant materials. It was shown recently, that NADES have a limited ability to coextract elements from the plant materials. NADES extracts from plants and seaweeds possess no health risk for both ingestion and topical application.

Response 1: We thank the honorable reviewer and have added more benefits of NADES in the introduction of the revised manuscript as follows:

“Therefore, NADESs offer a range of remarkable benefits that make them a compelling alternative to traditional solvents. Their environmentally friendly, biodegradable, and non-toxic nature, combined with their ability to dissolve a wide range of compounds, makes them highly useful across many industries. Whether in pharmaceuticals, food processing, biotechnology, or green chemistry, NADESs contribute to safer, more sustainable practices while maintaining high efficiency, performance, and improved stability of bioactive compounds[15][16]”.

Comments 2: The stabilizing effect of NADES on active metabolites in extracts was reported. The stability of total phlorotannins content and dynamic of antioxidant activity of F. vesiculosus NADES extracts was recently studied during 360 days of storage.

Response 2: We thank the honorable reviewer for this wonderful suggestion. However, the scope of the current work does not include NADES-induced stability of the extracted compounds. But the suggestion is amazing, and we may consider a full study to investigate such.

Comments 3: In Sect. 2.1.2: please indicate who have identified Monodora myristica and provide voucher of specimen.

Response 3: We thank the honorable reviewer and have provided the information in the revised manuscript as follows:

“African nutmeg (Monodora myristica (Gaertn.) Dunal was first described by Joseph Gaertner as Annona myristica in 1791 and was later transferred to the genus Monodora by Michael Felix Dunal in 1817 and retained the later genus till this day”. A specimen voucher is provided in the supplementary information as specimen 1.

Comments 4: In Sect. 2.2.1 please clarify which method was used ultrasound-assisted or microwave technique?

Response 4: We thank the honorable reviewer but noted that we clearly stated from the beginning that an ultrasound-assisted technique was used for the synthesis of the solvents.

Comments 5: All Tables/ Figures must be self-explanatory. Please explain all abbreviations after each Table/ Figure.

Response 5: We thank the honorable reviewer and applied the suggestions in the revised manuscript.

Comments 6: Table 8: please indicate a statistically significant difference between Physical Properties for different NADES.

Response 6: We have indicated in the revised manuscript that significant differences are observed where (p ≤ 0.5).

Comments 7: Table 6 is not clear. Please provide FTIR spectra for clarification.

Response 7: We thank the honorable reviewer and have provided the FTIR spectra for all the synthesized NADESs in the supplementary information documents as supplementary figure 1.

Comments 8: Figures 1-5: no statistics was provided. It looks as results of single experiments were provided. Statistically significant differences must be indicated.

Response 8: The results are presented as means and standard deviations of 30 experimental runs with statistically significant differences shown at p ≤ 0.5.

Comments 9: For comparison, DPPH scavenging activity must be calculated as IC50.

Response 9: We thanked the honorable reviewer for his views but noted that different techniques for DPPH analysis exist including IC50, EC50, %ARA, etc. In the present work, however, %ARA fulfilled the objectives of the study which is to test if the NADES extracts possess antiradical activity rather than the elucidation of concentration-based antiradical activity. The IC50 technique is more appropriate when investigating the concentration of molecules on a biological, cell, or organ (animal or human) model, and that objective is outside the scope of the present study. 

Comments 10: Positive control for all antioxidant methods must be used.

Response 10: We thank the honorable reviewer and stated that we used positive controls for all antioxidant studies and presented detailed controls in the methodology section.

Comments 11: The discussion is weak. Authors must compare own results with the results published in literature.  For example, physicochemical and antimicrobial properties of lactic acid-based natural deep eutectic solvents as a function of water content were were published recently..

Response 11: We thank the honorable reviewer for his views. We compared our work with the work of others where appropriate. However, the lack of literature where citric acid-based NADES was applied to extract polyphenols from African nutmeg peels made solvent-to-solvent and material-to-material comparisons much more difficult. To avoid misleading the readers, we compared the efficiency of our process and solvents to those in the literature rather than comparing the results.

Comments 12: Conclusions must be based on statistics. Please update conclusion accordingly.

Response 12: We agree with the honorable reviewer and have rewritten the conclusion, updating it accordingly in the revised manuscript.

Comments 13: 13, The limitation of the study must be indicated.

Response 13: We also agree with the honorable reviewer and have included the limitations of the study in the revised manuscript as suggested. The limitations are stated in the conclusion as follows:

“The current study is limited to the synthesis and application of citric acid-based NADESs to TPC and TFC extraction from ANP, and the evaluation of the crude extracts for antiradical scavenging activities using in vitro methods. The study does not involve isolation of individual compounds from the extracts, nor does it involve biological testing of the extract through animal or cell in vivo study”.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Firstly I would like to thank you very much for answer all of my questions. Secondly, I accept that an improvement has been done in the basis of clarification, I would like to advice some scientifical and presentation things. 

Firstly, regarding your answer about the particle size, where you claim that you did not insert it because you did not find any significance on preliminary experiments. I understand your argument, but in order for the study's reproducibility, which is a major issue generally, i don't think that it is a major issue for your research team to measure the particle size with mesh and inform us. Whether you agree with it or not, particle size is proved to be a significant factor on extraction process.

For the case of the presentation, I would like to thank you very much for inserting contour plots and photographs of your plant material in all forms.

Author Response

Comments 1:

Dear authors,

Firstly I would like to thank you very much for answer all of my questions. Secondly, I accept that an improvement has been done in the basis of clarification, I would like to advice some scientifical and presentation things.

Firstly, regarding your answer about the particle size, where you claim that you did not insert it because you did not find any significance on preliminary experiments. I understand your argument, but in order for the study's reproducibility, which is a major issue generally, i don't think that it is a major issue for your research team to measure the particle size with mesh and inform us. Whether you agree with it or not, particle size is proved to be a significant factor on extraction process.

For the case of the presentation, I would like to thank you very much for inserting contour plots and photographs of your plant material in all forms.

Response to comment 1: We are thankful to you, the honorable reviewer, for your general advice and insights from the first review phase to the second review phase. Your views greatly helped us to improve the readership of our manuscript, and we are grateful.

We agreed that particle size can be a significant factor in some experiments, however, after our preliminary studies, where we used three different sieve sizes (No 4: 4.75mm; No10: 2.00mm; & No 20: 850µm), and observed insignificant difference on extraction capacity, we stopped further consideration of particle size in the study used for the present manuscript. We felt it would be deceptive to report any one of the sieve sizes knowing that in the present work, we used the peels' powder without sieving into separate particle sizes.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

no further comments.

Author Response

Comments 1: no further comments

Response 1: We thank the honorable reviewer for the great insights provided in the first phase of the review process and are happy that the honorable reviewer was satisfied with our responses. We agreed that the work in general can be improved, as there is no perfection with human endeavors.  We will continue to improve and innovate the process more and more.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have revised and updated the manuscript, however, some my recommendations were not addressed completely.

1. My comment 1 from previous round is important especially for food products. The possible contamination of NADES extracts with trace elements is is essential for food industry. Since authors haven't studied this aspect, it must be discussed. The references to research articles are more appropriate than references to review articles in this case.
2. Figures 1-4 no statistics and statistically significant differences were provided.
3. Table 8 no statistically significant differences were provided.
4. My comment 9 from previous round was not completely addressed.

Author Response

Comments 1: My comment 1 from previous round is important especially for food products. The possible contamination of NADES extracts with trace elements is is essential for food industry. Since authors haven't studied this aspect, it must be discussed. The references to research articles are more appropriate than references to review articles in this case.

Response 1: We thank the honorable reviewer for considering this coextraction and contamination challenge, however, this aspect is outside the scope of the current study. As the honorable reviewer had already noted that we haven’t studied the aspect, we considered discussing it in this manuscript to be out of scope, premature, and misleading.

Comments 2: Figures 1-4 no statistics and statistically significant differences were provided.

Response 2: We stated that figures 1-4 represent the means of 30 replicates for each of the synthesized NADESs at the different experimental conditions. At this point of our study, we were discovering the conditions necessary for efficient responses and had not started comparing the NADES extraction results with one another to determine significant differences among them.

Comments 3: Table 8 no statistically significant differences were provided.

Response 3: We thank the honorable reviewer and provide that in Table 8, the results in mean±SD of 3 replicates at each experimental run are presented. The results have not been compared with one another to determine if there are statistically significant differences among NADES extracts just as in Figures 1-4. Statistical significance was provided in Table 12 where the NADES extracts were finally compared with one another.

Comments 4: My comment 9 from previous round was not completely addressed.

Response 4: Dear honorable reviewer, your comment 9 from the previous round was that “For comparison, DPPH scavenging activity must be calculated as IC50”, however, we did not completely agree with your suggestion (keyword “MUST”) that IC50 must be used for DPPH calculation. As we previously responded, there are different methods of DPPH analysis which are based on the objective of the assessment. The Objective of IC50 measurement is to determine the concentration of the substance that scavenges half of the radical species. That is not the same with the objective of our study which is to elucidate if the extracts have antiradical scavenging capacity measured by DPPH. In this case, percentage antiradical activity is the best approach.   

Author Response File: Author Response.pdf