Volatile Natural Deep Eutectic Solvents (VNADESs) for Extraction of Shikonin Derivatives from Echium vulgare Roots and Evaluation of Biological Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors  developed and evaluated a green chemistry-based extraction method to obtain shikonin derivatives from the roots of Echium vulgare L. using volatile natural deep eutectic solvents (VNADES). They also compared the extraction efficiency, selectivity, and biological potential (assessed by cytotoxic, antioxidant, anti-inflammatory, and antimicrobial assays) of this method with those of conventional hexane extraction.

I have an observation and some question for the manuscript:

1)  It is very important that the authors include the chemical structure of the compounds.

2) What was the reason for performing a single simple extraction using NADES systems?

3) How many extractions would be possible?

4) Is it possible to separate the compounds from VNADES?.

5) What was the purity of the compounds extracted with VNADES?.

6) Why were the VNADES formulated in terms of thymol, benzyl alcohol, camphor, and menthol?.

7) What were the criteria for selecting the VNADES and their composition?.

8) What were the criteria for selecting only 4 VNADES?.

9) On line 116 the authors wrote "...The main hypothesis of this study is that VNADES-based systems enable the more efficient recovery of shikonin derivatives than hexane...". What are the possible interactions that improve extraction with NADES compared to hexane?.

10) Since the authors conclude that VNADES extraction appears to be a promising method, and further research is needed to reduce shikonin losses during extraction using these methods. What is their proposals to improve extraction?.

Author Response

Reviewer 1.

We sincerely thank the Reviewer for the careful evaluation of our manuscript entitled “Volatile natural deep eutectic solvents (VNADESs) for the extraction of shikonin derivatives from Echium vulgare roots and evaluation of biological activity” and for the constructive comments aimed at improving its clarity, and scientific value. We have carefully considered all suggestions and revised the manuscript accordingly. Our detailed responses are provided below.

Comment 1

 It is very important that the authors include the chemical structure of the compounds.

Response:
We agree with the Reviewer that including the chemical structures is important for clarity. Therefore, we provided the general structures of shikonin derivatives in Figure 2.

Comment 2, 3

What was the reason for performing a single simple extraction using NADES systems?

How many extractions would be possible?

Response:

We thank the Reviewer for this insightful comment.

In the initial stage of this study, a single-step extraction was intentionally performed using different VNADES systems in order to screen their extraction efficiency and identify the most promising solvent system for shikonin derivatives. To clarify the rationale for performing a single-step extraction, an explanatory sentence has been added to the Methods section (Section 3.5). Based on these preliminary results, the thymol:benzyl alcohol system was selected as the most effective. Subsequently, a multi-step extraction ( 6 cycles) was performed for various molar ratios (T:Ba 1:9 to 6:4) to evaluate the extraction completeness. These results are presented in the Supplementary Materials (Tables S2–S6).

The results indicate that the majority of shikonin derivatives are extracted within the first 3 extraction steps, while 5 cycles are sufficient to achieve near-complete extraction. In the 6th step, no detectable shikonin derivatives were observed using T:Ba systems for extraction analyzed by HPLC under the applied conditions. Therefore, from a practical perspective, 3–5 extraction steps can be considered optimal.

Comment 4

Is it possible to separate the compounds from VNADES?

Response:

We thank the Reviewer for this important question.

The separation of target compounds from VNADES systems remains a significant challenge. This issue of has been addressed in the revised manuscript. In the Results section (Section 2.1.3) we indicate that freeze-drying can be used for the removal of VNADES; however, this approach is associated with significant losses of shikonin derivatives.

To the best of our knowledge, there is no universally established and efficient method for the isolation of compounds from VNADES.

We agree that this is an important limitation and have highlighted it in the revised manuscript as a perspective for future work in Conclusion section (Section 4).

Comment 5

What was the purity of the compounds extracted with VNADES?

Response

We thank the Reviewer for this important remark.

In the present study, the aim was not to isolate individual compounds, but rather to evaluate the efficiency of VNADES in extracting shikonin derivatives as a complex mixture, as well as to assess the biological activity of the obtained extracts.

Therefore, the purity of individual compounds was not determined, as no isolation step was performed. Instead, the composition of the extracts was characterized using HPLC analysis.

We acknowledge that determination of compound purity would require additional purification steps, which may be considered in future studies focused on isolation rather than extraction efficiency.

Comment 6

Why were the VNADES formulated in terms of thymol, benzyl alcohol, camphor, and menthol?

Response:

We thank the Reviewer for this important comment. The selection of VNADES components (thymol, benzyl alcohol, menthol, and camphor) was based on our previous study (Kulinowska, M.; Dresler, S.; Skalska-Kamińska, A.; Hanaka, A.; Strzemski, M. Methodological Aspects of Green Extraction of Usnic Acid Using Natural Deep Eutectic Solvents. Molecules 2023, 28, doi:10.3390/molecules28145321), in which their suitability for dissolving lipophilic natural products was demonstrated. Additionally, preliminary experiments indicated high extraction efficiency of thymol:benzyl alcohol systems for shikonin derivatives. This rationale has now been clarified in the revised manuscript

We agree that this rationale was not sufficiently clarified and have now added an explanation in the revised manuscript (Section 3.4).

Comment 7, 8

What were the criteria for selecting the VNADES and their composition?

What were the criteria for selecting only 4 VNADES?

Response:

We thank the Reviewer for this valuable comment.
The selection of VNADES systems was based on their previously demonstrated ability to dissolve lipophilic bioactive compounds, potential to form stable eutectic systems, volatility of components (facilitating removal), and preliminary experimental results indicating high extraction efficiency. Based on these criteria, four VNADES systems were selected as representative and promising candidates for further investigation. This rationale has now been clarified in the revised manuscript (Section 3.4).

Comment 9

On line 116 the authors wrote "...The main hypothesis of this study is that VNADES-based systems enable the more efficient recovery of shikonin derivatives than hexane...". What are the possible interactions that improve extraction with NADES compared to hexane?

Response:
We thank the Reviewer for this insightful comment.
The explanation of our hypothesis has been clarified in the revised manuscript (Introduction) where we now include that enhanced solubilisation of shikonin derivatives in VNADES may arise from hydrogen-bonding and other non-covalent interactions.

Comment 10

Since the authors conclude that VNADES extraction appears to be a promising method, and further research is needed to reduce shikonin losses during extraction using these methods. What is their proposals to improve extraction?

Response:

We thank the Reviewer for this important point.
The proposed strategies to improve extraction efficiency and reduce shikonin losses have been added to the revised manuscript (specially in Conclusion- Section 4). We believe that the added clarification satisfactorily addresses the Reviewer’s concerns.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript

Your manuscript entitled “Volatile natural deep eutectic solvents (VNADESs) for the extraction of shikonin derivatives from Echium vulgare roots and evaluation of biological activity” has been carefully evaluated.
Manuscript No.: molecules-4252183

Brief outline of the manuscript

Summary of the paper

The manuscript presents an interesting and timely study focused on the use of sustainable extraction media for the recovery of valuable bioactive compounds. The topic is relevant from both scientific and environmental perspectives, as it addresses the growing interest in greener extraction methodologies. The comparison between volatile natural deep eutectic solvents (VNADESs) and hexane for the extraction of shikonin derivatives from Echium vulgare roots is meaningful and well aligned with current trends in green chemistry. Overall, the study suggests that VNADES systems may offer a promising and more environmentally friendly alternative for obtaining these compounds.

Materials and methodology

The experimental work is extensive and supported by a suitable combination of analytical and biological methods used for characterization of the obtained extracts and assessment of their activity. The use of advanced analytical techniques, such as HPLC-ESI-QTOF-MS/MS for phytochemical profiling and HPLC-PDA for quantitative analysis, represents a strong aspect of the study. In addition, the comparison of hexane and VNADES extracts, together with cytotoxicity, antioxidant, anti-inflammatory, and antibacterial evaluations, provides a broad and useful view of the biological potential of the extracts. The methodology is generally well described and scientifically appropriate.

Results and discussion

The results are clearly presented and supported by relevant experimental findings. The manuscript provides valuable information on the chemical composition and biological activity of the extracts, and the experimental effort behind the work is evident. The study offers several useful observations, especially regarding extraction performance and biological effects. At the same time, the discussion could be further enriched by a somewhat deeper interpretation of the results and a more explicit connection to related published studies, which would help emphasize the broader significance of the findings.

Conclusion

The results indicate that VNADES are effective green solvents for the extraction of shikonins from E. vulgare, providing comparable or even higher yields than hexane, although noticeable losses occur during freeze-drying. The VNADES extracts showed lower cytotoxicity and statistically significant inhibition of ROS and NO, whereas the hexane extracts exhibited stronger antimicrobial activity. These findings make the study relevant and potentially useful for the development of greener extraction strategies. The manuscript is supported by relevant literature and contributes to the fields of green chemistry and circular economy.

 

Comments and suggestions for improvement

• The manuscript could be further strengthened by including a somewhat more detailed comparison and analysis of the components used in the VNADES systems, particularly with respect to their physicochemical characteristics.
• The final subsection of the Introduction may be improved by focusing more clearly on the research gap and the specific objectives of the study.
• It would be beneficial to discuss the possible applications and practical feasibility of VNADES systems somewhat more explicitly in the main text.
• Some of the reported improvements in relation to VNADES, especially in extraction efficiency, appear relatively modest and may be presented in a slightly more balanced manner.
• The manuscript would benefit from somewhat stronger integration with the existing literature in order to better position the study and clarify its specific contribution.
• A careful round of language editing and proofreading would improve the overall consistency and readability of the text.
• A clearer graphical presentation of the shikonin derivatives in Figure 2, especially dimethylacrylshikonin, would improve clarity and make the discussion easier to follow.
• The sentence regarding fragmentation pathways may need revision, since the acyl substituent is absent and therefore does not prevent McLafferty-type rearrangement. In addition, the proposed fragmentation pathway, including the molecular ion and the major fragments at m/z 219.0284 and 189, could be presented more clearly and in a form more consistent with the usual interpretation  of experimental data obtained in negative ionization mode.
• The main contribution of the study could be emphasized more clearly, especially in relation to the practical applicability of VNADES systems, including cost-related aspects, the use of biologically active components, and the role of freeze-drying. It may also be helpful if the authors briefly comment on the recyclability of the VNADES systems.
• The loss of shikonin derivatives during freeze-drying (Figures 4–7) appears to be an important aspect of the study and deserves somewhat more detailed discussion.
• It would be useful to clarify why Step 4 deviates from the general trend observed in Table 2.
• The interpretation of the results could be deepened in several places in order to make the conclusions even more convincing, particularly when comparing the extraction methodologies and discussing the influence of component structure.
• The link between objectives, methods, and findings could be made more explicit, which would improve the overall coherence and flow of the manuscript.

 

Final recommendation

In my opinion, the manuscript addresses an interesting and relevant topic and contains valuable experimental results. Subject to these minor revisions, it could be considered suitable for publication.

 

Author Response

Reviewer 2.

We sincerely thank the Reviewer for the careful evaluation of our manuscript entitled “Volatile natural deep eutectic solvents (VNADESs) for the extraction of shikonin derivatives from Echium vulgare roots and evaluation of biological activity” and for the constructive comments aimed at improving its clarity, and scientific value. We have carefully considered all suggestions and revised the manuscript accordingly. Our detailed responses are provided below. 

Comment 1

The manuscript could be further strengthened by including a somewhat more detailed comparison and analysis of the components used in the VNADES systems, particularly with respect to their physicochemical characteristics.

Response

We thank the Reviewer for this valuable suggestion.
The manuscript has been revised to include a more detailed description of the physicochemical rationale behind the selection of VNADES components (Section 3.4). Specifically, the choice of thymol, benzyl alcohol, menthol, and camphor is now discussed in terms of their lipophilicity, hydrogen-bonding and π–π interaction potential, as well as their ability to form low-melting eutectic systems and facilitate post-extraction removal.

Comment 2

The final subsection of the Introduction may be improved by focusing more clearly on the research gap and the specific objectives of the study.

Response

We thank the Reviewer for this valuable comment.

We would like to note that the final subsection of the Introduction has been structured to clearly present the research gap, followed by the hypothesis and study objectives, in a separate paragraph to improve clarity and logical organization of the manuscript.

Comment 3

It would be beneficial to discuss the possible applications and practical feasibility of VNADES systems somewhat more explicitly in the main text.

Response

We thank the Reviewer for this valuable suggestion.
A discussion of the possible applications and practical feasibility of VNADES systems has been added to the manuscript (Conclusions- Section 4). The revised text now more explicitly addresses their potential as green extraction media for shikonin derivatives and highlights practical aspects related to solvent volatility and removal.

Comment 4

Some of the reported improvements in relation to VNADES, especially in extraction efficiency, appear relatively modest and may be presented in a slightly more balanced manner.

Response

We thank the Reviewer for this valuable comment. In response, we have carefully revised the manuscript to ensure a more balanced presentation of the VNADES-related results. Specifically, we have moderated overly strong qualitative statements and replaced strong expressions with more neutral and data-reflective wording, particularly in Section 2.1.3, Section 2.1.4, Section 2.2.1, Section 2.2.5.

We believe these modifications improve the clarity and balance of the manuscript while preserving the scientific interpretation of the results.

Comment 5

The manuscript would benefit from somewhat stronger integration with the existing literature in order to better position the study and clarify its specific contribution.

Response

We thank the Reviewer for this helpful suggestion. The integration with the existing literature has been strengthened, particularly in Introduction, Section 2.1.3, Section 2.2.2, and in Conclusion where additional references and comparative discussion have been included.

Comment 6

A careful round of language editing and proofreading would improve the overall consistency and readability of the text.

Response

We thank the Reviewer for this comment. The manuscript has been carefully language-edited and proofread, and revisions have been applied throughout the abstract, Results and Discussion, and Conclusions sections to improve clarity and consistency.

Comment 7

A clearer graphical presentation of the shikonin derivatives in Figure 2, especially dimethylacrylshikonin, would improve clarity and make the discussion easier to follow.

Response

Thank you for this suggestion. Figure 2 has been revised to improve clarity. In particular, the substituents in shikonin derivatives, including dimethylacrylshikonin and isovalerylshikonin, are now clearly labeled, making the structures easier to interpret.

Comment 8

The sentence regarding fragmentation pathways may need revision, since the acyl substituent is absent and therefore does not prevent McLafferty-type rearrangement. In addition, the proposed fragmentation pathway, including the molecular ion and the major fragments at m/z 219.0284 and 189, could be presented more clearly and in a form more consistent with the usual interpretation  of experimental data obtained in negative ionization mode.

Response:

We thank the Reviewer for this important comment. The sentence describing the fragmentation behavior of shikonin has been revised to avoid overinterpretation regarding the role of the acyl substituent and McLafferty-type rearrangement. The text now more accurately reflects that the absence of an acyl substituent influences the fragmentation pattern rather than preventing a specific mechanism.

Additionally, the description of the proposed fragmentation pathway has been clarified and adapted to be more consistent with typical interpretation of MS/MS data obtained in negative ionization mode.

Comment 9

The main contribution of the study could be emphasized more clearly, especially in relation to the practical applicability of VNADES systems, including cost-related aspects, the use of biologically active components, and the role of freeze-drying. It may also be helpful if the authors briefly comment on the recyclability of the VNADES systems.

Response

We have expanded the Conclusion to better highlight the practical relevance of VNADES systems, including their reduced reliance on conventional volatile organic solvents, the potential sustainability benefits associated with their naturally derived components, and a brief comment on solvent recovery and recyclability.

Comment 10

The loss of shikonin derivatives during freeze-drying (Figures 4–7) appears to be an important aspect of the study and deserves somewhat more detailed discussion.

Response:

Thank you for this insightful comment. We agree that the loss of shikonin derivatives during freeze-drying is an important aspect of the study. In the revised manuscript, this issue has been discussed in greater detail. In particular, an additional final paragraph has been included in Section 2.1.3 to further elaborate on this phenomenon, and the Conclusions section (Section 4) has been expanded the discussion of freeze-drying-related losses, emphasizing this as a key limitation of NADES-based post-extraction processing and highlighting its impact on the final shikonin content in dried extracts, emphasiszes some future perspectives.

Comment 11

It would be useful to clarify why Step 4 deviates from the general trend observed in Table 2.

Response:

Thank you for this important observation. We have clarified the deviation observed in Step 4 of Table 2 (in Section 2.1.4). A similar pattern has also been observed in our previous extractions of other plant materials using conventional solvents, suggesting that this effect may be related to matrix-dependent extraction behavior rather than an isolated anomaly.

In the present study, this deviation is mainly visible for hexane, while no clear discontinuity between Steps 4 and 5 was observed for NADES systems. We cautiously attribute this effect to a combination of low analyte concentrations in later fractions and increased variability, as well as possible matrix effects. One possible explanation is that earlier extraction steps remove ballast compounds, thereby facilitating solvent penetration into less accessible regions of the plant matrix and enabling the release of additional shikonin derivatives in Step 4.

Additionally, the higher yield observed for TBa 2:8 may indicate more efficient matrix penetration, potentially leading to earlier extraction of internally localized compounds. However, these interpretations remain speculative and are not directly supported by literature data. We therefore chose to approach this observation with caution in the manuscript, although the reproducibility of this effect was verified experimentally.

Comment 12

The interpretation of the results could be deepened in several places in order to make the conclusions even more convincing, particularly when comparing the extraction methodologies and discussing the influence of component structure.

Response

Thank you for this valuable suggestion. We agree that a deeper interpretation of the results strengthens the overall conclusions of the study. In the revised manuscript, we have expanded the discussion in several sections to better highlight the relationships between extraction efficiency, solvent properties, and the chemical structure of shikonin derivatives.

In particular, additional clarification has been introduced in Sections 2.1.3 and 2.2.1–2.2.5 to more explicitly relate the observed extraction trends to solvent polarity, matrix interactions, and the structural features of shikonin derivatives. We have also emphasized how these factors may influence the biological activity of the extracts, including the potential contribution of synergistic effects and differences in compound stability during post-extraction processing.

These additions provide a more comprehensive interpretation of the results and strengthen the comparison between conventional solvents and NADES systems.

Comment 13

The link between objectives, methods, and findings could be made more explicit, which would improve the overall coherence and flow of the manuscript.

Response:

Thank you for this helpful suggestion. We agree that strengthening the connection between the study objectives, applied methods, and obtained findings improves the coherence and flow of the manuscript.

We have improved the manuscript, particularly in the Introduction, Results and Conclusion, to better guide the reader through the rationale of the study and integrate the main findings in relation to the original study objectives.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors developed and evaluated a green chemistry-based extraction method to obtain shikonin derivatives from the roots of Echium vulgare L. using volatile natural deep eutectic solvents (VNADES). They also compared the extraction efficiency, selectivity, and biological potential (assessed by cytotoxic, antioxidant, anti-inflammatory, and antimicrobial assays) of this method with those of conventional hexane extraction.

I had an observation and some question for the original manuscript:

 

1)  It is very important that the authors include the chemical structure of the compounds.

2) What was the reason for performing a single simple extraction using NADES systems?

3) How many extractions would be possible?

4) Is it possible to separate the compounds from VNADES?.

5) What was the purity of the compounds extracted with VNADES?.

6) Why were the VNADES formulated in terms of thymol, benzyl alcohol, camphor, and menthol?.

7) What were the criteria for selecting the VNADES and their composition?.

8) What were the criteria for selecting only 4 VNADES?.

9) On line 116 the authors wrote "...The main hypothesis of this study is that VNADES-based systems enable the more efficient recovery of shikonin derivatives than hexane...". What are the possible interactions that improve extraction with NADES compared to hexane?.

10) Since the authors conclude that VNADES extraction appears to be a promising method, and further research is needed to reduce shikonin losses during extraction using these methods. What is their proposals to improve extraction?.

The authors addressed the observation and questions raised.