Technological Optimization and Antioxidant Efficacy via the NRF-2-Mediated Defense Pathway of Corylus avellana L. Skin Extracts: A Sustainable Approach for Developing Health-Promoting Natural Products

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript needs major revision

Comments for author File: Comments.pdf

Author Response

1. What is the principal novelty of this study in comparison to prior research on the antioxidant properties of hazelnut skin extracts? Auth: We thank the reviewer for this question. The principal novelty of this study lies in the systematic optimisation of key extraction parameters within the extraction technique previously identified as the most effective. This approach enabled the identification of optimal extraction conditions to maximise antioxidant potential using green solvents and employing hazelnut skin from plants grown in Basilicata region and in the Viterbo area, highlighting the influence of the production site on extracts characteristics. In addition, the optimisation of the extraction process was coupled with the evaluation of NRF-2 pathway activation, providing an additional level of insight into the antioxidant mechanism and the potential cytoprotective effect of the extract. We have added this information in paragraph 1 introduction.

 

2. Numerous investigations have documented phenolic-rich extracts derived from hazelnut by-products. In what ways does the current optimization method markedly enhance existing knowledge? Auth: We thank the reviewer for this question. While previous research primarily focused on comparing different extraction methods, few studies have simultaneously assessed the effects of extraction time, temperature and solvent composition on hazelnut skin extracts. However, the present study investigated the entire experimental range of each parameter, including extreme values, thus facilitating a comprehensive understanding of how each factor influences phenolic compounds recovery and antioxidant activity.

 

3. The manuscript correlates extraction optimization with the activation of the NRF-2 pathway. Could the authors elucidate whether this mechanistic study is original concerning hazelnut skin extracts? We thank the reviewer for this insightful comment. Auth: To the best of our knowledge, while previous studies have explored the antioxidant properties of hazelnut skin extracts, none have directly investigated the activation of the NRF-2 pathway in this context. Moreover, the correlation between extraction optimization and the extent of NRF-2–mediated antioxidant responses has not been reported. Therefore, our study provides novel mechanistic insight into how bioactive compounds from hazelnut skin extracts can modulate NRF-2 signaling, highlighting the relevance of extraction conditions in maximizing their cytoprotective potential.

 

4. The authors employed a 3³ full factorial design (FFD) to optimize parameters for ultrasound-assisted extraction. What was the rationale for selecting this design over response surface approaches like Box–Behnken or Central Composite Design? Auth: We thank the reviewer for this comment. We chose a full 3³ factorial design because our primary objective was to explore the entire experimental range of UAE conditions (time, temperature, and solvent composition) within a compact range, while retaining the ability to fit a second-order polynomial model without excluding any extreme or intermediate combinations of the factors. In a three-level FFD, all combinations of low, medium, and high levels are tested, which allows for: (i) a comprehensive evaluation of main effects and two-factor interactions, (ii) direct detection of curvature across the middle levels, and (iii) a robust estimation of quadratic terms without relying on extrapolation from a reduced subset of points, as would be the case in Box-Behnken designs that omit the vertices of the cube.

 

5. The solvent system varied from 0% to 100% ethanol. What was the scientific justification for choosing this range, especially given that polyphenols often extract most effectively in mid-range hydroalcoholic solutions? Auth: We thank the reviewer for this insightful comment. The decision to examine the entire ethanol concentration range from 0 to 100% was made in order to evaluate all possible combinations of solvent composition with the other independent variables (temperature and extraction time) within the framework of the factorial design. As is well established, polyphenols are frequently optimally extracted in mid-range hydroalcoholic mixtures. However, the present study set out to comprehensively evaluate the influence of solvent polarity under all the specific experimental conditions adopted. This approach enabled the determination of the interaction effects between the solvent composition and the other process variables, confirming a progressive decrease in extraction efficiency for the recovery of compounds with antioxidant activity as the ethanol concentration increased. This finding aligns with the conclusions of earlier studies documented in the extant literature https://doi.org/10.1016/j.indcrop.2016.05.033.

 

6. The model predicted an optimal extraction time of 70.86 minutes, although the experimental design examined a duration of only 1 to 3 hours. What method was employed to extrapolate this result from the factorial model? Auth: We thank the reviewer for this comment. The value of 70.86 minutes was not obtained by extrapolating beyond the experimental domain, but rather directly from the multi-response optimization algorithm applied to the fitted second-order polynomial models. In the desirability-based optimization, the extraction time was treated as a continuous variable ranging from 1 to 3 hours (i.e., 60–180 minutes), and the software identified an optimum at 1.181 hours, which corresponds to 70.86 minutes. This value was therefore reported only in minutes to facilitate interpretation of the optimal condition and remains entirely within the time interval explored in the factorial design, without any extrapolation outside the studied experimental space.

 

7. Were the extraction yields (percentage recovery of extract from biomass) ascertained? How can extraction efficiency be thoroughly assessed if not? Auth: We thank thank the reviewer for this comment. Extraction yields were calculated and the related data were added in sections "2.1. Model adequacy" and "4.3. Optimization of the extraction procedure and extraction yield".

 

8. Did the authors assess the impact of ultrasound power or frequency, which are recognized to greatly affect ultrasound-assisted extraction? Auth: We thank thank the reviewer for this comment. Ultrasound power and frequency were maintained constant according to the instrument specifications, in order to reduce experimental variability and focus the optimization on solvent composition, temperature, and extraction time. We added this information in section “4.3. Optimization of the extraction procedure and extraction yield”.

 

9. The manuscript exhibits high R² values across many models. Were further diagnostics, such as lack-of-fit tests or residual analysis, performed to validate the model? Auth: We thank the reviewer for this comment. In addition to the high values of R², adj-R², and pred-R², we performed further diagnostic analyses of the models to verify the adequacy of the fitted second-order polynomial models. First, we assessed the lack of fit (Table S1 and S2) using the replicated center points, and no significant lack of fit was detected for the responses under consideration, indicating that the chosen model order was appropriate within the experimental domain. Second, Residual analysis was conducted through normal probability plots (Figure S1) showing that, the residuals are distributed around the center line, confirming that the model fits the experimental data adequately and follows a normal distribution.

 

10. Despite the proximity of adjusted and anticipated R² values, was cross-validation conducted to verify predictive resilience outside the experimental domain? Auth: We thank the reviewer for this comment. Cross-validation was not performed as a separate procedure because the experimental design included replicated central points, and model fit was thoroughly assessed using various complementary diagnostic analyses: high agreement between adjusted R² and predicted R² values (difference < 0.2), normal probability plots of the residuals (Figure S1), and high adequate precision indices (Table S3). Furthermore, predictive capability was experimentally verified by preparing the optimized extract under the specified conditions [30 °C, 70.86 min (1.181 h), 21.13% EtOH] and measuring its antioxidant activities, which fell within the 95% confidence intervals of the model predictions (Table 3). These results confirm the robustness and predictive accuracy of the model within the explored experimental domain.

 

11. The coefficient of variation (CV%) is indicated as <8% for the majority of replies. Could the authors specify the number of independent experimental replicates conducted for each condition? Auth: We thank the reviewer for the question.The 3³ full factorial design consisted of 27 experimental runs as detailed in Table 1, each performed as a single independent extraction, plus three additional independent extractions of the centre point (run 14), for a total of 30 independent extractions (Table S1). Antioxidant assays (TPC, ABTS, DPPH, FRAP, BCB) were conducted in triplicate on each extract (technical replicates, n=3). CV% values reflect analytical precision: 7.13%, 6.72%, 6.32%, 7.33%, and 20.48% for TPC, ABTS, DPPH, FRAP, and BCB, respectively (Table S3). On the other hand, centre point triplicates confirmed extraction reproducibility, enabling robust model diagnostics including pure error estimation and lack-of-fit assessment (Table S1 and S2).

 

12. Only 9 of the 25 identified compounds were validated using reference standards. What is the reliability of the provisional identifications for the remaining compounds? Auth: We thank the reviewer for raising this important point. In the present study, nine compounds were unambiguously identified using authentic reference standards. For the remaining compounds, a tentative identification approach was adopted based on a combination of analytical criteria, including accurate mass measurements, isotopic pattern consistency, MS/MS fragmentation behavior, comparison with literature-reported fragmentation patterns for hazelnut-derived phenolics and structurally related compounds. This multi-parameter approach is widely accepted in untargeted and semi-targeted phytochemical profiling when authentic standards are not available. While we acknowledge that these identifications cannot be considered fully confirmed, the convergence of these criteria provides a moderate-to-high level of confidence for structural assignment. The manuscript has been revised to clearly distinguish between confirmed and tentative identifications (section “2.4. Evaluation of the phytochemical profile”).

 

13. Could the authors furnish further validation (e.g., MS/MS spectrum comparison with databases or literature fragmentation patterns)? Auth: We appreciate the reviewer’s suggestion. The tentative identification of compounds was supported by comparison of MS/MS fragmentation patterns with literature data and publicly available spectral databases (e.g., MassBank, PubChem, and previously published papers). Diagnostic fragment ions and characteristic neutral losses (e.g., CO₂, H₂O, sugar moieties) were used to support structural assignments. Due to the lack of commercially available standards for several compounds, additional confirmation (e.g., NMR) was beyond the scope of the present study. This limitation has now been explicitly acknowledged in the revised manuscript (Section “4.9. Phytochemical profile analysis by UHPLC-MS”).

 

14. Given that 3,5-dihydroxybenzoic acid has been identified as the predominant constituent, may the authors elucidate its concentration in relation to prior studies on hazelnut skin extracts? Auth: We thank the reviewer for this question. In our study, 3,5-dihydroxybenzoic acid was confirmed as the predominant compound in the hazelnut skin optimised extract. Since this isomer is rarely reported in hazelnut skin, its amount was compared with values reported for the 3,4-dihydroxybenzoic acid, also called protocatechuic acid, one of the most frequently described hydroxybenzoic acids in hazelnut skin extracts (https://doi.org/10.3390/antiox8100460; http://dx.doi.org/10.1021/jf202449z). The higher levels observed may be attributed to differences in extraction conditions, solvent composition, and the geographical origin of the hazelnuts, highlighting the importance of the optimization process in enhancing phenolic recovery.

 

15. Numerous chemicals were designated as “unknown” in Table 4. Were efforts undertaken to further elucidate these compounds utilizing high-resolution mass spectrometry or molecular networking methodologies? Auth: We acknowledge the reviewer’s comment. Several signals were classified as “unknown” due to the absence of matching fragmentation patterns or insufficient structural information. High-resolution mass spectrometry was used in the present study; however, further structural elucidation (e.g., molecular networking, advanced database matching, or NMR) was beyond the scope of this work. These compounds have been retained in the dataset as they may represent minor or previously unreported constituents, potentially contributing to the overall bioactivity of the extract. We added this information in “Discussion” (Moreover, some detected compounds could not be structurally assigned and were classified as unknown. These compounds may represent minor or previously unreported constituents and could contribute to the overall biological activity. Further studies employing advanced tools such as molecular networking or NMR would be required for their characterization).

 

16. The research employs various antioxidant assays, including TPC, DPPH, ABTS, FRAP, and BCB. Were the associations between these assays subjected to statistical evaluation? Auth: We thank the reviewer for this suggestion. A correlation analysis among the different antioxidant assays was performed to evaluate the consistency of the results. The assays showed coherent trends, reflecting the contribution of phenolic compounds to the antioxidant activity, although differences were observed due to the distinct chemical mechanisms underlying each assay (electron transfer vs hydrogen atom transfer). We added “Correlation analysis among the different antioxidant assays (TPC, DPPH, ABTS, FRAP, and BCB) showed consistent trends, supporting the reliability of the antioxidant evaluation, although variations were observed due to the different reaction mechanisms involved.” in section “2.1. Model adequacy”.

 

17. Given that the Folin–Ciocalteu method identifies non-phenolic reducing chemicals, how did the authors ascertain that the TPC values accurately represent phenolic content? Auth: We acknowledge the limitation of the Folin–Ciocalteu assay, which may also react with non-phenolic reducing compounds. However, in the present study, the TPC results were interpreted in combination with chromatographic profiling and multiple antioxidant assays, which together support the predominance of phenolic compounds in the extract. Therefore, TPC values were not used as a standalone measure but as part of a multi-analytical approach.

 

18. The stated TPC value (~365 mg GAE/g extract) is somewhat elevated. Could matrix effects or extraction concentration affect these values? Auth: We thank the reviewer for this observation. The relatively high TPC value observed in this study may be attributed to several factors as the optimized extraction conditions, which enhance phenolic recovery or the concentration of the extract, expressed on an extract basis rather than raw material.

 

19. The antioxidant activity was assessed in HepG2 cells subjected to tert-butyl hydroperoxide (TBH). What was the rationale for selecting this particular oxidative stress model? Auth: We thank the reviewer for this comment. TBH (tert-butyl hydroperoxide) was selected as a model of oxidative stress because it is a well-characterized pro-oxidant that reliably induces intracellular ROS in HepG2 cells. This model is extensively used in our laboratory and in the literature to study cellular antioxidant defenses, allowing reproducible assessment of the protective effects of natural extracts under controlled oxidative stress conditions (https://www.sciencedirect.com/science/article/pii/S0308814622013632?casa_token=wKopYahZOwYAAAAA:azvWH9eh260uKNRCXdOKtYADxXWHQRdT3unOegbK8Iv7lFCn0nGf1mbvC6QV9A_5brjIHCz4-t8 - s0010; https://www.sciencedirect.com/science/article/pii/S0041008X05004217?via=ihub).

 

20. Were the activity of intracellular antioxidant enzymes assessed, or were only the levels of protein expression evaluated? Auth: We thank the reviewer for this comment. In the present study, the protein expression levels were assessed only by Western blot. Although the enzymatic activity of these proteins was not directly measured, the observed upregulation of their expression is widely recognized as an indicative marker of the activation of cellular antioxidant defense pathways.

 

21. The research asserts the activation of the NRF-2 signaling pathway. Was the nuclear translocation of NRF-2 experimentally validated, or merely inferred from elevated protein expression? Auth: We thank the reviewer for this comment. In the current study, NRF‑2 pathway activation was evaluated by Western blot analysis of total NRF‑2 protein levels, together with the expression of downstream antioxidant enzymes. While nuclear translocation of NRF‑2 was not directly assessed, it is well established in the literature that an increase in the expression of NRF‑2 and its downstream targets reflects activation of the NRF‑2/ARE antioxidant defense pathway (https://pmc.ncbi.nlm.nih.gov/articles/PMC4680839/).

 

22. Is it possible that the observed drop in ROS is due to direct radical scavenging instead of genuine activation of the NRF-2 pathway? Auth: We thank the reviewer for this important point. While a direct radical-scavenging effect of the hazelnut skin extract may contribute to the observed reduction in intracellular ROS, as also supported by the in vitro DPPH and ABTS assay, the modulation of NRF-2 protein levels and the upregulation of downstream antioxidant enzymes, including NQO1, SOD, and catalase, indicate the activation of a cellular antioxidant defense mechanism. Therefore, the observed decrease in ROS likely reflects a combination of direct scavenging and NRF-2–mediated enhancement of endogenous antioxidant pathways. An additional sentence has been included on lines 49–50 to clarify this point.

 

23. Were dose–response curves conducted to ascertain IC₅₀ or EC₅₀ values for ROS inhibition? Auth: We thank the reviewer for this comment. In the present study, ROS inhibition was assessed at selected non-cytotoxic concentrations of the hazelnut skin extract (25–200 µg/mL). The results clearly demonstrate a concentration-dependent reduction in intracellular ROS levels. Given the limited number of concentrations and the complex nature of the natural extract, the calculation of IC₅₀ or EC₅₀ values was not within the scope of the present study.

 

24. The research indicates possible uses in nutraceutical and pharmaceutical formulations. Have the stability or bioavailability aspects of the extract been assessed? Auth: We thank the reviewer for this important comment. In the present study, stability and bioavailability aspects were not directly assessed, as the main objective was to optimize the extraction process, characterize the phytochemical profile of the obtained extract, and evaluate its antioxidant potential through in vitro chemical assays and a cell-based model. We fully agree that stability and bioavailability are essential parameters for the actual development of nutraceutical or pharmaceutical formulations. However, these aspects fall beyond the scope of the current work and would require dedicated investigations, including evaluation of the extract under different storage and formulation conditions, as well as digestion, absorption, and metabolism studies.

 

25. Given that HepG2 cells serve as a model for hepatic cancer, to what extent may these findings be generalized to normal human cells or tissues? Auth: We thank the reviewer for this comment. HepG2 cells, although derived from hepatocellular carcinoma, are a well-established model for studying oxidative stress and NRF-2–mediated antioxidant responses (https://pubs.rsc.org/en/content/articlelanding/2020/fo/c9fo01466g; https://www.sciencedirect.com/science/article/pii/S0278691517304088?via=ihub). However, as a cancer-derived cell line, they differ from normal hepatocytes in terms of gene expression, metabolism, and signaling pathways. Consequently, the results obtained in HepG2 cells cannot be fully generalized to normal human cells or tissues and should be interpreted with caution. Nevertheless, the observed effects provide valuable mechanistic insights into the antioxidant and cytoprotective potential of the hazelnut skin extract.

 

26. Do long-term exposures to concentrated hazelnut skin extracts pose any toxicological concerns? Auth: We thank the reviewer for this question. Cytotoxicity was assessed at 4 and 24 hours of exposure. We considered it appropriate to limit cytotoxicity assessment to 24 hours since our primary interest was in the antioxidant effects occurring at the earlier 4-hour time point. Long-term toxicity beyond 24 hours was not assessed in this study; therefore, potential toxicological concerns related to prolonged exposure would require further investigation.

 

27. Biological action is often ascribed to flavonoids like catechin, luteolin, and naringenin. Were these chemicals present in adequate amounts to explain the observed effects? Auth: Based on our quantitative analysis, and considering the concentrations of extract used in the biological assays (100–200 µg/mL), the estimated concentrations of these compounds ranged from approximately 0.003 to 0.205 µg/mL. These levels are lower than the concentrations typically reported to exert biological effects when the compounds are tested individually. Therefore, while these flavonoids may contribute to the observed activity, the effects are likely due to the combined action of multiple constituents present in the extract (https://www.sciencedirect.com/science/article/pii/S1756464620304205; https://academic.oup.com/toxsci/article-abstract/69/1/149/1707390?redirectedFrom=fulltext&login=false).

 

28. The study proposes synergistic effects among phenolic substances. Can this theory be substantiated using fractionation or activity-guided assays? Auth: We thank the reviewer for this insightful comment. We agree that the demonstration of synergistic effects among phenolic compounds would ideally require fractionation approaches and activity-guided assays, allowing the contribution of individual components and their interactions to be systematically evaluated. In the present study, however, the hypothesis of synergistic effects is based on indirect evidence, including the complex phytochemical profile revealed by UHPLC–MS/MS analysis, the presence of multiple phenolic classes with known antioxidant activity, and the consistent results obtained across different antioxidant assays and cellular models. These observations are consistent with the widely recognized concept that plant extracts often exhibit biological activity resulting from the combined and potentially interactive effects of multiple constituents, rather than from a single dominant compound. We acknowledge that this work does not provide direct experimental validation of synergy. Future analyses involving fractionation, recombination experiments, and activity-guided analyses would be interesting for further studies to confirm and quantify synergistic interactions.

 

29. Numerous typographical and formatting issues exist, such as redundant journal header information and discrepancies in space. The manuscript requires meticulous proofreading. Auth: We thank the reviewer for highlighting these issues. The manuscript has been thoroughly proofread and carefully revised to correct typographical errors and formatting inconsistencies.

 

30. Latin binomial nomenclature, such as Corylus avellana, should be uniformly italicized throughout the text. Auth: We thank the reviewer for his comments. The Latin binomial nomenclature has been consistently italicized throughout the text.

 

31. Abbreviations (e.g., OE, TBH, NRF-2, BCB) must be explicitly described upon first usage in both the abstract and the main body. Auth: We thank the reviewer for this comment. The abbreviations have been explicitly defined upon first usage in both the Abstract and the main text.

 

32. Certain figure legends reiterate information previously detailed in the Results section and might be condensed for clarity. Auth: We thank the reviewer for this helpful suggestion. The figure legends have been revised and condensed to avoid redundancy with the Results section and to improve overall clarity.

 

33. Maintain uniformity in units throughout tables and figures (e.g., mg GAE/g, mg TE/g, μg/mL). Auth: We thank the reviewer for this comment. Units have been carefully revised and standardized throughout all tables and figures to ensure consistency.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a timely and relevant topic, focusing on the valorization of hazelnut skins as a phenolic-rich by-product of the food industry and their potential use as a source of functional ingredients. The integration of extraction optimization, phytochemical profiling, in vitro antioxidant assays, and cell-based evaluation in HepG2 cells adds value to the study. In particular, the combination of ultrasound-assisted extraction with factorial design, UHPLC–MS/MS-based chemical characterization, and the investigation of intracellular ROS levels and NRF2-related pathways represent notable strengths that enhance the depth of the work.

Nevertheless, although the antioxidant activity was assessed using multiple in vitro assays, the biological significance of these assays should be discussed more critically. While the extract demonstrated antioxidant potential and cellular compatibility, the implications regarding its possible nutraceutical and pharmaceutical applications may extend beyond the evidence currently presented. Therefore, the conclusions should be framed more cautiously, emphasizing the extract as a promising functional ingredient candidate rather than making broader application claims. In addition, the manuscript would benefit from careful language revision to improve clarity and scientific precision. Overall, the study appears potentially publishable and scientifically interesting; however, substantial revisions are needed with respect to methodological clarity, data interpretation, and the framing of the conclusions.

Author Response

The manuscript addresses a timely and relevant topic, focusing on the valorization of hazelnut skins as a phenolic-rich by-product of the food industry and their potential use as a source of functional ingredients. The integration of extraction optimization, phytochemical profiling, in vitro antioxidant assays, and cell-based evaluation in HepG2 cells adds value to the study. In particular, the combination of ultrasound-assisted extraction with factorial design, UHPLC–MS/MS-based chemical characterization, and the investigation of intracellular ROS levels and NRF2-related pathways represent notable strengths that enhance the depth of the work. Nevertheless, although the antioxidant activity was assessed using multiple in vitro assays, the biological significance of these assays should be discussed more critically. While the extract demonstrated antioxidant potential and cellular compatibility, the implications regarding its possible nutraceutical and pharmaceutical applications may extend beyond the evidence currently presented. Therefore, the conclusions should be framed more cautiously, emphasizing the extract as a promising functional ingredient candidate rather than making broader application claims. In addition, the manuscript would benefit from careful language revision to improve clarity and scientific precision. Overall, the study appears potentially publishable and scientifically interesting; however, substantial revisions are needed with respect to methodological clarity, data interpretation, and the framing of the conclusions.

 Auth: We thank the reviewer for this comment. The revised conclusions can be found in the relevant section.

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer Comments (Questions and Suggestions for Improvement)

1. Extraction parameters: Could the authors clarify why only temperature, extraction time, and solvent composition were selected as independent variables in the Full Factorial Design used for ultrasound-assisted extraction optimization? Other important parameters, such as ultrasound power or the solid-liquid ratio, can also significantly influence extraction efficiency. Please explain why these factors were not included in the experimental design.
2. Effect of solvent composition: The results indicate that ethanol concentration had a strong influence on the extraction efficiency and antioxidant activity. Could the authors further explain the mechanism behind the negative effect of higher ethanol concentrations, particularly in relation to the polarity of the phenolic compounds present in hazelnut skin?
3. Model validation and reproducibility: The optimal extraction conditions were predicted using the factorial design model. Could the authors provide more information on how the reproducibility of the optimized conditions was verified and how closely the experimental results matched the predicted values?
4. Phytochemical identification: UHPLC-MS/MS analysis revealed several compounds that were only tentatively identified. Could the authors clarify whether further analytical techniques (e.g., comparison with reference standards or NMR analysis) are planned to confirm the structures of these compounds?
5. Cellular antioxidant activity: The extract showed a significant reduction in intracellular ROS levels in HepG2 cells. Could the authors provide additional details regarding the dose-response relationship and the statistical comparison with the NAC positive control?
6. References and DOI formatting: Please verify whether the DOI numbers in the reference list comply with the journal’s formatting requirements and ensure that all DOIs are presented in a consistent format.
7. Technical Quality. There are minor stylistic repetitions in the text (for example, excessive repetition of the argument regarding the “circular economy”).

It is recommended to standardize the notation of TBH / t-butyl hydroperoxide.

Check the correctness of the units of measurement (mg TE/g, mg GAE/g) for consistency in formatting.

 

 

Author Response

1. Extraction parameters: Could the authors clarify why only temperature, extraction time, and solvent composition were selected as independent variables in the Full Factorial Design used for ultrasound-assisted extraction optimization? Other important parameters, such as ultrasound power or the solid-liquid ratio, can also significantly influence extraction efficiency. Please explain why these factors were not included in the experimental design. Auth: We thank the reviewer for this observation. The selection of temperature, extraction time and solvent composition as independent variables was based on their established role in influencing the efficiency of ultrasound-assisted extraction. The ultrasonic power and the solid-liquid ratio were not included in the factorial design, as evidenced by research findings in the literature. It has been demonstrated that high ultrasonic power can lead to the degradation of phenolic compounds, while low levels may prove insufficient to improve extraction. Moreover, when the extraction time and solvent are constant, its influence on the final yield is frequently limited. In a similar manner, the solid-liquid ratio was set to a high value (1:100) to avoid solvent saturation and ensure that no limitations occurred in the extraction process. In the given conditions, and under constant extraction time and ultrasonic power, this parameter does not appear to be a determining factor in extraction efficiency. Consequently, in order to reduce experimental complexity whilst maintaining a robust and meaningful design, the focus was directed towards the three variables that most directly influence the extraction process (https://doi.org/10.1007/s11947-025-03775-z, doi: 10.15237/gida.GD24025).

 

2. Effect of solvent composition: The results indicate that ethanol concentration had a strong influence on the extraction efficiency and antioxidant activity. Could the authors further explain the mechanism behind the negative effect of higher ethanol concentrations, particularly in relation to the polarity of the phenolic compounds present in hazelnut skin? Auth: We thanks the author for the question. The strong negative effect of higher ethanol concentrations on extraction efficiency is primarily due to solvent polarity mismatch with the phenolic compounds identified in hazelnut skin extracts. UHPLC-MS/MS analysis (Table 4) revealed that the main bioactive compound, the phenolic acid 3,5-dihydroxybenzoic acid, have a moderate polarity with a LogP of 0.934 making it optimally soluble in hydroalcoholic mixtures (~20% EtOH) rather than pure ethanol. This assumption is also confirmed by literature demonstrating that low concentration of ethanol (23 %) was more effective for extracting polar polyphenols, such as phenolic acids than high concentration of ethanol (85%) (https://doi.org/10.1016/j.lwt.2025.118481). Similarly, it was seen that the extraction of catechins like gallogatechin, which have a comparable LogP of (-)-catechin (LogP of 0.877 and 0.938, respectively), increased with 20% of ethanol (https://doi.org/10.1007/s11694-022-01722-6). Higher ethanol concentrations (>50%) decrease solvent polarity, reducing hydrogen bonding capacity and favouring precipitation/exclusion of polar phenolics, as evidenced by the highly significant negative quadratic term (A², p<0.0001, Table 2) in all response models. This explains why 100% EtOH runs systematically yielded the lowest TPC and antioxidant activities.

 

3. Model validation and reproducibility: The optimal extraction conditions were predicted using the factorial design model. Could the authors provide more information on how the reproducibility of the optimized conditions was verified and how closely the experimental results matched the predicted values? Auth: The reproducibility of the optimized conditions was verified experimentally by preparing the optimized hazelnut skin extract under the predicted UAE conditions (30 °C, 70.86 min, and 21.13% ethanol) and comparing the observed responses with the model predictions. The obtained values were in good agreement with the predicted ones and fell within the 95% confidence intervals for all response variables (Table 3).

 

4. Phytochemical identification: UHPLC-MS/MS analysis revealed several compounds that were only tentatively identified. Could the authors clarify whether further analytical techniques (e.g., comparison with reference standards or NMR analysis) are planned to confirm the structures of these compounds? Auth: We appreciate the reviewer’s suggestion. The tentative identification of compounds was supported by comparison of MS/MS fragmentation patterns with literature data and publicly available spectral databases (e.g., MassBank, PubChem, and previously published papers). Diagnostic fragment ions and characteristic neutral losses (e.g., CO₂, H₂O, sugar moieties) were used to support structural assignments. Due to the lack of commercially available standards for several compounds, additional confirmation (e.g., NMR) was beyond the scope of the present study. This limitation has now been explicitly acknowledged in the revised manuscript (Section “4.9. Phytochemical profile analysis by UHPLC-MS”).

 

5. Cellular antioxidant activity: The extract showed a significant reduction in intracellular ROS levels in HepG2 cells. Could the authors provide additional details regarding the dose-response relationship and the statistical comparison with the NAC positive control? Auth: The antioxidant activity of the extract was evaluated at different concentrations (25–200 µg/mL) in HepG2 cells exposed to TBH to assess the dose–response relationship. As shown in Figure 4, the extract reduced intracellular ROS levels in a concentration-dependent manner. In particular, the highest concentrations (200 and 100 µg/mL) produced a more pronounced decrease in ROS levels compared with the lower concentrations (50 and 25 µg/mL). NAC, used as positive control, significantly reduced ROS levels, and the effect observed with the highest concentrations of the extract (200 and 100 µg/mL) was comparable to that obtained with NAC. Additional details regarding the concentration-dependent antioxidant effect and the statistical analysis have been clarified in the revised manuscript (lines 26–29/Figure 4).

 

6. References and DOI formatting: Please verify whether the DOI numbers in the reference list comply with the journal’s formatting requirements and ensure that all DOIs are presented in a consistent format. Auth: Thanks for the suggestion, we have verified the the reference list according to the journal’s guidelines.

 

7. Technical Quality.There are minor stylistic repetitions in the text (for example, excessive repetition of the argument regarding the “circular economy”). It is recommended to standardize the notation of TBH / t-butyl hydroperoxide. Check the correctness of the units of measurement (mg TE/g, mg GAE/g) for consistency in formatting. Auth: We thank the reviewer for this observation. The abbreviation TBH has been standardized throughout the manuscript and is now written in full (tert-butyl hydroperoxide) only at its first occurrence in the Abstract. We checked the correctness units.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors carried out interesting research on optimizing the extraction process of secondary metabolites from Corylus avellana L. skin (by-product), selecting the appropriate temperature and extraction time as well as the composition of the solvent, and then identified the metabolites present in the extract and examined their antioxidant properties, concluding that the extract has a modulating effect on the endogenous defense system.

The paper is well-written, and the discussion and conclusions do not raise any objections. The paper cites 55 references, most of which were published after 2020. The cited publications are closely related to the topic of the study. Ref. 43 lacks the journal volume number.

The paper would be more interesting and understandable if the authors had included a photo of a hazelnut in the Introduction, with particular emphasis on the skin from which the bioactive compounds were extracted.

A weak point of the paper is the identification of bioactive compounds without the use of appropriate chemical standards. The authors are aware of this (last paragraph, page 8), but the lack of standards remains a fact.

The content of 9 bioactive compounds was quantified (Table 4), but there is no information on the calibration method and key validation parameters of the quantitative analysis methods developed (e.g., precision, recovery, LOD, LOQ).

The Materials and Methods section lacks information on skin particle size, and it is known that particle size significantly affects extraction efficiency.

All abbreviations should be explained where they first appear. This will make it easier to understand the text without having to look for explanations at the end of the manuscript. This applies to the abbreviations: NRF-2 (line 41), SOD2, CAT, NQ01 (line 96), EtOH (line 106), DPPH, ABTS, BCB (line 107), TPC, FRAP (line 116), OE (Section 2.5, line 1), PLE (Section 3. Discussion, line 89).

Author Response

• The paper would be more interesting and understandable if the authors had included a photo of a hazelnut in the Introduction, with particular emphasis on the skin from which the bioactive compounds were extracted. Auth: Thanks for the suggestion, we've added figure 1 to the introduction.

 

• A weak point of the paper is the identification of bioactive compounds without the use of appropriate chemical standards. The authors are aware of this (last paragraph, page 8), but the lack of standards remains a fact. Auth: We thank the reviewer for raising this important point. In the present study, nine compounds were unambiguously identified using authentic reference standards. For the remaining compounds, a tentative identification approach was adopted based on a combination of analytical criteria, including accurate mass measurements, isotopic pattern consistency, MS/MS fragmentation behavior, comparison with literature-reported fragmentation patterns for hazelnut-derived phenolics and structurally related compounds. This multi-parameter approach is widely accepted in untargeted and semi-targeted phytochemical profiling when authentic standards are not available. While we acknowledge that these identifications cannot be considered fully confirmed, the convergence of these criteria provides a moderate-to-high level of confidence for structural assignment. The manuscript has been revised to clearly distinguish between confirmed and tentative identifications (section “2.4. Evaluation of the phytochemical profile”). Several signals were classified as “unknown” due to the absence of matching fragmentation patterns or insufficient structural information. High-resolution mass spectrometry was used in the present study; however, further structural elucidation (e.g., molecular networking, advanced database matching, or NMR) was beyond the scope of this work. These compounds have been retained in the dataset as they may represent minor or previously unreported constituents, potentially contributing to the overall bioactivity of the extract. We added this information in “Discussion” (Moreover, some detected compounds could not be structurally assigned and were classified as unknown. These compounds may represent minor or previously unreported constituents and could contribute to the overall biological activity. Further studies employing advanced tools such as molecular networking or NMR would be required for their characterization).

 

• The content of 9 bioactive compounds was quantified (Table 4), but there is no information on the calibration method and key validation parameters of the quantitative analysis methods developed (e.g., precision, recovery, LOD, LOQ). Auth: We thank the reviewer for this important comment. We agree that clearer information on the calibration approach and the main analytical performance parameters is necessary to improve the transparency of the quantitative UHPLC–MS/MS analysis. In the revised manuscript, we have clarified that the quantification of the nine compounds reported in Table 4 was performed using external calibration curves prepared with the corresponding available authentic reference standards. The analytical response was evaluated within the selected concentration ranges, and the quantitative results should be interpreted as referring to the compounds for which standard-based calibration was available.

 

• The Materials and Methods section lacks information on skin particle size, and it is known that particle size significantly affects extraction efficiency. Auth: We thank the reviewer for the comment. The requested information was added in section 4.2.

 

• All abbreviations should be explained where they first appear. This will make it easier to understand the text without having to look for explanations at the end of the manuscript. This applies to the abbreviations: NRF-2 (line 41), SOD2, CAT, NQ01 (line 96), EtOH (line 106), DPPH, ABTS, BCB (line 107), TPC, FRAP (line 116), OE (Section 2.5, line 1), PLE (Section 3. Discussion, line 89). Auth: We thank the reviewer for this comment. Units have been carefully revised the text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Revised version of manuscript accepeted for publication