Valorization of the Bioactive Potential of Juniperus communis L. Berry Extracts Using a Box–Behnken Design and Characterization of Kernel Oil Compounds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Holistic Valorization of Juniperus communis L. Berries Extracts 2 Using Box-Behnken Design and Characterization of Berry Kernel Oil Compounds" presents an in-depth investigation of the bioactive compounds present in Juniperus communis berries and kernels, through a combination of modern analytical methods and optimized extraction parameters. The study is notable for its rigorous methodological approach and the valorization of a natural resource with potential pharmaceutical and food applications.
However, certain aspects require further investigation or clarification to strengthen the scientific significance of the study. The following questions address this objective:

  1. Could the authors clarify how the selected ranges for temperature, solvent concentration, and time were chosen? 
2. Polyphenol yield is reported per gram of defatted dry weight. Could the authors justify the relevance of using defatted material for extraction and how this might affect polyphenol bioavailability in a whole-matrix application?
3. Why was magnetic stirring chosen over other extraction methods, such as microwave-assisted extraction? Have you compared the efficiency or selectivity of magnetic stirring to this technique?

4. Were any toxicity or cytotoxicity tests performed or referenced for the kernel oil or polyphenol-rich extract? 
5. Could the authors elaborate on any limitations regarding scalability or potential interactions when used as additives?

Author Response

The manuscript "Holistic Valorization of Juniperus communis L. Berries Extracts 2 Using Box-Behnken Design and Characterization of Berry Kernel Oil Compounds" presents an in-depth investigation of the bioactive compounds present in Juniperus communis berries and kernels, through a combination of modern analytical methods and optimized extraction parameters. The study is notable for its rigorous methodological approach and the valorization of a natural resource with potential pharmaceutical and food applications.

We would like to thank the reviewer for the positive feedback regarding our manuscript.

However, certain aspects require further investigation or clarification to strengthen the scientific significance of the study. The following questions address this objective:

1. Could the authors clarify how the selected ranges for temperature, solvent concentration, and time were chosen?

• Solvent polarity was tuned with water and ethanol to target polar and less-polar bioactives, respectively.
• Temperature ranges (20–80 °C) were chosen to enhance solubility and diffusion without degrading thermolabile compounds.
• Extraction times (30–90 min) were set based on preliminary trials showing no significant gains beyond 30 min (p > 0.05); thus, we limited heating duration to minimize energy input. These details are now explicitly described in Section 2.2.2 with supporting citations.

2. Polyphenol yield is reported per gram of defatted dry weight. Could the authors justify the relevance of using defatted material for extraction and how this might affect polyphenol bioavailability in a whole-matrix application?

• We defatted the kernels to isolate the oil and resin components separately, preventing oil interference in antioxidant assays.
• Removing lipids enhances assay accuracy and focuses on polyphenol content.
• We discuss potential matrix effects on bioavailability in Section 2.2.1.

3. Why was magnetic stirring chosen over other extraction methods, such as microwave-assisted extraction? Have you compared the efficiency or selectivity of magnetic stirring to this technique?

• Magnetic stirring was selected for its simplicity, cost-effectiveness, and ease of scale-up.
• We discuss prospects for future comparisons with green methods (ultrasound, microwave, PLE, PEF) in the Conclusion.

4. Were any toxicity or cytotoxicity tests performed or referenced for the kernel oil or polyphenol-rich extract?

• No cytotoxicity tests were conducted; we now acknowledge this as a limitation and propose it for future work (see Conclusion).

5. Could the authors elaborate on any limitations regarding scalability or potential interactions when used as additives?

• The stirring method can be readily scaled; however, ultrasonication or microwave may offer efficiency gains.
• Cytotoxicity of polyphenol extracts and the intense resinous aroma of the kernel oil may limit direct food applications.
• These points are now detailed in Section 3.6.1 and the Conclusion.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is well presented and can be accepted after some revisions. Here are some suggestions.

1. The innovations and advantages of this manuscript should be further described in the Abstract, Introduction and Conclusion section. Please improve.
2. Extraction method should be briefly described in the Introduction section.
3. For Figure 5, how were the 11 compounds determined? If they were determined by HPLC-MS, please provide some MS evidence in the Supplementary Information. If they were determined by standard samples, please provide HPLC of standard samples and prove them.
4. Please make a comparison of this method with other method of extraction  of berry kernel oil compounds as a subsection.

Author Response

This manuscript is well presented and can be accepted after some revisions. Here are some suggestions.

We would like to acknowledge the reviewer for the thorough examination of our manuscript in order to increase its readability.

1. The innovations and advantages of this manuscript should be further described in the Abstract, Introduction and Conclusion section. Please improve.

We have enriched these sections by clearly stating our methodological novelty (Box-Behnken optimization of stirring extraction) and the valorization of both extract and kernel oil for functional applications.

2. Extraction method should be briefly described in the Introduction section.

A concise description of our stirring-based extraction has been added to the Introduction, citing a foundational work on homogeneous magnetic stirring for plant polyphenols.

3. For Figure 5, how were the 11 compounds determined? If they were determined by HPLC-MS, please provide some MS evidence in the Supplementary Information. If they were determined by standard samples, please provide HPLC of standard samples and prove them.

We clarified that compounds were identified via HPLC-DAD against authentic standards (λmax, retention time), with calibration curves (R² > 0.996), LOD/LOQ data in Table A1, and UV-visible spectral characteristics (λmax) in Figure S1.

4. Please make a comparison of this method with other method of extraction of berry kernel oil compounds as a subsection.

We have strengthened the discussion in Section 3.6.1 rather than introducing a separate subsection to maintain focus on our study’s objectives.

Reviewer 3 Report

Comments and Suggestions for Authors

This paper is potentially interesting but there are some issues that should be carefully addressed by authors before making the paper suitable for publication in the Separations.

Specific comments and suggestions are given below.

Title: ‘’Holistic Valorization’’ is a rather undefined term. I suggest replacing with ‘’Valorization of Bioactive Potential’’.

Line 17: Please add tested parameters and delete ‘’magnetic stirring’’.

Line 21: How many phenolics were identified in berries and kernel oil?

I suggest to add a photo of both plant and berries.

Lines 83-84: Please move to the Introduction.

Line 92: How did you store samples until analysis? Please add.

Line 95: How did you isolate seeds? Please describe.

Line 100: In the experimental part it is not clear what part of plant authors used to made extract. What did you mean by ‘’defatted grounded powder’’? Of what?

Line 108: What method? This part is unclear.

Line 109: Please explain ‘’Stirring Extraction (STE) process’’.

Line 109: ‘’targeting the solid residue of J. communis L. after berry oil extraction’’. This part is confusing. Please explain better which methods you applied to which samples.

Line 152: How did you prepare samples for HPLC analysis? What was the volume of the extract injected?

Line 157: SPME procedure is poorly described. How did you prepare samples? Incubation and extraction temperature and time are missing. What was the thickness of the finer coating? What was the volume of the headspace vial?

Line 171: Since authors analysed different type of samples in previous study, at least brief description is needed. The same comment is also for other spectrophotometric assays.

In tables and figures, authors should indicate that the results refer to fruit extracts.

Author Response

This paper is potentially interesting but there are some issues that should be carefully addressed by authors before making the paper suitable for publication in the Separations.

We would like to thank the reviewer for the thorough examination of our manuscript.

Specific comments and suggestions are given below.

Title: ‘’Holistic Valorization’’ is a rather undefined term. I suggest replacing with ‘’Valorization of Bioactive Potential’’.

The title has been changed.

Line 17: Please add tested parameters and delete ‘’magnetic stirring’’.

We rephrased to list solvent ratio, temperature, and time explicitly; magnetic stirring is detailed in the Methods.

Line 21: How many phenolics were identified in berries and kernel oil?

We clarified 5.41 mg/g for berry polyphenols in the Abstract; kernel oil phenolics were not quantified.

I suggest to add a photo of both plant and berries.

Figure 1 now shows berries before and after grinding.

Lines 83-84: Please move to the Introduction.

This sentence has been moved to the Introduction section.

Line 92: How did you store samples until analysis? Please add.

We deep-freeze defatted berry powder at –30 °C until further analysis. Berry kernel oil was immediately analyzed to prevent possible oxidation phenomena.

Line 95: How did you isolate seeds? Please describe.

We describe the defatting pretreatment in detail, as suggested by the reviewer.

Line 100: In the experimental part it is not clear what part of plant authors used to made extract. What did you mean by ‘’defatted grounded powder’’? Of what?

We have revised subsection 2.2.1. which describes the process in detail.

Line 108: What method? This part is unclear.

We have revised the specific sentence to provide clarity on the extraction optimization process.

Line 109: Please explain ‘’Stirring Extraction (STE) process’’.

Added maceration at controlled temperature with magnetic stirring description.

Line 109: ‘’targeting the solid residue of J. communis L. after berry oil extraction’’. This part is confusing. Please explain better which methods you applied to which samples.

We rephrased this sentence as suggested by the reviewer. We specify that the bioactive compound and antioxidant capacity assays were used to evaluate the capabilities of defatted berry powder. Berry kernel oil was separately analyzed from berry powder.

Line 152: How did you prepare samples for HPLC analysis? What was the volume of the extract injected?

We have revised subsection 2.5.1. which now explains the pretreatment of samples and HPLC analysis in more detail.

Line 157: SPME procedure is poorly described. How did you prepare samples? Incubation and extraction temperature and time are missing. What was the thickness of the finer coating? What was the volume of the headspace vial?

Detailed information about the SPME procedure and its finer coating have been provided in subsections 2.4. and 2.6.4.

Line 171: Since authors analysed different type of samples in previous study, at least brief description is needed. The same comment is also for other spectrophotometric assays.

These assays are not interfered by the type of samples; however, a brief description of each assay has been added in the manuscript which sheds more light on the methodology and aligning with the reviewer’s comment.

In tables and figures, authors should indicate that the results refer to fruit extracts.

All labels now specify fruit extract or kernel oil as appropriate.

Reviewer 4 Report

Comments and Suggestions for Authors

The article titled "Holistic Valorization of Juniperus communis L. Berries Extracts Using Box-Behnken Design and Characterization of Berry Kernel Oil Compounds" focuses on the integral valorization of Juniperus communis L. fruits. The study optimized the extraction of bioactive compounds by magnetic stirring and response surface methodology (Box–Behnken) and characterized the volatile and lipid constituents of the oil extracted from the kernel. The ideal extraction conditions (55% ethanol, 80°C, 30 min) provided a high polyphenol content (53.11 mg/g). The extracts showed high antioxidant capacity, and the oil revealed a high oleic acid content (58.75%), in addition to a complex terpene profile. The authors conclude that both the extracts and the oil have potential for use as natural preservatives and functional ingredients in the food and pharmaceutical industries. Regarding clarity and structure, the abstract is clear, concise (<300 words), self-explanatory, and covers objectives, methods, main results, and conclusions—without references. The article contextualizes the medicinal value of J. communis well and justifies the need to explore its seeds, not just the fruits.

The description of sample collection and preparation is adequate and reproducible.

In terms of statistical methodology, the use of the Box-Behnken Design (BBD) is considered robust, and the response surface methodology (RSM) to optimize extraction is appropriate. Statistical tests include ANOVA with model validation (F-tests, R², RMSE, CV, PRESS)—all well reported (Lines 93-121, 226-232). Models with R² > 0.97 reveal no significant lack of fit (p > 0.05), and adjusted regressions are well described (Table 3, Line 274). The polynomial equations are presented correctly, with justification for removing insignificant terms (p > 0.05) (Lines 282-294), and Pareto plots are well used to visualize the impact of each variable.

In terms of reproducibility, the instrumentation and reagents are fully specified, including brands and models (Lines 140-167), and replicates are available. Normality testing (Kolmogorov–Smirnov), and the use of ANOVA + Tukey-HSD are solid practices.

Despite the good methodological designs, some methodological detail was expected for the analyses using the TPC, AAC, FRAP, and DPPH methods, and some conclusions lack support, as outlined below. #1_Lines 509–520 (Conclusions): They state that the extracts and oil “can replace synthetic preservatives” and that there is “therapeutic potential” for the compounds, but no in vivo bioactivity tests were performed, nor were any comparisons with synthetic preservatives. As a suggestion to the authors, we suggest that the conclusions be reformulated to better reflect the in vitro data and highlight that this is preliminary evidence.

#2_Lines 171 and 185: Lack of methodological detail in some analyses. The methods for TPC, AAC, FRAP, and DPPH are described as “previously discussed” (ref. [16]), but are not reproduced here. To ensure full reproducibility, briefly describe the main steps (volumes, times, concentrations) or move the description of ref. [16] to the Appendix.

#3_Lines 390–392: Limitation in the identification of phenolic compounds. The authors themselves acknowledge the failure to identify other polyphenols as a limitation. This point should be included in the Discussion section as a clear limitation of the work and justified based on the instrumental methods used.

#4_ Lines 285–294: Results inflated by extrapolation. The polynomial equation and RSM plots extrapolate to values outside the original experimental range (e.g., 55% solvent, 80°C temperature, 30 min time), even with only three central points. Although the fit is statistically sound, direct experimental validation under these optimized conditions should be reported separately, with the associated error—which is only done in Table 6, but could have come earlier or been explicitly stated in the Discussion.

#5_ Lines 323–324 (Pareto): Slightly biased statistical interpretation. They state that the parameter X3 (time) had “no impact,” but in Table 3, X3 showed an influence on DPPH (β = -17.6), even though p > 0.05. It is recommended to state that "X3 had a minor and non-statistically significant impact (p > 0.05)" rather than generalizing its irrelevance.

The manuscript presents a technically sound study, with good application of RSM, well-conducted statistics, and sufficient description of most methods. However, there are some flaws in logical reasoning in the discussion and conclusions, with extrapolations that are not directly supported by in vivo tests or efficacy comparisons with synthetic compounds. With adjustments to these points and greater interpretative caution, the manuscript has scientific merit and is close to being publishable.

Author Response

The article titled "Holistic Valorization of Juniperus communis L. Berries Extracts Using Box-Behnken Design and Characterization of Berry Kernel Oil Compounds" focuses on the integral valorization of Juniperus communis L. fruits. The study optimized the extraction of bioactive compounds by magnetic stirring and response surface methodology (Box–Behnken) and characterized the volatile and lipid constituents of the oil extracted from the kernel. The ideal extraction conditions (55% ethanol, 80°C, 30 min) provided a high polyphenol content (53.11 mg/g). The extracts showed high antioxidant capacity, and the oil revealed a high oleic acid content (58.75%), in addition to a complex terpene profile. The authors conclude that both the extracts and the oil have potential for use as natural preservatives and functional ingredients in the food and pharmaceutical industries. Regarding clarity and structure, the abstract is clear, concise (<300 words), self-explanatory, and covers objectives, methods, main results, and conclusions—without references. The article contextualizes the medicinal value of J. communis well and justifies the need to explore its seeds, not just the fruits.

The description of sample collection and preparation is adequate and reproducible.

In terms of statistical methodology, the use of the Box-Behnken Design (BBD) is considered robust, and the response surface methodology (RSM) to optimize extraction is appropriate. Statistical tests include ANOVA with model validation (F-tests, R², RMSE, CV, PRESS)—all well reported (Lines 93-121, 226-232). Models with R² > 0.97 reveal no significant lack of fit (p > 0.05), and adjusted regressions are well described (Table 3, Line 274). The polynomial equations are presented correctly, with justification for removing insignificant terms (p > 0.05) (Lines 282-294), and Pareto plots are well used to visualize the impact of each variable.

In terms of reproducibility, the instrumentation and reagents are fully specified, including brands and models (Lines 140-167), and replicates are available. Normality testing (Kolmogorov–Smirnov), and the use of ANOVA + Tukey-HSD are solid practices.

Despite the good methodological designs, some methodological detail was expected for the analyses using the TPC, AAC, FRAP, and DPPH methods, and some conclusions lack support, as outlined below.

We would like to sincerely thank the reviewer for his detailed feedback and positive observations on our manuscript.

#1_Lines 509–520 (Conclusions): They state that the extracts and oil “can replace synthetic preservatives” and that there is “therapeutic potential” for the compounds, but no in vivo bioactivity tests were performed, nor were any comparisons with synthetic preservatives. As a suggestion to the authors, we suggest that the conclusions be reformulated to better reflect the in vitro data and highlight that this is preliminary evidence.

We have tempered our Conclusions to reflect that these are in vitro results only and removed overreaching claims.

#2_Lines 171 and 185: Lack of methodological detail in some analyses. The methods for TPC, AAC, FRAP, and DPPH are described as “previously discussed” (ref. [16]), but are not reproduced here. To ensure full reproducibility, briefly describe the main steps (volumes, times, concentrations) or move the description of ref. [16] to the Appendix.

Detailed steps for each assay are now in the relevant Methods subsections.

#3_Lines 390–392: Limitation in the identification of phenolic compounds. The authors themselves acknowledge the failure to identify other polyphenols as a limitation. This point should be included in the Discussion section as a clear limitation of the work and justified based on the instrumental methods used.

We now discuss this in the Discussion, noting that additional MS-based analyses in future work would refine our polyphenol profile.

#4_ Lines 285–294: Results inflated by extrapolation. The polynomial equation and RSM plots extrapolate to values outside the original experimental range (e.g., 55% solvent, 80°C temperature, 30 min time), even with only three central points. Although the fit is statistically sound, direct experimental validation under these optimized conditions should be reported separately, with the associated error—which is only done in Table 6, but could have come earlier or been explicitly stated in the Discussion.

Added paragraph in Discussion with three confirmatory extractions at 55% EtOH, 80 °C, 30 min demonstrating close agreement between predicted and observed values (Table 6).

#5_ Lines 323–324 (Pareto): Slightly biased statistical interpretation. They state that the parameter X3 (time) had “no impact,” but in Table 3, X3 showed an influence on DPPH (β = -17.6), even though p > 0.05. It is recommended to state that "X3 had a minor and non-statistically significant impact (p > 0.05)" rather than generalizing its irrelevance.

We have corrected the language to state that X₃ exhibited a “minor and non-statistically significant effect (p > 0.05).”

The manuscript presents a technically sound study, with good application of RSM, well-conducted statistics, and sufficient description of most methods. However, there are some flaws in logical reasoning in the discussion and conclusions, with extrapolations that are not directly supported by in vivo tests or efficacy comparisons with synthetic compounds. With adjustments to these points and greater interpretative caution, the manuscript has scientific merit and is close to being publishable.

We have adjusted the manuscript to align with the reviewer’s request to increase its readability.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

All the recommendations and issues raised have been answered and amended accordingly.

Minor comments:

Line 18: Authors have not listed extraction parameters such as solvent ratio, temperature, and time.

Line 22: Please add number of detected phenolics.

Author Response

All the recommendations and issues raised have been answered and amended accordingly.

We thank the reviewer for the constructive comments.

Minor comments:

Line 18: Authors have not listed extraction parameters such as solvent ratio, temperature, and time.

We have added the key extraction parameters (solid-to-solvent ratio, ethanol concentration, temperature, and time) directly into the Abstract.

Line 22: Please add number of detected phenolics.

We have now specified that 11 individual polyphenols were detected and quantified.