The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript is generally well written and structured. It provides valuable insights into black pepper ethanolic extracts and their antioxidant properties. However, certain methodological aspects (e.g., statistical analysis, ultrasound temperature) and presentation choices (e.g., figure formatting, full terminology in tables) need refinement. Additionally, inconsistencies in data correlations require clarification. Please see detailed comments in document attached.
Comments for author File: Comments.pdf
Author Response
Title: The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts (Manuscript ID: horticulturae-3377156)
Response to Reviewer 1 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes and some highlighted in the re-submitted files. [This is only a recommended summary. Please feel free to adjust it. We do suggest maintaining a neutral tone and thanking the reviewers for their contribution although the comments may be negative or off-target. If you disagree with the reviewer's comments please include any concerns you may have in the letter to the Academic Editor.]
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
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Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
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Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
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Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: Tables: Ensure that the full name for Total Extractive Matter (TEM) is used throughout titles in all tables instead of abbreviations. This will enhance clarity for readers.
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Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have changed it in the tables captions and the changes are saved in track changes
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Comments 2: Statistical Analysis: The manuscript does not include any statistical analysis, such as a oneway ANOVA, to compare the results across different extraction methods. Incorporating |
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Response 2: We appreciate the suggestion to incorporate statistical analyses, such as one-way ANOVA, to compare results across different extraction methods. However, we have conducted a statistical comparison of the TEM yields using one-way ANOVA and post-hoc tests, which allowed us to identify significant differences between the tested conditions. These results are reflected in the tables, where statistical groups (e.g., a, b, c) have been included for clarity. |
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Comments 3: Figure 1: While the decision to present extraction methods in separate graphs (due to differences in extraction times) is understandable, combining them into a single graph with clear markers for each method could make comparisons easier and more intuitive for readers. |
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Response 3: We appreciate this suggestion, but we think there would not be any major difference, so we will leave it as is. |
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Comments 4: Ultrasound Extraction Temperature: It is unclear if the temperature during ultrasound extraction was measured. Since ultrasonic waves can cause significant heating, monitoring and reporting the temperature during the extraction process is critical to ensure reproducibility and to account for any effects that heat might have on extraction efficiency and compound stability. This aspect should be addressed and discussed in the methods and results sections. |
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Response 4: Thank you for this observation. The temperature was carefully controlled throughout the ultrasonic extraction process, which was conducted in 15-minute intervals, with a 5-minute pause between each interval, for a total duration of up to 60 minutes. |
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Comments 5: Correlation: The results show a weak or inconsistent correlation between phenolic/flavonoid content and antioxidant activity in some assays. This needs further analysis or acknowledgment. For example, include a brief discussion of how non-phenolic compounds |
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Response 5: We appreciate your suggestion, but we would like to point out that this aspect has already been addressed in the manuscript. Specifically, for each antioxidant assay, we have discussed whether the observed activity correlates with the phenolic and flavonoid content or if it might depend on other compounds present in the extracts. These explanations are provided in detail and highlighted: Page [15], Lines [531–534], [558–563] Page [16], Lines [564–569], [574–583], [583–593] |
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4. Response to Comments on the Quality of English Language |
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Point 1:/ |
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Response 1: / |
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5. Additional clarifications |
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Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors
The similarity report is high; for your manuscript to be considered for publication, you need to make an effort to reduce it to 15% or less and change majors in your writing style.
I also take the liberty of making the following other observations.
Mentioning plant material is incorrect, as they only work with one tissue type.
Using an alternative expression to solvemodule, is confusing.
And statistical analysis? For comparing treatments before using the model mathematics.
Use current references.
Line 26: Include how the particle size was determined and whether other sizes were obtained.
Line 115: write down what the boiling point is.
Lines 130-136 and lines 112-120 are recurring. Why estimate the optimal concentration if you first extracted with a 70% ethanol ratio?
Line 142: por que realizar la extracción durante 120 minutos, mientras en las líneas 112-120 utilizaron 360 minutos.
Line 151: Now, 180 minutes, the extraction time can be another variable to evaluate. How can we ensure that the time extraction does not affect the process extraction?
Line 155: it should have done this for each type of extraction.
Line 189: must mention the equipment specifications
Author Response
Title: The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts (Manuscript ID: horticulturae-3377156)
Response to Reviewer 2 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: The similarity report is high; for your manuscript to be considered for publication, you need to make an effort to reduce it to 15% or less and change majors in your writing style. |
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Response 1: Thanks for this comment, but we would then be grateful if you could point out to us which parts of the text we should pay attention to, so that the text itself does not lose its meaning. |
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Comments 2: Mentioning plant material is incorrect, as they only work with one tissue type. |
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Response 2: We have indicated the place of purchase of the plant material, as well as the country of origin. It is also mentioned how the material was stored and processed before the analysis began, so it is not clear to us what you mean when you say that the mention is incorrect. |
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Comments 3: Using an alternative expression to solvemodule, is confusing. |
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Response 3: The term "solvomodule" was clarified immediately after it was first mentioned, and is used in scientific papers, so we think there is no need to change it in this paper. |
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Comments 4: And statistical analysis? For comparing treatments before using the model mathematics. |
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Response 4: Thanks for this suggestion. We have added the statistical analysis, page[6] which was applied at the beginning of the manuscript (tables 1 and 2). When it comes to antioxidant tests, they all have different mechanisms although the comparison of activities is done based on the same parameters such as EC50 values. So ANOVA in this case would not be reliable for all tests, only if it were used for each test individually. |
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Comments 5: Line 26: Include how the particle size was determined and whether other sizes were obtained. |
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Response 5: This part was clarified in the subtitle “2.1. Plant material”, because of the word limit in the abstract. The mean particle diameter (0.258 mm) of the grounded plant material was determined by the sieve analysis. For granulometric analysis, a set of laboratory sieves with a mechanical shaker (LABDEX LTD., UK) were used. The plant material was placed in a thin layer on the first sieve with the largest mesh size (1.32 mm) and covered with a lid. Below this, a series of sieves with progressively smaller mesh sizes (1 mm, 0.8 mm, 0.63 mm, 0.5 mm, 0.4 mm, and 0.1 mm) were arranged, with a collection pan at the bottom. The shaker was activated, and the sieving process was set for 15 minutes. After sieving, the mass of each fraction was measured, the mass fractions were calculated, and the mean particle diameter was determined by equation: inserted new equation (1) Old (1) became (2) Old (2)à new(3) Old (3)à new (4) Old (4)à new (5) inserted new equation (6) Old (5)à new (7) Old (6)à new (8) Old (7)à new (9) |
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Comments 6: Line 115: write down what the boiling point is. |
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Response 6: In the experiment, the term boiling point refers specifically to the temperature at which the mixture of the plant material and the solvent begins to boil. This value does not represent the boiling point of the pure solvent but rather the boiling behavior of the mixture under the experimental conditions. It should be emphasized that the boiling point was not measured continuously during the process but was determined only as the temperature at which boiling commenced. |
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Comments 7: Lines 130-136 and lines 112-120 are recurring. Why estimate the optimal concentration if you first extracted with a 70% ethanol ratio? Line 142: por que realizar la extracción durante 120 minutos, mientras en las líneas 112-120 utilizaron 360 minutos. Line 151: Now, 180 minutes, the extraction time can be another variable to evaluate. How can we ensure that the time extraction does not affect the process extraction? |
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Response 7: The section entitled "2.3. Determining of total extractive matter content (TEM) in the plant material" refers to the determination of the maximum total extractable matter (qo) content present in the plant material. This parameter is fundamental for the mathematical modeling of the extraction process and, as such, is introduced at the beginning of the manuscript after the description of the origin of the plant material.
In contrast, the discussion related to ethanol concentration explores the optimization of extraction conditions. Specifically, it was examined which of the given ethanol concentrations at a solvomodule of 1:15 m/V and an extraction time of 120 minutes gives the highest yield of total extractive matter. (Table 1). After the ethanol concentration was determined, the effect of the solvomodule was also analyzed, also for an extraction time of 120 minutes, after which 1:10 m/V was adopted as the optimal solvomodule. (Table 2). Although there may seem to be an overlap in content, these sections serve different purposes: the first provides basic data for modeling the process, while the second focuses on experimental optimization to achieve maximum extraction efficiency.
Additional extraction time (line 151) The mention of an extraction time of 180 minutes in "The influence of temperature and time" reflects additional research under previously optimized conditions (70% ethanol V/V and 1:10 m/V). The purpose was to investigate whether increasing the extraction time beyond 120 minutes significantly increases the yield or whether the process reaches a plateau. The results, summarized in Table 3, show how extended extraction times affect the total extractive matter. |
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Comments 8: Line 155: it should have done this for each type of extraction. |
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Response 8: Extraction kinetics of TEM were monitored at specified periods (5–180 minutes) for maceration (25°C), reflux extraction (40°C, 50°C and boiling point), (5-240 min) for Soxhlet extraction and (5-60 min) for ultrasonic extraction (see the figures 2 and 3). |
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Comments 9: Line 189: must mention the equipment specifications |
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Response 9: Added in the text “Cole Parmer Spectrophotometer”. Page[5] |
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4. Response to Comments on the Quality of English Language |
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Point 1:/ |
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Response 1: / |
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5. Additional clarifications |
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article ‘The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts’ is interesting although there are many studies in the literature. The form of the article is good, but it needs to be supplemented with a few refinements to be considered for publication in this journal. I will pass on some of my suggestions to the authors:
Some doubts such as the reliability of considering only 2mL to determine total extractive matter. Considering 250 mL of solvent and 25 g of sample, 2 mL of extract is not representative of the sample. During maceration, was the temperature monitored and maintained at 25°C for 120 min?
Explain more about the solvomodule;
Statistical analysis to verify the best TEM yield?
To determine the extraction kinetics, were several extractions conducted for each time considered?
The reflux (up to 180 min) and Soxhlet (240 min) methods are obsolete and are too expensive economically, energetically and in terms of time considering the sustainability of the research;
Gallic no galic; rutin no routine;
Implement the description of DPPH and ABTS and define how the results were expressed;
Arrange table 1 as well as table 2
Insert statistical analysis between samples; insert multivariate statistical analysis to check the effect of different variables (extraction method, time etc.).
The results should be commented and described better. Comments are very small and without explanation and discussion;
Do not state in the header of the table (Tab 7 +- sd).Insert in materials and methods the explanation of the replications conducted and that the data are reported as mean + ds;
Improve the conclusion;
There are too many self-citations;
Revise English form (many mistakes and form) (the results was expressed as...);
Comments on the Quality of English LanguageThe English form must be revised
Author Response
Title: The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts (Manuscript ID: horticulturae-3377156)
Response to Reviewer 3 Comments
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1. Summary |
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Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections in track changes in the re-submitted files.
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2. Questions for General Evaluation |
Reviewer’s Evaluation |
Response and Revisions |
Does the introduction provide sufficient background and include all relevant references? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are all the cited references relevant to the research? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Is the research design appropriate? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the methods adequately described? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the results clearly presented? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
Are the conclusions supported by the results? |
Yes/Can be improved/Must be improved/Not applicable |
/ |
3. Point-by-point response to Comments and Suggestions for Authors |
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Comments 1: Some doubts such as the reliability of considering only 2mL to determine total extractive matter. Considering 250 mL of solvent and 25 g of sample, 2 mL of extract is not representative of the sample. |
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Response 1: We appreciate your observation regarding the reliability of considering only 2 mL of extract to determine the total extractive matter. However, we would like to clarify that the extract obtained during our procedure is homogenous due to the thorough mixing and extraction process applied. The 2 mL aliquot accurately reflects the composition of the total extract, ensuring the reliability and reproducibility of the results. |
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Comments 2: During maceration, was the temperature monitored and maintained at 25°C for 120 min? |
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Response 2: Yes, the temperature was monitored and maintained at 25°C throughout the entire extraction process. |
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Comments 3: Explain more about the solvomodule; |
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Response 3: Thank you for your comment regarding the discussion of solvomodule, and if you believe this aspect requires further elaboration, we kindly ask for guidance on where and how to expand this discussion. |
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Comments 4: And statistical analysis? For comparing treatments before using the model mathematics. |
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Response 4: Thanks for this suggestion. We have added the statistical analysis, page[6] which was applied at the beginning of the manuscript (tables 1 and 2). When it comes to antioxidant tests, they all have different mechanisms although the comparison of activities is done based on the same parameters such as EC50 values. So ANOVA in this case would not be reliable for all tests, only if it were used for each test individually. |
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Comments 5: To determine the extraction kinetics, were several extractions conducted for each time considered? |
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Response 5: The extraction kinetics were monitored for all extraction techniques in triplicate to ensure accuracy and reproducibility. However, the modeling extraction kinetics was performed without incorporating standard deviations. |
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Comments 6: The reflux (up to 180 min) and Soxhlet (240 min) methods are obsolete and are too expensive economically, energetically and in terms of time considering the sustainability of the research; |
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Response 6: The primary factor we monitored and used to select the optimal extraction technique was the yield of total extractive matter. According to the literature, classical extraction techniques often result in higher yields of total extractive matter. However, this does not necessarily imply that these extracts are always of higher quality compared to those obtained using modern extraction techniques. |
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Comments 7: Gallic no galic; rutin no routine; |
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Response 7: Thank you. We corrected this throught the text in track changes. |
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Comments 8: Implement the description of DPPH and ABTS and define how the results were expressed; |
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Response 8: We have revised and expanded this section of the methods for determining antioxidant activity, specifically describing the DPPH and ABTS assays and defining how the results were expressed. |
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Comments 9: Arrange table 1 as well as table 2 Insert statistical analysis between samples; insert multivariate statistical analysis to check the effect of different variables (extraction method, time etc.). |
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Response 9: We would like to clarify that classical optimization of extraction conditions was not conducted in this study. Instead, the focus was on evaluating the TEM yield under various experimental conditions. As such, multivariate analysis would not be applicable in this case due to the absence of systematically designed experiments for optimization.
However, we have conducted a statistical comparison of the TEM yields using one-way ANOVA and post-hoc tests, which allowed us to identify significant differences between the tested conditions. These results are reflected in the tables, where statistical groups (e.g., a, b, c) have been included for clarity. |
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Comments 10: The results should be commented and described better. Comments are very small and without explanation and discussion. |
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Response 10: Thank you for your feedback regarding the comments on the results. The core findings are briefly outlined in the "Results" section, while detailed explanations, interpretations, and comparisons with existing literature are thoroughly addressed in the "Discussion" section. |
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Comments 11: Do not state in the header of the table (Tab 7 +- sd).Insert in materials and methods the explanation of the replications conducted and that the data are reported as mean + ds; |
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Response 11: Thank you for your comment. This suggestion has been accepted, and the changes have been implemented accordingly. |
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Comments 12: Improve the conclusion
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Response 12: This has been accepted and conclusion is corrected in track changes.
“Applied extraction techniques have an impact on TEM yield, kinetics and activity. The extract obtained by Soxhlet extraction yielded the highest total extractive matter (TEM), due to its continuous solvent circulation. Ultrasonic extraction significantly reduced time by disrupting cell walls and enhancing mass transfer. The application of kinetic extraction models as a novelty when it comes to ethanolic extracts of black pepper was significant for this research. The extract obtained by reflux extraction at boiling point temperature showed the highest antioxidant activity according to all applied tests, except for the reducing power method, where the extract obtained by ultrasonic extraction showed the higher activity. The correlation between phenolic and flavonoid content and antioxidant activity was presented for each test, leading to the conclusion that the antioxidant activity of BPFEEs does not only originate from these compounds but is also a result of synergistic effects with other extracted components.rTherefore, future analyzes will focus on determining the chemical composition of BPFEEs, in order to better understand the antioxidant activity of the obtained extracts.“ |
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Comments 13: There are too many self-citations; |
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Response 13: We appreciate this observation. The self-citations reflect our team's extensive research in this field and are included to provide relevant context and build upon our prior findings. We will ensure all references are balanced and justified in their relevance to the study. |
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4. Response to Comments on the Quality of English Language |
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Point 1: Revise English form (many mistakes and form) (the results was expressed as...); |
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Response 1: This suggestion has been accepted, and we have made efforts to address the issues and correct the errors accordingly. |
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5. Additional clarifications |
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
Comments 1: The similarity report is high; for your manuscript to be considered for publication, you need to make an effort to reduce it to 15% or less and change majors in your writing style. |
Response 1: Thanks for this comment, but we would then be grateful if you could point out to us which parts of the text we should pay attention to, so that the text itself does not lose its meaning. Comments 1b: Attached is the document with the similarity report. 15% or less is satisfactory. |
Comments 2: Mentioning plant material is incorrect, as they only work with one tissue type. |
Response 2: We have indicated the place of purchase of the plant material, as well as the country of origin. It is also mentioned how the material was stored and processed before the analysis began, so it is not clear to us what you mean when you say that the mention is incorrect. Comments 2b:Plant material can include roots, stems, leaves, flowers, fruits, and seeds. This plant material can be obtained in various forms such as fresh, dehydrated, or powdered. |
Comments 3: Using an alternative expression to solvemodule, is confusing. |
Response 3: The term "solvomodule" was clarified immediately after it was first mentioned, and is used in scientific papers, so we think there is no need to change it in this paper. Comments 3b: ok. |
Comments 4: And statistical analysis? For comparing treatments before using the model mathematics. |
Response 4: Thanks for this suggestion. We have added the statistical analysis, page[6] which was applied at the beginning of the manuscript (tables 1 and 2). When it comes to antioxidant tests, they all have different mechanisms although the comparison of activities is done based on the same parameters such as EC50 values. So ANOVA in this case would not be reliable for all tests, only if it were used for each test individually. Comments 4b: Even so, there is a descriptive statistic to consider. |
Comments 5: Line 26: Include how the particle size was determined and whether other sizes were obtained. |
Response 5: This part was clarified in the subtitle “2.1. Plant material”, because of the word limit in the abstract. The mean particle diameter (0.258 mm) of the grounded plant material was determined by the sieve analysis. For granulometric analysis, a set of laboratory sieves with a mechanical shaker (LABDEX LTD., UK) were used. The plant material was placed in a thin layer on the first sieve with the largest mesh size (1.32 mm) and covered with a lid. Below this, a series of sieves with progressively smaller mesh sizes (1 mm, 0.8 mm, 0.63 mm, 0.5 mm, 0.4 mm, and 0.1 mm) were arranged, with a collection pan at the bottom. The shaker was activated, and the sieving process was set for 15 minutes. After sieving, the mass of each fraction was measured, the mass fractions were calculated, and the mean particle diameter was determined by equation: Comments 5b: ok |
Comments 6: Line 115: write down what the boiling point is. |
Response 6: In the experiment, the term boiling point refers specifically to the temperature at which the mixture of the plant material and the solvent begins to boil. This value does not represent the boiling point of the pure solvent but rather the boiling behavior of the mixture under the experimental conditions. It should be emphasized that the boiling point was not measured continuously during the process but was determined only as the temperature at which boiling commenced. Comments 6b: then this initial temperature must be indicated |
Comments 7: Lines 130-136 and lines 112-120 are recurring. Why estimate the optimal concentration if you first extracted with a 70% ethanol ratio? Line 151: Now, 180 minutes, the extraction time can be another variable to evaluate. How can we ensure that the time extraction does not affect the process extraction? |
Response 7: The section entitled "2.3. Determining of total extractive matter content (TEM) in the plant material" refers to the determination of the maximum total extractable matter (qo) content present in the plant material. This parameter is fundamental for the mathematical modeling of the extraction process and, as such, is introduced at the beginning of the manuscript after the description of the origin of the plant material. In contrast, the discussion related to ethanol concentration explores the optimization of extraction conditions. Specifically, it was examined which of the given ethanol concentrations at a solvomodule of 1:15 m/V and an extraction time of 120 minutes gives the highest yield of total extractive matter. (Table 1). After the ethanol concentration was determined, the effect of the solvomodule was also analyzed, also for an extraction time of 120 minutes, after which 1:10 m/V was adopted as the optimal solvomodule. (Table 2). Although there may seem to be an overlap in content, these sections serve different purposes: the first provides basic data for modeling the process, while the second focuses on experimental optimization to achieve maximum extraction efficiency.
Additional extraction time (line 151) The mention of an extraction time of 180 minutes in "The influence of temperature and time" reflects additional research under previously optimized conditions (70% ethanol V/V and 1:10 m/V). The purpose was to investigate whether increasing the extraction time beyond 120 minutes significantly increases the yield or whether the process reaches a plateau. The results, summarized in Table 3, show how extended extraction times affect the total extractive matter. Comments 7b: ok |
Comments 8: Line 155: it should have done this for each type of extraction. |
Response 8: Extraction kinetics of TEM were monitored at specified periods (5–180 minutes) for maceration (25°C), reflux extraction (40°C, 50°C and boiling point), (5-240 min) for Soxhlet extraction and (5-60 min) for ultrasonic extraction (see the figures 2 and 3). Comments 8b: ok |
Comments 9: Line 189: must mention the equipment specifications |
Comments 9b: ok
Comments for author File: Comments.pdf
Author Response
Comments 1: The similarity report is high; for your manuscript to be considered for publication, you need to make an effort to reduce it to 15% or less and change majors in your writing style. |
Response 1: Thanks for this comment, but we would then be grateful if you could point out to us which parts of the text we should pay attention to, so that the text itself does not lose its meaning. Comments 1b: Attached is the document with the similarity report. 15% or less is satisfactory. |
Response 1b: We have made efforts to reduce the similarity index by revising portions of the manuscript. The updated version of the manuscript has been uploaded, and the modified sections have been highlighted for clarity and need to accept changes in track changes. We would like to emphasize that certain sentences and phrases recognized as similar cannot be changed, such as descriptions of equipment, the source and description of plant material (as this material has already been used in other published studies), or standard, unaltered procedures in, for example, antioxidant tests. Significant changes to these expressions and phrases could distort the true meaning of the sentences.
Comments 2: Mentioning plant material is incorrect, as they only work with one tissue type. |
Response 2: We have indicated the place of purchase of the plant material, as well as the country of origin. It is also mentioned how the material was stored and processed before the analysis began, so it is not clear to us what you mean when you say that the mention is incorrect. Comments 2b:Plant material can include roots, stems, leaves, flowers, fruits, and seeds. This plant material can be obtained in various forms such as fresh, dehydrated, or powdered. Response 2b: Our study specifically focused on the fruit of black pepper (Piper nigrum) due to its bioactive compounds, such as piperine, which are key to its pharmacological and antioxidant properties. Additionally, black pepper is not cultivated in our country (Serbia, Europe) and is predominantly imported in the form of dried fruits, making this material the most practical and relevant choice for our analysis. This clarification has been added to the manuscript (section „Plant material“), and changes are highlighted. |
Comments 4: And statistical analysis? For comparing treatments before using the model mathematics. |
Response 4: Thanks for this suggestion. We have added the statistical analysis, page[6] which was applied at the beginning of the manuscript (tables 1 and 2). When it comes to antioxidant tests, they all have different mechanisms although the comparison of activities is done based on the same parameters such as EC50 values. So ANOVA in this case would not be reliable for all tests, only if it were used for each test individually. Comments 4b: Even so, there is a descriptive statistic to consider. |
Response 4b: All our results are already presented as mean values with standard deviations (mean ± SD) based on three independent experimental repetitions. This approach provides a clear representation of the central tendency and variability of our data. We hope this adequately addresses your concerns and we remain open to further suggestions.
Comments 6: Line 115: write down what the boiling point is. |
Response 6: In the experiment, the term boiling point refers specifically to the temperature at which the mixture of the plant material and the solvent begins to boil. This value does not represent the boiling point of the pure solvent but rather the boiling behavior of the mixture under the experimental conditions. It should be emphasized that the boiling point was not measured continuously during the process but was determined only as the temperature at which boiling commenced. Comments 6b: then this initial temperature must be indicated |
Response 6b: The experiments were repeated, and the boiling point of the mixtures in the flask was measured. The temperature at which boiling commenced for the mixture of the plant material (reflux extraction) and the 70% ethanol solvent was observed to be 80.2°C under the experimental conditions. For the Soxhlet extraction this temeprature was (79.4°C).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have improved the manuscript and it can be considered for publication. I recommend revising the English form, which can be improved (e.g. The results were expressed and many other sentences).
Comments on the Quality of English LanguagePlease revise English form
Author Response
Thank you for all the comments. All changes have been made in track changes in the latest version of the manuscript, where we also focused on improving the English language and reducing the similarity index.
Author Response File: Author Response.pdf