Polyphenolic Profile and Antioxidant Activity of Juglans regia L. Leaves and Husk Extracts
Round 1
Reviewer 1 Report
The subject is interesting, by being focusing on food waste and using advanced techniques, but the description of methodologies and results should be improved. The type of paper should be changed from "Article" to "Short Communication".
In Introduction, the profile of bioactive components of walnuts and related wastes should be better described. Moreover some additional lines on definition of antioxidant properties and update of research approach on antioxidant properties should be inserted with updated references. The advantage of FTIR technique should be inserted.
Major details should be given of number and type of samples and a scheme of sampling procedure should be inserted.
The paragraph 2.2. Preparation of the extracts should be implemented.
The paper should be better organized. Looking at the discussion of results and the conclusion, they don't reflect the aim of paper. In this order, the aim of paper should be rewritten and the discussion of results should be implemented: the results in table 1 should be better described in the text and compared with previous data of literature. After that the authors should explain the effect of different techniques.
In the Conclusion the authors should mark the limits and advantages of this research.
The linguistic revision of whole manuscript should be carried out.
The sentence in lines 21-23 should be clarified.
Check line 65.
Rewrite the sentence at line 71-72.
Rewrite Line 96-97
Lines 195: Specify better that the analysis were carried out on whole sample and extracts.
The sentence in 243-246 should be clarified.
Author Response
Response to Review Comments
Forests
Manuscript ID: forests-623979
Title: Analysis of composition and properties of extracts of walnut (Juglans regia L.) leaves and husk
Article Type: Article
Authors: Anna Masek, Malgorzata Latos-Brozio, Ewa Chrzescijanska, Anna Podsedek
Dear Editor and Reviewers,
We would like to thank the Editor and Reviewers for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Corrections in manuscript have been marked in yellow. Details of our responses to each comment are shown below:
Answers to reviewer #1 comments
Reviewer #1: The subject is interesting, by being focusing on food waste and using advanced techniques, but the description of methodologies and results should be improved. The type of paper should be changed from "Article" to "Short Communication".
Answer: Thank you for your comment. We ask you to leave the manuscript as a form of article due to reorganization of the text. For us it is very important that the form of the article stays, because for our university only this form is suitable.
Reviewer #1: In Introduction, the profile of bioactive components of walnuts and related wastes should be better described. Moreover some additional lines on definition of antioxidant properties and update of research approach on antioxidant properties should be inserted with updated references. The advantage of FTIR technique should be inserted.
Answer: The introduction has been corrected. Supplementary information on substances present in the extract have been added. he advantage of FTIR technique has been inserted.
Reviewer #1: Major details should be given of number and type of samples and a scheme of sampling procedure should be inserted.
Answer: Samples of leaves and green walnut husks of walnut were obtained from trees growing on the farm in central Poland. Plant materials were taken from seven different walnut trees, variety: non-grafted, age 16-years. Material from the seven different walnut trees was collected for testing: leaves and green husks. From the husks and leaves, collected from each of the seven trees, 3 leaves and husks extracts from one tree were prepared. Concentration of all extracts were 50 mg/ml.
Leaves and husks of the walnut were cut into pieces and then ground in a ball mill. The particle size of the shredded plant materials was less than 1mm. Plant materials were extracted using a 5-fold volume of 70% ethanol under continuous mixing conditions (200 RPM, 25°C). The extraction was carried out at 25°C and at dark for 7 days. The final extracts of leaves and husks of the walnut were concentrated to constant weight using a rotary evaporator under reduced pressure conditions at 30°C.
In all considered analysis, the tests were repeated three times on samples of husks and leaf extracts obtained from seven trees. The average results are presented in the manuscript.
Reviewer #1: The paragraph 2.2. Preparation of the extracts should be implemented.
Answer: Preparation of the extracts has been implemented.
Reviewer #1: The paper should be better organized. Looking at the discussion of results and the conclusion, they don't reflect the aim of paper. In this order, the aim of paper should be rewritten and the discussion of results should be implemented: the results in table 1 should be better described in the text and compared with previous data of literature. After that the authors should explain the effect of different techniques.
Answer: The manuscript structure has been improved. Test results were presented and discussed in the following order: characteristics of the thermal stability of raw plant materials (thermal analysis - TGA); analysis of polyphenolic profile of extracts (FTIR and UV-VIS spectroscopy and HPLC chromatography); analysis of properties of extracts (Cyclic and differential pulse voltammetry and ABTS, DPPH, FRAP and CUPRAC methods). Result in table 1 was improved.
Reviewer #1: In the Conclusion the authors should mark the limits and advantages of this research.
Answer: Conclusion was corrected
Reviewer #1: The linguistic revision of whole manuscript should be carried out.
Answer: The linguistic revision has been done.
Reviewer #1: The sentence in lines 21-23 should be clarified.
Answer: The sentence has been corrected: A high affinity for scavenging free radicals ABTS and DPPH was found for natural substances present in leaves and green husks.
Reviewer #1: Check line 65.
Answer: It has been corrected.
Reviewer #1: Rewrite the sentence at line 71-72.
Answer: The sentence has been corrected.
Reviewer #1: Rewrite Line 96-97
Answer: The sentence has been improved.
Reviewer #1: Lines 195: Specify better that the analysis were carried out on whole sample and extracts.
Answer: It has been improved.
Reviewer #1: The sentence in 243-246 should be clarified.
Answer: The sentence has been improved.
Yield of extraction of walnut appears to depend on temperature. Higher temperature reduces the efficiency of extraction.; the higher the temperature, the lower the yield. Juglone degrades in certain solvents and aquatic conditions that include acetonitrile, methanol, acidic solutions, alkaline solutions, and saline water [32, 33], but juglon is stable in acidic conditions.
Reviewer 2 Report
Using a mass detector hyphenated with the HPLC will be useful to characterize more precisely the different phenolic compounds present in the extract (70% are not identified) Using the electrochemical detector is quite interesting to assess the antiox properties of the extract, instead of classical testing. Nevertheless, we have to keep in mind this is only an vitro testing, and therefore not representative of the in vivo antiox properties : is this kind of detector usable to perform measurement on biological material (plasma, urine...)?Author Response
Answers to Reviewer #4 comments
Reviewer #4: Using a mass detector hyphenated with the HPLC will be useful to characterize more precisely the different phenolic compounds present in the extract (70% are not identified) Using the electrochemical detector is quite interesting to assess the antiox properties of the extract, instead of classical testing. Nevertheless, we have to keep in mind this is only an vitro testing, and therefore not representative of the in vivo antiox properties: is this kind of detector usable to perform measurement on biological material (plasma, urine...)?
Answer: The authors agree with the reviewer that the use of liquid chromatography coupled with mass spectrometer will allow more accurate determination of the composition of the extracts tested. In the presented work, we determined the content of 15 individual phenolic compounds. Amaral and colleagues (Food Chemistry, 2004, 88, 373-379) conducted an analysis of the phenolic profile in walnut leaf by HPLC-DAD / MS and identified only 7 phenolic compounds. Due to the lack of literature data, determining the phenolic profile of various morphological parts of the walnut is important and necessary.
Reviewer 3 Report
The authors have to improve the introduction and the references list.
- Regarding lines 28-30 "Polyphenols, flavonoids and tannins are widely distributed in plants, mainly in fruits and vegetables, which contribute to their taste and color. They constitute a large group of compounds with different activities that also have antioxidant and/or antibacterial properties.", the authors have to modify this paragraph as follow:
Polyphenols represent a wide group of secondary plant metabolites, arising from phenylalanine or shikimic acid, playing a pivotal role in counteracting different type of stress (ultraviolet irradiation, aggression by pathogens, parasites and plant predators), other than contributing to organoleptic
properties of plants and plant-derived food (Di Mauro et al., 2017). In addition, these compounds are well‐known for their beneficial effects on human health, due to their antioxidant, cardioprotective, anticancer, anti‐inflammatory, and antimicrobial properties (Di Mauro et al., 2019; Fraga et al., 2019).
- Regarding lines 34-37 "The walnut is a deciduous tree in the genus Juglans (Family Juglandaceae). The walnut is grown for food because of its edible seeds, called walnuts, as well as for medicinal and cosmetic purposes. Both the walnut leaf and its products show, among others, astringent, anti-microbial and anti-inflammatory properties [1, 2].", the authors have to modify as follow:
Juglans regia L. (common walnut), a deciduous tree belonging to Juglandaceae family, was widely used in traditional medicine for its antioxidant, antidiabetic, antimicrobial, anti-inflammatory, anti-atherogenic and liver-protective effects (Acquaviva et al., 2019, ref.2).
Please add the suggested articles and delete reference 1.
- Di Mauro et al., Polyphenolic profile and antioxidant activity of olive mill wastewater from two Sicilian olive cultivars: Cerasuola and Nocellara etnea. Eur. Food Res. Technol. 2017, 243, 1895–1903
- Di Mauro et al., Antioxidants 2019, 8(10),462
- Fraga et al., The effects of polyphenols and other bioactives on human health. Food Funct. 2019, 10, 514–528.
- Acquaviva et al. (2019). Natural Product Research, DOI:10.1080/14786419.2019.1650352
Regarding the results, the authors have to show the different chromatograms.
Finally I suggest to the authors to modify the title as follow:
"Polyphenolic profile and antioxidant activity of Juglans regia L. leaves and husk extracts."
Author Response
Response to Review Comments
Forests
Manuscript ID: forests-623979
Title: Analysis of composition and properties of extracts of walnut (Juglans regia L.) leaves and husk
Article Type: Article
Authors: Anna Masek, Malgorzata Latos-Brozio, Ewa Chrzescijanska, Anna Podsedek
Dear Editor and Reviewers,
We would like to thank the Editor and Reviewers for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Corrections in manuscript have been marked in yellow. Details of our responses to each comment are shown below:
Reviewer #2: The authors have to improve the introduction and the references list.
- Regarding lines 28-30 "Polyphenols, flavonoids and tannins are widely distributed in plants, mainly in fruits and vegetables, which contribute to their taste and color. They constitute a large group of compounds with different activities that also have antioxidant and/or antibacterial properties.", the authors have to modify this paragraph as follow:
Polyphenols represent a wide group of secondary plant metabolites, arising from phenylalanine or shikimic acid, playing a pivotal role in counteracting different type of stress (ultraviolet irradiation, aggression by pathogens, parasites and plant predators), other than contributing to organoleptic properties of plants and plant-derived food (Di Mauro et al., 2017). In addition, these compounds are well‐known for their beneficial effects on human health, due to their antioxidant, cardioprotective, anticancer, anti‐inflammatory, and antimicrobial properties (Di Mauro et al., 2019; Fraga et al., 2019).
Answer: The introduction has been improved.
Reviewer #2: - Regarding lines 34-37 "The walnut is a deciduous tree in the genus Juglans (Family Juglandaceae). The walnut is grown for food because of its edible seeds, called walnuts, as well as for medicinal and cosmetic purposes. Both the walnut leaf and its products show, among others, astringent, anti-microbial and anti-inflammatory properties [1, 2].", the authors have to modify as follow: Juglans regia L. (common walnut), a deciduous tree belonging to Juglandaceae family, was widely used in traditional medicine for its antioxidant, antidiabetic, antimicrobial, anti-inflammatory, anti-atherogenic and liver-protective effects (Acquaviva et al., 2019, ref.2).
Answer: The introduction has been improved.
Reviewer #2: Please add the suggested articles and delete reference 1.
- Di Mauro et al., Polyphenolic profile and antioxidant activity of olive mill wastewater from two Sicilian olive cultivars: Cerasuola and Nocellara etnea. Eur. Food Res. Technol. 2017, 243, 1895–1903
- Di Mauro et al., Antioxidants 2019, 8(10),462
- Fraga et al., The effects of polyphenols and other bioactives on human health. Food Funct. 2019, 10, 514–528.
- Acquaviva et al. (2019). Natural Product Research, DOI:10.1080/14786419.2019.1650352
Answer: Articles has been added. Reference 1 has been delete.
Reviewer #2: Regarding the results, the authors have to show the different chromatograms.
Answer: Chromatograms has been added.
Reviewer #2: Finally I suggest to the authors to modify the title as follow: "Polyphenolic profile and antioxidant activity of Juglans regia L. leaves and husk extracts."
Answer: The title has been modify.
Reviewer 4 Report
The manuscript by Masek et al. is focused on the antioxidant activity of extracts of walnut leaves and husk. In my opinion, this paper in the present form has a severe lack of novelty and scientific rigor. However, I do think the idea may be publishable after doing a lot of new experiments and a more in-depth analysis of the results.
The paper does not present properly the novelty and the motivation behind this research. The use of cyclic voltammetry and differential pulse voltammetry to determine the antioxidant capacity for plant extracts, if intended to be the main aspect, should be much more profound. I recommend the authors to rethink their work and to resubmit after seriously doing more experiments to validate proposed electrochemical methods (using appropriate statistical methods) and to obtain quantitative results on antioxidant activity. It is necessary to show the advantages and limitations of the electrochemical techniques in assessing the antioxidant activity of plant extracts. Just doing a set of characterizations without regarding the information that they may provide is no serious research.
The authors in the Introduction (line 67) and Conclusion (line 342) point to the correlation of electrochemical methods with spectrophotometric methods used for the determination of antioxidant capacity. However, I do not see any correlation since no quantitative data on electrochemical methods are given.
The paper also contains many typos, errors, and wrong constructions, which sometimes drastically affect scientific meaning, making the paper difficult for reading and understanding. A native English speaker with a scientific background should carefully revise the manuscript before its resubmission.
Here are some of the errors:
Line 16: “Innovative” is not a suitable word for describing standard widely used methods.
Line 109: Replace “light” to “visible”
Line 111: Replace “spectrophotometer” to “spectrometer”
Lines 138, 143, 239, 252: Missing references.
Lines 158-158, 163: UV-Vis spectrum is a plot of the absorbance vs. the wavelength, and it is not recorded at a specific wavelength.
Line 160: Wrong units for TEAC. Also, TEAC is not used in the paper at all.
Figure 1 A and B, X-axis: It is not “Wavelength”.
Line 264: Replace “basic” to “supporting”.
Author Response
Response to Review Comments
Forests
Manuscript ID: forests-623979
Title: Analysis of composition and properties of extracts of walnut (Juglans regia L.) leaves and husk
Article Type: Article
Authors: Anna Masek, Malgorzata Latos-Brozio, Ewa Chrzescijanska, Anna Podsedek
Dear Editor and Reviewers,
We would like to thank the Editor and Reviewers for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Corrections in manuscript have been marked in yellow. Details of our responses to each comment are shown below:
Answers to reviewer #3 comments
Reviewer #3: The manuscript by Masek et al. is focused on the antioxidant activity of extracts of walnut leaves and husk. In my opinion, this paper in the present form has a severe lack of novelty and scientific rigor. However, I do think the idea may be publishable after doing a lot of new experiments and a more in-depth analysis of the results.
The paper does not present properly the novelty and the motivation behind this research. The use of cyclic voltammetry and differential pulse voltammetry to determine the antioxidant capacity for plant extracts, if intended to be the main aspect, should be much more profound. I recommend the authors to rethink their work and to resubmit after seriously doing more experiments to validate proposed electrochemical methods (using appropriate statistical methods) and to obtain quantitative results on antioxidant activity. It is necessary to show the advantages and limitations of the electrochemical techniques in assessing the antioxidant activity of plant extracts. Just doing a set of characterizations without regarding the information that they may provide is no serious research.
Answer: Manuscript has been reorganized, much of it has been improved according to the Reviewer's guidelines. The description of the electrochemical part has been corrected. Unfortunately, due to the limitations in the access to the apparatus, the results of the research could not be added.
Reviewer #3: The authors in the Introduction (line 67) and Conclusion (line 342) point to the correlation of electrochemical methods with spectrophotometric methods used for the determination of antioxidant capacity. However, I do not see any correlation since no quantitative data on electrochemical methods are given.
Answer: The electrochemical index (EI) was calculated taking into account to the main voltammetric parameters, peak potential (Epa), and peak current (Ipa). Based on the fact that the lower the Epa (thermodynamic parameter), the higher is the electron donor ability, and the higher the Ipa (kinetic parameter), the higher is the amount of electroactive species, EI was calculated and results present in Table 2.
Reviewer #3: The paper also contains many typos, errors, and wrong constructions, which sometimes drastically affect scientific meaning, making the paper difficult for reading and understanding. A native English speaker with a scientific background should carefully revise the manuscript before its resubmission.
Answer: The linguistic revision has been done. Manuscript was corrected by Brownhill Ruyton XI Towns Shrewsbury SY4 1LR Tel: +44 (0)1939 261 121
email: [email protected] http://www.eleventowns.co
Reviewer #3: Here are some of the errors:
Line 16: “Innovative” is not a suitable word for describing standard widely used methods.
Answer: It has been corrected.
Line 109: Replace “light” to “visible”
Answer: It has been corrected.
Line 111: Replace “spectrophotometer” to “spectrometer”
Answer: It has been corrected.
Lines 138, 143, 239, 252: Missing references.
Answer: It has been added.
Lines 158-158, 163: UV-Vis spectrum is a plot of the absorbance vs. the wavelength, and it is not recorded at a specific wavelength.
Answer: It has been improved.
Line 160: Wrong units for TEAC. Also, TEAC is not used in the paper at all.
Answer: The sentence has been deleted.
Figure 1 A and B, X-axis: It is not “Wavelength”.
Answer: X-axis on Figure 1 A and B has been corrected.
Line 264: Replace “basic” to “supporting”.
Answer: It has been corrected.
Round 2
Reviewer 1 Report
The authors have improved the manuscript that it is now suitable for publication
Author Response
Thank you very much for the reviewers' comments.
Reviewer 3 Report
The authors have improved the manuscript.
Author Response
Thank you very much for the reviewers' comments.
Reviewer 4 Report
The authors have addressed most of the reviewers’ concerns, improving the manuscript with their edits. However, there are still some points for authors to consider:
*Lines 77-80: “A specific analytical procedure including sample preparation was developed and validated for the detection of phenolic compounds. HPLC-UV method has been developed and validated for the quantification of phenolic content in walnut leaf extract and walnut husk extract”. Consider either removing this phrase or adding some discussion with references.
*Line 165: Consider citing here the recent paper on HPLC-UV determination of phenolic acids (Journal of Analytical Chemistry 2015 70(11): 1406-1411, DOI: 10.1134/S1061934815110143).
*Line 181: Define the value indicated in parentheses (t = 10 mV s-1).
*Tables 2 and 4: The standard deviation should be expressed as ONE significant figure; that is, unless the number is between 11 and 19 times some power of ten, in which case you can use two significant figures. The mean value should be rounded off at the decimal place corresponding to the last significant digit of its standard deviation. For example, 50.25 ± 6.15 (Table 4) should be presented as 50 ± 6; and 17.68 ± 1.47 (Table 4) should be presented as 17.7 ± 1.5, etc.
*Сheck once again your paper for grammatical errors, wrong spellings, and word usage. Here are SOME examples that need to be corrected:
--Across the text: Use either “FTIR” or “FT-IR”
--Lines 103-104: Remove Trolox from the list of reagents since it was not used.
--Lines 161-162: Please check and correct the mobile phase gradient.
--Line 181: Specify the value given in parentheses.
--Line 214: Replace “mol” to “mol.L-1”.
--Figures 2a, 2b, 2c: Replace “spectra” to “spectrum” (4 times).
--Line 322: Replace “0.1M” to “0.1 mol.L-1” (for uniformity).
--Lines 353-354: Replace “w 0.1 M” to “in 0.1 mol.L-1”.
--Line 370: Replace “extarks” to “extracts”.
Author Response
Manuscript ID: forests-623979
Title: Polyphenolic profile and antioxidant activity of Juglans regia L. leaves and husk extracts
Article Type: Article
Authors: Anna Masek, Malgorzata Latos-Brozio, Ewa Chrzescijanska, Anna Podsedek
Dear Editor and Reviewer,
We would like to thank the Editor and Reviewer for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Corrections in manuscript have been marked in yellow. Details of our responses to each comment are shown below:
Answers to reviewer comments
Reviewer: The authors have addressed most of the reviewers’ concerns, improving the manuscript with their edits. However, there are still some points for authors to consider:
*Lines 77-80: “A specific analytical procedure including sample preparation was developed and validated for the detection of phenolic compounds. HPLC-UV method has been developed and validated for the quantification of phenolic content in walnut leaf extract and walnut husk extract”. Consider either removing this phrase or adding some discussion with references.
Answer: The sentence has been deleted.
Reviewer: *Line 165: Consider citing here the recent paper on HPLC-UV determination of phenolic acids (Journal of Analytical Chemistry 2015 70(11): 1406-1411, DOI: 10.1134/S1061934815110143).
Answer: It has been added.
Reviewer: *Line 181: Define the value indicated in parentheses (t = 10 mV s-1).
Answer: The sentence “The DPV was recorded with a modulation potential of 25 mV and a pulse width of 50 ms t = 10 mV s-1)” was corrected to: ”DPV were recorded in the same potential range with modulation amplitude 25 mV, pulse width 50 ms (scan rate 0.01 V s-1).”
Reviewer: *Tables 2 and 4: The standard deviation should be expressed as ONE significant figure; that is, unless the number is between 11 and 19 times some power of ten, in which case you can use two significant figures. The mean value should be rounded off at the decimal place corresponding to the last significant digit of its standard deviation. For example, 50.25 ± 6.15 (Table 4) should be presented as 50 ± 6; and 17.68 ± 1.47 (Table 4) should be presented as 17.7 ± 1.5, etc.
Answer: Please leave the format of the average value and standard deviations unchanged. The instructions for authors do not specify the requirements for writing numerical values (the need to include significant digits). Numerical values in publications from the last issue of the journal (for example 46.85 ± 1.93 in Forests 2019, 10 (10), 928; https://doi.org/10.3390/f10100928) are in a format with an accuracy of two decimal places.
Reviewer: *Сheck once again your paper for grammatical errors, wrong spellings, and word usage. Here are SOME examples that need to be corrected:
-Across the text: Use either “FTIR” or “FT-IR”
Answer: It has been corrected.
-Lines 103-104: Remove Trolox from the list of reagents since it was not used.
Answer: It has been removed.
-Lines 161-162: Please check and correct the mobile phase gradient.
Answer: The mobile phase gradient has been corrected.
-Line 181: Specify the value given in parentheses.
Answer: The sentence “The DPV was recorded with a modulation potential of 25 mV and a pulse width of 50 ms t = 10 mV s-1)” was corrected to: ”DPV were recorded in the same potential range with modulation amplitude 25 mV, pulse width 50 ms (scan rate 0.01 V s-1).”
-Line 214: Replace “mol” to “mol.L-1”.
Answer: It has been corrected.
-Figures 2a, 2b, 2c: Replace “spectra” to “spectrum” (4 times).
Answer: Figure 2 has been corrected.
-Line 322: Replace “0.1M” to “0.1 mol.L-1” (for uniformity).
Answer: It has been corrected.
-Lines 353-354: Replace “w 0.1 M” to “in 0.1 mol.L-1”.
Answer: It has been replaced.
-Line 370: Replace “extarks” to “extracts”.
Answer: It has been replaced.
