Cytoprotection of Antioxidant Biocompounds from Grape Pomace: Further Exfoliant Phytoactive Ingredients for Cosmetic Products
, Melissa M. Gonçalves 2, Rebeca W. O. D’Angelo 2, Ana B. Girassol 1, Ana P. Tulio 1, Yasmine M. Pupo 3 and Paulo V. Farago 4Round 1
Reviewer 1 Report
Authors tested grape pomace as antioxidant cosmetic material. Authors presented several safety(MTT assay and 3T3 cell data) and efficacy data. However, more safety test data are needed to provide sufficient information as possible cosmetic agent.
Author Response
The authors are grateful to the reviewers for their careful reading of this manuscript and the insightful comments.
We agree that more tests are needed to consider grape pomace safe for use as a cosmetic ingredient, including in vivo studies. However, these additional tests depend on the ethics committee approval and they are beyond the scope of this paper. Further studies can be carried out in the continuity of this work, considering that tests of cytotoxicity and cytoprotection demonstrated promising results.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript describes the study of the efficacy and the safety of grape pomace (Vitis labrusca L.) obtained from winemaking process as antioxidant raw material for cosmetic formulations. The authors performed bioactivity/cytotoxicity assays and metabolite determination. The concept if the work is interesting, however there are a few points which need to be addressed by the authors:
1) Why do authors chose to determine the antioxidant activity using only one assay (DPPH method). To my opinion other methods (ORAC, FRAP) are more informative.
2) The extraction was performed using 75% organic-water system. The authors should explain why.
3) The authors should explain and discuss (Figure 4), the absence of concentration dependence since all concentrations used protect the cells to the same degree.
Author Response
Point 1: Why do authors chose to determine the antioxidant activity using only one assay (DPPH method). To my opinion other methods (ORAC, FRAP) are more informative.
Response 1: The authors are grateful to the reviewers for their careful reading of this manuscript and the insightful comments. The DPPH was the method of choice for being considered accurate, easy and extensively reported in the literature for total antioxidant activity determination. It would be interesting to compare results obtained by DPPH to those obtained by other methods such as ORAC and FRAP, however such comparisons not rarely generate inconclusive results and these additional experiments were beyond the scope of this work.
Point 2: The extraction was performed using 75% organic-water system. The authors should explain why.
Response 2: Extraction was performed using 75% organic-water system because this proportion revealed better results of extraction yield considering the polarity of antioxidants biomolecules.
Point 3: The authors should explain and discuss (Figure 4), the absence of concentration dependence since all concentrations used protect the cells to the same degree.
Response 3: Done (line 224).
Author Response File: Author Response.pdf
Reviewer 3 Report
The aim of the study was to determine some biological properties of grape pomace as a substitute for microplastics used as exfoliants in cosmetic products.
Major point :
1. It is not clear how the parameters assessed in this study might be useful to replace microplastics as exfoliants. If I understand properly, only the safety and the cytoprotection against oxidative stress of grape pomace is assessed. In this case this should be clearly explained; in particular, it should be indicated why the parameters assessed in this study are hepful for the use of grape pomace as a substitute for microplastics.
Minor points :
2. Introduction, line 72 : add references regarding the exfoliant properties of grape pomace.
3. Methods : add references for the following assays : phenolic content, DPPH, MTT, cytoprotection (IC50of 600 µM for hydrogen peroxide).
4. Methods : DPPH assay : (1) explain how you determined the EC50for DPPH assay; (2) did you anaylse a kinetic parameter, since the antioxidant capacity is mostly determined by the reactivity toward oxidants ?
5. Statistics : please indicate how you calculated the statistics mentioned in the text.
6. Phenolic compounds : (1) : Do you have data for the determination of phenolic compounds (absorbance values, table or graph) ? (2) The unit indicated for phenolic compounds leaching-out rate is not a rate (mg GAE/g) : to have a rate, you must add the time-dependence (mg GAE/g/min).
7. Figure 4 : (1) How did you define the cell viability using the MTT assay ? (2) Did you apply a statistical test to compare the values of the samples ?
Author Response
Point 1: It is not clear how the parameters assessed in this study might be useful to replace microplastics as exfoliants. If I understand properly, only the safety and the cytoprotection against oxidative stress of grape pomace is assessed. In this case this should be clearly explained; in particular, it should be indicated why the parameters assessed in this study are hepful for the use of grape pomace as a substitute for microplastics.
Response 1: Microplastics use as exfoliant ingredient in cosmetic products has been banned in countries such as Canada and the United States because of its environment impact. They can overcome the filtering system of industrial plants being dumped in the river bed and ingested by marine animals. The cosmetic industry trend is to employ biodegradable raw materials like fruit seeds as natural exfoliants. In this context, grape pomace is an example of natural exfoliant with additional antioxidant activity for skin anti-aging treatments. As an active ingredient in cosmetic products, grape pomace needs to be submitted to safety and efficacy studies.
We have added additional information to improve clarity and completeness.
Point 2: Introduction, line 72 : add references regarding the exfoliant properties of grape pomace.
Response 2: Added.
Point 3: Methods : add references for the following assays : phenolic content, DPPH, MTT, cytoprotection (IC50of 600 µM for hydrogen peroxide).
Response 3: Added.
Point 4: Methods : DPPH assay : (1) explain how you determined the EC50for DPPH assay;
Response 4: We thank the reviewer for this important suggestion. This explanation has been added to the text.
(2) did you anaylse a kinetic parameter, since the antioxidant capacity is mostly determined by the reactivity toward oxidants ?
Response 4: We did not performed a kinetic analysis. We found that the EC50 determination was a suitable parameter.
Point 5: Statistics: please indicate how you calculated the statistics mentioned in the text.
Response 5: Done.
Point 6: Phenolic compounds : (1) : Do you have data for the determination of phenolic compounds (absorbance values, table or graph) ?
Response 6: Yes, we have absorbance values, but we found that these data were not so relevant to be demonstrated in the manuscript.
(2) The unit indicated for phenolic compounds leaching-out rate is not a rate (mg GAE/g) : to have a rate, you must add the time-dependence (mg GAE/g/min).
Response 6: Agree, this sentence has been modified into the text.
Point 7: Figure 4 : (1) How did you define the cell viability using the MTT assay?
Response 7: We thank the reviewer for this relevant suggestion. This explanation has been added to the text.
(2) Did you apply a statistical test to compare the values of the samples?
Response 7: No, we did not. We calculated standard deviation that for cell culture assays is more usual.
The authors are grateful to the reviewers for their careful reading of this
manuscript and the insightful comments.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript was improved
Author Response
Curitiba, July 18th 2018.
Dear Editors-in-chief,
Please consider our revised manuscript, “Cytoprotection of antioxidant biocompounds from grape pomace: further exfoliant phytoactive ingredient for cosmetic products”, for publication in the Cosmetics.
We appreciate the interest that the editors and reviewers have taken in our manuscript and the constructive criticism they have given. We also greatly appreciate the reviewers for their complimentary comments and suggestions. Please find attached a point-by-point response to reviewer’s concerns. We hope that you find our responses satisfactory and that the manuscript is now acceptable for publication.
We look forward to hearing from you,
Best regards,
Daniela Florencio Maluf, Ph D.
Department of Pharmacy
Universidade Federal do Paraná
Av. Prefeito Lothário Meissner 632, 80210-170. Curitiba/PR Brazil.
Tel:+55(41)3360-4077.
e-mail: [email protected]
Author Response File: Author Response.pdf
Reviewer 3 Report
1. References for the methods used :
a. for the phenolic content (Ref 24), it is better to cite a reference with the complete description rather than a reference that cites a reference...
b. for the DPPH assay, in the cited reference (25), it is the ABTS method, not that with DPPH, which is used to assess the antioxidant capacity. A reference for the DPPH assay is still missing.
c. the reference 26 could be cited in the paragraph 2.8 (MTT assay) too.
2. DPPH assay : The equation (1) (line 130) gives a sigmoid curve, so I don't understand how the authors got a straight line (y = ax + b)... Please clarify.
3. Statistics : a description of the error bars (SD, SE/SEM ?) is still missing. It is not explained how the ANOVA was performed and the graphs (or anywere else) don't indicate the values statistically significant from a control.
4. Phenolic compounds : data are still missing. In line 199, the word "rate" has been replaced by "value", so the unit is now correct. This modification could have been highlighted in yellow to find it easily.
Author Response
Curitiba, July 18th 2018.
Dear Editors-in-chief,
Please consider our revised manuscript, “Cytoprotection of antioxidant biocompounds from grape pomace: further exfoliant phytoactive ingredient for cosmetic products”, for publication in the Cosmetics.
We appreciate the interest that the editors and reviewers have taken in our manuscript and the constructive criticism they have given. We also greatly appreciate the reviewers for their complimentary comments and suggestions. Please find attached a point-by-point response to reviewer’s concerns. We hope that you find our responses satisfactory and that the manuscript is now acceptable for publication.
We look forward to hearing from you,
Best regards,
Daniela Florencio Maluf, Ph D.
Department of Pharmacy
Universidade Federal do Paraná
Av. Prefeito Lothário Meissner 632, 80210-170. Curitiba/PR Brazil.
Tel:+55(41)3360-4077.
e-mail: [email protected]
Author Response File: Author Response.pdf
