Anti-Inflammatory and Anti-Quorum Sensing Effect of Camellia sinensis Callus Lysate for Treatment of Acne

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors are presenting their work regarding efficacy against Acne for a developed callus lysate form Camellia sinensis. The work has scientific importance, however there are several improvements that are needed both in methodology and result presentation before this manuscript can be accepted for publication.

Below are my specific comments and suggestions for improvement.

Throughout the manuscript, please correct the line breaks so that they do not occur in the middle of words and so that if brackets are used, they will be as a pair of brackets and never alone. I am missing many references and discussions regarding previous studies within the area. I also advice to review language and grammar more carefully in order to increase the quality of the current work. I started to make grammatical suggestions, however since there are several improvements that are necessary to the content and how to present the results I focused on this part and can look more into language in a future submission, however I feel that application a more formal language would improve this manuscript. This does not mean that a “story telling mode” should not be used, but to select more formal wordings throughout the manuscript.

Row 29-30, I suggest to remove “…,previously called Propionibacterium acnes),…”.

Row 31-32, I suggest to write “On the skin, C. acne will proliferate until it has reached a high population density and will thereafter form a biofilm through excretion of extracellular matrix….” if one aspiration timepoint was used or “C. acnes proliferates and arrives at a specific population density, forming biofilms through the excretion of the extracellular matrix…..”  

Row 32, I suggest to write “….which will decrease their sensitivity to….” or similar instead of “….limit bacterial exposure to….”.

Row 36, I suggest to write “…quorum sensing pathway,...” or similar instead of “…quorum sensing production,….”.

Row 37, I suggest to write “…and widespread for several…” or similar instead of “…and is widespread in several…”.

Row 38-40, I suggest to write “Some of these pathways can be induced to prevent bacterial overgrowth and virulence induction, without using wide-spectrum antibiotics that kill pathogenic as well as commensal bacteria” or similar instead of “This is an interesting approach with which to prevent bacterial overgrowth and virulence induction without using wide-spectrum antibiotics that kill pathogenic and commensal bacteria”.

Row 40, I suggest to write “Even though several promising anti-aging active molecules have been identified …” or similar instead of “Even though several researches reported promising anti-aging active molecules …”.

Row 41-52, In this section all standard treatments and research should be summarised. I am missing information here and have therefore not investigated the language and grammar here.

Row 53-57, In this section I am also missing information. I advise to do a more in-depth presentation regarding plant-based actives that are having a general antimicrobial effect, are preventing growth of certain species, that can strengthen the physical properties of the skin and therefore can help to prevent colonisation, or that are having a beneficial effect of the overall microbial composition of the skin. Also remember that in pharma many of the actives used have plant origin. I therefore advise not to write “pharmacological and nonpharmacological approaches”. It is correct that polyphenolic compounds have interesting properties, however in regards to plant actives more actives should also be mentioned beyond phenolic components. Furthermore, for phenolics a broader range of information regarding these are needed in my opinion and to mention more examples within this group of actives apart from catechins. In regards to cultivation of plant cells and cell line development, here I am missing a more general information regarding what is controlling production of actives, what is important when developing cell lines and what is the reason for using a callus cell line in the current study. To just write GMP is not at all sufficient since it does not say anything apart from that the protocols and guidelines are followed within the laboratory. There also need to be a description regarding callus cell lines, how they differ from differentiated or partially differentiated cell lines, and to add references to previous work from this area.

Row 64-73, describe why an undefined lysate is used instead of a characterised extract. Since this is a scientific publication a general description of previous work within the area is always needed and refer to published articles within the field.

Material and methods, Row 76-264

1)      include more information regarding the cell line, such as scientific name, origin, cell line description and details regarding cell line development, cultivation and stimulation for the production of actives.

2)      Provide details regarding how the lysate was produced, how it was standardised and characterised. For the chemical analysis provide more information regarding concentrations, primers for PCR, conditions and maintenance.

3)      What concentration and how was the lysate standardised for the quantification of terpenes, catechin.

4)      Define how many technical replications that were used for the analysis and how many times these were repeated in time.

Results

Throughout the manuscript

In this work a botanical lysate is used. It is important to determine the concentration of the lysate. This can be achieved through determination of the dry matter content of the filtrated lysate/extract and to express the concentration as dry extract (or lysate) content/g lysate. Thereafter for all analysis performed, correlate the results to dry extract (or lysate) matter. It will be a large difference if the lysate is 10% or 40% in concentration. Furthermore, dry matter standardisation is crucial for efficacy determination and presentation.  

Terpene content, present the data as mean of terpenes / dry extract (or lysate) content +/- standard derivation. Also compare the content of terpenes in the lysate to that of other plants, and to Camellia sinensis leaf extract.

HPLC analysis, For figure 1 it is important to use the same scale for the y axis in a and b. Clarify the naming of the x and y axis appropriate, such as densitometric units (AU) and retention time (min). Furthermore, improve the resolution for the chromatogram and use the same number for the presented marker substances in a and b. Make sure that the same loading concentration is used for the analysis (same amount of dry extract (lysate) content and express the results correlated to marker substance correlated to dry extract (or lysate) content. With a known loading amount of marker substance, the amount of this can be correlated to the area for this peak in the densitogram and with the area or the corresponding peak of the botanical lysate and be presented as e.g. ng epicatechin/g dry extract (or lysate). Also be more clear in the presentation which catechins tat you were able to identify in the botanical lysate, since to my understanding only epicatechin was identified. When performing compound identification was wave-length scan mode for the specified retention time applied and did these scans match? Include these scans in figure 1. When comparing the obtained results to those of others, be more specific, describe more details from what has previously been published and include more publications to have a more appropriate results comparisons other than writing “This value is approximately within the range previously reported by other authors.

Anti-inflammatory response, For figure 4 the resolution is not sufficient and I am not able to see details and numbers for this figure. Improve this figure. It could benefit to create relative comparisons in a graph. Also compare these results to when lipopolysaccharide (LPS) is used to induce inflammation to prove that both can be used as triggers or present this comparison if performed by others. In the result presentation, give details from previously published results and how there can be related to the results obtained in the current study. Include more publications from other groups. In figure 5, these results are not clear and need to be supported by a complementary analysis or signal intensity quantification and calculations. In figure 6, here a plot is showed, however if these would be further processed and quantified a clearer presentation could be performed.

Anti-biofilm activity, correlate the results to absorbance as well as cfu. Also present the d0, d5 as well as end point value dX values for control as well as treatment. Also include a positive control to compare with a known inhibitor and correlate the effectiveness of the lysate in comparisment to know inhibitor and no treatment controls. When presenting statistical significance, it is important to clarify which treatments that this correlates to and which significance level, both in text and in figures. Include more publications from other groups.

Anti-lipase activity, Include a positive control in this study and correlate the obtained results to the positive as well as no treatment control. Include more publications from other groups. Add statistical comparisons and conclusions.

Quantification of autoinducer 2, Add statistical comparisons and conclusions. Include more publications from other groups when discussing the results.

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper is well organaised and written in decent scientific style. The experiments are reliable and the results are statistically processed. The topic is interesting and fall into the journal's scope. I have the following recommendations:

1. please improve the quality of figure 5;

2. Add statistics to figure 7.

 

Author Response

Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This work discussed the anti-inflammatory and anti-quorum sensing effect of Camellia sinensis callus lysate for treatment of acne. There are some issues in this manuscript that should be addressed as follows:

·         Abstract:

- The aim of the study should be mentioned clearly in the Abstract.

- Page 1 Line 17: The word “nucleus” should be replaced with “nuclear”.

- Page 1 Line 19: The word “bactericide” should be replaced with “bactericidal”.

- Key words: The number of the key words should be decreased.

·         Introduction:

1.    The novel aspects in this study should be clarified as there are other articles that discussed similar issues; e.g. https://pubmed.ncbi.nlm.nih.gov/32812270/; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9027544/; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384166/ .

2.    Page 1 Line 38-40: The sentence “This is an interesting approach with which to prevent bacterial overgrowth and virulence induction without using wide-spectrum antibiotics that kill pathogenic and commensal bacteria” should be revised grammatically.

3.    Page 2 Line 58: [15,16,17,18] should be written as [15-18].

4.    More data about the anti-inflammatory effect of Camellia sinensis callus lysate should be added.

·         Materials and methods:

1.    The numbering of the subheadings “4.1. Materials and 4.1. Methods” should be revised.

2.    The exact source, concentrations and the catalogue numbers of the used kits, and chemicals should be mentioned.

3.    The numbering of the references should be revised. Although the last reference in the “Introduction” is [20], the first reference in the “Materials and Methods” is [41]. Please, revise.

4.    Page 3: A reference for Catechin quantification should be added.

5.     Page 3 Line 139: [25, 26, 27] should be written as [25-27].

6.    Page 4 Line 188: The sentence “in Section 4.2.6.” should be corrected to “in Section 2.2.6.”

7.    Page 4: A reference for the method of NF-kB nuclear translocation evaluation should be added.

8.    Page 5 Line 210: The numbering “2.2.1.” should be corrected.

9.    Page 5: References for equations (1, 2, 3, 4, and 5) should be added.

10. The methods of statistical analysis should be added.

·         Results:

- Figures 7 and 9: The marks of statistical significance (*) should be added in the figure.

- It is recommended to add a collective diagram that summarizes the results.

·         Discussion: It is recommended to separate the discussion from the results and re-write the discussion to focus on analysis of the results of the present study.

·         Conclusion. I think that the conclusion is not sufficient. The possible clinical implications of the results of the present study should be added.

·         General comments:  

1. The manuscript should be revised by English-naïve speaker to improve the quality of the language.

2. The manuscript should be checked regarding the grammatical errors and plagiarism.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

The authors had addressed most of my comments. However, it is advisable to separate the results from the discussion.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors are presenting their work regarding efficacy against Acne for a developed callus lysate form Camellia sinensis. Extracts from cell cultures, differentiated of undifferentiated, and their application areas are highly relevant. Once the current study is presented in a clearer way it will be highly relevant for the scientific community. However, it is important to know that we are not comparing apples and pears, standardisation of extracts is crucial to ensure that the same metabolic content and efficacy is obtained. The work has scientific importance, however there are several improvements that are needed both in methodology and result presentation before this manuscript can be accepted for publication.

In point 12, answer from authors, it is fully accurate to use botanical extracts and this can even be beneficial, since there may be additive benefits from the complexity, however the extract needs to be standardised to dry extract content and to selected marker substances, or it will not be possible to know if the observed efficacy will be the same.

In point Point 14: Provide details regarding how the lysate was produced, how it was standardised and characterised. Here I am referring to how the botanical extract/lysate was produced and backround information regarding cultivation conditions, since this has a great impact in quality of extract/lysate and presence of actives.

In point Point 14: For the chemical analysis provide more information regarding concentrations, primers for PCR, conditions and maintenance. Here I was asking that this information should be added to the manuscript of to be provided as supplementary information.

In point 15, based on the response from the authors it seems like the current study is based on one extract batch, see my further questions and clarifications below. Please clarify this part since it important when addressing chemical characterisation and efficacy.

Below are my specific comments and suggestions for improvement.

In the initial review, due to the number of changes that we felt were necessary there were some critical points that were not discovered but that we feels need to be corrected now in the second round of reviewing process.

1)      In regards to the use of extract from in vitro cultivated cells. It is not always the case that in vitro produced biomass will be higher in production of bioactives and that less amount of biomass is required. The first publications regarding in vitro cultivation of plants/plant cells originates from 1910. Since then, substantial work has been carried out. I am not suggesting to summarise all this work, however select a few articles that could represent application areas of in vitro cultivation of plantlets, plant organs and plant cells regarding production of bioactives. The production of actives will also depend of cell maturation grade/cell type, which is something that is also something that should be addressed and to add a few references. Furthermore, it is not correct that callus cultures generally will be high in production of actives. This will depend on which type of active and statements such as that needs to be supported by previous published research.

2)      In the current project a callus lysate was used. Does this callus lysate consist of biomass/callus cells alone or biomass/callus cells + nutrient medium? Was the lysate/extract filtered to ensure that no cell remains are present in the solution used to perform the further characterisation? Please clarify this. If there is no filtration but only centrifugation performed to produce the lysate, verification that no cells are present can be performed by checking that the dry matter content does not decrease after filtration, unless there are formation of precipitates in the lysate.

Author Response

Dear Reviewer 1,

Please, we think that it has been some mistake or misunderstanding regarding the last referee report. The referee report stated in the CIMB link as the Reviewer 1 (Round 3) Report is exactly the same report as one described a few weeks ago as the Reviewer 1 (Round 2) Report. Indeed, the text is identical, although now there is only the first page of the previous document.

For this reason, we would appreciate it if you could clarify us this issue in order to let us know in which point our submission is.

Yours sincerely,

Dr. Laia Montell

Author Response File: Author Response.pdf