Effects of Three Different Brazilian Green Propolis Extract Formulations on Pro- and Anti-Inflammatory Cytokine Secretion by Macrophages

Round 1

Reviewer 1 Report

1)The phenolic equipment of the propolis samples used in the study was tested with HPLC-PDA. However, the calibration and validation values are not given. If the 39th article has been cited but has not yet been published, LOD and LOQ values must be given.

2) The units used for the total amount of phenolic substance and the total amount of flavonoid substance should be given in mg GAE/g or mgQUE/g.

3) There are many grammatical errors in the article, they should be corrected.

4) The "Preparation of The Three Propolis Extracts" part is very complex and should be simpler and more understandable.It should be separate and understandable for each product.

5)The recommended source (67.) for baccharin isolation is not sufficient, and the isolation method should be given.

6) HPLC chromatograms of three different propolis samples should be given.

7) Were the standards used in the cell culture study (caffeic acid, p-coumaric acid, artepillin C and baccharin) isolated from the propolis or used as commercially purchased standards. This is not clear from the explanation in Fig 3.

8) It is seen that propolis formulations and their ingredients suppressed some of the anti-inflammatory agents (cytokines IL-6, IL-10, and TNF-α) and induced others. In this situation, is it possible to say that they provide an immunomodulatory effect exactly? However, these are not clearly understood from the writing style of the article.

 

9) In the phenolic profile of propolis formulations, very few components were identified in hPLC, and there are other phenolic acids and flavonoids that may be present in their ingredients. More accurate results can be obtained if a wider phenolic component profile is studied.

 

 

 

The article needs to be revised grammatically.

Author Response

Thank you very much for all contributions. It helped a lot in the improvement of the quality of this work. Follow attached all points answered.

Kind regards, Andresa A. Berretta

Author Response File: Author Response.pdf

Reviewer 2 Report

I have reviewed the article entitled "Immunomodulatory effects of three different Brazilian green propolis extract formulations in a macrophage model" for Applied Sciences. Overall, the study provides interesting insights into the effects of Brazilian green propolis extract on macrophages. However, there are several minor and major issues that need to be addressed.

Minor issues:

1. The introduction should provide more information about the role of macrophages in immune regulation, as this will help readers to better understand the significance of the study.

2. The composition of propolis and the differences between Brazilian green propolis extract and other regional propolis extracts should be explained in more detail.

3. The authors should discuss the increase in cell viability observed in all groups in more detail, as this is not well-explained in the current manuscript.

Major issues:

1. The authors should explain why they isolated bone marrow-derived macrophages (BMDMs) from normal mice and did not generate a model of chronic inflammatory disease to test the effects of the extracts. This would help to better contextualize the study and provide more insight into the potential clinical relevance of the findings.

2. The authors should investigate the effects of propolis on BMDMs while they are differentiated into macrophages, as this could have other effects on macrophage function.

3. Since evaluating the function of macrophages is important, I suggest adding a phagocytosis assay to the study to better understand the effect of the extracts on this function.

4. Assessing the expression of macrophage polarization (M1 or M2) markers by flow cytometry is a must as it will allow a more detailed understanding of the effect of the extracts on macrophage function.

 

I suggest sending the article to a native speaker or an editing company for additional review and feedback.

Author Response

Thank you very much for all contributions. It helped a lot in the improvement of the quality of this work. Follow attached all points answered.

Kind regards, Andresa A. Berretta

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors, 

Your study consists one of the greatest works that have been contacted about the propolis. 

Here are my minor comments about it: 

1) Please check how many tables and figures you can have in your manuscript. The rest of them give them as supplementary material

2) The legends of the tables and figures should be shorter 

3) Please provide information about the propolis production 

your English are fine but please revise minor changes 

Author Response

Thank you very much for all contributions. It helped a lot in the improvement of the quality of this work. Follow attached all points answered.

Kind regards, Andresa A. Berretta

Author Response File: Author Response.pdf

Reviewer 4 Report

Authors in the present study investigated Immunomodulatory effects of three different Brazilian green propolis extract formulations on the cytokines secreted by bone marrow cell-derived macrophages (BMDM) in cell culture. In addition, isolated propolis components namely: caffeic acid, p-coumaric acid, artepillin C and baccharin were also investigated.

The manuscript is comprehensive, well organized, and valuable.  I consider this document as an interesting contribution to the valorization of propolis for a potential application in pharmaceutical industries or in complementary and alternative medicine. However, the following concerns need to be addressed .

1-      Abstract:

Abstract need to be improved. Overall it is quite clear but it does not describe elements which in my opinion are of capital importance. The concentrations used (propolis formulations and isolated compounds), the stimulation of the cells studied (agent, dose, duration of treatment). However, the parameters studied are very clear.

2-      Introduction:

The introduction is well explained but in my opinion the objectives deserve to be more developed. Except for the last sentence, there is nothing that describes them

3-      Material and methods:

·         Different sections lack references. Kindly add your reference to the following section:

2.4.3. Macrophage Treatments and LPS Stimulation

2. 5. Cell Viability Assessment

·         In the section Macrophage Treatments and LPS Stimulation (2.4.3):

-There is a lot of confusion regarding the LSP concentration used to stimulate macrophages. In this section, it is 1 µg/mL. On the other hand in the section Quantification of Cytokine Levels (2. 6), the used concentration is 6%. Finally, in figures 2 and 3 in the result section, it is mentioned 200 µg/ml. Which of these concentrations was used?. All the parts must be checked again. If the concentration used is the same, this will have to be explained. If different concentrations were used. Why this difference? and it must be mentioned and justified.

-Macrophages were treated with differents propolis formulations and isolated compounds for 20 hours. Why exactly 20 hours and not 24 hours or 48 hours?

-Propolis extracts samples PPF, MPE, and PSDE concentrations were 1, 10, 25, 50, 100, or 300 μg/mL. and isolated components (caffeic acid, p-coumaric acid, artepillin C, or baccharin) were tested at concentrations of 10, 25, 50 or 100 μg/mL. Why did the authors chose to perform the tested using these concentrations? Is there a report about this?. Is it based on previous investigation not mentioned in the present study?. This choice need to be explained.

- The author chose to test as isolated compounds caffeic acid, p-coumaric acid, artepillin C, or baccharin and they mention in line 210-221 why they test the cited compounds:  « These compounds were selected to include two from the more polar fraction and two that are prenylated compounds, which is characteristic of green propolis and more apolar than the phenolic acids”. However, I do not agree with this choice and I do not find it in line with the chemical composition of the formulations tested. It would have been preferable to test all the products identified, therefore the compounds responsible for the activity would have been identified. The second choice is to include in addition to the tested products the Drupanin and 4,5 Dicaffeoylquinic acid which are a predominant compound of PSDE and MPE, respectively associated to artepillin C.

 4- Results and discussion:

The results of the present study are in contradiction with the other studies concerning the extracts or formulations tested but also the isolated compounds.

Author Response

Thank you very much for all questions and comments. Every suggestion was carefully evaluated and the manuscript were updated accordingly. Certainly, with this support, the quality of the manuscript was greatly improved.

Kind regards, Andresa A. Berretta

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Recommendations are completed.

Reviewer 2 Report

After thoroughly reviewing the revised version of the manuscript, I am pleased to report that the authors have adequately addressed all of the concerns raised in my previous review. The revisions have significantly improved the quality of the manuscript, and it is now suitable for publication in the Applied Sciences Journal.

The manuscript's English is of an acceptable standard and does not detract from the clarity or readability of the text.