Anti-Inflammatory and Antioxidant Properties of Ethanol Extracts of Raw, Blanched, Steamed, and Sous-Vide Cooked Okra (Abelmoschus esculentus L.) in LPS or H₂O₂-Treated RAW264.7 Cells

Round 1

Reviewer 1 Report

The manuscript describes a set of in vitro experiments which evaluate alcoholic extracts of okra preparations in murine macrophage cell line. The purpose of this study is not clear. The authors do not supply any credible literature evidence that okra intake modulates inflammatory processes in humans. The study lacks a clearly formulated research question. A mere fact of the anti-inflammatory effects of okra extracts has been previously established in both cell culture and animal studies.

Introduction. Provide the following information that will help readers to understand the rationale: i) clearly state, are there any intervention human studies, such as randomized clinical trials, any prospective human studies, such as cohort studies, or any observational case-control studies that have established a relationship between consumption of okra and risk of the inflammatory disease or related physiological responses. References to laboratory experiments in animals, cell culture or anecdotal and folk tales will not satisfy the case. If no such studies are available, then clearly state it; ii) provide a summary of laboratory studies on anti-inflammatory effects of okra in animal and cell culture models. What is not known that you are going to reveal; iii) provide a justification of using mouse cells, rather than human isolated macrophages. Explain here, why RAW264.7 line is a good model for human gut resident macrophages

Line 31: The references 1-3 are in vitro studies and thus are irrelevant to the statement, which must be supplied by references to clinical and epidemiological human studies.

Line 36: The references 4-5 are irrelevant to the statement

Line 39: The references 7-8 are irrelevant to the statement

Line 42: The references 10 is irrelevant to the statement

Experimental part: i) Provide chemical analysis of the okra extracts, specifically rutin/quercetin and mucilage polysaccharide, as these are thought to be the principal anti-inflammatory components of okra preparations; ii) Perform a comparative experiment using human isolated or immortalized (like THP-1) macrophages. Such experiment will provide for some relevance to your study.

Discussion: Analyze correlations between chemical composition of the extracts and their activities in your experiments.

Author Response

"Please see the attachment"

 Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this study, the anti-inflammatory effect of okra extracts, prepared using four different cooking methods, namely raw, blanching, steaming, and sous-vide, were investigated using lipopolysaccharide (LPS)-induced macrophages.

It was demonstrated that all cooking methods inhibited nitric oxide production (indicator of inflammatory responses), thus confirming the anti-inflammatory and antioxidant effect of raw and multi-method cooked okra. 

The work is conducted with adequate means. The paper is overall well-written.

My only concern is about the Introduction. It should be more extended and a critical comparison with previously published papers on the topic need to be reported. 

Author Response

"Please see the attachment"

 Thank you.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors discussed anti-inflammatory and antioxidant properties of Okra (Abelmoschus esculentus L.), a plant grown in many countries with tropical climate. The work has a great cognitive value due to the importance of determining the health-promoting properties of food raw materials, and its high scientific value also results from the using of technically advanced analytical methods. The article is written in clear, specific and concise language, and authors should only make a few minor corrections before publishing it. They are indicated in details in the attached revised manuscript, but here I will mention only the most important of them:

1. Table 1 is scattered (pages 5 and 6) and it contains an unclear way of marking statistically significant differences between individual values – all values in the table should be marked with specific letters. Different sized letters should be used to denote differences between the values in the rows (differences in the method of heat treatment) and in the columns (differences between the applied amount of the additive) The table also lacks the units in which all values are expressed.
2. Table 2 and 3 also should contain the units in which the given values are expressed, and additionally all values given in Table 2 should be marked with the appropriate letters.
3. The title of Figure 4 and the beginning of its description are not visible.
4. Literature is cited inconsistently - all names of journals should be written in the same way, preferably in the form of permitted abbreviations given in italics, obligatorily with the appropriate DOI number.

Comments for author File: Comments.pdf

Author Response

"Please see the attachment"

 Thank you.

Author Response File: Author Response.pdf

Reviewer 4 Report

1. The title should be rewritten. The title does not need to list all the parameters measured in the experiment.
2. English should be checked and corrected in the manuscript. 
3. Minor typing mistakes should be corrected (eg. some full stops and spaces have been omitted).
4. The Introduction should be rewritten. There is no information about the chemical composition of the okra parts. Is there anything known about the most important active compounds present in this plant? What type of processes may occur in the plant during the presented type of cooking? It should be explained in the Introduction or in the Discussion.
5. Line 41: aging is not a disease, the term "unsuccesful aging" could be used.
6. I have some doubts about the design of the study. The control experiments are not described enough. Why 0.1% DMSO was used as a vehicle control - the okra extracts were in ethanol and there is no information if DMSO was used in the extraction of okra? Was the solution of DMSO done in ethanol? If not, We cannot be sure if the inhibitory effects come from okra or from ethanol itself.
7. In Table 1, the results of control should be added.
8. Subchapters 2.4 and 2.8 - the chemical background of the methods, not only the procedures, should be described.
9. Some parts of Results discusses the experiment results with some other studies, it should be moved to the Discussion section.
10. Figure 2: I suppose there is an error in the column captions. The second column is: 200ug/ml (what solution?}. In the description the group of 25ug/ml of okra is mentioned - it is not included in the Figure 2. The control group is not explained in the description.
11. ROS means reactive oxygen species, not radical oxygen species.
12. The research manuscript should have a section Discussion. It should be extracted from the Results section. The Results section should be carefully rewritten and Discussion should be added.
13. The Conclusions section should conclude the results, not only summarize them. It could be more general.
14. I do not understand why, in the figures, control groups have four bars, separately for every type of cooking. I suppose, the control was one for the experiment.

Author Response

"Please see the attachment"

 Thank you.

Author Response File: Author Response.pdf

Reviewer 5 Report

The article entitled "Anti-inflammatory (COX-2, iNOS, TNF-α, IL-6, IL-1β) and Antioxidant (ROS) Properties of Okra (Abelmoschus esculentus L.) Extracts in LPS / H2O2-treated RAW264.7 Cells "presents very interesting results and the authors have done a lot of experimental work. In general, the results are well organized, although a detailed review is necessary covering the following aspects:

Authors must be more careful with the way of writing, a detailed review of English must be carried out

Further comparison with the bibliography should be incorporated into the revised manuscript.

The introduction should be improved, highlighting in more detail the importance of this study and indicating what is already done in the bibliography.

The authors should provide data on the composition and antioxidant profile of the extracts used.

Author Response

"Please see the attachment"

 Thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

In the revised version of this manuscript, unfortunately, the authors have not satisfactorily addressed any of the criticism and suggestions expressed by this Reviewer.  The anti-inflammatory effects of okra have been established previously in experimental animals, and recommendations of nutritionists in regard with inclusion/exclusion of okra in diets of patients with inflammatory disorders are available, as well. With such background in place, it is expected that any new in vitro studies would supplement existing knowledge with novel information, such as molecular mechanisms of the phenomenon. This manuscript deals with evaluating effects of okra extracts at the in vitro level, and its main conclusion is that different cooking methods of okra provide different “anti-inflammatory activities”.

In this work, only one cell line, mouse macrophage, has been employed, so that it is not known whether the results would be reproducible in human cells, let alone any organism. No need to remind that inflammation is a phenomenon at the organism level, so any in vitro studies by no means can claim more than what has been observed. Yet, in this manuscript, the authors go as far as “sous-vide cooking could be recommended method to prepare okra, as this improves the plants anti-inflammatory and anti-oxidant properties” (page 15, line 468). Clearly, this is an irresponsible and misleading statement.

In this work, no attempt was made to control composition of the okra preparations, so that the observed differences in their effect on cells could be a pure chance. Meanwhile, a number of studies that have identified chemical entities responsible for the anti-inflammatory activities of okra are available and should be guidance for establishing any relationship between okra preparation and its biological activity.

Referencing to irrelevant citations is yet another weak point of this manuscript. The authors’ explanation that they are short on time to improve the manuscript is unacceptable.

All in all, data presented in this manuscript could be a useful supplement to a truly novel, relevant and meaningful study. This Reviewer cannot recommend its publication as a separate paper. 

Author Response

Dear Reviewer,

We are sincerely grateful for your meticulous advice.

We revised this manuscript according to reviewer's comment.

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In the revised version of this manuscript, unfortunately, the authors have not satisfactorily addressed any of the criticism and suggestions expressed by this Reviewer. The anti-inflammatory effects of okra have been established previously in experimental animals, and recommendations of nutritionists in regard with inclusion/exclusion of okra in diets of patients with inflammatory disorders are available, as well. With such background in place, it is expected that any new in vitro studies would supplement existing knowledge with novel information, such as molecular mechanisms of the phenomenon. This manuscript deals with evaluating effects of okra extracts at the in vitro level, and its main conclusion is that different cooking methods of okra provide different “anti-inflammatory activities”.

-->I agree with this opinion. The effect of Okra has been well known, therefore, we focused the effective cooking methods for its real supplement. And SVOE showed better anti-inflammatory (and anti-oxidant) activities via COX-2 and iNOS expressions. Its detail molecular mechanism should be studied further, and this description was added in Discussion.

In this work, only one cell line, mouse macrophage, has been employed, so that it is not known whether the results would be reproducible in human cells, let alone any organism. No need to remind that inflammation is a phenomenon at the organism level, so any in vitro studies by no means can claim more than what has been observed. Yet, in this manuscript, the authors go as far as “sous-vide cooking could be recommended method to prepare okra, as this improves the plants anti-inflammatory and anti-oxidant properties” (page 15, line 468). Clearly, this is an irresponsible and misleading statement.

--> As preliminary study, we studied the effect of Okra in Raw 264.7 cells, as representative cell for anti-inflammatory effect. And the results suggested its anti-inflammatory and anti-oxidant properties. Therefore, we deleted irresponsible and misleading statement that you pointed out. And we add the description about the necessary of further study on different cell lines.

 

In this work, no attempt was made to control composition of the okra preparations, so that the observed differences in their effect on cells could be a pure chance. Meanwhile, a number of studies that have identified chemical entities responsible for the anti-inflammatory activities of okra are available and should be guidance for establishing any relationship between okra preparation and its biological activity.

-->I agree with this opinion. Biological activities of okra is extremely important. It should be confirmed further study.

 

Referencing to irrelevant citations is yet another weak point of this manuscript. The authors’ explanation that they are short on time to improve the manuscript is unacceptable.

--> Reference style was revised.

 

All in all, data presented in this manuscript could be a useful supplement to a truly novel, relevant and meaningful study. This Reviewer cannot recommend its publication as a separate paper.

--> Thank you for your kind review.

Reviewer 4 Report

After the revision, I recommend the manuscript to be published after minor revision done.

1. New title is ok for me, please change the old title to the new one.
2. The sentence in the abstract in yellow color should be rewritten (the verb is omitted in the second part of this sentence).
3.  The subchapter 3.5 - the title should be: "Reactive oxygen species (ROS)". 

Author Response

Dear Reviewer, 

Thank you for your kind check. I revised this manuscript according to the comments. 

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After the revision, I recommend the manuscript to be published after minor revision done.

1. New title is ok for me, please change the old title to the new one.

--> It was revised. 

1. The sentence in the abstract in yellow color should be rewritten (the verb is omitted in the second part of this sentence).

--> It was revised. 

1. The subchapter 3.5 - the title should be: "Reactive oxygen species (ROS)". 

--> It was revised.