Antibacterial and Anti-Inflammatory Potential of Mouthwash Composition Based on Natural Extracts

Round 1

Reviewer 1 Report

On request of Applied Science, I have revised the manuscript titled “Anti-bacterial and anti-inflammatory potential of mouthwash composition based on natural extracts” by Sung-Ho Lee et al.

By reading the abstract, it appears clear that this study aimed at confirming the antibacterial, and anti-inflammatory activity of three natural extract and at evaluating those of home-made mixtures having a different composition in the natural extracts singularly evaluated and other natural compounds, and at comparing the results with those by the same experiments on commercial mouthwashes made of chemical compounds. To achieve their goal, the authors tested the natural extracts, PG (Platycodon grandiflorum), CSK (Chaenomeles sinensis Koehne) and SG (Siraitia grosvenorii), the natural mixtures (M1-M5) and the chemical mouthwashes (GA, LIS) on eleven oral pathogens and two non-pathogenic oral bacteria, and by performing the NO essay. In addition, the same compounds were tested on macrophages Raw 264.7 cells, to evaluate their cytotoxic effects. Regarding this, the authors explain clearly, why they want to investigate natural products. They wanted to evaluate the feasibility of proposing them as ingredients for an innovative and safer mouthwash. Indeed, they have reported about the drawbacks associated to the spread use of conventional mouthwashes, containing chlorhexidine, triclosan, cetylpyridinium chloride, benzethonium chloride and fluoride which, while possessing excellent antibacterial activity, can provoke both oral and systemic diseases due to their poor selectivity for pathogenic microorganisms. On this consideration, and on the current trend to recover to natural compounds for replacing chemicals, they developed the idea to test mixture of natural products to be proposed as safer ingredients of an innovative mouthwash, which could maintain the capabilities of conventional mouthwashes, but which could be more courteous of oral environment. Anyway, it is not clear if they aim at a mouthwash totally natural or to a new a product merging the traditional ingredients and the natural extracts or mixture herein reported. The latter hypothesis seems validated at the beginning of the abstract where the authors report “in this study, the potentials of natural extract to cope with the chemical mixture mouthwash were confirmed”, while it seems confirmed the first hypothesis at the end of the abstract, when they report “It is considered that appropriately formulated natural extracts can maintain a healthy oral environment, and further replace chemical mixtures of mouthwash”. Saying “To cope with” is very different than saying “to replace”. The authors must explain better this question.

Collectively, all the manuscript is rather confused, the description of the experiments and the presentation of the results are very poor and must be improved.

As an example, according to the fact that when macrophages are stimulated with LPS, inducible nitric oxide synthase (iNOS) is expressed, producing excessive NO, which is known to induce tissue damage by promoting inflammatory response [90], the dissertation on results obtained by NO essays contained in lines 268-278, is the contrary of that correct. NO cannot be 100% before LPS treatment and 0% after LPS treatment. The authors are very confused.

Concerning the evaluation of the antibacterial activity, I believe that the evaluation of the inhibition zone, caused by the exposure of the bacteria to the sample to be tested, is an extremely preliminary test, even unreliable, quantitatively speaking. A good microbiological work involves the evaluation of the minimum inhibitory concentration (MIC), performed for example with the microdilution method, to establish the precise concentration of the tested compound or mixture capable to inhibit the bacterial growth. Authors should carry out these investigations and include them in their work for both the experimental part, the presentation of the results (using a new table) and for discussion.

 

Lines 38-39. It is a strange concept, or it has been badly expressed. Please, clarify better.

Line 56. What did the authors mean with “problems such as human body”?

Line 70. Please, remove “In”.

Line 146. Please, remove the dot after Table 1, and apply the same along all the manuscript, each time that a Table is mentioned.

Line 164. Please, replace “&” with “and”.

Line 170. For the Table title, I would suggest using “Table 3. Ingredients Contained in the natural mixture series (M1~M5).

Section 2.1. More details concerning the extraction procedures are necessary. Please, insert detailed procedures for the extraction of PG, CSK, and SG, including also extractions yields.

Line 185. Please, replace “suggested” with “reported”.

Line 207. Please, change “sample were each added” with the more correct form “each sample was added”.

Lines 204 and 215. In my opinion, to say “cells regeneration” is misleading, while a more correct form could be “cells viability”.

Line 218. Please, insert a space before nm.

Line 228. Please, remove the space after the slash.

Line 230. Please, use Figure and not Fig. Apply the same correction along all the manuscript.

Line 237. Did the authors say “tested” in place of “treated”???

Table 4. Please, reproduce the table in pages with a horizontal orientation.

Line 248. The footnote is useless.

Line 270. I think that “before” is incorrect and has to be replaced with “after”, and make the contrary in the subsequent sentence.

Lines 284-286. Badly expressed. Non suitable words have been used. Please, rewrite.

Line 300. According to the content of this sentence, it seems that oral diseases are evoked by natural compounds, which, on the contrary, should be beneficial. I suggest reformulating.

Concerning all discussion section, for avoiding further confusions or tedious repetitions, I would suggest merging it with the results section and to comment results from every experiment separately. In addition, a careful check of the English grammar is to be made, if possible, with the help of an expert.

Conclusions must be improved.

Line 366. In what experiment was the oral stability of the tested natural products evaluated?

Lines 366-368. The sentence expresses incorrect concepts. On macrophages the authors have evaluated the cytotoxicity of tested compounds and not the “phagocytosis”, as well as the NO assay was used to determine the anti-inflammatory properties of the tested compounds and not to evaluate the phagocytosis, as well.

Lines 380-382. Confused.

The references list does not respect completely the template provided by App. Sci. Please, check and correct accordingly.

The quality of this manuscript is very poor and the results concerning the antibacterial properties are rather questionable without the MICs determination. This aspect influences the relevance of the research as well as the interest for the scientific community in a very negative way.  I am forced to ask for major revisions.

Author Response

Please confirm the attached file.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is well-conducted ad -written. The results can be of interest for the readers. Generally, the authors have cited so many references, which is not recommended in original researches. However, I have some comments, suggestions, and questions to improve its quality:

 

Abstract

L29: "NO" should be defined for the first use

L30,205,256,...: revise "Raw 264.7 cells" to "RAW 264.7 cells", please

L30-32: the results can be evidenced by mentioning the values, in brief

 

Introduction

- Overall, the introduction is too long, in my opinion, it should be shorten

L51-55: please revise/rewrite "The ingredients of mouthwash .... of the teeth [4]."

L131,132: please correct "It is also known to inhibit 1,1-diphenyl-2-picrydrazyl (DPPH) radical scavenging activity [83]."

- please shortly explain reason for choosing these plants, since according to authors there is no folk medicinal application and related bioassays for oral health cases

 

Methods

- Table 2: I cannot see any difference in the mixture samples M2, M3, M4, and M5 in the text, however it is described in Table S1, please also mention in brief in the text as well

 

L155: the ratio of plant extracts in the mixtures (M1-M5) are not specified 

L 215: please format "㎕"

 

Results

- Figure 1a should be replaced with a higher quality one

L242: please mention Fig. 2 is a representative 

Table 4, please mention what are the values (units), are they "growth inhibition"? 

 

Discussion

- Since the phytochemicals are responsible for plants' bioactivities, this note is also suggested to briefly mention; on the other hand, oral antibacterial effects of the plants studied can be referred to major constituents of them, e.g. flavonoids, terpenes, phenolic acids, etc.

 

Good luck!

Author Response

Please confirm the attached file.

Author Response File: Author Response.docx

Reviewer 3 Report

This is an interesting study, with topic highly on demand, as there is a growing concern in terms of CHX safety. However, some criticism should be raised:

Keywords: they are very general, add a few more wisely according to MeSH in terms of study design and outcome

Introduction:

This section is very well written, authors provided good rationale and study hypothesis. However, there are many self-citations in here and large part of a section reads like an oral microbiology handbook (L73-L132) - I feel that cutting this part to more brief and concise style will improve the general manuscript readability, therefore self citations number would be limited, either. Yet, the most important article published recently seems missing https://pubmed.ncbi.nlm.nih.gov/33067640/ Also NO pathway should be briefly elaborated here, as there is none.

Results

Fig 1 quality must be improved, letters are barely visible now

Fig 2 is difficult to read - I suggest replacing bracketed letters on top with bacterial culture names e.g. (A) for S.constellatus and so on. Also, higher quality pictures should be used.

L279 - 'All experimental groups...' should start with a new paragraph

Discussion

This is well written and conducted study. Authors seemingly put a lot of effort into conducting this research. However, some points are missing and should be discussed here. Firstly, CBD oil as a part of mouthwash formulation - this should be addressed and discussed with relevant citation e.g. https://pubmed.ncbi.nlm.nih.gov/33526124/. Secondly, authors failed to mention Spirulina plant - there were successful attempts to formulate mouthwashes before https://innovareacademics.in/journals/index.php/ijpps/article/view/18415 also, it is a natural source of Fluoride https://www.fluorideresearch.online/epub/files/092.pdf - please incorporate and discuss at least these three articles, possibly some more as this will improve entire section.

L295 - do the authors mean microhardness?

Patents

There is some generic description here, either remove it or provide relevant information

References

There are a lot of self citations within this section, most of them may be successfully removed without declining overall manuscript quality - this stands again MDPI citation policy and good publishing practice

 

Author Response

Please confirm the attached file.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I have now completed the second-round revision for the manuscript by Sung-Ho Lee et al.

My first concern regarding this second version of the manuscript is about the title. Two different titles have been provided by the authors. In the new version (pdf) of the manuscript, the old title “Anti-bacterial and anti-inflammatory potential of mouthwash composition based on natural extracts” has been reported, while in the point-by-point response form, a new title: " Anti-bacterial and anti-inflammatory effect of new natural extracts" has been provided. What is the actual title of the manuscript???

The authors did not explain if their scope was to test and suggest natural extracts to cope with the conventional synthetic chemicals present in current mouthwashes, or to replace them. Indeed, even if the authors have modified the abstract, the points concerning my requests have not been modified. 

In the abstract, the authors say that the anti-inflammatory properties of the compounds have been evaluated by the NO assay tested with LPS. It is strongly incorrect. LPS has been used to induce NO production and create a model of inflammation. The anti-inflammatory properties were evaluated by the Nitrate colorimetric assay. 

Line 83. Please, remove the dot in the name of bacterium.

Lines 123-124. The authors have added a sentence which, as expressed, provides a concept strongly incorrect. They said, "It is also known to inhibit 1,1-diphenyl-2-picrydrazyl (DPPH) radical scavenging activity [40]". The DPPH has not a radical scavenging activity. Anyway, a compound that inhibits the radical scavenging activity is a prooxidant one. I think authors refer to the evaluation of the antioxidant properties of one of the considered extracts by using the DPPH essay, with which the radical scavenging activity of a compound is evaluated by quantifying its potency in reducing the number of the DPPH radicals. To say that a substance inhibit the DPPH radical scavenging activity is non-sense. 

Along all manuscript, please use the form “µL” to abbreviate microliters.

Please remove Section 6.

Even if I have notified that the references list did not respect the App. Sci. template, and the authors asserted that they have modified it accordingly, it has not been modified adequately. The years are not in bold, all titles of journal must be abbreviated and those abbreviated must contain dots.

 I recognize that some work has been done by the authors to satisfy my requests and compensate for the shortcomings and confusion present in the original version, however even in this form the manuscript is still a long way from being publishable. What then left me perplexed is that, in adding new parts, probably at the request of other reviewers, the authors provided incorrect information and generated further confusion. 

 

Author Response

Thank you so much for your important and valuable opinion.

Please confirm the attached file.

Author Response File: Author Response.docx

Reviewer 2 Report

The revised version of manuscript can be considered for further publication procedure, in my opinion.

 

Good luck!

Author Response

Thank you for your comment.

Reviewer 3 Report

An entire manuscript was very accurately edited, therefore improved greatly. I am more than satisfied now. Just one more point:

Reference 51 should be cited as follows: Kałduńska, J.; Jakubczak, K.;  Gutowska, I.; Dalewski, B.; Janda, K.; Fluoride content in dietary supplements of spirulina (Arthrospira spp) from conventional and organic cultivation. Fluoride, 2020 : vol. 53, nr 3 pt 1, pp. 469-476, p-ISSN: 0015-4725

Good luck with publication and patents though!

Author Response

Thank you for your important point. This was corrected as indicated. (Line 522-523)

Round 3

Reviewer 1 Report

Even if my opinion is similar to the previous I think that this article could be published on Applied Sciences. 

Author Response

Thank you for your comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The article “Anti-bacterial and anti-inflammatory effect of mouthwash composition based on natural extracts” by Sung-Ho Lee and Won-Hyeon Kim et al. describes a study on the antimicrobial properties of a mouthwash composed of natural extracts as the active ingredients and comparing it to commercially available similar products.

The subject is important as frequent use of antimicrobials and disinfectants of synthetic origin is often associated with increase of the microbial resistance, as well as the ecology-related factors are beneficial. What is more, the possibility to develop a product that would enable elimination of pathogenic bacteria without affecting the natural microbiome of the oral cavity would be greatly welcome.

 

Compared to the previous version of the manuscript some improvements were implemented but certain comments still remain unanswered and thus the article is still unsuitable for publication in its current state. The most significant comments that would have to be addressed include:

1. Based on the presented materials and methods only M1 can be distinguished from M2-M5, as the given composition differs qualitatively. Currently, presenting the results for five quantitatively different gives the reader no information and the experiments could not be reproduced by any other scientific group. This is in contrast to the policy of the Materials and can be found in the Aims & Scope of the journal: “The full experimental details must be provided so that the results can be reproduced.”, see https://www.mdpi.com/journal/materials/about. If you are reluctant to share such information, you should first file a patent for the composition or release a commercial product that would be available to compare for the others.
2. What type of extracts (PG, CSK, SG) were used – what was the solvent for the extraction? What was the plant fragment? Without this information it would be difficult to assess the group of phytochemicals responsible for the studied activity.
3. Did you perform a control experiment using only the adjuvants (vehicle without the extracts)? I noticed that you mentioned that all the extract mixtures M1-M5 do contain a large portion of “Mint extract powder”, most probably as a colorant/fragrance. What was the genus of the mint? Such information should be checked, as i.e. Mentha x piperita exhibits not only antimicrobial but also anti-inflammatory properties which could affect the obtained results (see: doi.org/10.3390/antiox7120184 and doi.org/10.1016/j.arabjc.2011.01.019). Similar effect can be ascribed to propolis, although at higher concentrations.
4. Why do you suppose the natural microbiota was not affected while the pathogenic strains were susceptible to the tested compositions?
5. It strikes me that the microbial viability values are given as %, while you were using disc diffusion method. It would be more appropriate to give it as mm of clear zones.
6. The MTT test is a standard in cell viability calculations, but it is known that certain types of compounds, including especially plant extracts and polyphenols present in them, can interfere with the results (doi.org/10.1055/s-2002-32073; doi.org/10.5246/jcps.2010.03.027). Did you consider such phenomenon in your studies?
7. You mention MBC in line 298 but no information of calculation of these parameters can be found in-text. In case such calculations were performed, wouldn’t it be better to include it in the manuscript?

Less significant remarks:

1. Starting from line 73 – when a cell line is mentioned for the first time, its full name should be given.
2. What do you mean by “enzyme salt”?
3. You write that “Long-term use of mouthwash develops resistance due to various kinds of antibiotics contained for effective inhibition of oral microbes” which would imply that OTC mouthwashes contain antibiotics.
4. The Figure 1 might be misleading as the scales in a) and b) are rising in the opposite directions.
5. Please refer to “acidic” rather than to “were acids” (lines 213 and 214).
6. In table 4 you mention that each number represents a percentage. Do you mean the percentage of the control experiment? It should be specified.
7. Please rewrite the conclusion 2 in lines 338-341 as it is unclear.
8. There are many typos and English/style errors throughout the text. Most striking are: mouthwash (line 21); synthetic materials (22); its mixtures (29); sterilize bacteria (49); lead to cause (58); colors (colorants) (61); phosphated-buffered (186); 100 mg/ml of sample were each added 100 μl (line 192); bacterial instead of bacteria (220 x2, 221 x2, 226, 290 x2, 330); observed with a chemical mixture (228); italic in names of the bacteria (230); due to natural extracts (260); tis is considered that it does (300-301); had been observed (332).

Author Response

Please confirm the attached file.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors reported the evaluation of several natural extracts for the anti-bacterial activity, anti-inflammatory effect and cell viability. This article shows interesting results, but more aspect needs to be improved before I recommend its publication in Materials.

• Please write the number of replicates for each test in the legend of figures and in the Materials and methods section.
• Please write with more details the procedure for the antibacterial test.
• Please specify that the numbers in Table 4 represent the percentage of bacterial growth inhibition. Please specify which is the positive control and negative control.
• Please correlate the obtained results with the properties derived from the composition of each sample.
• The last phrase at lines 344-346 is not correct. You have to correct it. It was an antibacterial effect against pathogenic oral microorganisms and an anti-inflammatory activity revealed on macrophages.

Author Response

Please confirm the attached file.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript, although improved at some extent, is not improved significantly. The first four comment were not addressed appropriately. The added supplement is a huge block of text – probably a fragment of the patent claim, as the page numbers can be found it-text – describing the technical aspects of preparation of certain components of the mouthwash and many information that is not supported by citations. It is still however unknown how M2-M4 differ from each other.

In my opinion it is still unfit to publish in Materials, as it presents little scientific value.

Reviewer 2 Report

The authors addressed all the comments and the manuscript has improved.

I recommend its publication in Materials.