Peer Review

Dear Ms. Celine Wan,

We thank the editor and the reviewers for the careful reading of our manuscript and their insightful suggestions. We have addressed the various issues raised and have revised the manuscript accordingly. We hope you will find the revised version suitable for publication in Pharmaceuticals.

Reviewer #1

Comments and Suggestions for Authors

In this paper, it is entitled "Inhibition of respiratory RNA viruses by a composition of ionophoric polyphenols with metal ions ".In vitro experiments were demonstrated that ionic phenols in complex with metal ions can attenuate respiratory RNA virus replication.The component cytotoxicity was mainly assessed by MTT cell viability test, and the results verified that the selected compounds were safe. Then, the real-time reverse recording effect of PCR (qRT-PCR) was tested. The study results showed the reduction of respiratory RNA virus replication.The significance of this research is that a mouth can be developed Taking bioavailable therapies to attenuate the replication of this pathogen and other respiratory RNA viruses provides a novel approach for the treatment of respiratory RNA viruses. However, there are some errors in the manuscript in this paper, and the comments that we summarize are shown below.

We thank the reviewer for the thorough reading of the manuscript and for appreciating the novelty of our work. We are also thankful for the important remarks on the structure of the manuscript.

Main points:

1、Abstract, Content

The purpose, method, results and conclusion of the article are not expressed in the abstract, and the results part lack detailed data support;

In the English statement is lengthy, example the second sentence, suggested modification;

 

We thank the reviewer for the suggestion. We have revised the abstract to better provide the description of purpose, method, results and conclusion and provided in it more detailed data support.

Alongside several additional small textual changes that are evident in the track changes version, The revised parts are as follows: 

Lines 16-29: 

“However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50-95%) decrease in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.”

2、Introduction

The individual sentences in the introduction are not accurately expressed. For example in the third sentence in the introduction, " Since its first outbreak in Wuhan, China, in December 2019, it has spread worldwide at an unprecedented rate, causing the COVID-19 disease."this statement is misstated and its content is not causal.Examples include " Patients infected by SARS-CoV-2 may exhibit either no symptoms or a spectrum of symptoms including fever, dry cough, muscle weakness, and pneumonia."In this statement, pneumonia is not a symptom associated with fever, dry cough, and so on.

 

We thank the reviewer for raising this issue. Both sentences are now revised to reflect more accurate statements. The revised introductory parts read as follows:

Lines 35-37:

“This disease, caused by a novel coronavirus - the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally during the past two years at an unprecedented rate[2].”

Lines 41-44:

“While some patients infected by SARS-CoV-2 exhibit no symptoms, others present a spectrum of symptoms including fever, dry cough, muscle weakness, and some may experience pneumonia[4–6], comparable to the manifestation of flu[7].”

The quote content uses errors in Chinese and English symbols, for example, the fifth sentence semicolon.

 

We double-checked for the absence of incorrect symbols. However, we could not find the above-mentioned symbols and we assume that it was the technical result of file uploads/format conversions. If nevertheless some symbols require modifications – we will appreciate letting us know.

The first paragraph of the introduction is confusing, such as the disease pandemic and mortality repeat; such as the absence of the pathogenesis of the COVID-19 disease, with less discussion of experiment-related respiratory RNA virus diseases.

 

As described above, we have revised the first paragraph of the Introduction to meet these comments.

3、Experimental part

Only in vitro experiments, no in vivo experiments show, the scientific arguments are not sufficient.

The serial number was not marked, and the structure arrangement was chaotic.

 

We thank the reviewer for the comments on the experimental part. Indeed, our results are in vitro and cell-based. Clinical verification of efficacy in humans is pending. However, given the current status of the pandemic and the spread of related respiratory viruses, and since all of the compounds used in this work are GRAS and have been used extensively as dietary supplements for years, we believe it is in the interest of the scientific and general community to be aware of our present findings. 

Nevertheless, to better emphasize the reviewer’s comment, we concluded the Discussion section with the sentence: “The value of these combinations awaits in vivo and human trials”. 

We corrected the experimental part to reflect the results more accurately. We have provided a better edit for all of the figures. 

We do not understand the remark regarding the serial number. The structural arrangement of the manuscript was performed as descried.

4、conclusion

The conclusion part is chaotic, such as in the last paragraph of the conclusion, the first and second sentence has no obvious correlation;

Conclusion part lacks their own thinking and outlook.

 

We revised the conclusion part to be more organized. We have, however, written "we infer that viral inhibition by the combinations examined is rather general and thus should be helpful against a variety of viruses, particularly RNA viruses associated with winter respiratory infections". In addition and as a cautious outlook (mentioned above) we have added that:" The value of these combinations awaits in vivo and human trials".

In words， there are some syntax errors. Please reexamine the manuscript; For the introduction and the conclusion of the related content discussion, please pay attention to the content logic.

We have carefully proofread the manuscript to correct any grammatical issues.

Reviewer #2

Comments and Suggestions for Authors

                This manuscript reports the impact of combinations of zinc picolinate, copper sulfate, EGCG, quercetin, and taxifolin  +/- naringenin on cell viability and the growth of several RNA viruses on a variety of cell lines.  Overall the experimental design is straightforward and much of the data supports the conclusions drawn.  However as noted below, there are several points that need to be addressed to provide rigorous support for the conclusions that are made.

We thank the reviewer for the comprehensive reading, important suggestions and positive evaluation of the manuscript

Major Points:

1:  Since an MTT assay assesses an aspect of cell metabolism rather than cell proliferation per se, it might be worthwhile for the authors to also directly assess cell growth in the presence of these compounds in order to provide additional support for their conclusion that they are ‘safe’.  In addition, establishing safety of a potential drug requires more than cell culture assays, so I would encourage the authors to tone down their conclusion of ‘safe for use’ from this one experiment. 

 

Following the advice of the reviewer, we have toned down the statement. The revised text is as follows:

Lines 134-137:

”The tested compounds did not impair the viability of either A549 cells (Figure 1A-E) H1299 cells (Figure 1F), Vero cells (Figure 1G), and SH-SY5Y cells (Figure S1A) upon treatment with the indicated selected compounds and concentrations. The results verified that the selected compounds did not possess a significant cytotoxic effect as determined by the MTT assay.

3E:  The authors conclude that combination 1 shows significant inhibition of hMPV viral replication – but I do not see an asterisk on the graph indicated a significant p value for combo 1.  Please address this discrepancy.

4D: The statement in the text ‘Vero cells that were treated with the combinations, especially with Combination 2, showed reduced levels of the hMPV Phosphoprotein (P) gene (Figure 4D).’ is simply not supported by the data shown.  Only one combination surprisingly had an effect on P gene expression.  Thus the data do not support the conclusion that is drawn.

 

We thank the reviewer for these comments. Since Comments 2 and 3 are related, we provided a combined reply. Both Figures 3E and 4D represent the evaluation of viral replication of hMPV using FACS and qRT-PCR (respectively); we see that Combination 1 has little to no effect on inhibiting viral replication. The only difference between Combination 1 and Combination 2 is the presence of naringenin in the latter. This may imply the importance of naringenin and the additive effect in the case of hMPV infection.  

The revised text is the following:

Lines 220-223

“Vero cells that were treated with the combinations exhibited a different trend, with only Combination 2 reducing the levels of hMPV Phosphoprotein (P) gene (Figure 4D) and this correlates with Figure 3E, indicating the importance of naringenin for halting hMPV replication.”

Do the combinations need to be added 4 hrs prior to viral infection to see an effect or can they be give at or after viral infection?

 

We thank the reviewer for this comment. In all the experiments described in the manuscript, the combinations were added 4 hours prior to the infection This mimics a scenario where such GRAS compounds are taken as prophylactics. We haven’t tested adding the combinations at or after infection. 

The revised text is the following:

Lines 399-401

“Furthermore, we mimicked a scenario where such GRAS compounds are taken as prophylactics and demonstrated that the combinations are highly effective against various RNA viruses in vitro.”

Minor Points:

The introduction of the manuscript begins with a very long paragraph that would be much more effective if it were broken up into two or three paragraphs to ensure that the various points that it is making land with the reader.

 

As described above, we revised the beginning of the Introduction.

2C: Viability is misspelled on the y axis of the graph.

 

We have corrected the spelling

Fig 4: Combination 1 reads ‘combinatic’ on all of the x axes.  Please correct the spacing of the labels so that all are readable.

 

We have corrected the typo

Fig S3: given the importance of these data to a mechanistic interpretation of the action of the combinations used, I would strongly encourage the authors to move these data into the main body of the manuscript.  In addition, I’m surprised that the last combo tested is not also significant (i.e.  why is there no asterisk on that bar?)

 

Figure 3S was corrected and added to the manuscript (now Figure 5)