PONE-D-24-51141Assessing the biomedical applicability of biogenically synthesized AuNPs using Salvia splendens extractPLOS ONE

Dear Dr. Tiwari,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I reviewed this paper; Therefore, I cannot recommend this manuscript to be published unless the authors answer all comments. It needs "major revisions" before acceptance for publication.

1- Synthesis green mechanism of Au-NPs must be added to text.

2- FTIR spectra of Au-NPs and Salvia splendens extract must be added to text.

3- PSA curve of Au-NPs must be added to text according TEM image.

4- XRD pattern of Au-NPs must be added to text.

5- Last paragraph of the introduction is not appropriately written. Explain the main objective of this work.

6- Overall, the discussions are not enough and the different sections must be described scientifically.

7- Authors should be appropriate to carefully check the compliance of the article with the English grammar rules by an academic whose native language is English. The English of the entire article should be improved. With more caution, many mistakes can be avoided.

8- References should be reviewed, and referring to more up-to-date references would be better.

9- The abbreviation should be mentioned completely for the first time and mentioned after that as an abbreviation. Please revise throughout the manuscript.

10- To give a better view to this work, authors are suggested to improve the introduction and other sections using recent and related articles.

DOI: 10.1016/j.inoche.2024.113399

DOI: 10.1109/TNB.2023.3287805

DOI: 10.1016/j.ceramint.2023.03.234

DOI: 10.1007/s12010-023-04407-y

DOI: 10.1007/s12257-024-00004-w

Reviewer #2: Comments to authors

The authors have studied the in vitro antioxidant, anticancer, and anti-inflammatory activities of biogenically synthesized AuNPs using Salvia splendens extract. The research work is informative and interesting. However, some inadequacy was noted in the manuscript which needs to be addressed for the improvement of the quality of the research article considering the publication criteria of the highly esteemed journal like Plos One. The comments are as follows:

1. The introduction seems to be too preliminary and there is no significant literature support for the natural products-based antioxidant, anti-inflammatory, and anticancer activities. In addition, there needs a better description on the bioactive molecules in the plant extract/ similar plant species which could be responsible for their plausible potential.

2. What is the significance of oxidative stress and inflammation in the development and progression of lung cancer? Authors could stress these details in the introduction part.

3. Incorporate the machine generated data in the figure 1d instead of incorporating the data manually.

3. Incorporate a size distribution graph in the TEM results.

4. The authors should explain the basis for keeping treatment duration for 24 h in MTT/ cytotoxicity assay.

6. In vivo work would have been strengthening the present findings.

7. There are numerous published reports demonstrating the potential of synthesized plant-based nanoformulations. Hence, what is the novelty and exclusiveness of this work needs to be explained.

8. How can the authors corroborate the antioxidant and anticancer/ cytotoxicity of the extract?

9. The concentration of DPPH solution (100 mM) used in this study is well beyond the recommended level. It should be in micromole (µM) concentration.

10. The absorbance of formazan is a peak at 570 nm. The authors need to clarify why they have used 490 nm for the MTT assay.

11. Annexin V-FITC/PI apoptosis assay and immunoblotting of apoptosis markers would have further strengthened the results of present investigation.

12. Why didn’t the authors determine the cytotoxic effect of SSLE-AuNPs on J774A.1 cells? The decrease in cytokine levels is due to the anti-inflammatory activity or cytotoxicity of SSLE-AuNPs?

13. Authors are suggested to include one inclusive section in the introduction part citing some recent references on the usage of natural compounds and green synthesized nano-formulation as an anticancer agent and their application so that the horizon of the current article could be improved further. Below articles could be covered in that recommended section:

i. Ahmad et al. (2024) Cell Biochemistry & Function, 42: e3911.

ii. Suhail et al. (2023) Frontiers in Pharmacology, 14:1236173.

iii. Ahmad et al. (2023) International Journal of Molecular Sciences, 24 (7): 6651.

iv. Suhail et al. (2023) Frontiers in Nutrition, 9: 1078642.

v. Nazam et al. (2023) Pharmaceuticals, 16: 274.

vi. Zughaibi et al. (2023) Cell Biochemistry & Function, 41: 1174-1187.

vii. Alafaleq et al. (2023) Nanomaterials, 13 (7): 1201.

viii. Alserihi et al. (2022) Nanotechnology Review, 11: 298-311.

ix. Tabrez et al. (2022) Nanotechnology Review, 11: 2714-2725.

x. Tabrez et al. (2022) Nanotechnology Review, 11: 1322-1331.

xi. Tabrez et al. (2022) Frontiers in Chemistry, 10: 970193.

Reviewer #3: The manuscript introduces interesting and valuable information on a multiple application of green-approach synthesized AuNPs. However, since the manuscript contained a lot of information, the authors must carefully check all method and discussion to control the quality of the manuscript. I believe the manuscript can be published in the journal with following modification.

1. “S. splenders” should be uniformly written in Italic

2. What is “S. spp” in page 3, line 75-76 stand for?

3. The introduction was well arranged and written. However, I suggest that the authors should expand this section with more literature review. For e.g., based on the main content of this paper, I recommend that the authors could write a review paragraph focusing on the progress of other authors’ contribution to treating lung cancer cells. Introduction of the relationship of all the studied factors in the manuscript to lung cancer would give better comprehension to readers.

4. Considering that the references used in this paper were only before 2024, the authors should update the references published in 2024 for “up-to-date” purpose. Combing this comment with my previous recommendation would be reasonable.

5. In section 2.2.3, specific ratio of dried leaves and DI water (weight/volume) should be presented. In addition, if the extraction was only conducted with crushing the dried leaves with DI water at room temperature without any heating, then specific extraction time should be noted. Furthermore, practical images of DI water and leaves extract (before and after extraction) should be included in the paper for better visual and reliability.

6. The authors used the terms “enzymes”, “proteins” throughout the manuscript. Due to the different of expertise, I could not confirm whether using these terms would be appropriate or not. Nonetheless, with the information the authors mentioned in the introduction section, I think “phytochemicals” would be a better and more reasonable interpretation.

7. In section 2.2.4.1, the authors should specifically mention how they prepared AuNPs suspension? For e.g., how would they prepare AuNPs suspension with a content of 50 µg/mL in section 2.2.5.1? Would the nanoparticles be centrifuged, dried and redispersed in water or the weight/volume were used as theoretical values (considering all Au3+ ions were converted into AuNPs)? This information should be clearly confirmed in the manuscript.

8. In page 7, line 161, should “bank” be “blank”?

9. The citation of references 15, 16, and 17 should be carefully revised by the authors, considering that the term “slight modification” would not be appropriate comparing the authors’ methods with the ones in references. As for reference 17, in this literature, there was no information about the ABTS activity assay method? If the authors still want to use these references, I suggest that these reports could be mentioned in the introduction section. Otherwise, in the response letter, the authors should clarify why those references was used and cited in those positions?

10. Usually, in a ABTS assay method, K2S2O8 would be introduced to react with ABTS to produce free radical ABTS·+ cations, then the antioxidant activity of the sample was measured by the ability to convert free radical ABTS·+ cations into ABTS (monitoring by the reduction of UV-vis absorbance at 734 nm, characteristic for the existence of ABTS·+). Herein this work, according to the experimental information in section 2.2.5.2, why the authors let the AuNPs colloid react with ABTS (no free radical ions)? Why was a 50% methanolic ABTS used as blank but not a solution containing ABTS·+? The authors must carefully revise this problem because this would induce question about the reliability of this work.

11. The authors should clearly mention that ABTS was used as solution form or solid form (diammonium salt) in the Material section. In the 2.2.5.2 section, I suspect that the ABTS was directly used as commercial solution since there was no specific concentration mentioned. On the other hand, in page 15, line 318-319, ABTS was mentioned as diammonium salt. This is confusing, the authors must clarify this problem.

12. In section 2.2.6.1, the volume of sample injected in each well should be specifically noted.

13. Additional analysis to confirm the formation of AuNPs (e.g. XRD, SAED, etc.) should be carried out.

14. The statement in page 13, line 279-281 was suggested to not written if there was no evidence (UV-vis spectra of AuNPs colloid with different storage duration) attached in this manuscript.

15. According to reference 32, the absolute zeta potential value needs to be higher than 30 to indicate a stable colloidal system: “Conversely, a high zeta potential (either positive or negative), typically more than 30 mV, maintains a stable system”. Even though the comment on the zeta potential for a stable colloidal system would be controversial, it was commonly concluded that an absolute zeta potential higher than 30 mV would be corresponding to a stable colloidal system (Surendra Nimesh, Ramesh Chandra, Nidhi Gupta, Advances in Nanomedicine for the Delivery of Therapeutic Nucleic Acids, 2017; https://doi.org/10.1002/cjce.23914). Hence, the discussion stating that the AuNPs colloidal system having a zeta potential value of 21 mV was stable should be carefully reclaimed.

16. How the authors analyzed the average particle size from TEM image (Fig. 1D)? As the Fig. 1D illustrated, the largest particle diameter would be around 80 nm, which indicated that the average size would be a value that is smaller than 80 nm. The authors can consider use another TEM image with larger scale to observe more particles, which might induce better average particle size value.

17. In addition, particle size distribution graph should be included together with TEM image. From this analysis, the difference or the relationship between average particle size obtained from TEM image and DLS analysis should be discussed.

17. p-value is used for hypothesis testing in statistic study. Therefore, p-value should not be used in this work to interpret for average value of 3 measurements.

18. Based on the results studying DPPH and ABTS assays, IC50 values of SSLE-AuNPs (µg/mL) could be determined and compared with IC50 value of ascorbic acid (standard in authors’ study). This result could be more valuable and replaced for simple statement of “The SSLE-AuNPs exhibited a dose dependent anti-oxidant activity as manifested by substantial neutralization of DPPH and ABTS radicals” in the abstract.

19. If the MIC50 values corresponding to the antibacterial activity were determined from the graphs in Figure 3, then the graphs should be illustrated in form of line + symbol instead of curve + symbol.

20. If the authors aimed to provide information about the as-synthesized AuNPs for medicinal purpose (treating lung cancer cells), then the cytotoxicity of SSLE-AuNPs on human cells should also be studied. A limit of using AuNPs concentration could help authors determined specific parameters in lung cancer cells treating, instead of just introducing relationship of colloidal concentration to the viability assay study.

21. In the cytotoxicity assay study on lung cancer cells of SSLE-AuNPs, authors should provide the cell viability of SSLE sample (without AuNPs).

22. The error line in graphs should be illustrated with black color.

23. There are spelling mistakes in the manuscript, the authors should carefully check the manuscript again.

24. The images’ quality (resolution) should be improved for better visual.

25. The results in Table 2 and 3 should interpreted in form of graphs for better comparison and visuallization.

Reviewer #4: Comments:

1. The article is nicely written and easily understandable.

2. It would be better if the authors clarified the functional compounds that were responsible for reducing the agent from Au+ to Au in their study. By providing specific details about these compounds, readers would gain a clearer understanding of the mechanisms at play and could more effectively evaluate the implications of the research. Otherwise, the article is understandable.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes:  Mst. Sanjida Akhter

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Assessing the biomedical applicability of biogenically synthesized AuNPs using Salvia splendens extract

PONE-D-24-51141R1

Dear Dr. Tiwari,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Thanh-Danh Nguyen, PhD

Academic Editor

PLOS ONE

Comments from PLOS Editorial Office:

We note that one or more reviewers has recommended that you cite specific previously published works in an earlier round of revision. As always, we recommend that you please review and evaluate the requested works to determine whether they are relevant and should be cited. It is not a requirement to cite these works and you may remove them before the manuscript proceeds to publication. We appreciate your attention to this request.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I reviewed the revised paper. After evaluating the revisions made by the authors, I am pleased to report that they have adequately addressed the concerns and suggestions raised in the initial review.

I recommend that this manuscript be accepted for publication in journal PLOS One.

Reviewer #2: The authors have responded to my queries satisfactorily. Hence, I recommend the Acceptance of this article in its present form.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes:  Majid Darroudi

Reviewer #2: No

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