Green Synthesis of Cocos nucifera-Based Nanomaterials and Mechanistic Basis of Their Antimicrobial Action

Round 1

Reviewer 1 Report

The article entitled ”Green synthesis of Cocos nucifera-based nanomaterials and mechanistic basis of their antimicrobial action” tackles an interesting issue, developing new materials with a potential antimicrobial effect. It is a type of research very necessary in our current context. However, I have some concerns that I will address as follows:

-        Abstract -  The abstract is way too long. It should generally provide only some information regarding the aims, the research protocol and perhaps some results. In current state it has way too many technical details. Essentially, it is a summary of the article not a proper abstract. Please rephrase it so it becomes more clear, easy to read and proper for a reader to tackle this article. Also, the first sentence is very complicated and needs to be better phrased.

-        Introduction – Line 54-55 . The first sentence presents basically the definition of an infectious disease. It is not necessary. Please keep only the relevant information. It should either be rephrased or just eliminated.

-        Line 71-72 – you mentioned 5 pathogens but gave only 4 examples.

-        Line 77-87 – the presence of the oxygen reactive species in this context seems irrelevant. Why did you choose to mention it? If there is a reason ply explain in the text, why that information.

-        Line 98 – what do you mean by severe uncomplicated infection? Please address in the text further explanations. Also, you gave only some examples but there are many other so please give more examples or keep it as a general information.

-        Line 138 – what do you mean by green synthesis. Please explain.

-        Table 1 – It is not necessary since you only repeat the information that are already available in the text. Keep either the table or the information in the text.

-        Please explain why you chose ciprofloxacin and fluconazole as controls.

-        Please explain why you chose to use only Escherichia coli for the time-kill kinetics test.

-        Table 2 and Table 3 – Please use the mean and standard deviation for the 3 determinations.

-        In limitations and perspectives, you mentioned that this study only summarizes the preliminary screening of the antimicrobial action. However, the article was not advertised as so up until this sentence. Please rephrase or add in introduction a clear presentation of the aims of these studies.

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 1

The article entitled ”Green synthesis of Cocos nucifera-based nanomaterials and mechanistic basis of their antimicrobial action” tackles an interesting issue, developing new materials with a potential antimicrobial effect. It is a type of research very necessary in our current context. However, I have some concerns that I will address as follows:

Reply: We thank you for the positive view of the reviewer toward our manuscript.

-        Abstract -  The abstract is way too long. It should generally provide only some information regarding the aims, the research protocol and perhaps some results. In current state it has way too many technical details. Essentially, it is a summary of the article not a proper abstract. Please rephrase it so it becomes more clear, easy to read and proper for a reader to tackle this article. Also, the first sentence is very complicated and needs to be better phrased.

      Reply: Thank you very much for the remark. We have now rewritten the abstract to reduce its length for the reader. The changes have been colored red with yellow stripes in the abstract.

-        Introduction – Line 54-55 . The first sentence presents basically the definition of an infectious disease. It is not necessary. Please keep only the relevant information. It should either be rephrased or just eliminated.

      Reply: The changes have been amended as suggested. The definition of the term infectious disease has now been omitted.

-        Line 71-72 – you mentioned 5 pathogens but gave only 4 examples.

     Reply: We agree with the reviewer’s inquisitiveness. The fifth pathogen as now been added as suggested.

-        Line 77-87 – the presence of the oxygen reactive species in this context seems irrelevant. Why did you choose to mention it? If there is a reason ply explain in the text, why that information.

Reply: We agree with the reviewer’s apprehension. We have now rewritten the statement to consider only relevant information across the introductory part of the manuscript. The changes have been colored red with yellow stripes.

-        Line 98 – what do you mean by severe uncomplicated infection? Please address in the text further explanations. Also, you gave only some examples but there are many other so please give more examples or keep it as a general information.

Reply: We agree with the reviewer’s concern. The sentence has now been rewritten while naming other examples as suggested.

-        Line 138 – what do you mean by green synthesis. Please explain.

Reply: Green synthesis is an environmentally friendly method presenting a different way of thinking in chemistry intended to eliminate toxic waste, reduce energy consumption, and to use ecological solvents (water, ethanol, ethyl acetate, etc.) and biological material (plants, etc.) (Bedlovičová, 2022). In the green synthesis approach, plant extracts are used for the preparation of nanoparticles to substitute the use of hazardous chemicals (Abuzeid et al., 2023). All in all, it is an eco-friendly, cost-effective, non-toxic, and more stable method to synthesize nanoparticles (Vijayaram et al., 2024).

-        Table 1 – It is not necessary since you only repeat the information that are already available in the text. Keep either the table or the information in the text.

      Reply: Table 1 has now been omitted as suggested.

-        Please explain why you chose ciprofloxacin and fluconazole as controls.

Reply: Ciprofloxacin is a broad-spectrum antibiotic, which is active against a number of both Gram-positive and Gram-negative bacteria (Sharma et al., 2017). In this investigation, the studied bacterial strains (Escherichia coli, Shigella sonnei, Staphylococcus aureus, Shigella flexneri, methicillin resistant Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae) were susceptible to ciprofloxacin with MIC values of 0.078, 0.078, 0.039, 0.078, 0.078, 0.156, 0.078, 0.039 µg/ml, respectively. These results might help to compare those obtained from the experimental groups treated with the nanomaterials.

On the other hand, fluconazole is used to treat invasive fungal or yeast infections, including vaginal candidiasis, oropharyngeal candidiasis (thrush, oral thrush), esophageal candidiasis (candida esophagitis), other candida infections (including urinary tract infections, peritonitis [inflammation of the lining of the stomach], and infections that may occur in different parts of the body) (Charlier et al., 2006; Partha et al., 2022). Although there are reports on the fluconazole resistance in a number of clinical strains of Candida (Berkow and Lockhart, 2017), fluconazole susceptibility to Candida species is well known (Maenchantrarath et al., 2022). In this study, fluconazole showed susceptibility to the studied Candida species at a micromolar range [Candida albicans (CA NR-29456), Candida glabrata, Candida krusei, Candida parapsilosis, and Candida albicans (CA ATCC-14516); MIC values: 0.078, 0.156, 0.078 and 0.039 µg/ml, respectively]. Thus, it is not unreasonable to consider that fluconazole might be an appropriate positive control in in vitro tests involving these Candida species.

-        Please explain why you chose to use only Escherichia coli for the time-kill kinetics test.

Reply: Thank you. Among the bacterial strains tested, Escherichia coli was chosen for the time-kill kinetics test, because this was the most susceptible strain, and the only that exhibited MIC values’ range as low as 7.8125-62.5 µg/ml.

-        Table 2 and Table 3 – Please use the mean and standard deviation for the 3 determinations.

Reply: Thank you. For the determination of the minimum inhibitory concentrations, the test was performed in triplicate. Details have now been incorporated in the concerned tables. It is worth noting that the MIC values from the three determinations did not changed for each sample.

-        In limitations and perspectives, you mentioned that this study only summarizes the preliminary screening of the antimicrobial action. However, the article was not advertised as so up until this sentence. Please rephrase or add in introduction a clear presentation of the aims of these studies.

Reply: Thank you for the remark. We have now rewritten the sentence as recommended. The changes have been colored red with yellow stripes.

We request the reviewer for re-evaluation and kind consideration.

References

-Abuzeid, H.M.; Julien, C.M.; Zhu, L.; Hashem, A.M. Green synthesis of nanoparticles and their energy storage, environmental, and biomedical applications. Crystals 2023, 13, 1576. https://doi.org/10.3390/cryst13111576

-Bedlovičová, Z. 2022. Chapter 21-Green synthesis of silver nanoparticles using actinomycetes. Green Synthesis of Silver Nanomaterials. Nanobiotechnology for Plant Protection. 2022, Pages 547-569.

-Berkow, E.L.; Lockhart, S.R. Fluconazole resistance in Candida species: a current perspective. Infect. Drug Resist. 2017, 10, 237-245.

-Charlier, C.; Hart, E.; Lefort A.; Ribaud P.; F. Dromer, F.; Denning, D.W.; Lortholary O. Fluconazole for the management of invasive candidiasis: where do we stand after 15 years? J. Antimicrob. Chemother. 57 (3), 2006, 384-410.

-Maenchantrarath, C.; Khumdee, P.; Samosornsuk, S.; Mungkornkaew, N.; Samosornsuk, W. Investigation of fluconazole susceptibility to Candida albicans by MALDI-TOF MS and real-time PCR for CDR1, CDR2, MDR1 and ERG11. BMC Microbiol. 2022, 22 (1) 153. doi: 10.1186/s12866-022-02564-4.

-Partha, A.D.S.L.; Widodo, A.D.W.; Endraswari, P.D. Evaluation of fluconazole, itraconazole, and voriconazole activity on Candida albicans: A case control study. Ann. Med. Surg. (Lond). 2022, 84, 104882. doi: 10.1016/j.amsu.2022.104882.

-Sharma, D.; Patel, R.P.; Zaidi, S.T.R.; Sarker, M.M.R.; Lean, Q.Y.; Ming, L.C. Interplay of the quality of ciprofloxacin and antibiotic resistance in developing countries. Front. Pharmacol. 2017, 8, 546. doi: 10.3389/fphar.2017.00546.

-Vijayaram, S.; Razafindralambo, H.; Sun, Y.Z.; Vasantharaj, S.; Ghafarifarsani, H.; Hoseinifar, S.H.; Raeeszadeh, M. Applications of green synthesized metal nanoparticles - a Review. Biol Trace Elem Res. 2024, 202 (1) 360-386.

Author Response File: Author Response.pdf

Reviewer 2 Report

The study of Zuriatou Yajeh Tanka, Naphtali Odogu Ankoro, Vincent Ngouana, Franklin Loïc Tchinda Taghu, Abongta Lum Mforbesi, Branly-nathalien Nguena-Dongue, Julius Nsami Ndi, Boniface Pone Kamdem, Paul Keilah Lunga, Fabrice Fekam Boyom named “Green synthesis of Cocos nucifera-based nanomaterials and mechanistic basis of their antimicrobial action” is devoted to the undoubtedly important problem of overcoming microbial multidrug resistance to available drugs. The use of green synthesis of metal nanoparticles is considered as a promising candidate for the role of new antimicrobial drugs of a wide range of action. The work was carried out at a good methodological level. The conclusions logically follow from the results obtained. A fundamentally new result is the development of a nanocomposite based on activated carbon, Cocos nucifera extract and Ag-NPs with extremely low cytotoxicity. I believe that this article can be published after a number of additions.

Comments

The introduction is written in a very detailed and clear manner. However, there is a lack of justification for the choice of metal nanoparticles and silver nanoparticles in particular. Detailed descriptions of the prospects of silver nanoparticles can be found in the reviews (doi:10.3390/ph15080968, doi: 10.2147/IJN.S246764, doi: 10.3390/ijms22137202).

Materials and Methods:

At the leaf grinding stage, it is desirable to specify the grinder power used and the grinding time.

Line 177: double bracket

Line 230: The description of the culture medium is atypical and makes it difficult to clearly understand the culture protocol. It is desirable to replace it with a more standard one, e.g. "for the cultivation of Vero cells we used DMEM medium supplemented with 10% fetal bovine serum... 1% penicillin/streptomycin antibiotics", etc. It is sufficient to indicate the mass fractions of the additives without specifying the volumes (line 232). Two basic media, MEM and DMEM, are specified. Usually only one is used. Less commonly, mixtures are used, e.g. DMEM/F12. In the case of mixtures, the proportions in which the media were mixed should be given, e.g. for DMEM/F12 it is 1:1. If MEM and DMEM were used at different stages of the study, it is also desirable to specify and clarify this. For example, "DMEM was used for pre-experimental cell growth and MEM was used during the cytotoxic assay".

It is desirable to specify how the cell counting was performed: in a Neubauer chamber (or other) or with an automated counter.

Lines 244: The use of the term optical density is incorrect for fluorescence. It should be replaced with "fluorescence intensity".

Figures 2 and 3: Major and minor ticks should be added to the all scales (as in Figures 4-5).

Figures 4 and 5: Error bars are not visible on the graphs. You will need to add them or modify the graph so that the error bars are visible. The caption says that stars highlight data with statistical differences. However, I could not find any stars in Figure 5. You should add the missing stars to Figure 5 or state that there are no differences. The caption should clearly state that * corresponds to p<0.05 and *** corresponds to p<0.001.

Lines 441 and 453: Duplicate table numbers

Tables 4 and 5: The letters "a" and "b" in the footnote below the table should be explained.

In section 3.5. For completeness, it is desirable to provide microphotographs of cell morphology in positive (podophyllotoxin) and negative (no additives) controls and at least after incubation with the highest concentrations of nanoparticles and nanocomposites. An example can be found in the paper (doi: 10.1016/j.sjbs.2020.11.022).

Lines 552-558: Links to relevant sources required.

Best regards

Author Response

RESPONSE TO THE REVIEWER’S COMMENTS POINT BY POINT

Reviewer 2

The study of Zuriatou Yajeh Tanka, Naphtali Odogu Ankoro, Vincent Ngouana, Franklin Loïc Tchinda Taghu, Abongta Lum Mforbesi, Branly-nathalien Nguena-Dongue, Julius Nsami Ndi, Boniface Pone Kamdem, Paul Keilah Lunga, Fabrice Fekam Boyom named “Green synthesis of Cocos nucifera-based nanomaterials and mechanistic basis of their antimicrobial action” is devoted to the undoubtedly important problem of overcoming microbial multidrug resistance to available drugs. The use of green synthesis of metal nanoparticles is considered as a promising candidate for the role of new antimicrobial drugs of a wide range of action. The work was carried out at a good methodological level. The conclusions logically follow from the results obtained. A fundamentally new result is the development of a nanocomposite based on activated carbon, Cocos nucifera extract and Ag-NPs with extremely low cytotoxicity. I believe that this article can be published after a number of additions.

Reply: Thank you for the positive view of the reviewer toward our manuscript.

Comments

The introduction is written in a very detailed and clear manner. However, there is a lack of justification for the choice of metal nanoparticles and silver nanoparticles in particular. Detailed descriptions of the prospects of silver nanoparticles can be found in the reviews (doi:10.3390/ph15080968, doi: 10.2147/IJN.S246764, doi: 10.3390/ijms22137202).

Reply: The prospects of silver nanoparticles as antimicrobials have now been incorporated in the introductory part of the manuscript and duly referenced as suggested. The changes have been colored red with yellow stripes.

Materials and Methods:

At the leaf grinding stage, it is desirable to specify the grinder power used and the grinding time.

Reply: Thank you. As suggested, the power of the grinder has now been incorporated in the material and methods’ section.

Line 177: double bracket

Reply: The additional bracket has now been omitted.

Line 230: The description of the culture medium is atypical and makes it difficult to clearly understand the culture protocol. It is desirable to replace it with a more standard one, e.g. "for the cultivation of Vero cells we used DMEM medium supplemented with 10% fetal bovine serum... 1% penicillin/streptomycin antibiotics", etc. It is sufficient to indicate the mass fractions of the additives without specifying the volumes (line 232). Two basic media, MEM and DMEM, are specified. Usually only one is used. Less commonly, mixtures are used, e.g. DMEM/F12. In the case of mixtures, the proportions in which the media were mixed should be given, e.g. for DMEM/F12 it is 1:1. If MEM and DMEM were used at different stages of the study, it is also desirable to specify and clarify this. For example, "DMEM was used for pre-experimental cell growth and MEM was used during the cytotoxic assay".

Reply: We agree with the reviewer’s inquisitiveness. The change has now been amended as recommended.

It is desirable to specify how the cell counting was performed: in a Neubauer chamber (or other) or with an automated counter.

Reply: We agree with the reviewer’s suggestion. We have now specified how the cell counting was performed as “in a Neubauer chamber”.

Lines 244: The use of the term optical density is incorrect for fluorescence. It should be replaced with "fluorescence intensity".

Reply: The change has been amended as recommended. “optical density” has now been replaced by “fluorescence intensity”

Figures 2 and 3: Major and minor ticks should be added to the all scales (as in Figures 4-5).

Reply: The ticks have now been added to the scales in Figure 2 and 3.

Figures 4 and 5: Error bars are not visible on the graphs. You will need to add them or modify the graph so that the error bars are visible. The caption says that stars highlight data with statistical differences. However, I could not find any stars in Figure 5. You should add the missing stars to Figure 5 or state that there are no differences. The caption should clearly state that * corresponds to p<0.05 and *** corresponds to p<0.001.

Reply: We agree with the reviewer’s suggestions. We have now amended the changes in the captions of Figure 4 and 5, as recommended. The changes have been colored red with yellow stripes.

Lines 441 and 453: Duplicate table numbers

Reply: We agree with the reviewer’s apprehension. The tables have now been re-numbered to avoid duplicate table numbers.

Tables 4 and 5: The letters "a" and "b" in the footnote below the table should be explained.

Reply: The letters “a” and “b” have now been explained in the foot notes right below tables 4 and 5.

In section 3.5. For completeness, it is desirable to provide microphotographs of cell morphology in positive (podophyllotoxin) and negative (no additives) controls and at least after incubation with the highest concentrations of nanoparticles and nanocomposites. An example can be found in the paper (doi: 10.1016/j.sjbs.2020.11.022).

Reply: We acknowledge that. However, all relevant information is already available in the text.

Lines 552-558: Links to relevant sources required.

Reply: We agree with the reviewer’s proposition. The statement has now been duly referenced as suggested. The reference numbers incorporated have been colored red with green stripes.

Best regards

We request the Reviewer for re-evaluation and kind consideration.

With kind regards,

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors really took into consideration the suggestions. The quality of the article improved. It is suited for publication.

Reviewer 2 Report

For the future, I would like to wish the authors to evaluate in more detail the dependence of antibacterial properties on the physicochemical properties of nanoparticles (elemental composition, shape, size, etc.).

Best regards