Biotechnological Potential of Algerian Saffron Floral Residues: Recycling Phytochemicals with Antimicrobial Activity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

i. Authors introduction section should be well-paragraphed.

ii. In the methodology section, authors collected the plants and started working on it without detailed authentication. No voucher number was mentioned. The implication is that the identity of the plant  under study is questionable.

iii. Authors should clarify this "The solvent of this fraction was completely evaporated using a rotary evaporator and then replaced with dimethyl sulfoxide (DMSO) at the indicated concentrations to give the dichloromethane fraction." 

iv. It seems as though successive extraction was done. It is not correct to refer to extracts from successive extraction as fractions. They still remain extract. It is when you separate a whole extract into different fractions using solvents of increasing polarity that you use the word fraction. This error should be reworked.

v. In the results and discussion section, authors should emphasize the key role of interactions established between the ligands and proteins in the biological activity studied.

vi. Authors should perform time-kill assay to improve their antimicrobial study

vii. No scientific/botanical name of plants and microbial species was italicized throughout the manuscript. 

viii. Authors should contact a native English language editor to assist in improving the quality of English usage.

Comments on the Quality of English Language

The quality of English language must be improved

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study by Meliani et al. presents a comprehensive investigation of the bioactivity of Algerian saffron floral residue extracts through phytochemical analysis, bioactivity assays, and molecular docking simulations. The work is substantial and adequately supports the conclusions. However, I have outlined a few observations below that require careful attention. I recommend minor revision of the manuscript.

1. Were any reference compounds (known inhibitors) with established binding affinities used to validate the AutoDock Vina docking protocol?

2. Although binding interactions are described, their biological relevance is not sufficiently explained. Specifically, how does epicatechin binding to P. aeruginosa aminopeptidase translate into an antimicrobial effect?

3. Why are 3-hydroxytyrosol, chrysin, and caffeic acid phenethyl ester included in Table 2, despite not being detected in the samples? Were these compounds previously reported in Algerian saffron? If so, this should be clearly stated. Additionally, why are these compounds discussed in the manuscript (Line 375) despite their absence in the current analysis?

4. Is Table 9 absolutely necessary in the main text? If possible, moving it to the Supporting Information would improve the readability of the manuscript.

5. The reported concentration of petunidin-3-glucoside (3.890 ± 0.233 µg/mg) is more than 100-fold higher than that reported in a previous study (0.035 ± 0.018 µg/mg; Line 380). The authors should provide a clear explanation for this substantial change.

6. The HPLC-DAD method developed for phytochemical quantification is described without any reference, which suggests that the method may be newly developed. In such a case, appropriate validation parameters should be provided. Given that the levels of quantification are in the order of ng/mg, the authors should report the R² values for each standard compound, as well as the Limit of Detection (LOD) and Limit of Quantification (LOQ). If inclusion in the main text is not feasible, these data may be provided as Supporting Information.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. All scientific names of plants and microorganisms should be italicized throughout the manuscript. After the first full mention, the genus name should be abbreviated in subsequent occurrences (e.g.,Crocus sativus, Enterococcus faecalis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans).
2. The keyword “antiradical effect” has been revised to “antioxidant” to ensure clearer and more widely accepted terminology.
3. The title has been revised to clearly specify the antimicrobial focus of the study.
4. In the Experimental section, the Materials subsection should specify the sources and suppliers of the key chemicals and reagents used in this study, such as DPPH radical, standard ascorbic acid, standard gallic acid, and antibiotics (e.g., ampicillin).
5. The profiles of phenolic compounds should be arranged in descending order based on their abundance, from the most abundant to the least abundant compounds. Compounds that were not detected should be reported as “N.D.” instead of “0 ± 0,” with a footnote in the table indicating that “N.D.” denotes not detected.
6. The similarity index is relatively high (33%) and requires further improvement.

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

In this Manuscript, Meliani et. al. reported the “phytochemical profiling and in-silico analysis of bioactive compounds of Algerian Saffron Floral residues”. They have performed sequential organic solvent extraction and evaluated their antioxidant and antimicrobial activity. Furthermore, they carried out an ADMET profile and molecular docking analysis of the selected bioactive compounds. However, the present study overlaps previously published research in Waste and Biomass Valorization (2021) journal, which also reported the detailed phytochemical composition of the stigma and flower residue extracts (obtained using a methanol:water solvent mixture) and assessed their antioxidant and antibacterial activities. The C. sativus flower is also collected from the same geographical location, but at a different time period.
It is unclear what the paper contributes to the existing academic literature. The only change is the extraction procedure and inclusion of an in silico analysis of selected phytochemicals, specifically the ADMET profile, which has already been reported in the literature for most of the identified compounds. This does not yield any new information. The proteins used for molecular docking studies, 8ACR and 2NRK, are not commonly used in docking studies. However, the authors selected these proteins without explaining the reason behind this choice. 

Overall, the present work lacks novelty and has serious structural issues. Hence, it cannot be recommended for publication.

Comments on the Quality of English Language

The plant species name should be italicized throughout the manuscript.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

While the authors have improved their manuscript, the following comments should be addressed before acceptance.

i. If it is impossible to perform a time-kill assay, then authors should run a 100 ns simulation for the top-ranked molecule for each enzyme

Comments on the Quality of English Language

The quality of English language must be improved

Author Response

Reviewer 1

R1: While the authors have improved their manuscript, the following comments should be addressed before acceptance. i. If it is impossible to perform a time-kill assay, then authors should run a 100 ns simulation for the top-ranked molecule for each enzyme.

A: We thank the reviewer for this insightful and scientifically sound suggestion. We fully understand the rationale behind the request for a 100 ns molecular dynamics simulation, as it represents a valuable tool for assessing the dynamic stability of protein-ligand complexes. However, after careful consideration, we believe that adding a new full-scale simulation at this stage would not only require a significant and independent computational investigation but would also shift the focus of the manuscript toward a dynamic-based study that was not part of our initial research design. Given lack of specific bioinformatic resources and expertise for this type of analysis, we would like to ask the reviewer to understand that such a substantial experiment is arduous to be reached for us now. However, we want to say that it could be a very interesting suggestion to carry out in our future studies, also including some human proteins involved in inflammation as targets of the studied phytochemical. Nevertheless, we decided to enrich our manuscript with a further paragraph in which the importance of the proposed docking analysis is valorised (lines 738-744). We greatly hope that the reviewer can help us, considering the paper satisfactory to date. Thanks again in advance.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you for submitting the revised version of the manuscript and for addressing my previous major comments.

The revised manuscript is well-written, and the study's aim is clearly stated. This research work demonstrates how the yield of extracts is significantly enhanced by adopting an appropriate extraction method, thereby improving their bioactivity. Additionally, the impact of plant harvesting time might be responsible for the variation in extract yield, which is also discussed. The inclusion of in silico work provides a putative understanding of the role of individual metabolites in inhibiting microbial activity. Overall, this manuscript contributes to enhancing our understanding of the phytochemicals in C. sativus tepals and their role in antimicrobial activity, and presents a framework for future studies.
Therefore, I recommend that this manuscript be considered for publication. However, the Authors need to make some minor changes.
Minor revision:
1. Authors mention: “The selection of bioactive compounds for ADMET and molecular docking analyses was based on the highest concentrations and significant biological activity of specific phytochemicals.” As written, this can be interpreted as activity demonstrated in the present study for the individual phytochemicals. However, this manuscript reports antimicrobial experiments for the extracts, not for the individual compounds.
My recommendation is to rewrite this line as: “The selection of bioactive compounds for ADMET and molecular docking analyses was based on the highest concentrations and reported significant bioactivity of specific phytochemicals in prior research.

Author Response

Reviewer 4

R4: Thank you for submitting the revised version of the manuscript and for addressing my previous major comments. The revised manuscript is well-written, and the study's aim is clearly stated. This research work demonstrates how the yield of extracts is significantly enhanced by adopting an appropriate extraction method, thereby improving their bioactivity. Additionally, the impact of plant harvesting time might be responsible for the variation in extract yield, which is also discussed. The inclusion of in silico work provides a putative understanding of the role of individual metabolites in inhibiting microbial activity. Overall, this manuscript contributes to enhancing our understanding of the phytochemicals in C. sativus tepals and their role in antimicrobial activity and presents a framework for future studies. Therefore, I recommend that this manuscript be considered for publication. However, the Authors need to make some minor changes.

A: We thank the reviewer for having captured the main aims of our work and for his/her good comment about it.

R4: Minor revision - 1. Authors mention: “The selection of bioactive compounds for ADMET and molecular docking analyses was based on the highest concentrations and significant biological activity of specific phytochemicals.” As written, this can be interpreted as activity demonstrated in the present study for the individual phytochemicals. However, this manuscript reports antimicrobial experiments for the extracts, not for the individual compounds. My recommendation is to rewrite this line as: “The selection of bioactive compounds for ADMET and molecular docking analyses was based on the highest concentrations and reported significant bioactivity of specific phytochemicals in prior research”.

A: We would like to thank the reviewer for this constructive comment. We modified this part in the experimental section (lines 282-284).