Antioxidant and Antimicrobial Potential of Malva neglecta Wallr. Extracts Prepared by “Green” Solvents
, Plamena Staleva 2,3, Mariana Kamenova-Nacheva 2,3, Zvezdelina Yaneva 4, Nikolina Rusenova 5, Neli Grozeva 1, Stela Ginin 1 and Milena Tzanova 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsResearch article on the pharmacological potential of Malva neglecta Wallr. extracts, specifically looking into the extraction methods, antioxidant, antimicrobial, and antifungal activities of the plant using various solvents, including natural deep eutectic solvents, the manuscript is well-structured but some points need to be improve before the publishing.
The abstract does not explain what is new compared to previous research on Malva neglecta. “explain novelty”
The abstract shoul contains excessive methodological detail for example “spectrophotometric assays and HPLC-PDA-MS profiling, DPPH, FRAP, and ABTS assays”
Line 39-40 ““which may be attributed to their slightly higher alkaloid content and lower pH” this is speculative and belongs in the Discussion, not the Abstract.
The intorduction provides useful information about M. neglecta, its medicinal relevance, and prior phytochemical studies, but does not clearly articulate: What specific knowledge is missing? Why previous extraction approaches are insufficient? What is unknown regarding NADES extraction of M. neglecta? What challenge this study aims to solve?
The study uses multiple extraction methods (water, ethanol, three NADES), but key procedural details are missing or incomplete, Exact extraction yield formula is not provided.
Sample-to-solvent ratios are described, but extraction time and conditions need more precision. Ultrasonication parameters (frequency, temperature, bath vs. probe) are missing.
It is unclear whether all extracts were filtered, centrifuged, or evaporated before use.
Provide complete extraction protocols including time, temperature, ultrasonication settings, and post-extraction processing.
The NADES mixtures are described (e.g., citric acid + β-alanine, choline chloride + urea), but important information is missing. Exact molar ratios used (w/w or molar basis?).
Heating/stirring conditions (temperature, duration, stirring speed).
Water content % in each NADES. Whether mixtures were filtered or stored, and their stability across experiments.
Please describe the NADES preparation method with detail or provide the reference you followed, so that another lab can reproduce the solvents accurately.
Please mention the post-hoc tests after ANOVA.
Author Response
Dear Reviewer,
First of all, we are thankful for your remarks, which resulted in improved quality of the manuscript. Here are our responses:
#1 “The abstract does not explain what is new compared to previous research on Malva neglecta. “Explain novelty”.
Response: We have clarified the “novelty”. Please, see lines 22-23.
#2 “The abstract should contains excessive methodological detail for example “spectrophotometric assays and HPLC-PDA-MS profiling, DPPH, FRAP, and ABTS assays”
Response: In the Abstract, all methods used were listed. We are limited in the number of words and unfortunately cannot provide many details in the abstract. Since the journal offers "open access," we will take advantage of this offer (if this article is not rejected), and readers will be able to read all the details.
#3 “Line 39-40 ““which may be attributed to their slightly higher alkaloid content and lower pH” this is speculative and belongs in the Discussion, not the Abstract.”
Response: We removed this part of the sentence.
#4 “The introduction provides useful information about M. neglecta, its medicinal relevance, and prior phytochemical studies, but does not clearly articulate: What specific knowledge is missing? Why previous extraction approaches are insufficient? What is unknown regarding NADES extraction of M. neglecta? What challenge this study aims to solve?”
Response: We clarified that at the end of the introduction and set a better explained experimental goal. Please, see lines 87-95.
#5 “The study uses multiple extraction methods (water, ethanol, three NADES), but key procedural details are missing or incomplete”.
Response: Please, see lines 119-139, and Table 1.
#6 “Sample-to-solvent ratios are described, but extraction time and conditions need more precision. Ultrasonication parameters (frequency, temperature, bath vs. probe) are missing.”
Response: Please, see lines 121-126.
#7 “It is unclear whether all extracts were filtered, centrifuged, or evaporated before use.”
Provide complete extraction protocols including time, temperature, ultrasonication settings, and post-extraction processing.”
Response: Please, see Lines 121-126, and 135-139.
#8 “The NADES mixtures are described (e.g., citric acid + β-alanine, choline chloride + urea), but important information is missing. Exact molar ratios used (w/w or molar basis?). Heating/stirring conditions (temperature, duration, stirring speed). Water content % in each NADES. Whether mixtures were filtered or stored, and their stability across experiments. Please describe the NADES preparation method with detail or provide the reference you followed, so that another lab can reproduce the solvents accurately.”
Response: Extraction details are given. Please, see lines 121-139, and Table 1.
#9 “Please mention the post-hoc tests after ANOVA.”
Response: It is done.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript ″ Pharmacological Potential of Malva neglecta Wallr. Extracts 2 Prepared by Green” Solvents″ provides a comprehensive comparative analysis of three Malva neglecta extracts, obtained with ethanol and two NADES systems, revealing their distinct phytochemical profiles, antioxidant activities, and variable antimicrobial effects. The study is meticulously conducted and holds significant pharmacognostic interest. Here are my observations:
- Abstract
Line 32: The text should describe the organs where the alkaloids were detected in low quantities.
- Introduction
Few morphological characteristics of the species should be described in the second paragraph.
The phrase from line 61 to 62 has no sense in the context of the paragraph.
Line 80: replace the word ″against″ with ″for″.
The aim and scope of study should be stated at the end of Introduction.
- Materials and methods
For Determination of Total Flavonoid Content, why didn’t you use rutin or quercetin as standard?
Line 246: I think it’s not catechin hydrate, but atropine.
- Results and discussions
Tables 2, 3, and 4 should have a legend for abbreviations.
Regarding the pH of NADES 1 extract, the discussion should take into account limitations and precautions related to its use, given its extremely low pH. In this context, is the antimicrobial activity of the extract driven by its acidity, or by the bioactive compounds present within it?
Author Response
Dear Reviewer,
First of all, we are thankful for your remarks, which resulted in improved quality of the manuscript. Here are our responses:
#1 “Line 32: The text should describe the organs where the alkaloids were detected in low quantities.”
Response: The info is added.
#2 “Few morphological characteristics of the species should be described in the second paragraph.”
Response: The info is added. Please, see lines 57-60.
#3 “The phrase from line 61 to 62 has no sense in the context of the paragraph.”
Response: The phrase is corrected.
#4 “Line 80: replace the word ″against″ with ″for″.”
Response: The sentence was rewritten.
#5 “The aim and scope of study should be stated at the end of Introduction.”
Response: It is done.
#6 “For Determination of Total Flavonoid Content, why didn’t you use rutin or quercetin as standard?”
Response: Ðœany research teams used catechin as a standard in this method for quantitative determination of Total Flavonoid Content. Please, see https://doi.org/10.7324/JAPS.2015.5011; https://www.academia.edu/1238146/Determination_of_total_phenol_condensed_tannin_and_flavonoid_contents_and_antioxidant_activity_of_Uncaria_gambir_extracts ; DOI: 10.3390/pr9020248, etc. Furthermore, it is easy, economical, and convenient to use one substance as a standard for two methods (TFC and TCT).
#7 “Line 246: I think it’s not catechin hydrate, but atropine.”
Response: The technical error is corrected.
#8 “Tables 2, 3, and 4 should have a legend for abbreviations.”
Response: All abbreviations used in the text have been listed at the end of the main text, Please, see paragraph “Abbreviations”.
#9 “Regarding the pH of NADES 1 extract, the discussion should take into account limitations and precautions related to its use, given its extremely low pH. In this context, is the antimicrobial activity of the extract driven by its acidity, or by the bioactive compounds present within it?”
Response: Up to date, there is a lack of comprehensive toxicity (including ecotoxicity) data for acidic NADESs. Of course, a careful handling of low pH extracts and pure NADES solutions, as well as the use of personal protective equipment is mandatory (Please, see lines 371-372). The antimicrobial activity of NADES extracts is driven both by the acidity and by the bioactive compounds content of them, but acidity is the dominant factor. This is further clarified by lines 558-575 which we corrected/added in the text.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe study investigates the biological activity of conventional extracts and extracts prepared using deep eutectic solvents from Malva neglecta (dwarf mallow). Below are my comments, which I believe will help improve the quality of the manuscript.
I suggest replacing "Pharmacological Potential" with "Antioxidant and Antimicrobial Potential", as this wording more accurately reflects the scope of the study.
Please define the abbreviation "FA" at its first mention (line 157).
The authors should verify the correctness of the formula for ABTS (%) presented in line 184.
Regarding total flavonoid content: why was catechin used as the standard? Considering the HPLC-PDA-MS results, using a flavonoid actually present in Malva extracts would provide more relevant quantitative data.
In line 246, it is incorrectly stated that catechin hydrate was used as the standard for total alkaloid determination. This should be corrected.
Lines 314-315 state: "In total, thirty-one secondary metabolites were detected and characterized". While 31 metabolites were detected, only a subset appears to have been characterized. This should be clarified.
My main concern relates to compound identification. p-Coumaroyl hexose, as a hydroxycinnamic acid derivative, should show a UV absorption maximum around 320 nm, but this is not reported. Compounds 10, 12, 14, and 16 are assigned as di- or triglucosides, yet only molecular ion information is provided; without fragment ions, it is unclear how identity of sugar units were determined. Compounds 12 and 16 contain the term "flavanoid", which should be corrected to "flavonoid". Compound 27, although quercetin derivative, shows only one absorption maximum at 247 nm, with no expected band around 350 nm. Similarly, compound 29, a hydroxycinnamic acid derivative, lacks a UV maximum near 320 nm. These identifications should be reconsidered or justified more clearly.
For reader convenience, the authors should provide full names for all abbreviations used in Table 3.
Line 554 states: "NADES1 extracts showed similar activity to ethanolic extracts". The basis for this conclusion should be clarified.
Author Response
Dear Reviewer,
We would like to thank you for your in-depth review, which resulted in greatly improved quality of the manuscript. We made the corrections to the best of our ability. Here are our responses:
#1 “I suggest replacing "Pharmacological Potential" with "Antioxidant and Antimicrobial Potential", as this wording more accurately reflects the scope of the study.”
Response: It is done.
#2 “Please define the abbreviation "FA" at its first mention (line 157).”
Response: It is defined in line 150.
#3“The authors should verify the correctness of the formula for ABTS (%) presented in line 184.”
Response: The technical error in the parameters explanation after equation (1) was corrected.
#4 “Regarding total flavonoid content: why was catechin used as the standard? Considering the HPLC-PDA-MS results, using a flavonoid actually present in Malva extracts would provide more relevant quantitative data.”
Response: Ðœany research teams used catechin as a standard in this method for quantitative determination of Total Flavonoid Content. Please, see https://doi.org/10.7324/JAPS.2015.5011; https://www.academia.edu/1238146/Determination_of_total_phenol_condensed_tannin_and_flavonoid_contents_and_antioxidant_activity_of_Uncaria_gambir_extracts ; DOI: 10.3390/pr9020248, etc. Furthermore, it is easy, economical, and convenient to use one substance as a standard for two methods (TFC and TCT).
#5 “In line 246, it is incorrectly stated that catechin hydrate was used as the standard for total alkaloid determination. This should be corrected.”
Response: The technical error is corrected.
#6 “Lines 314-315 state: "In total, thirty-one secondary metabolites were detected and characterized". While 31 metabolites were detected, only a subset appears to have been characterized. This should be clarified.”
Response: Thank you for your comment. We agree that the original wording was misleading. We have revised the manuscript to clarify that thirty-one metabolites were detected: seven confirmed with authentic standards, fifteen tentatively identified by MS and UV data, and nine remaining unidentified.
#7 “My main concern relates to compound identification. p-Coumaroyl hexose, as a hydroxycinnamic acid derivative, should show a UV absorption maximum around 320 nm, but this is not reported. Compounds 10, 12, 14, and 16 are assigned as di- or triglucosides, yet only molecular ion information is provided; without fragment ions, it is unclear how identity of sugar units were determined. Compounds 12 and 16 contain the term "flavanoid", which should be corrected to "flavonoid". Compound 27, although quercetin derivative, shows only one absorption maximum at 247 nm, with no expected band around 350 nm. Similarly, compound 29, a hydroxycinnamic acid derivative, lacks a UV maximum near 320 nm. These identifications should be reconsidered or justified more clearly.”
Response: Thank you for these valuable comments regarding compound identification. We acknowledge the omission of the expected UV absorption maxima—approximately 320 nm for p-coumaroyl hexose and compound 29, and around 350 nm for compound 27. These compounds exhibited very low signal intensity, leading to limited spectral quality. Following careful re-evaluation, the missing spectral details have been added to the revised manuscript. Regarding compounds 10, 12, 14, and 16, we based sugar unit assignments primarily on molecular ion data, UV spectra and comparison with literature. We agree that fragment ions would provide stronger evidence; however, MS/MS data were not available in this study. We have revised the text and the table to indicate these are tentative assignments and corrected “triglucoside” to “triglycoside” as intended. The term “flavanoid” has been corrected to “flavonoid” for compounds 12 and 16.
#8 “For reader convenience, the authors should provide full names for all abbreviations used in Table 3.”
Response: The full names are provided. Please, see the footnote of Table 3.
#9 “Line 554 states: "NADES1 extracts showed similar activity to ethanolic extracts". The basis for this conclusion should be clarified.”
Response: The basis for this conclusion is clarified at the end of the sentence. The full sentence is: “In the present study, NADES1 extracts demonstrated similar antibacterial activity to ethanolic extracts against S. aureus, B. cereus, and P. aeruginosa, as well as higher activity against E. coli (against which ethanolic extracts were not active), compared to the negative controls.” That is, the activity of NADES1 extracts was similar to that of ethanolic extracts when compared to the negative controls.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsagree with the author response
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have improved the manuscript, and I now consider it suitable for publication in its current form.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn my opinion, the manuscript can be accepted in its current form.
