Exploring the Multi-Faceted Potential of Carob (Ceratonia siliqua var. Rahma) Leaves from Morocco: A Comprehensive Analysis of Polyphenols Profile, Antimicrobial Activity, Cytotoxicity against Breast Cancer Cell Lines, and Genotoxicity

Round 1

Reviewer 1 Report

Title: Phytochemical Analysis and ADME Properties of Carob Extract Summary: The article presents a study on the phytochemical composition and ADME (absorption, distribution, metabolism, and excretion) properties of carob extract.

The authors used HPLC-DAD technique to analyze the chemical composition of ethanolic extracts of carob and identified several compounds, including naringin, succinic acid, 2-hydroxycinnamic acid, flavone, phloridzin dihydrate, 3-hydroxybenzoic acid, orcinol, and syringic acid. The study also assessed the physiochemical and pharmacokinetic properties of these compounds, including solubility, permeability, absorption, distribution, metabolism, and excretion. The bioavailability and potential toxicity of the compounds were also evaluated using computational models. Review: Overall, the article provides valuable information on the phytochemical composition and ADME properties of carob extract. The use of HPLC-DAD technique to identify and quantify the compounds is appropriate and reliable. The study detected several important compounds, such as naringin, which has been associated with anti-inflammatory and antioxidant effects, and quercetin and kaempferol, which have shown potential anti-cancer properties. The assessment of the physiochemical and pharmacokinetic properties of the compounds is thorough and provides insights into their potential therapeutic applications. The evaluation of solubility, permeability, absorption, distribution, metabolism, and excretion parameters is important for predicting the compounds' behavior in the body. The adherence to Lipinski's Rule of Five and Veber filter criteria suggests that most of the compounds have favorable drug-likeness properties. The bioavailability analysis, including the bioavailability radars and the BOILED-Egg model, provides a visual representation of the compounds' potential for oral bioavailability. The identification of compounds with high absorption and ability to cross the blood-brain barrier is noteworthy. However, it is important to note that the bioavailability predictions are based on computational models and should be validated through further in vitro and in vivo investigations. The assessment of potential toxicity using computational models is a valuable addition to the study. The prediction of hepatotoxicity, mutagenicity, carcinogenicity, immunotoxicity, and cytotoxicity endpoints provides insights into the possible health effects associated with exposure to the carob extract. However, it is crucial to validate these predictions through empirical research. Overall, the article is well-written and presents a comprehensive analysis of the phytochemical composition and ADME properties of carob extract. The findings contribute to the understanding of the potential therapeutic applications and safety considerations of the identified compounds. 

experimental studies were necessary to confirm the observed properties and to explore the effectiveness and safety of carob extract in various therapeutic applications.

The article maintains the scientific language and terminology appropriate for an academic article.

Author Response

Reviewer 1.

Title: Phytochemical Analysis and ADME Properties of Carob Extract Summary: The article presents a study on the phytochemical composition and ADME (absorption, distribution, metabolism, and excretion) properties of carob extract.

 

The authors used HPLC-DAD technique to analyze the chemical composition of ethanolic extracts of carob and identified several compounds, including naringin, succinic acid, 2-hydroxycinnamic acid, flavone, phloridzin dihydrate, 3-hydroxybenzoic acid, orcinol, and syringic acid. The study also assessed the physiochemical and pharmacokinetic properties of these compounds, including solubility, permeability, absorption, distribution, metabolism, and excretion. The bioavailability and potential toxicity of the compounds were also evaluated using computational models. Review: Overall, the article provides valuable information on the phytochemical composition and ADME properties of carob extract. The use of HPLC-DAD technique to identify and quantify the compounds is appropriate and reliable. The study detected several important compounds, such as naringin, which has been associated with anti-inflammatory and antioxidant effects, and quercetin and kaempferol, which have shown potential anti-cancer properties. The assessment of the physiochemical and pharmacokinetic properties of the compounds is thorough and provides insights into their potential therapeutic applications. The evaluation of solubility, permeability, absorption, distribution, metabolism, and excretion parameters is important for predicting the compounds' behavior in the body. The adherence to Lipinski's Rule of Five and Veber filter criteria suggests that most of the compounds have favorable drug-likeness properties. The bioavailability analysis, including the bioavailability radars and the BOILED-Egg model, provides a visual representation of the compounds' potential for oral bioavailability. The identification of compounds with high absorption and ability to cross the blood-brain barrier is noteworthy. However, it is important to note that the bioavailability predictions are based on computational models and should be validated through further in vitro and in vivo investigations. The assessment of potential toxicity using computational models is a valuable addition to the study. The prediction of hepatotoxicity, mutagenicity, carcinogenicity, immunotoxicity, and cytotoxicity endpoints provides insights into the possible health effects associated with exposure to the carob extract. However, it is crucial to validate these predictions through empirical research. Overall, the article is well-written and presents a comprehensive analysis of the phytochemical composition and ADME properties of carob extract. The findings contribute to the understanding of the potential therapeutic applications and safety considerations of the identified compounds.

 

experimental studies were necessary to confirm the observed properties and to explore the effectiveness and safety of carob extract in various therapeutic applications.

Response:

Dear Editors and Reviewers,

We would like to express our gratitude for providing us with the opportunity to enhance our manuscript through the revised version, and we sincerely appreciate your valuable comments.

We are particularly thankful to the Reviewers for their comprehensive review, which has greatly contributed to the improvement of our work. We have made significant efforts to address the queries and concerns raised by the Reviewers in this revised version. Our aim is to ensure that our manuscript meets the required standards for publication in Pharmaceuticals.

In response to the requested changes, we have carefully addressed each query and weakness, either by incorporating the suggested modifications or by providing a detailed response. The major changes made in the revised manuscript have been highlighted in yellow text for easier identification. We would like to assure you that all minor concerns and typos have been rectified in the manuscript, although they may not be specifically mentioned in this response.

Once again, we sincerely appreciate your time, effort, and constructive feedback. We hope that our revised manuscript successfully addresses all the reviewers' comments and meets the necessary criteria for publication in Pharmaceuticals.

Thank you and kind regards,

Dr. Addi and the co-Authors

 

Author Response File: Author Response.docx

Reviewer 2 Report

The article “Exploring the Multi-Faceted Potential of Carob Leaves from Morocco: A Comprehensive Analysis of Polyphenols Profile, Antimicrobial Activity, Cytotoxicity Against Breast Cancer Cell Lines, and Genotoxicity” investigates the antioxidant, antimicrobial, and cytotoxic properties of the ethanolic extract of Ceratonia siliqua leaves (CSEE). Chemical composition analysis revealed phenolic acids and flavonoids as the primary constituents. The extract exhibited potent antioxidant activity, as demonstrated by its DPPH scavenging capacity and ability to inhibit oxidative damage. It also showed strong antimicrobial activity against tested bacterial strains but only moderate activity against fungi. In vitro tests indicated dose-dependent inhibitory activity of the extract against breast cancer cell lines. At lower concentrations, the extract did not induce DNA damage, but a higher concentration showed genotoxic effects. Computational analysis was conducted to determine the physicochemical and pharmacokinetic characteristics of the extract's constituent molecules and to predict potential biological activities. The study provides valuable insights into the medicinal properties of CSEE, highlighting its antioxidant and antimicrobial potential, as well as its cytotoxic effects on cancer cells.

The text is clear and aligns perfectly with the scope of Pharmaceuticals.

Regarding specific changes, I propose:

- Abstract: it is too long, exceeds the 200-word limit set by Pharmaceuticals. Try to summarize it. Line 32 and 53 needs italics (C. siliqua and in vitro, respectively).

- Line 104: C. siliqua L. should be changed to Ceratonia siliqua L.

- Table 1: In the text accompanying the Table there should be an explanation of why there is bold text and colored cells.

Line 172 and Table 2: 96.98 ± 172 1.15 g GAE/100 g DW is an extremely high TPC value. Other studies say that "The phenolic content of carob fruit extracts showed considerable variation as a function of the solvent systems used and ranged from 7.1 ± 0.9 mg GAE 100 g−1 to 382.0 ± 23.8 mg GAE 100 g−1." Goulas V, Georgiou E. Utilization of Carob Fruit as Sources of Phenolic Compounds with Antioxidant Potential: Extraction Optimization and Application in Food Models. Foods. 2019 Dec 24;9(1):20. doi: 10.3390/foods9010020. PMID: 31878230; PMCID: PMC7022565. The value exposed in this article is several orders of magnitude higher than that.

Please review the calculations and units to verify the results.

Table 6: Explain the meaning of red text.

- Line 527: Abbreviate Ceratonia siliqua L. Check the rest of uses throughout the article.

- References: There are references with DOI and others without it. Please homogenize this section.

Overall, I think the article is good and should be accepted for Pharmaceuticals publication after addressing the changes proposed above.

The use of English is correct throughout the entire manuscript. However, some formatting is required (italics, capitalization, etc.) 

Author Response

Reviewer 2.

The article “Exploring the Multi-Faceted Potential of Carob Leaves from Morocco: A Comprehensive Analysis of Polyphenols Profile, Antimicrobial Activity, Cytotoxicity Against Breast Cancer Cell Lines, and Genotoxicity” investigates the antioxidant, antimicrobial, and cytotoxic properties of the ethanolic extract of Ceratonia siliqua leaves (CSEE). Chemical composition analysis revealed phenolic acids and flavonoids as the primary constituents. The extract exhibited potent antioxidant activity, as demonstrated by its DPPH scavenging capacity and ability to inhibit oxidative damage. It also showed strong antimicrobial activity against tested bacterial strains but only moderate activity against fungi. In vitro tests indicated dose-dependent inhibitory activity of the extract against breast cancer cell lines. At lower concentrations, the extract did not induce DNA damage, but a higher concentration showed genotoxic effects. Computational analysis was conducted to determine the physicochemical and pharmacokinetic characteristics of the extract's constituent molecules and to predict potential biological activities. The study provides valuable insights into the medicinal properties of CSEE, highlighting its antioxidant and antimicrobial potential, as well as its cytotoxic effects on cancer cells.

The text is clear and aligns perfectly with the scope of Pharmaceuticals.

Dear Editors and Reviewers,

We would like to express our gratitude for providing us with the opportunity to enhance our manuscript through the revised version, and we sincerely appreciate your valuable comments.

We are particularly thankful to the Reviewers for their comprehensive review, which has greatly contributed to the improvement of our work. We have made significant efforts to address the queries and concerns raised by the Reviewers in this revised version. Our aim is to ensure that our manuscript meets the required standards for publication in Pharmaceuticals.

In response to the requested changes, we have carefully addressed each query and weakness, either by incorporating the suggested modifications or by providing a detailed response. The major changes made in the revised manuscript have been highlighted in yellow text for easier identification. We would like to assure you that all minor concerns and typos have been rectified in the manuscript, although they may not be specifically mentioned in this response.

Once again, we sincerely appreciate your time, effort, and constructive feedback. We hope that our revised manuscript successfully addresses all the reviewers' comments and meets the necessary criteria for publication in Pharmaceuticals.

Thank you and kind regards,

Dr. Addi and the co-Authors

 

Regarding specific changes, I propose:

- Abstract: it is too long, exceeds the 200-word limit set by Pharmaceuticals. Try to summarize it. Line 32 and 53 needs italics (C. siliqua and in vitro, respectively).

We thank the reviewer for his/her pertinent remark; however, it is not possible to summarize the abstract as it provides a concise overview of the main points and findings of the research, and this valuable information cannot be easily condensed. Summarizing such an abstract may result in the loss of important details, context, and key findings, and may lead to an incomplete representation of the research, potentially misleading readers or omitting vital aspects of the study. On the other hands, there is papers published in Pharmaceuticals that exceed the 200-word limit set by the journal itself, for the same abovementioned reasons. Thank you again for your remark, and understanding.

- Line 104: C. siliqua L. should be changed to Ceratonia siliqua L.

Thank you for you remark, we have rectified it in the main text.

- Table 1: In the text accompanying the Table there should be an explanation of why there is bold text and colored cells.

We removed the color coding of cells and included textual explanations within the table to clarify the sections that are emphasized in bold. Thank you for you remark.

Line 172 and Table 2: 96.98 ± 172 1.15 g GAE/100 g DW is an extremely high TPC value. Other studies say that "The phenolic content of carob fruit extracts showed considerable variation as a function of the solvent systems used and ranged from 7.1 ± 0.9 mg GAE 100 g−1 to 382.0 ± 23.8 mg GAE 100 g−1." Goulas V, Georgiou E. Utilization of Carob Fruit as Sources of Phenolic Compounds with Antioxidant Potential: Extraction Optimization and Application in Food Models. Foods. 2019 Dec 24;9(1):20. doi: 10.3390/foods9010020. PMID: 31878230; PMCID: PMC7022565. The value exposed in this article is several orders of magnitude higher than that.

Please review the calculations and units to verify the results.

We are very grateful to the reviewer for his/her sharp remark, indeed after reviewing the calculations it was found that the values are in mg GAE/ 100 g DW for TPC, in mg RE/100 g DW for TFC, and mg CE/100 g DW for TCT. We have rectified them in the main manuscript.

Table 6: Explain the meaning of red text.

To prevent any potential misunderstanding, we have removed the use of red text that was initially employed to distinguish active molecules from non-active molecules in terms of the studied toxicological end-points. This modification has been made to ensure clarity and to avoid any confusion. Thank you for your valuable remark.

- Line 527: Abbreviate Ceratonia siliqua L. Check the rest of uses throughout the article.

We have implemented the requested change to abbreviate "Ceratonia siliqua L." as instructed. Furthermore, we have thoroughly reviewed the entire article to ensure consistent usage of the abbreviation throughout. Thank you for bringing this to our attention, and we appreciate your careful review.

- References: There are references with DOI and others without it. Please homogenize this section.

We have added the missing DOIs for each reference, however some references are with no DOI.

Overall, I think the article is good and should be accepted for Pharmaceuticals publication after addressing the changes proposed above.

- We extend our sincerest appreciation for the invaluable insights you have shared with us.

 

Author Response File: Author Response.docx

Reviewer 3 Report

This paper titled as “Exploring the Multi-Faceted Potential of Carob Leaves from 2 Morocco: A Comprehensive Analysis of Polyphenols Profile, 3 Antimicrobial Activity, Cytotoxicity Against Breast Cancer Cell 4 Lines, and Genotoxicity” studies biological application of the Carob Leaves extracts .  The study aims to ascertain the 31 antioxidant, antimicrobial, and cytotoxic properties of the ethanolic extract of C. siliqua leaves 32 (CSEE). The current draft of the manuscript, in its present form, is not ready for publication. I welcome the author to consider the following comments and resubmit the manuscript.

 

Comments:

 

Comment 1: The study used only one extraction method (99% ethanol) to obtain the carob leaf extract, which may limit the generalizability of the results to other extraction methods. There should be the comparison of the water, methanol etc extracts.

Comment 2: spectrophotometric measurements were taken at 280 nm, although this wavelength is commonly used for phenolic compounds, it may not be optimal for detecting certain compounds with different absorption characteristics.

Comment 3: HPLC-DAD method is not validated. Method validation is essential to ensure the accuracy, precision, selectivity, and reliability of the analytical results. Without validation data, the quality and robustness of the method cannot be adequately assessed.

Comment 4: 3.3. TPC, TFC, and TCT Contents: colorimetric assays described for TFC and TCT measurements rely on the formation of colored complexes. However, these assays may be susceptible to interferences from other components present in the extract, leading to inaccurate results. The potential interferences and specificity of the assays should be considered and addressed to ensure reliable measurements.

Comment 5: validation of the colorimetric assays used for TPC, TFC, and TCT measurements is not provided.

Comment 6: The calibration curves for TPC and TCT were constructed using gallic acid and catechin, respectively, over a concentration range of 0-0.1 mg/mL. The narrow concentration range may limit the accuracy and precision of the quantification, particularly if the samples have higher or lower phenolic concentrations.

Comment 7: 3.4. PASS, ADME, and the prediction of the Toxicity Analysis (Pro-Tox II) : Pharmacokinetics is a complex process influenced by various factors, and the predictions obtained from web servers may not capture the full range of factors affecting drug absorption, distribution, metabolism, and excretion. Authors should consider performing NCA analysis .

Comment 8: 3.5.4. Total Antioxidant Capacity: The phosphor-molybdenum methodology relies on the reaction of antioxidants with the reagent solution containing sulfuric acid, sodium phosphate, and ammonium molybdate. However, the reaction can also be influenced by other reducing substances present in the sample, such as sugars, polyols, and other non-antioxidant compounds. These substances may contribute to the absorbance at 695 nm, leading to an overestimation or underestimation of the actual antioxidant capacity. Author should provide proper explanation about the specificity of this assay.

Comment 8: 3.4. PASS, ADME, and the prediction of the Toxicity Analysis (Pro-Tox II) : Detailed comparison of the predicted results with experimental data or literature references is required

Comment 9: 3.5.4. Total Antioxidant Capacity: What precautions does author took to make sure that there is no loss or degradation of antioxidants during sample preparation? How does the author studied or compare the degradation of antioxidants compounds during sample preparation?

Comment 10: What is the clinical relevance of the selected strains of bacteria?

Comment 11: 3.9.2. Comet Assay: author briefly mentions the visualization of comets using the ethidium bromide method but does not provide any details regarding the staining procedure, observation parameters, or imaging technique.

Comment 12: The passage briefly mentions the potential therapeutic applications of the detected compounds, such as anti-inflammatory, antioxidant, and anti-cancer effects. However, it does not provide a detailed discussion of these implications or cite specific studies supporting these claims.

Comment 13: 2.3. Physiochemical and Pharmacokinetic Properties (ADME) of CSEE :This analysis relies on predictive models and software tools While these tools provide valuable insights, their accuracy is dependent on the quality of the input data and the assumptions made by the models. Experimental validation of alteast one or two compounds is necessary to confirm the predicted properties.

Comment 14: Three compounds, namely naringin,and phloridzin dihydrate, do not comply with Lipinski's Rule of Five. These compounds have violations related to molecular weight, the number of oxygen atoms, and the number of hydrogen-bond donors, respectively.

 

 

 

 

 Minor editing of English language required

Author Response

Reviewer 3.

This paper titled as “Exploring the Multi-Faceted Potential of Carob Leaves from 2 Morocco: A Comprehensive Analysis of Polyphenols Profile, 3 Antimicrobial Activity, Cytotoxicity Against Breast Cancer Cell 4 Lines, and Genotoxicity” studies biological application of the Carob Leaves extracts.  The study aims to ascertain the 31 antioxidant, antimicrobial, and cytotoxic properties of the ethanolic extract of C. siliqua leaves 32 (CSEE). The current draft of the manuscript, in its present form, is not ready for publication. I welcome the author to consider the following comments and resubmit the manuscript.

Dear Editors and Reviewers,

We would like to express our gratitude for providing us with the opportunity to enhance our manuscript through the revised version, and we sincerely appreciate your valuable comments.

 

We are particularly thankful to the Reviewers for their comprehensive review, which has greatly contributed to the improvement of our work. We have made significant efforts to address the queries and concerns raised by the Reviewers in this revised version. Our aim is to ensure that our manuscript meets the required standards for publication in Pharmaceuticals.

In response to the requested changes, we have carefully addressed each query and weakness, either by incorporating the suggested modifications or by providing a detailed response. The major changes made in the revised manuscript have been highlighted in yellow text for easier identification. We would like to assure you that all minor concerns and typos have been rectified in the manuscript, although they may not be specifically mentioned in this response.

Once again, we sincerely appreciate your time, effort, and constructive feedback. We hope that our revised manuscript successfully addresses all the reviewers' comments and meets the necessary criteria for publication in Pharmaceuticals.

Thank you and kind regards,

Dr. Addi and the co-Authors

 

Comments:

Comment 1: The study used only one extraction method (99% ethanol) to obtain the carob leaf extract, which may limit the generalizability of the results to other extraction methods. There should be the comparison of the water, methanol etc extracts.

Thank you for your valuable feedback. We acknowledge that using only one extraction method, specifically 99% ethanol, to obtain the carob leaf extract might limit the generalizability of the results to other extraction methods. We understand the importance of comparing different extraction solvents such as water, methanol, and others, as they can yield variations in the composition and bioactivity of the extract. Including a comparison of multiple extraction methods would provide a more comprehensive understanding of the carob leaf extract and its potential applications. This aspect could be considered in future research to explore the effects of various solvents on the extraction efficiency, phytochemical profile, and biological activities of the carob leaf extract. However, the aim of our study was to investigate the phytochemical profile, and the biological activities of the ethanolic extract of carob leaves, as ethanol can dissolve both polar and non-polar metabolites found in the plant material. Ethanol is the second most important solvent after water, and is the least toxic of the alcohols (only poisonous in large amounts), which makes it more suitable for use for such experiments.

 

Comment 2: spectrophotometric measurements were taken at 280 nm, although this wavelength is commonly used for phenolic compounds, it may not be optimal for detecting certain compounds with different absorption characteristics.

Thank you for raising an important point. The primary aim of the phytochemical analysis in our study was to determine the phenolic composition of the extract. To achieve this, spectrophotometric measurements were conducted at a wavelength of 280 nm, which is commonly employed for the detection of phenolic compounds due to their characteristic absorption at this wavelength. The authors totally agree with the reviewer, a paragraph has been added on line XXXX giving a detailed description of compound identification. “Spectrophotometric detection was carried out at 254, 280, 320, 350, and 540 nm. While spectrophotometric identification was carried out at 280 nm, and the compounds were identified by comparing their retention times to those of authentic standards”.

 

Comment 3: HPLC-DAD method is not validated. Method validation is essential to ensure the accuracy, precision, selectivity, and reliability of the analytical results. Without validation data, the quality and robustness of the method cannot be adequately assessed.

Thank you for your insightful comment about the HPLC-DAD method used in our work to separate and identify phenolic compounds. This method is a protocol of our laboratory already published in the literature (Benkirane et al. (2023) DOI: 10.1039/D2RA04081F) and validated according to the AFNOR standard, NFV03-110 NFT90-210. The protocol was validated by determining its linearity, accuracy for ethanolic plant extracts, limits of detection and quantification, and the yield from supplemented matrix before extraction. We remain available for any proposal that you consider helpful for enhancing the scientific quality of this work.

 

Comment 4: 3.3. TPC, TFC, and TCT Contents: colorimetric assays described for TFC and TCT measurements rely on the formation of colored complexes. However, these assays may be susceptible to interferences from other components present in the extract, leading to inaccurate results. The potential interferences and specificity of the assays should be considered and addressed to ensure reliable measurements.

Thank you for your insightful comment and concern regarding the colorimetric assays used for Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Total Condensed Tannin Content (TCT) measurements in our study. We want to assure you that we have taken all the necessary precautions to address potential interferences and ensure reliable measurements. The protocols we adopted for these colorimetric assays are well-known and widely used in the literature. These methods have been extensively validated and employed in numerous studies focusing on phenolic compounds and plant extracts. We carefully followed established protocols to minimize interferences and maximize the specificity of the assays.

Comment 5: validation of the colorimetric assays used for TPC, TFC, and TCT measurements is not provided.

We greatly appreciate the reviewer's pertinent remark. We want to assure you that the protocols adopted for the colorimetric tests used to measure TPC, TFC, and TCT in our study have already been validated. We followed the protocols described by Ghanemi et al. (2017) [1], and Zrouri et al. (2021) [2], Frond et al. (2019) [3], Mohti et al. (2020) [4], for the validation of these colorimetric tests TPC, TFC, and TCT, respectively. These protocols are well-established in the literature and have been widely used and validated by numerous researchers in the field. The validation process involved rigorous evaluation of the assays to ensure their accuracy, precision, and specificity. We adhered to the recommended procedures and parameters outlined in the protocols, enabling us to confidently measure TPC, TFC, and TCT in our samples. To ensure accurate and reproducible results in our study, all measurements were performed in triplicate.

1. Ghanemi, F.Z.; Belarbi, M.; Fluckiger, A.; Nani, A.; Dumont, A.; De Rosny, C.; Aboura, I.; Khan, A.S.; Murtaza, B.; Benammar, C.; et al. Carob Leaf Polyphenols Trigger Intrinsic Apoptotic Pathway and Induce Cell Cycle Arrest in Colon Cancer Cells. J. Funct. Foods 2017, 33, 112–121, doi:10.1016/j.jff.2017.03.032.
2. Zrouri, H.; Elbouzidi, A.; Bouhrim, M.; Bencheikh, N.; Kharchoufa, L.; Ouahhoud, S.; Ouassou, H.; El Assri, S.; Choukri, M. Phytochemical Analysis, Antioxidant Activity, and Nephroprotective Effect of the Raphanus Sativus Aqueous Extract. Mediterr. J. Chem. 2021, 11, 84, doi:10.13171/mjc02101211565lk.
3. Frond, A.D.; Iuhas, C.I.; Stirbu, I.; Leopold, L.; Socaci, S.; Andreea, S.; Ayvaz, H.; Andreea, S.; Mihai, S.; Diaconeasa, Z. Phytochemical Characterization of Five Edible Purple-Reddish Vegetables: Anthocyanins, Flavonoids, and Phenolic Acid Derivatives. Molecules 2019, 24, 1536, doi:https://doi.org/10.3390/molecules24081536.
4. Mohti, H.; Taviano, M.F.; Cacciola, F.; Dugo, P.; Mondello, L.; Zaid, A.; Cavò, E.; Miceli, N. Silene Vulgaris Subsp. Macrocarpa Leaves and Roots from Morocco: Assessment of the Efficiency of Different Extraction Techniques and Solvents on Their Antioxidant Capacity, Brine Shrimp Toxicity and Phenolic Characterization. Plant Biosyst. - An Int. J. Deal. with all Asp. Plant Biol. 2020, 154, 692–699, doi:10.1080/11263504.2019.1674404.

 

Comment 6: The calibration curves for TPC and TCT were constructed using gallic acid and catechin, respectively, over a concentration range of 0-0.1 mg/mL. The narrow concentration range may limit the accuracy and precision of the quantification, particularly if the samples have higher or lower phenolic concentrations.

We wholeheartedly agree with the reviewer's comment. It was indeed a mistake on our part to state that the calibration curve range was from 0 - 0.1 mg/mL. We apologize for this error and appreciate your keen observation. The correct calibration curve range used in our study was 0 - 5 mg/mL. We deeply regret any confusion that may have arisen due to this oversight.

Comment 7: 3.4. PASS, ADME, and the prediction of the Toxicity Analysis (Pro-Tox II): Pharmacokinetics is a complex process influenced by various factors, and the predictions obtained from web servers may not capture the full range of factors affecting drug absorption, distribution, metabolism, and excretion. Authors should consider performing NCA analysis.

Thank you for your comment and suggestion. We understand the importance of performing Non-compartmental Analysis (NCA) in pharmacokinetic studies to assess drug absorption, distribution, metabolism, and excretion. However, in our specific study, the focus was on utilizing PASS (Prediction of Activity Spectra for Substances) and ADME (Absorption, Distribution, Metabolism, and Excretion) predictions, as well as Toxicity Analysis (Pro-Tox II), to gather preliminary insights into the potential pharmacological activity, pharmacokinetic behavior, and toxicity of the tested substances.

It is worth noting that while web server predictions can provide valuable initial information, they have limitations and may not capture the full complexity of pharmacokinetics. NCA analysis involves extensive data collection, modeling, and specific measurements that go beyond the scope of our current study. We acknowledge that performing NCA analysis would provide a more comprehensive understanding of the pharmacokinetic parameters of the tested substances. In future studies or subsequent research, we will consider incorporating NCA analysis if the scope and objectives of the study warrant it, and if the necessary resources and data are available. Thank you for your suggestion, and we appreciate your consideration of NCA analysis in our study.

 

Comment 8: 3.5.4. Total Antioxidant Capacity: The phosphor-molybdenum methodology relies on the reaction of antioxidants with the reagent solution containing sulfuric acid, sodium phosphate, and ammonium molybdate. However, the reaction can also be influenced by other reducing substances present in the sample, such as sugars, polyols, and other non-antioxidant compounds. These substances may contribute to the absorbance at 695 nm, leading to an overestimation or underestimation of the actual antioxidant capacity. Author should provide proper explanation about the specificity of this assay.

We appreciate the reviewer's comment regarding the phosphor-molybdenum methodology used for assessing the total antioxidant capacity in our study. The protocol for this assay was adopted from the literature (Chaudhary et al. (2015), and Prieto et al. (1999)) and is widely recognized in the field.

We acknowledge that the reaction can potentially be influenced by other reducing substances present in the sample, such as sugars, polyols, and non-antioxidant compounds. These substances may contribute to the absorbance at 695 nm, which could lead to an overestimation or underestimation of the actual antioxidant capacity. While our study did not specifically address the specificity of this assay, it is important to note that the phosphor-molybdenum method is a widely accepted and commonly employed technique for assessing total antioxidant capacity. Its limitations and potential interferences are acknowledged in the literature.

 

• Chaudhary, S.; Chandrashekar, K.S.; Pai, K.S.R.; Setty, M.M.; Devkar, R.A.; Reddy, N.D.; Shoja, M.H. Evaluation of Antioxidant and Anticancer Activity of Extract and Fractions of Nardostachys Jatamansi DC in Breast Carcinoma. BMC Complement. Altern. Med. 2015, 15, 1–13, doi:10.1186/s12906-015-0563-1.
• Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Biochem. 1999, 269, 337–341.

 

Comment 8: 3.4. PASS, ADME, and the prediction of the Toxicity Analysis (Pro-Tox II) : Detailed comparison of the predicted results with experimental data or literature references is required

We respectfully disagree with the suggestion that a detailed comparison of the predicted results with experimental data or literature references is necessary for the section on PASS, ADME, and toxicity analysis using Pro-Tox II. The objective of this section was to employ predictive models and software tools to evaluate the physiochemical and pharmacokinetic properties, as well as potential toxicity, of the investigated compounds. Although experimental validation holds value, it may not always be practical or feasible for every study. Additionally, the scarcity of experimental data on all the compounds examined in our research makes it challenging to conduct a thorough and comprehensive comparison. Thus, we acknowledge the limitations in conducting a precise and complete comparison due to the absence of extensive experimental findings on all the studied compounds in the current literature.

 

Comment 9: 3.5.4. Total Antioxidant Capacity: What precautions does author took to make sure that there is no loss or degradation of antioxidants during sample preparation? How does the author studied or compare the degradation of antioxidants compounds during sample preparation?

Thank you for your valuable comments and questions regarding the Total Antioxidant Capacity (TAC) analysis in our study. We appreciate your concern about the potential loss or degradation of antioxidants during sample preparation and the need to assess and compare any degradation that may occur. To address these concerns, we took several precautions during sample preparation to minimize the loss or degradation of antioxidants. These precautions include:

Standardized sample handling: We followed standardized protocols for sample collection, storage, and processing to ensure consistency and minimize variations in antioxidant content.

Minimization of light and oxygen exposure: We protected the samples from excessive light and oxygen exposure, as these factors can contribute to the degradation of antioxidants. Sample storage was performed in dark, airtight containers to reduce the potential for degradation.

Regarding the study and comparison of antioxidant degradation during sample preparation, it is important to note that our specific focus in this study was on assessing the total antioxidant capacity rather than investigating degradation rates. While we did not explicitly compare degradation of antioxidants during sample preparation, our methodology aimed to minimize such degradation by following established protocols and using appropriate storage and extraction methods.

Comment 10: What is the clinical relevance of the selected strains of bacteria?

Microbial resistance is a serious threat to global public health. Thus, there has been a growing interest in researching and developing new antimicrobial agents mainly from medicinal and aromatic plants. While investigating the antimicrobial potential C. siliqua Ethanolic Extract against, we tried to cover a broad spectrum of microorganisms including Gram positive bacteria, Gram negative bacteria and fungal strains. S. aureus and E. faecalis have been chosen to represent Gram positive bacteria, E. coli, E. vekanda and P. aeruginosa have been chosen to represent Gram negative bacteria, C. albicans have been chosen to represent yeasts and G. candidum have been chosen to represent molds.

Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus have been highlighted in the clique of microorganisms – acronymically termed ‘the ESKAPE pathogens’ – capable of ‘escaping’ the biocidal action of antibiotics and collectively representing new paradigms in pathogenesis, transmission and resistance. These bacteria cause multiple infections including pneumonia, bacteremia, urinary tract, ear, skin, and soft tissue infections and have numerous virulence factors and the ability to acquire antibiotic resistance determinants.

1. faecalis represents Enterococci which are important nosocomial pathogens. They are uniquely armed for the antibiotic era and express intrinsic reduced susceptibility to major classes of antimicrobial agents and biocides. The high propensity of enterococci to acquire and express new resistance determinants further enhances their ability to sustain antibiotic selection, promoting gastrointestinal colonization and nosocomial infections by antibiotic-resistant enterococci.

Geotrichum candidum is an extremely common fungus with a worldwide distribution and is the causative agent of geotrichosis . Pulmonary involvement is the most frequently reported form of the disease, but bronchial, oral, vaginal, cutaneous and alimentary infections have also been reported. Geotrichosis affects mainly the patients who are immunocompromised due to some underlying disease such as neoplasms, diabetes mellitus, leucosis, renal transplant and HIV.

Since the 1970s, the death rate from Candida disease has risen in conjunction with increasing numbers of patients at risk for serious fungal infections, such as those immunocompromised because of tissue or organ transplantation, chemotherapy, acquired immunodeficiency syndrome (AIDS), or advanced age. Candidal infections are not only prevalent but also associated with considerable mortality and morbidity. The majority of candidiasis cases, regardless of clinical setting and geographical location, are caused by C. albicans. C. albicans is capable of causing a wide spectrum of infections, from superficial thrush to life-threatening systemic candidiasis, making it the most prevalent fungal pathogen in humans.

Comment 11: 3.9.2. Comet Assay: author briefly mentions the visualization of comets using the ethidium bromide method but does not provide any details regarding the staining procedure, observation parameters, or imaging technique.

Thank you for your valuable comment. We have incorporated additional details into the main text of our study's protocol, which are now highlighted in yellow. We greatly appreciate your relevant observation and contribution.

 

Comment 12: The passage briefly mentions the potential therapeutic applications of the detected compounds, such as anti-inflammatory, antioxidant, and anti-cancer effects. However, it does not provide a detailed discussion of these implications or cite specific studies supporting these claims.

Thank you for your valuable feedback on our manuscript. We appreciate your thorough evaluation of the passage discussing the potential therapeutic applications of the compounds detected in carob extracts. We understand your concern regarding the need for specific studies supporting these claims.   In response, we would like to point out that we have indeed cited relevant studies to support our statements. For example, we cited the study by Eldahshan (2010) [22], which identified several phenolic compounds in the ethanolic extract of carob leaves, including gallic acid, quercetin 3-O-β-D-glucoside (isoquercetin), kaempferol 3-O-α-L-rhamnoside (afzelin), quercetin 3-O-α-L-rhamnoside (quercitrin), 1,2,6 tri-O-galloyl-β-D-glucopyranose, (-)-epigallocatechin-3-O-gallate, kaempferol, and quercetin. This study provides evidence for the presence of these compounds in carob extracts, suggesting their potential therapeutic applications. Additionally, we referenced the study by Goulas et al. (2019) [23], which identified gallic acid and rutin as the major phenolic compounds in carob powder. These findings further support the notion that carob extract possesses therapeutic potential.   Furthermore, we mentioned the detection of naringin as the most abundant constituent in our extract, highlighting its association with various health benefits, such as anti-inflammatory and antioxidant effects [24,25]. These effects have been demonstrated in studies investigating the properties of naringin. Moreover, the presence of quercetin and kaempferol in our extract suggests the potential anti-cancer properties of carob, as these compounds have been shown to possess anti-cancer effects in previous studies [26–31]. While we did not cite specific studies for these compounds in the passage, there is a substantial body of literature available supporting their anti-cancer properties.   We appreciate your insightful feedback, which will undoubtedly enhance the quality of our manuscript. Should you have any further suggestions or concerns, please do not hesitate to let us know.

Comment 13: 2.3. Physiochemical and Pharmacokinetic Properties (ADME) of CSEE : This analysis relies on predictive models and software tools While these tools provide valuable insights, their accuracy is dependent on the quality of the input data and the assumptions made by the models. Experimental validation of alteast one or two compounds is necessary to confirm the predicted properties.

Thank you for your feedback and suggestion regarding the physiochemical and pharmacokinetic properties (ADME) analysis of C. siliqua Ethanolic Extract (CSEE) in our study. We acknowledge that predictive models and software tools used in this analysis provide valuable insights, but their accuracy relies on the quality of input data and the underlying assumptions of the models. While experimental validation of the predicted properties through the testing of one or two compounds within the CSEE would indeed enhance the credibility of the findings, it is important to note that performing such experimental validation is beyond the scope of our current study.

Our study primarily focused on utilizing available predictive models and software tools to gather initial insights into the physiochemical and pharmacokinetic properties of the CSEE. The purpose was to provide a preliminary understanding of its potential ADME characteristics and aid in the formulation of future research directions.

Comment 14: Three compounds, namely naringin, and phloridzin dihydrate, do not comply with Lipinski's Rule of Five. These compounds have violations related to molecular weight, the number of oxygen atoms, and the number of hydrogen-bond donors, respectively.

Thank you for your feedback and observation regarding Lipinski's Rule of Five analysis in our study. We acknowledge that three compounds, namely naringin and phloridzin dihydrate, exhibit violations of Lipinski's Rule of Five criteria. Naringin exceeds the threshold for molecular weight, while phloridzin dihydrate has violations related to the number of oxygen atoms and the number of hydrogen-bond donors. These violations indicate that these compounds may possess physicochemical properties that could potentially affect their drug-like characteristics. While Lipinski's Rule of Five is a widely used guideline for predicting drug-likeness, it is important to note that its application is not absolute. There are instances where compounds that violate these criteria still demonstrate desirable pharmacological properties and therapeutic potential.

In our study, the focus was to investigate the activity and potential applications of these compounds, taking into account their specific structural features and target interactions. The violations of Lipinski's Rule of Five criteria were acknowledged and considered as factors that may influence their drug-like properties. It is worth noting that further analyses and considerations beyond Lipinski's Rule of Five can provide a more comprehensive assessment of the compounds' drug-like characteristics, such as exploring other molecular descriptors, conducting in vitro and in vivo studies, and evaluating their pharmacokinetic profiles.

We appreciate your insightful comment regarding the violations of Lipinski's Rule of Five in these compounds. We will ensure to provide a clear discussion on the implications of these violations and consider additional analyses to enhance the understanding of their pharmacological potential and suitability for further development.

 

Author Response File: Author Response.docx