Interactions of Oleanolic Acid, Apigenin, Rutin, Resveratrol and Ferulic Acid with Phosphatidylcholine Lipid Membranes—A Spectroscopic and Machine Learning Study

Round 1

Reviewer 1 Report

This work investigates the in vitro interactions of oleanolic acid and phenolic compounds: apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine membranes/liposomes and their effect on the structural properties of the membranes. The combination of oleanolic acid and phenolic compounds is characteristic for the Mediterranean diet.

Likewise, the results of structural studies were compared with antioxidant activity. The antioxidant effects of oleanolic acid on the peroxidation of liposomes, as well as the antioxidant effect of phenolic compounds on the peroxidation of liposomes containing oleanolic acid, were analyzed. The topic is relevant in this field. The conclusions are consistent with the evidence presented

 Here are some additional/specific comments to the article:

1. Introduction

The introduction does not clearly indicate the purpose and goals of the research. The introduction is also too long and needs to be rewritten.

The aim of the research is stated in lines 80-87 and again in lines 110-113. It should be in one paragraph at the end of this section.

In the introduction, it is necessary to clearly explain and emphasize that food components can interact with biological membranes, e.g. their components such as lipids, proteins, etc. Next, the importance of secondary plant metabolites, their potential biological activity, influence on cell signaling pathways and metabolism should be emphasized.

For the purpose of research, it should be stated why only interactions with lipids were observed, e.g. for the purpose of bioavailability of secondary metabolites.

 2. Lines 219-221: Full name for the NBD-PE is already mentioned.

 3. Conclusion. It should be noted whether further structural investigations of the tested phenolic compounds present in the natural/original form in plants are planned. It is well known that free phenols are relatively rare in plants. They are bounded as glycosides or esters, and also occur as condensation products.

Author Response

Dear Reviewer 1,

thank you very much for your valuable comments. I have made the changes as you recommended. This certainly improved and strengthened the substantive aspects of my study.

Response to Reviewer 1 Comments:

This work investigates the in vitro interactions of oleanolic acid and phenolic compounds: apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine membranes/liposomes and their effect on the structural properties of the membranes. The combination of oleanolic acid and phenolic compounds is characteristic for the Mediterranean diet.

Likewise, the results of structural studies were compared with antioxidant activity. The antioxidant effects of oleanolic acid on the peroxidation of liposomes, as well as the antioxidant effect of phenolic compounds on the peroxidation of liposomes containing oleanolic acid, were analyzed. The topic is relevant in this field. The conclusions are consistent with the evidence presented

 Here are some additional/specific comments to the article:

1. Introduction

The introduction does not clearly indicate the purpose and goals of the research. The introduction is also too long and needs to be rewritten.

We shortened the text in the introduction. We correct into the text. 

The aim of the research is stated in lines 80-87 and again in lines 110-113. It should be in one paragraph at the end of this section.

We removed repetitive content accordingly and improved the purpose of the work.:

The aim of this study was to evaluate the effects of the co-presence of oleanolic acid and phenolic compounds (apigenin, rutin, resveratrol, ferulic acid) on the properties of model liposome membranes by analyzing interactions with the membrane surface and hydrophobic core. In addition, the effects of these compounds on the process of lipid peroxidation in the membrane were examined, which may indeed support with a better understanding of their activity in the presence of oleanolic acid and in predicting these interactions using artificial intelligence.

We correct into the text.

In the introduction, it is necessary to clearly explain and emphasize that food components can interact with biological membranes, e.g. their components such as lipids, proteins, etc. Next, the importance of secondary plant metabolites, their potential biological activity, influence on cell signaling pathways and metabolism should be emphasized.

We correct into the text. 

For the purpose of research, it should be stated why only interactions with lipids were observed, e.g. for the purpose of bioavailability of secondary metabolites.

This is since most nutrients and biologically active ones penetrate cell membranes in the process of bioavailability. Mechanisms of absorption of these substances, e.g. is its ability to penetrate lipid membranes. As a result, secondary metabolites that can interact with lipid membranes may indeed have a greater chance of achieving higher bioavailability, i.e. availability for the body to assimilate and use.

2. Lines 219-221: Full name for the NBD-PE is already mentioned.

We've removed duplicates. We corrected into the text.

3. Conclusion. It should be noted whether further structural investigations of the tested phenolic compounds present in the natural/original form in plants are planned. It is well known that free phenols are relatively rare in plants. They are bounded as glycosides or esters, and also occur as condensation products.

In conclusion we add this sentence. We corrected into the text.

 

 

Kind regards,

Krzysztof Przybył

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The idea of the article is novel and interesting. However, there are some comments.

·         Why didn’t you study membrane stability?

·         Why didn’t you study more activities of oleanolic acid and phenolic compounds: apigenin, rutin, resveratrol and ferulic acid in addition to the antioxidant activities??

·         Why didn’t you study other characteristics of liposomes such as their morphology?

·         English language needs improvement

 

 

Moderate editing of English language required

Author Response

Dear Reviewer 2,

thank you very much for your valuable comments. I have made the changes as you recommended. This certainly improved and strengthened the substantive aspects of my study.

Response to Reviewer 2 Comments:

The idea of the article is novel and interesting. However, there are some comments.

• Why didn’t you study membrane stability?

These studies focused on examining the effect of the co-presence of oleanolic acid and phenolic compounds (apigenin, rutin, resveratrol, ferulic acid) on the properties of model liposomal membranes by analyzing the interaction with the membrane surface and the hydrophobic core. In addition, the effects of these compounds on the lipid peroxidation process in the membrane were tested, which may significantly help in better understanding their activity in the presence of oleanolic acid and in predicting these interactions using artificial intelligence. Membrane stability was not the immediate goal of these studies. We improved the purpose of the work under transparency and clarity.

• Why didn’t you study more activities of oleanolic acid and phenolic compounds: apigenin, rutin, resveratrol and ferulic acid in addition to the antioxidant activities??

Particular emphasis was placed mainly on the interactions between these compounds and membranes. The aim of the work was not to provide a comprehensive analysis of the activity of each substance.

• Why didn’t you study other characteristics of liposomes such as their morphology?

Morphology was not planned at this stage of the research. The authors will take this into account in the future. Thank you for your feedback.

• English language needs improvement

We corrected into the text.

 

Kind regards,

Krzysztof Przybył

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments to the Authors

The manuscript “Interactions of oleanolic acid, apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine lipid membrane – spectroscopic and machine learning study” is quite well written, and the results provided are scientifically sound. However, the language needs to be revised throughout the manuscript, as well as the aim and the methods used should be further improved.

Here are my detailed comments:

Abstract and keywords

- Line 17: substitute “:” with “such as”

- Line 26: substitute: ferulic acid

- Line 26: interactions

- Line 27: compared to the investigated flavonoids

-Line 27-29: I suggest to add some results related to the application of machine learning models, since this statement only stated that these models have been developed.

-Keywords: uniform with “;” or “,”

Introduction

-Line 44: substitute “link” with “correlations”

-Lines 54-57: Add references

 - Lines 88-98: I suggest to move this part, in which the main advancements with respect to the present state of the art and the main gaps to be filled were assessed, before the Line 80, where the purpose of the study was reported, highlighting how you want to fill these gaps.

Materials and Methods

- 2.3. Steady-state fluorescence and anisotropy fluorescence measurements: there is no need to repeat again all the acronyms, since already reported before in the section 2.1.

- 2.6. Statistical analysis: were the experiments and analyses conducted in duplicate or triplicate? Please add these info in the statistical analysis since it affects the standard deviations.

- Line 194: W?

Results and discussion

-        Lines 219-220: remove the meaning of  NBD-PE and DPH since already reported before.

-        Line 228: causes

-        Figures 2 - 5: uniform the letters under each graph.

-        (apigenin (AP), rutin (RT), resveratrol (RS), ferulic acid (FA)): add these acronyms at the beginning of the manuscript, only the first time that these compounds were cited, then only the acronyms can be reported.

-        Lines 305-306: Add reference.

-        Line 312: change “Rigidifying” with a synonym.

-        Line 336: “the obtained effect consisted in a reduced hydrodynamic diameter of liposomes”.

-        Figure 6: Uniform the X and Y axes of the graphs.

-        Line 413: add the dot.

-        Line 498: achieved by [39]. Add the surname of the author.

-        Line 505: “In order to compare,”. This statement seems incomplete. To compare what?

-        As a general suggestion: uniform the verb tenses throughout all the manuscript. Moreover, I suggest to substitute the personal form “we, our,…” in impersonal.

-        Line 512: Ocampo et al. 2021. Uniform all the references cited in the text according to the journal requirements. In the manuscript, the references have been always reported as “surname and co-workers” without the year. Please check this issue.

-        Table 1: arrange the layout by uniforming the width of the columns.

-        Better explain the meaning of the name MLP (e.g.1:1-100-50-50-6:1).

Conclusions

-        “under the influence of this compound, an increased exposure of the hydrophilic membrane region to the water environment was observed.”

-        Uniform the verb tenses, some of them were expressed in the present form, others in the past.

 

 

 

 

 

 

Comments for author File: Comments.docx

The English language needs to be revised throughout the manuscript.

Some statements seem to be incomplete and not clearly argued and discussed. Moreover, the verb tenses were not consistent and coherently reported in the text.

Author Response

Dear Reviewer 3,

thank you very much for your valuable comments. I have made the changes as you recommended. This certainly improved and strengthened the substantive aspects of my study.

Response to Reviewer 3 Comments:

 

 

Comments to the Authors

The manuscript “Interactions of oleanolic acid, apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine lipid membrane – spectroscopic and machine learning study” is quite well written, and the results provided are scientifically sound. However, the language needs to be revised throughout the manuscript, as well as the aim and the methods used should be further improved.

Here are my detailed comments:

Abstract and keywords

- Line 17: substitute “:” with “such as”

We correct into the text. 

- Line 26: substitute: ferulic acid

We correct into the text. 

- Line 26: interactions

We correct into the text. 

- Line 27: compared to the investigated flavonoids

We correct into the text. 

-Line 27-29: I suggest to add some results related to the application of machine learning models, since this statement only stated that these models have been developed.

We add information about the results of machine learning model. We correct into the text. 

-Keywords: uniform with “;” or “,”

We correct into the text. 

Introduction

-Line 44: substitute “link” with “correlations”

We deleted this sentence. We correct into the text. 

 

-Lines 54-57: Add references

We deleted this sentence. We correct into the text. 

 - Lines 88-98: I suggest to move this part, in which the main advancements with respect to the present state of the art and the main gaps to be filled were assessed, before the Line 80, where the purpose of the study was reported, highlighting how you want to fill these gaps.

We revised the introduction to maintain continuity and clarity as other reviewers have suggested. After the changes, this paragraph is now in the right place. Thank you for your suggestion. We correct into the text.

Materials and Methods

- 2.3. Steady-state fluorescence and anisotropy fluorescence measurements: there is no need to repeat again all the acronyms, since already reported before in the section 2.1.

We corrected as suggested another reviewer too. We correct into the text. 

- 2.6. Statistical analysis: were the experiments and analyses conducted in duplicate or triplicate? Please add these info in the statistical analysis since it affects the standard deviations.

The experiments and analyses conducted in triplicate. We correct into the text. 

- Line 194: W?

We correct into the text. 

 

Results and discussion

-        Lines 219-220: remove the meaning of  NBD-PE and DPH since already reported before.

We corrected as suggested another reviewer too. We correct into the text. 

-        Line 228: causes

We correct into the text. 

-        Figures 2 - 5: uniform the letters under each graph.

The authors think it's ok. Thank you for the suggestion.

-        (apigenin (AP), rutin (RT), resveratrol (RS), ferulic acid (FA)): add these acronyms at the beginning of the manuscript, only the first time that these compounds were cited, then only the acronyms can be reported.

The authors think it's ok. Thank you for the suggestion.

-        Lines 305-306: Add reference.

We correct into the text. 

-        Line 312: change “Rigidifying” with a synonym.

We correct into the text. 

-        Line 336: “the obtained effect consisted in a reduced hydrodynamic diameter of liposomes”.

We correct into the text. 

-        Figure 6: Uniform the X and Y axes of the graphs.

The authors think it's ok. Thank you for the suggestion

-        Line 413: add the dot.

We correct into the text. 

-        Line 498: achieved by [39]. Add the surname of the author.

We correct into the text. 

-        Line 505: “In order to compare,”. This statement seems incomplete. To compare what?

We correct this sentence. We correct into the text. 

-        As a general suggestion: uniform the verb tenses throughout all the manuscript. Moreover, I suggest to substitute the personal form “we, our,…” in impersonal.

We correct into the text. 

-        Line 512: Ocampo et al. 2021. Uniform all the references cited in the text according to the journal requirements. In the manuscript, the references have been always reported as “surname and co-workers” without the year. Please check this issue.

We correct into the text. 

-        Table 1: arrange the layout by uniforming the width of the columns.

We correct into the text. 

-        Better explain the meaning of the name MLP (e.g.1:1-100-50-50-6:1).

We correct into the text. 

Conclusions

-        “under the influence of this compound, an increased exposure of the hydrophilic membrane region to the water environment was observed.”

We correct into the text. 

-        Uniform the verb tenses, some of them were expressed in the present form, others in the past.

We correct into the text

Kind regards,

Krzysztof Przybył

 

Author Response File: Author Response.docx

Reviewer 4 Report

Manuscript Number: applsci-2555017

Title: Interactions of oleanolic acid, apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine lipid membrane-spectroscopic and machine learning study

This manuscript developed models to predict concentrations for selected compounds based on liposome size. In my opinion, the manuscript is acceptable in terms of technical aspects. However, the manuscript requires additional work in the form of minor revision before it is published in the journal. To improve the manuscript, I recommend the authors to address the following points:

1. The manuscript should be carefully checked to avoid grammatical errors.

2. Conclusion: Authors should add some necessary data.

3. References: Authors should cite the recent five years literature.

For example: Castellano, J. M., Ramos-Romero, S., & Perona, J. S. (2022). Oleanolic acid: Extraction, characterization and biological activity. Nutrients, 14(3), 623. https://doi.org/10.3390/nu14030623

Rabbani, M. , Pezeshki, A. , Ahmadi, R. , Mohammadi, M. , & Ghorbani, M. . (2021). Phytosomal nanocarriers for encapsulation and delivery of resveratrol- preparation, characterization, and application in mayonnaise. LWT- Food Science and Technology, 151(1–2), 112093. https://doi.org/10.1016/j.lwt.2021.112093.

The manuscript should be carefully checked to avoid grammatical errors.

Author Response

Dear Reviewer 4,

thank you very much for your valuable comments. I have made the changes as you recommended. This certainly improved and strengthened the substantive aspects of my study.

Response to Reviewer 4 Comments:

Title: Interactions of oleanolic acid, apigenin, rutin, resveratrol and ferulic acid with phosphatidylcholine lipid membrane-spectroscopic and machine learning study

This manuscript developed models to predict concentrations for selected compounds based on liposome size. In my opinion, the manuscript is acceptable in terms of technical aspects. However, the manuscript requires additional work in the form of minor revision before it is published in the journal. To improve the manuscript, I recommend the authors to address the following points:

1. The manuscript should be carefully checked to avoid grammatical errors.

We corrected into the text.

2. Conclusion: Authors should add some necessary data.

We corrected into the text.

3. References: Authors should cite the recent five years literature.

For example: Castellano, J. M., Ramos-Romero, S., & Perona, J. S. (2022). Oleanolic acid: Extraction, characterization and biological activity. Nutrients, 14(3), 623. https://doi.org/10.3390/nu14030623

Rabbani, M. , Pezeshki, A. , Ahmadi, R. , Mohammadi, M. , & Ghorbani, M. . (2021). Phytosomal nanocarriers for encapsulation and delivery of resveratrol- preparation, characterization, and application in mayonnaise. LWT- Food Science and Technology, 151(1–2), 112093. https://doi.org/10.1016/j.lwt.2021.112093.

We add citation as reviewer suggested. We corrected into the text.

 

 

Kind regards,

Krzysztof Przybył

 

 

Author Response File: Author Response.docx