Review Reports - A Study of the Bioactive Compounds, Antioxidant Capabilities, Antibacterial Effectiveness, and Cytotoxic Effects on Breast Cancer Cell Lines Using an Ethanolic Extract from the Aerial Parts of the Indigenous Plant <i>Anabasis aretioïdes</i> Coss. & Moq.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study carried out by Elfazazi and co-workers is well written but doesn't seem to me to be significantly different from Hamid Toufik's study referenced in (1), which notably studied the antioxidant power of this plant. It seems to me that there is a repetition of results already published and that it would be at least necessary to reduce the antioxidant part. Furthermore, as the products mentioned are part of the Anabasis aretioïdes Coss. & Moq. are also present in other plants, the authors' reference to other plants makes the article a little confusing. In my opinion, the focus should be on Anabasis aretioïdes Coss. & Moq. For a better understanding of the substances that could be responsible for the effects listed in the ancestral use of this endemic plant, it would be desirable to add a figure with all the substances present and identified from this plant in their developed form. This would give a more precise idea of the actions potentially expected depending on the structures and by analogy with the actions identified for other plants.

The article is interesting, but it needs to focus on the really new results, so I think it should be reworked in this direction.

Author Response

Review Report round 1: responses

Article: Unveiling the bioactive compounds, antioxidant capabilities, antibacterial effectiveness, and cytotoxic effects on breast cancer cell lines of the aerial part of the Anabasis aretioïdes Coss. & Moq.

Dear Editors and Reviewers,

We are grateful for the opportunity to improve our manuscript and sincerely thank you for the constructive feedback.

We extend our heartfelt thanks to the Reviewers for their thorough and insightful evaluation. We have carefully considered each comment and made substantial revisions to address the concerns raised. We hope that these modifications satisfactorily address the Reviewers’ suggestions and enhance the quality of the manuscript.

In this revision, we have either acknowledged or provided a detailed response to each requested adjustment or identified limitation. To facilitate review, major changes are highlighted in yellow. All typographical errors and minor issues have been corrected throughout the document, and as such, are not individually addressed in this response.

We kindly request that the revised manuscript be considered for publication in Current Issues in Molecular Biology (CIMB).

Sincerely,

Review Report (Reviewer 1)

We sincerely thank you for dedicating your time to thoroughly read and evaluate our article, as well as for your support and keen insights into its subject matter. We greatly appreciate your encouraging and constructive feedback. Please be assured that we are fully committed to making every possible improvement to enhance the manuscript’s quality and conducting a comprehensive review to ensure its readiness for publication.

Comment:

The study carried out by Elfazazi and co-workers is well written but doesn't seem to me to be significantly different from Hamid Toufik's study referenced in (1), which notably studied the antioxidant power of this plant. It seems to me that there is a repetition of results already published and that it would be at least necessary to reduce the antioxidant part. Furthermore, as the products mentioned are part of the Anabasis aretioïdes Coss. & Moq. are also present in other plants, the authors' reference to other plants makes the article a little confusing. In my opinion, the focus should be on Anabasis aretioïdes Coss. & Moq. For a better understanding of the substances that could be responsible for the effects listed in the ancestral use of this endemic plant, it would be desirable to add a figure with all the substances present and identified from this plant in their developed form. This would give a more precise idea of the actions potentially expected depending on the structures and by analogy with the actions identified for other plants.

The article is interesting, but it needs to focus on the really new results, so I think it should be reworked in this direction.

Response:

The objective of Hamid Toufik’s study was to perform a phytochemical analysis of the aerial parts of Anabasis aretioïdes Coss. & Moq. from the Figuig region in Morocco. This involved preparing various extracts: aqueous (decoction, infusion, maceration), Soxhlet extracts using four solvents with increasing polarity (methanol, ethyl acetate, chloroform, and petroleum ether), and cold methanol maceration for screening and quantification of total polyphenols and catechin tannins. The pharmacological assessment focused on evaluating in vitro antioxidant and radical-scavenging activities using five analytical methods: hydrogen peroxide (H₂O₂) scavenging, DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) assay, FRAP (Ferric Reducing/Antioxidant Power), and the reducing power (RP) method. Antibacterial activity was evaluated in vitro using the agar disc diffusion method, with minimum inhibitory (MIC) and bactericidal (MBC) concentrations determined for strains sensitive to the tested extracts.

Research has repeatedly proven that water or acetone effectively extracts flavanols with higher molecular weights, whereas methanol is more adept in extracting polyphenols with lower molecular weights. Ethanol, a substance considered non-toxic for human ingestion, is recognized as an effective solvent for the extraction of polyphenols. Following data analysis, we opted to employ a water-ethanol mixture to improve the extraction process's efficiency and guarantee the safety of the resultant extract. This decision was taken based on its suitability for human use, contingent upon the ensuing results being advantageous.

Our study aims to explore the potential of Anabasis aretiodes Coss. & Moq's aerial parts as a valuable source of specific compounds for use in food and phytopharmaceuticals. We aim to enhance the use and assessment of these endimic plants, thereby increasing their reputation and value. The study's main goal is to look at the bioactive compounds that are in the hydroethanol aerial parts of Anabasis aretioïdes Coss. & Moq extract. The objective is to assess the potential therapeutic applications of these compounds, specifically focusing on their bioactive phenolic properties. The objective of this work was to analyze the bioactive components present in the hydro-ethanol extracts obtained. During the analysis, we measured the amounts of total polyphenols, flavonoids, and condensed tannins using spectrophotometric methods. The extracts were also subjected to high-performance liquid chromatography (HPLC-DAD) analysis to find out the quality characteristics of the phenolic components that gave them their bioactive properties. The study aims to quantify the antioxidant capacity of the extracts to ascertain their biological impact. We will achieve this by utilizing various methods, including the DPPH scavenging IC50, beta-carotene bleaching assay, ABTS scavenging techniques, and total antioxidant capacity. Furthermore, the investigation seeks to establish linear correlations that demonstrate the importance of bioactive molecules in this antioxidant activity. In addition, we will evaluate the antibacterial efficacy of the extracts against specific strains and investigate their potential toxicity when administered to breast cancer cell lines. We used a computer model to show and confirm the biological activities of the hydroethanol extract from the aerial parts of the Anabasis plant using specific examples. The model showed how the biologically active compounds in the extract work by interacting with active sites in the target proteins.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to authors:

1- The authors would change the word unveiling in the title as the bioactive compounds are not novel, also add graphic abstract.

2- The authors should do English editing to the manuscript.

Abstract:

1- Line 38; “Staphylococcus aureus and Enterococcus faecalis”. Please in Italic as “Staphylococcus aureus and Enterococcus faecalis”. The same applies for “Escherichia coli and Pseudomonas aeruginosa”.

2- Line 40; “A computer study of the bioactive parts”. Please clarify the meaning of computer study and explain in more details the study.

3- The authors should mention the possible applications in medicine, food and so on.

Introduction:

1- Please give examples of the traditional treatments and their health hazards.

2- From line 118 to 129; replace this part from introduction to material and method.

3- Please highlight the aim of the study.

Materials and Methods

1- Plant origin and extraction process; please mention the date of plant collection. and the method of extraction.

Results and discussion

1- Please mention the possible uses of antibacterial activity results in fighting bacterial resistance to highlight importance.

2- Line 601; the authors linked oxidative stress with COPD. The authors should clarify the pathological and epidemiological connections between oxidative stress and COPD more clearly.

Comments on the Quality of English Language

The authors should do English editing to the manuscript.

Author Response

Review Report round 1: responses

Dear Editors and Reviewers,

We are grateful for the opportunity to improve our manuscript and sincerely thank you for the constructive feedback.

We kindly request that the revised manuscript be considered for publication in Current Issues in Molecular Biology (CIMB).

Sincerely,

Review Report (Reviewer 2)

We sincerely thank you for your thorough evaluation of our article and for your support and attention to its subject matter. We are grateful for the time and effort you invested in reviewing the manuscript and for providing valuable insights. We have carefully considered your feedback and recommendations, implementing substantial changes to address the issues you highlighted.

1- The authors would change the word unveiling in the title as the bioactive compounds are not novel, also add graphic abstract.

Thank you very much. The title has been changed to the following “A study of the bioactive compounds, antioxidant capabilities, antibacterial effectiveness, and cytotoxic effects on breast cancer cell lines using an ethanolic extract from the aerial segments of the indigenous plant Anabasis aretioïdes Coss. & Moq”. The graphical abstract has also been added

2- The authors should do English editing to the manuscript.

Thank you .Work has been done on improving the language of the manuscript, and if necessary, the option to edit the manuscript in English will be approved upon acceptance of the manuscript.

Abstract:

1- Line 38; “Staphylococcus aureus and Enterococcus faecalis”. Please in Italic as “Staphylococcus aureus and Enterococcus faecalis”. The same applies for “Escherichia coli and Pseudomonas aeruginosa”.

The microorganism's name is written in italics.

2- Line 40; “A computer study of the bioactive parts”. Please clarify the meaning of computer study and explain in more details the study.

Thank you. The meaning of computer study has been clarified and the study explained in more detail

3- The authors should mention the possible applications in medicine, food and so on.

Possible applications in medicine, food, etc. are listed in the introduction. This is due to the limited words allowed to be used in the abstract

Introduction:

1- Please give examples of the traditional treatments and their health hazards.

Examples of traditional treatments and their health hazards are given.

2- From line 118 to 129; replace this part from introduction to material and method.

The paragraph from lines 118 to 129 has been moved from the introduction to material and method.

3- Please highlight the aim of the study.

Work was done to highlight the aim of the study

Materials and Methods

1- Plant origin and extraction process; please mention the date of plant collection. and the method of extraction.

Plant origin and extraction process:

The date of plant collection, and the extraction method was determined.

Results and discussion

1- Please mention the possible uses of antibacterial activity results in fighting bacterial resistance to highlight importance.

The potential uses of antibacterial activity that leads to fighting bacterial resistance are mentioned to highlight its importance

2- Line 601; the authors linked oxidative stress with COPD. The authors should clarify the pathological and epidemiological connections between oxidative stress and COPD more clearly.

The pathological and epidemiological links between oxidative stress and COPD have been more clearly demonstrated.

“There is a marked increase in oxidative stress in the lungs of patients with COPD, as measured by increased exhaled 8-isoprostane, ethane, and hydrogen peroxide in the breath. The lung may be exposed to exogenous oxidative stress from cigarette smoking and indoor or outdoor air pollution and to endogenous oxidative stress from reactive oxygen species released from activated inflammatory cells, particularly neutrophils and macrophages, in the lungs. Oxidative stress in COPD may be amplified by a reduction in endogenous antioxidants and poor intake of dietary antioxidants. Oxidative stress is a major driving mechanism of COPD through the induction of chronic inflammation, induction of cellular senescence and impaired autophagy, reduced DNA repair, increased autoimmunity, increased mucus secretion, and impaired anti-inflammatory response to corticosteroids. Oxidative stress, therefore, drives the pathology of COPD and may increase disease progression, amplify exacerbations, and increase comorbidities through systemic oxidative stress. This suggests that antioxidants may be effective as disease-modifying treatments. Unfortunately, thiol-based antioxidants, such as N-acetylcysteine, have been poorly effective, as they are inactivated by oxidative stress in the lungs, so there is a search for more effective and safer antioxidants. New antioxidants in development include mitochondria-targeted antioxidants, NOX inhibitors, and activators of the transcription factor Nrf2, which regulates several antioxidant genes.Exogenous oxidative stress from cigarette and biomass smoke exposure and air pollution, endogenous oxidative stress from activated inflammatory cells in the lungs, and reduced antioxidants all lead to a high level of oxidative stress in the lungs. This is a major factor driving the pathophysiology of COPD and its progression, as well as amplifying acute exacerbations. Increased oxidative stress in the lungs of COPD patients results in chronic inflammation, reduced anti-inflammatory effects of corticosteroids, cellular senescence and accelerated lung ageing, autoimmunity, fibrosis of peripheral airways, and mucus hypersecretion. Systemic oxidative stress may contribute to many of the comorbidities associated with COPD”.

Reference: Oxidative Stress in Chronic Obstructive Pulmonary Disease, Antioxidants 2022, 11(5), 965; https://doi.org/10.3390/antiox11050965 . By Peter J. Barnes “National Heart and Lung Institute, Imperial College London, London SW5 9LH, UK”.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The corrections and explanations provided by the authors improve the manuscript, which can now be published as is.