Influence of the Extraction Solvent and of the Altitude on the Anticancer Activity of Lebanese Eucalyptus camaldulensis Extract Alone or in Combination with Low Dose of Cisplatin in A549 Human Lung Adenocarcinoma Cells

Round 1

Reviewer 1 Report

Introduction

Please include evidence of altitude influence towards phytochemical contents and bioactivity extensively to support the rationale of the study.

The scientific name must be written correctly, please refer to the correct style

Some photography of Eucalyptus used in this study need to be included.

Materials and Methods

Information about plant authentication and voucher specimen must be provided when doing research with plant sample. This is to avoid miss identification of plant species.

Explain how the combination ratios were chosen

The combination treatment supposed to be tested on normal cells as claimed to reduced toxicity of cisplatin.

Result

Table presentation need to be improved. Is there any dose response curve result which used to get the IC50? If possible, please include.

Discussion

Depth discussion on the influence of altitude towards antiproliferative activity and the combination treatments.

Comments for author File: Comments.pdf

Author Response

Dear Editor, dear Reviewers,

We would like to thank you and the reviewers for the valuable comments. We have replied by the following point-by-point response to all of the reviewers’ comments. The article had been improved substantially.

Reviewer 1 :

Introduction

Please include evidence of altitude influence towards phytochemical contents and bioactivity extensively to support the rationale of the study.

Thank you for your comments:

We have demonstrated in the following article the influence of altitude on phytochemical contents and bioactivity. The sentence has been added as follow: “Previous study had shown that Lebanese plants are subjected to ingredients’ change with altitude and therefore may lose their medical values” (Rammal et al, 2015).(S.Kapoor et al, 2018) (A. Giorgi et al, 2010). Lines 58-65.

The scientific name must be written correctly, please refer to the correct style

Thank you for this point.

Scientific names and styles had been reviewed and amended accordingly.

Some photography of Eucalyptus used in this study need to be included.

We thank the reviewer for this comment.

A photography of the Eucalyptus plant and leaves has been added. Lines 410-411.

Materials and Methods

Information about plant authentication and voucher specimen must be provided when doing research with plant sample. This is to avoid miss identification of plant species.

We thank the reviewer for this comment.

Specimen Voucher and authentication of the plant has been added. Lines 400-401.

 Explain how the combination ratios were chosen

Thank you for this comment.

We have added the following sentence to the section. “Combination ratios of Eucalyptus extracts and cisplatin were chosen based on a previous study” (Nasser M. 2020). Lines 630-632.

The combination treatment supposed to be tested on normal cells as claimed to reduced toxicity of cisplatin.

We thank the reviewer for this valuable comment.

In fact, we have previously studied the effect of eucalyptus and cisplatin combination on normal cells. We found that eucalyptus extract played a protective role on normal cells (PBMC) as cell viability had increased when the former was added to cisplatin. We have added this sentence to the article “ as our previous study showed that eucalyptus extract had a protective role on normal cells when treated with cisplatin, combination treatment was not tested in this study on normal cells”.Lines 379-382.

Result

Table presentation need to be improved. Is there any dose response curve result which used to get the IC50? If possible, please include.

Thank you for this comment.

The presentation of the tables had been changed.

Regards

Reviewer 2 Report

1.      The topic is too long so that the research focus is not prominent.

2.      Tables need to be reflected in the form of three-line tables.

3.      Most of the figures have poor definition, especially Figure 3 and Figure 4.

4.      There are many formatting problems in the full text. For example, E. Camaldulensis needs italics, Na ₂CO ₃ instead of Na 2 CO 3,

5.      There are also many problems in the second and third level titles.

6.      The manuscript seems to lack the identification of structure, composition and purity.

7.      One column of data in Table 6 lacks errors.

8.      The abbreviations of AE and EE are given, but they are not used in the following text.

As a whole, I think this manuscript was prepared hastily.

Author Response

Dear Editor, dear Reviewers,

We would like to thank you and the reviewers for the valuable comments. We have replied by the following point-by-point response to all of the reviewers’ comments. The article had been improved substantially.

Reviewer 2

1. The topic is too long so that the research focus is not prominent.

We thank the reviewer for the comment.

Indeed, we agree with the reviewer that the topic is long. However, in this study, we tried to focus only on the effect of the altitude change of eucalyptus changes in order not to confuse the readers.

2. Tables need to be reflected in the form of three-line tables.

Thank you for this point. Tables have been changed accordingly. Lines: 99, 111, 117, 126 and 136.

3. Most of the figures have poor definition, especially Figure 3 and Figure 4.

Thank you for the comment.

We have improved the definition of figure 3 and 4. Lines160-162.

4. There are many formatting problems in the full text. For example, E. Camaldulensis needs italics, Na₂CO ₃ instead of Na 2 CO 3,

Thank you for the comment.

Formatting has been reviewed and changed throughout the manuscript.

5. There are also many problems in the second and third level titles.

Again, thank you for the comments.

Titles have been reformatted.

6. The manuscript seems to lack the identification of structure, composition and purity.

Thank you for this comment.

Some chemical composition and have been added. Lines 302-307.

7. One column of data in Table 6 lacks errors.

Thank you for this comment.

Standard errors have been added to table 6. Line 136.

8. The abbreviations of AE and EE are given, but they are not used in the following text.

Again, thank you for the comments.

9. The abbreviations have been changed

regards

Reviewer 3 Report

This manuscript could be accepted for publication in Processes. The topic of this manuscript is relevant to medicine. Lung cancer is the second most common cancer worldwilde. Eucalyptus plant extract had been shown to have anti-neoplastic effects. Authors investigated the antitumor effect of ethanolic (EE) and aqueous (AE) extracts of Eucalyptus Camaldulensis collected at different altitudes on A549. In addition, authors evaluated the additif effect of its combination with low-dose cisplatin (CDDP). Qualitative and quantitative analyses of secondary metabolites present in the plants were carried out. Antioxidant and cytotoxic activities of the different extracts on A549 were evaluated using the 2.2-diphenyl-1-picrylhydrazyl radical scavenging activity and neutral red assay, respectively. Cytotoxic effect of the combination of certain extract concentrations with low dose CDDP on A549 cells was studied. Higher level of active constituents and higher antioxidant activity were observed in the EE. The four E. Camaldulensis extracts showed cytotoxic activity on A549 cells with a higher extent for the EE and the sea-level altitude species. Moreover, the combination of the E. Camaldulensis with low dose CDDP exhibits a additif inhibitory effect at certain levels on A549 cells.  The introduction provide sufficient background. The research methodology is adequate. The results are clearly presented. The conclusions supported by the data. The manuscript good illustrated and interesting to read. The style is fine. It would be a good idea to mention in the manuscript not only cis-platin, but also some other organometallic compounds with potential anti-tumor activity and cite appropriate references: Inorg. Chim. Acta 2012. V. 380. P. 78.; Dalton Trans. 2015. V. 44. P. 6003.; Inorg. Chem. 2015. V. 54. P. 4039.; J. Mol. Struct. 2016, V. 1111. P. 142.; Organometallics 2016, V. 35. P. 3612.; New J. Chem. 2017, V. 41. P. 1940.; ACS Omega 2017, V. 2. P. 1380.; New J. Chem. 2018, V. 42. P. 8693. Finally, some polishing of English language is required. 

Author Response

Dear Editor, dear Reviewers,

We would like to thank you and the reviewers for the valuable comments. We have replied by the following point-by-point response to all of the reviewers’ comments. The article had been improved substantially.

This manuscript could be accepted for publication in Processes. The topic of this manuscript is relevant to medicine. Lung cancer is the second most common cancer worldwilde. Eucalyptus plant extract had been shown to have anti-neoplastic effects. Authors investigated the antitumor effect of ethanolic (EE) and aqueous (AE) extracts of Eucalyptus Camaldulensis collected at different altitudes on A549. In addition, authors evaluated the additif effect of its combination with low-dose cisplatin (CDDP). Qualitative and quantitative analyses of secondary metabolites present in the plants were carried out. Antioxidant and cytotoxic activities of the different extracts on A549 were evaluated using the 2.2-diphenyl-1-picrylhydrazyl radical scavenging activity and neutral red assay, respectively. Cytotoxic effect of the combination of certain extract concentrations with low dose CDDP on A549 cells was studied. Higher level of active constituents and higher antioxidant activity were observed in the EE. The four E. Camaldulensis extracts showed cytotoxic activity on A549 cells with a higher extent for the EE and the sea-level altitude species. Moreover, the combination of the E. Camaldulensis with low dose CDDP exhibits a additif inhibitory effect at certain levels on A549 cells.  The introduction provide sufficient background. The research methodology is adequate. The results are clearly presented. The conclusions supported by the data. The manuscript good illustrated and interesting to read. The style is fine. It would be a good idea to mention in the manuscript not only cis-platin, but also some other organometallic compounds with potential anti-tumor activity and cite appropriate references: Inorg. Chim. Acta 2012. V. 380. P. 78.; Dalton Trans. 2015. V. 44. P. 6003.; Inorg. Chem. 2015. V. 54. P. 4039.; J. Mol. Struct. 2016, V. 1111. P. 142.; Organometallics 2016, V. 35. P. 3612.; New J. Chem. 2017, V. 41. P. 1940.; ACS Omega 2017, V. 2. P. 1380.; New J. Chem. 2018, V. 42. P. 8693. Finally, some polishing of English language is required. 

We would like to thank you and the reviewers for the valuable comments.

We have mentioned the effects of organometallic compounds and their fewer side effects in the discussion with references. Lines 353-357.

regards

Round 2

Reviewer 2 Report

At present, this manuscript has basically solved the problems I raised.