Chemical Composition, Antioxidant, Antimicrobial and Anti-Proliferative Activities of Essential Oils of Rosmarinus officinalis from five Different Sites in Palestine

Round 1

Reviewer 1 Report

The current study, GC-MS were used to provide a quantitative and qualitative analysis and comparison of the leaves essential oil extracted from the aerial parts of Rosmarinus officinalis from five different sites in Palestine. In addition, the antioxidant activity, antimicrobial activity, and anti-proliferative activities of these essential oils was compared. These results reveal that the presence of a chemical variability of leaves essential oils extracted at five governorates in Palestine and the difference of these activities. The experimental content has certain value.

Several issues as follows:

1: Page 1: Lines 32: “Keywords:” “Antimicrobial, cytotoxicity.” Notice the punctuation mark between the two, and  the first letter of “cytotoxicity” should be capitalized.

2: Page 1: Lines 36: “It is widely used in cooking and healing across the world.” Please provide references. Similar advice is applied to Page 2: Lines 49-50: “Lung, colon and rectum, liver, stomach, and breast cancers are the leading causes of mortality worldwide, accounting for around 10 million fatalities in 2020.” etc.

Lines 66-70: “In the recent two decades, there has been a surge in the research of natural EOs as antibacterial, antioxidant, and anticancer agents for the treatment and prevention of various disorders.” Supplement the latest relevant literature, such as: ①Chemical composition, anticancer activities and related mechanisms of the essential oil from Alpinia coriandriodora rhizome. Industrial Crops and Products, 2022, 176: 114328; ②Antioxidant, Antibacterial, Enzyme Inhibitory, and Anticancer Activities and Chemical Composition of Alpinia galanga Flower Essential Oil. Pharmaceuticals, 2022, 15:1069.

3: Line 43-55:  Re-organize the relevant content of oxidative stress, cancer, and microorganism, it seems very messy.

4: Page 2：Lines 68-70: “The phytochemical contents of EOs are influenced not only by the plant species, but also by the stocking time, agrochemicals used in preparation, soil type, and climate, as well as the harvested plant portion, variety, and age, among other factors” and Page 2: Lines 71-72: “The phytochemical contents of EOs are influenced not only by the plant species, but also by the stocking period, agrochemicals used in preparation, soil type, climate, as well as the age and harvested portion of the plant.” are repeated.

5. Page 2: Line 76: “EOs collected from five distinct Palestinian governorates against multiple microbial strains and cancer cell lines.” According to Page2: Line 77: “2. Materials and Methods” the EOs here should be specified as “leaves EOs.”

6: Page 3: Line 101: “A UV-Vis spectrophotometer set at 517 nm was used to measure absorption.” The “A UV-Vis spectrophotometer” mentioned here should indicate the manufacturer and model.

7: Page 3: Line 104: The “ABS” in the formula should be replaced by “Abs”.

8: Page 3: Line 113: “each EO was dissolved in DMSO and distilled water.” The ratio of DMSO to distilled water should be indicated.

9: Page 3: “2.5. Cell culture and cytotoxicity assay”, please provide relevant references for the tests. Line129: “RPMI-1640 media and supplemented with 1% mixture of Streptomycin and Penicillin, 1% l-glutamine,” Streptomycin, penicillin, 1% l-glutamine are solid, using 1% to indicate a concentration is inappropriate.

10: Line 141-142: “Medicinal plants from semi-arid and arid parts of Palestine's West Bank have become key sources of bioactive compounds with anti-proliferative, antioxidant, hypoglycemic, anti-obesity, and antibacterial activities in recent years [22]”. Delete this sentence.

11: Line 166: “R. officinalis EOs” instead of “ROEO”.

12: Page 7: “Figure 1. DPPH inhibitory potentials by Rosemarinus officinalis essential oils from five Palestinian regions and Trolox.” Pay close attention to the ordinate's format in the figure.

13: Page12: “4. Conclusions” Line 286-287: “The findings of the present study reveal that r. officinalis EO collected from five distinct places in Palestine had strong antibacterial and antioxidant activities.” There are spelling errors in the reference to "r. officinalis EO."

14: Line 254: “Our findings indicate that R. officinalis EO has no cytotoxic impact and may hence be regarded harmless”. What is the criterion for non-cytotoxicity? The IC₅₀ is greater than how much to have no cytotoxicity. Provide appropriate references. Usually, a certain dose range in our experiment has no effect on cell proliferation, which only means that this concentration range has no cytotoxicity.

15: In Figure 2, was the MTS assay only repeated once? Why are there no error bars. Table 4 has no standard deviation. In line 245, “Figure 2A-H”, mark A to H in Figure 4.

16: In References, the format of references in this paper is inconsistent. In particular, plant names need to be italicized.

Author Response

Dear Reviewer,

I want to express my deep gratitude to your insightful comments and feedback that helped us enhance our article.  I hope my responses were adequate.

Several issues as follows:

1: Page 1: Lines 32: “Keywords:” “Antimicrobial, cytotoxicity.” Notice the punctuation mark between the two, and the first letter of “cytotoxicity” should be capitalized.

Done

2: Page 1: Lines 36: “It is widely used in cooking and healing across the world.” Please provide references. Similar advice is applied to Page 2: Lines 49-50: “Lung, colon and rectum, liver, stomach, and breast cancers are the leading causes of mortality worldwide, accounting for around 10 million fatalities in 2020.” etc.

Done

ines 66-70: “In the recent two decades, there has been a surge in the research of natural EOs as antibacterial, antioxidant, and anticancer agents for the treatment and prevention of various disorders.” Supplement the latest relevant literature, such as: ①Chemical composition, anticancer activities and related mechanisms of the essential oil from Alpinia coriandriodora rhizome. Industrial Crops and Products, 2022, 176: 114328; ②Antioxidant, Antibacterial, Enzyme Inhibitory, and Anticancer Activities and Chemical Composition of Alpinia galanga Flower Essential Oil. Pharmaceuticals, 2022, 15:1069.

Done

3: Line 43-55:  Re-organize the relevant content of oxidative stress, cancer, and microorganism, it seems very messy.

Done

4: Page 2：Lines 68-70: “The phytochemical contents of EOs are influenced not only by the plant species, but also by the stocking time, agrochemicals used in preparation, soil type, and climate, as well as the harvested plant portion, variety, and age, among other factors” and Page 2: Lines 71-72: “The phytochemical contents of EOs are influenced not only by the plant species, but also by the stocking period, agrochemicals used in preparation, soil type, climate, as well as the age and harvested portion of the plant.” are repeated.

Done

5. Page 2: Line 76:“EOs collected from five distinct Palestinian governorates against multiple microbial strains and cancer cell lines.” According to Page2: Line 77: “2. Materials and Methods” the EOs here should be specified as “leaves EOs.”

Done

6: Page 3: Line 101: “A UV-Vis spectrophotometer set at 517 nm was used to measure absorption.” The “A UV-Vis spectrophotometer” mentioned here should indicate the manufacturer and model.

Done

7: Page 3: Line 104: The “ABS” in the formula should be replaced by “Abs”.

Done

8: Page 3: Line 113: “each EO was dissolved in DMSO and distilled water.” The ratio of DMSO to distilled water should be indicated.

Done

9: Page 3: “2.5. Cell culture and cytotoxicity assay”, please provide relevant references for the tests. Line129: “RPMI-1640 media and supplemented with 1% mixture of Streptomycin and Penicillin, 1% l-glutamine,” Streptomycin, penicillin, 1% l-glutamine are solid, using 1% to indicate a concentration is inappropriate.

A suitable ref. was added to this method accordingly

10: Line 141-142: “Medicinal plants from semi-arid and arid parts of Palestine's West Bank have become key sources of bioactive compounds with anti-proliferative, antioxidant, hypoglycemic, anti-obesity, and antibacterial activities in recent years [22]”. Delete this sentence.

Deleted

11: Line 166: “R. officinalis EOs” instead of “ROEO”.

Done

12: Page 7: “Figure 1. DPPH inhibitory potentials by Rosemarinus officinalis essential oils from five Palestinian regions and Trolox.” Pay close attention to the ordinate's format in the figure.

We apologize for the poor layout of the figure; we have redesigned it accordingly.

13: Page12: “4. Conclusions” Line 286-287: “The findings of the present study reveal that r. officinalis EO collected from five distinct places in Palestine had strong antibacterial and antioxidant activities.” There are spelling errors in the reference to "r. officinalis EO."

Done

14: Line 254: “Our findings indicate that R. officinalis EO has no cytotoxic impact and may hence be regarded harmless”. What is the criterion for non-cytotoxicity? The IC₅₀ is greater than how much to have no cytotoxicity. Provide appropriate references. Usually, a certain dose range in our experiment has no effect on cell proliferation, which only means that this concentration range has no cytotoxicity.

Various references were added according to this issue, as well as the IC50 values to make toxicity were too high in comparison with positive control doxorubicin and the other biological targets, for example the effective dose to be active against most of bacterial strains were less than 50 µg/mL while the effective dose on normal and cancer cell lines were higher and in mg/mL.

15: In Figure 2, was the MTS assay only repeated once? Why are there no error bars. Table 4 has no standard deviation. In line 245, “Figure 2A-H”, mark A to H in Figure 4.

The MTS assay was done triplicate and we added the error bars, the SD also is added to the table 4, the figure was marked accordingly  

16: In References, the format of references in this paper is inconsistent. In particular, plant names need to be italicized.

Done

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript describes the chemical profiles of essential oils of Rosmarinus officinalis from five different governorates in Palestine by GC-MS technique, as well as the antioxidant activity using the DPPH test, the antimicrobial activity using the microdilution broth technique, and the antiproliferative activity using the MTS assay.

I have some comments for the paper, which must be taken into account to improve the article before it will be suitable for acceptance for publication:

-          The authors made a brief introduction on the therapeutic benefits of the plant and explaining the different diseases. Some readers are already quite familiar with this information. The introduction should emphasize progress in the field of research rather than providing background information. For example, nothing is mentioned about the numerous extraction techniques for essential oils, the methods used in their analysis, etc.

-          The information on lines 68-70 is repeated on lines 71-72.

-     In the “Materials and Methods” section: please include a section dedicated to all the reagents used in the study (methanol, DMSO, Trolox, etc.) with their purities, commercial companies, etc.

-          Line 79: please indicate the meaning of “RO”.

-  The authors must indicate the acquisition mode, software and fragmentation used in the GC-MS method. I imagine that the acquisition is in full scan (indicate it). Please, the authors should indicate the m/z range used.

-          The injection volume of 0.2L is correct?

-     Section 2.3. Free radical scavenging activity: Was a negative control (blank) prepared in parallel? Please indicate the spectrophotometer used (also in section 2.5).

-     The first time that an acronym is used it should be explained. For example, DMSO (line 114), DPPH, etc.

-          Line 115: authors should include the name of the specie in italics.

-          Section 2.6. Statistical analysis: The mean and standard deviation are the base for statistical analysis of the data, but they are not enough. Did the authors an ANOVA?

-          Table 1: authors may include m/z identified for each compound. Please indicate the meaning of RI_calculated in the table.

-          Line 166: please indicate the meaning of “ROEO”.

-          Authors may include a GC-MS chromatogram as an example.

-          The information on lines 193-195 is repeated on lines 196-198.

-          Table 2: authors should indicate the unit of the concentrations used.

-      Figure 1: the use of tables and figures serves to facilitate the reader's understanding, but results presented in figures should not be repeated in tables. In my opinion Figure 1 could be eliminated because the information is already available in Table 2.

-       The information displayed on lines 245-253 does not contribute anything beyond what is already presented in Table 4.

-          Line 275: doxorubicin should be included.

-          Table 4: please indicate the meaning of “DOX”.

-          The figure corresponding to cell viability is the number 3 (line 279) and should be referenced and discussed in the text.

-          The authors conclude that “the molecular profile of the essential oils of R. officinalis grown in Palestine differs somewhat depending on where the plant was harvested”, but no statistical analysis is indicated at any time to verify whether or not this difference is significant.

-       Line 286: authors should include the name of the specie in italics and capital letter.

Author Response

Dear Reviewer,

I want to express my deep gratitude to you a for the insightful comments and feedback that helped us enhance our article. . I hope my responses were adequate.

I have some comments for the paper, which must be taken into account to improve the article before it will be suitable for acceptance for publication:

-          The authors made a brief introduction on the therapeutic benefits of the plant and explaining the different diseases. Some readers are already quite familiar with this information. The introduction should emphasize progress in the field of research rather than providing background information. For example, nothing is mentioned about the numerous extraction techniques for essential oils, the methods used in their analysis, etc.

I completely agree, however our goal was not to compare extraction techniques, but to identify EO constituents and investigate their biological activities, and then to analyze the biological activities of their mixtures. Furthermore, we cannot afford to purchase all of the technologies essential for our work. 

-          The information on lines 68-70 is repeated on lines 71-72.

Done

-     In the “Materials and Methods” section: please include a section dedicated to all the reagents used in the study (methanol, DMSO, Trolox, etc.) with their purities, commercial companies, etc.

Done

-          Line 79: please indicate the meaning of “RO”.

Done

-  The authors must indicate the acquisition mode, software and fragmentation used in the GC-MS method. I imagine that the acquisition is in full scan (indicate it). Please, the authors should indicate the m/z range used.

Done

-          The injection volume of 0.2L is correct?

Corrected

-     Section 2.3. Free radical scavenging activity: Was a negative control (blank) prepared in parallel? Please indicate the spectrophotometer used (also in section 2.5).

the Negative control was prepared in parallel as well as the spectrophotometer was added to this section  

-     The first time that an acronym is used it should be explained. For example, DMSO (line 114), DPPH,

etc.

Done

-          Line 115: authors should include the name of the specie in italics.

Done

-          Section 2.6. Statistical analysis: The mean and standard deviation are the base for statistical analysis of the data, but they are not enough. Did the authors an ANOVA?

No we didn’t do that.

-          Table 1: authors may include m/z identified for each compound. Please indicate the meaning of RI_calculated in the table.

Done

-          Line 166: please indicate the meaning of “ROEO”.

Done

-          Authors may include a GC-MS chromatogram as an example.

-          The information on lines 193-195 is repeated on lines 196-198.

Done

-          Table 2: authors should indicate the unit of the concentrations used.

Done

-      Figure 1: the use of tables and figures serves to facilitate the reader's understanding, but results presented in figures should not be repeated in tables. In my opinion Figure 1 could be eliminated because the information is already available in Table 2.

the table was deleted. 

-       The information displayed on lines 245-253 does not contribute anything beyond what is already presented in Table 4.

the information in these lines is an explanation of Table 2 antimicrobial results, we improved them accordingly.

-          Line 275: doxorubicin should be included.

Done

-          Table 4: please indicate the meaning of “DOX”.

Done

-          The figure corresponding to cell viability is the number 3 (line 279) and should be referenced and discussed in the text.

we added a description regarding this figure

-          The authors conclude that “the molecular profile of the essential oils of R. officinalis grown in Palestine differs somewhat depending on where the plant was harvested”, but no statistical analysis is indicated at any time to verify whether or not this difference is significant.

Unfortunately we didn’t do that.

-       Line 286: authors should include the name of the specie in italics and capital letter.

Done

Author Response File: Author Response.docx

Reviewer 3 Report

The article titled chemical composition, antioxidant, antimicrobial and anti-prolifer-2 ative activities of essential oils of Rosmarinus officinalis from five 3 different sites in Palestine seems to be interesting. An article attempted to evaluate the changes in chemical compositions, antibacterial, antioxidant, and cytotoxic effects of R. officinalis EOs collected from five distinct Palestinian governorates against multiple microbial strains and cancer cell lines. I don't think it was written by paying attention to the journal writing rules. I think it will be more successful if edits are made in terms of writing and content. The article can be accepted after corrections. The areas that need to be corrected are given below

Check typographical errors, e.g. spacing, capitalization, italics, punctuation.

English and text construction in the manuscript needs refinement.  

The abstract needs to be improved

Keywords should not be chosen from the title.

More recent references can be given instead of very old references

Plant material authentication report from the expert should be attached or the accession no. of the plant shall be provided.

Statistical significance is missing from the tables.

Error bar and  Statistical significance is missing from the figure.

If abbreviations or letters are used in tables and figures, their explanation should be given under the table

Author Response

Dear Editor,

I want to express my deep gratitude to your insightful comments and feedback that helped us enhance our article.  I hope my responses were adequate.

The article titled chemical composition, antioxidant, antimicrobial and anti-prolifer-2 ative activities of essential oils of Rosmarinus officinalis from five 3 different sites in Palestine seems to be interesting. An article attempted to evaluate the changes in chemical compositions, antibacterial, antioxidant, and cytotoxic effects of R. officinalis EOs collected from five distinct Palestinian governorates against multiple microbial strains and cancer cell lines. I don't think it was written by paying attention to the journal writing rules. I think it will be more successful if edits are made in terms of writing and content. The article can be accepted after corrections. The areas that need to be corrected are given below

Check typographical errors, e.g. spacing, capitalization, italics, punctuation.

Done

English and text construction in the manuscript needs refinement.  

Done

The abstract needs to be improved

Done

Keywords should not be chosen from the title.

Nidal

More recent references can be given instead of very old references

We completely agree with the reviewer, and a new references were added

Plant material authentication report from the expert should be attached or the accession no. of the plant shall be provided.

done

Statistical significance is missing from the tables.

 The GC-MS study was repeated twice, and the findings were almost identical both times.

Error bar and Statistical significance is missing from the figure.

we added the Error bars and SD to the tables and figures

If abbreviations or letters are used in tables and figures, their explanation should be given under the table

Done

Reviewer 4 Report

The authors report the Chemical composition, antioxidant, antimicrobial and anti-prolifer-active activities of essential oils of Rosmarinus officinalis from five different sites in Palestine, I consider that minimal things should be revised.

Line 3: is it possible to know descriptor or variety?

Line 21: it is not necessary to repeat that you are working with five species, you have already said it before

line 79-84: Materials and Methods, provide conditions of collection sites; coordinates and altitudes. 

lines 145-149: can be compared with the yiel of other EOs in other countries or studies?

Line 154: please provide the RI of bibliography

Author Response

Dear Reviewer,

I want to express my deep gratitude to you for the insightful comments and feedback that helped us enhance our article. I hope my responses were adequate.

Comments and Suggestions for Authors

The authors report the Chemical composition, antioxidant, antimicrobial and anti-prolifer-active activities of essential oils of Rosmarinus officinalis from five different sites in Palestine, I consider that minimal things should be revised.

Line 3: is it possible to know descriptor or variety?

I'm not sure what the reviewers are saying.

Line 21: it is not necessary to repeat that you are working with five species, you have already said it before

Done

line 79-84: Materials and Methods, provide conditions of collection sites; coordinates and altitudes. 

Done

lines 145-149: can be compared with the yiel of other EOs in other countries or studies?

Done

Line 154: please provide the RI of bibliography

We compare the computed RI to those recorded in the NIST database and the pherobase database, and since the same chemical has several RI, I choose not to include the literature RI in the table.

Round 2

Reviewer 3 Report

I have critically reviewed the revised version of the manuscript Chemical composition, antioxidant, antimicrobial and anti-proliferative activities of essential oils of Rosmarinus officinalis from five different sites in Palestine. I have found that the authors well-addressed the major concerns and the revised version is improved significantly.