Comparison Study of Different Extracts of Plectranthus madagascariensis, P. neochilus and the Rare P. porcatus (Lamiaceae): Chemical Characterization, Antioxidant, Antimicrobial and Cytotoxic Activities

Round  1

Reviewer 1 Report

The manuscript "Comparison study of different extracts of Plectranthus madagascariensis, P. neochilus and the rare P. porcatus (Lamiaceae): Chemical characterization, antioxidant, antimicrobial and cytotoxic activities" is interesting, reports significant findings and it is written in a good English.

However, presentation needs to be improved. I suggest authors to revise the following concerns:

1) Line 56: references 1, 2, 3 are not in order (written 10,23). Please revise.

2) The compounds discussed in the introduction can be numbered to ease its citation without needing to write the full name each time. 

3) The figure with the structure of the compounds can be split in two, and then the structures could be moved to the introduction, to make them more visual after the first citation of the compounds in the introduction. The HPLC fingerprint would remain as a separate figure.

4) Please check carefully the manuscript to find possible typos. I have found "bellow" in line 138 and "K. pneumonia" (should be K. pneumoniae) in Table 2. 

5) Format of tables clearly requires to be improved. Table 1 has too big interlining space for the usual standards in Molecules. So it can be reduced, Table can occupy all the width of the page and the good values could be highlighted using bold or italics. This table and its legend and footnotes should fit in one page, to avoid being splitted between two pages. Interlining space of table footnote should be reduced also.

6) Similar changes need to be applied to the remaining tables 2 and 3. In table 2, the good activity values (that justify the MIC determination for selected extracts) could be highlighted (bold or italics). 

7) Paragraph in lines 243-251 (just after Table 1) needs to be rewritten. Methanolic extracts do not show higher activity than ascorbic acid, it should be stated something like that the methanolic extract show more antioxidant activity than the extracts obtaining using the remaining solvents, but that the antioxidant activity of the methanolic extracts is slightly lower than the one of the ascorbic acid. 

8) It is not clear which positive control is used in Table 3 for each bacterial strain. Three positive controls are used (vancomycin, rifampicin and norfloxacin). Please denote each one somehow (as example, a, b, c as superscripts) and in the MIC value, denote the respective superscript in function of the positive control used. 

9) Format of table 4 clearly needs to be improved. Please center left column and upper rows, and provide a more standardized scientific format, with less lines between cells. 

10) Last paragraph of the body of the article (lines 352-368; "In summary..." could be put as Conclusion, as this section is missed. 

Author Response

Manuscript ID: biomolecules-489425 entitled “Comparison study of different extracts of Plectranthus madagascariensis, P. neochilus and the rare P. porcatus (Lamiaceae): Chemical characterization and cytotoxic activity”

Dear Reviewer 1:

We appreciate the reviewer1 comments, which have helped us to improve the manuscript. We have carefully considered the suggestions, addressing and incorporating them in the manuscript as detailed below. The modifications in the corrected paper have a yellow background.

Reviewer #1: The manuscript "Comparison study of different extracts of Plectranthus madagascariensis, P. neochilus and the rare P. porcatus (Lamiaceae): Chemical characterization, antioxidant, antimicrobial and cytotoxic activities" is interesting, reports significant findings and it is written in a good English.

However, presentation needs to be improved. I suggest authors to revise the following concerns:

Comment 1) Line 56: references 1, 2, 3 are not in order (written 10,23). Please revise.

Authors answer: We thank the reviewer for the comment. Indeed the references are not in order and we delete these references.

Comment 2) The compounds discussed in the introduction can be numbered to ease its citation without needing to write the full name each time.

Authors answer: We thank the reviewer for the suggestion and we include the number for each compound in the manuscript.

Comment 3) The figure with the structure of the compounds can be split in two, and then the structures could be moved to the introduction, to make them more visual after the first citation of the compounds in the introduction. The HPLC fingerprint would remain as a separate figure.

Authors answer: We thank the reviewer for the comments and suggestions. We moved the figure with the structures of the compounds to the introduction (Figure 1) and kept the HPLC fingerprint in figure 2 in the same place (Figure 3).

Comment 4) Please check carefully the manuscript to find possible typos. I have found "bellow" in line 138 and "K. pneumonia" (should be K. pneumoniae) in Table 2.

Authors answer: We thank the reviewer for the comments. We corrected in two more places in the manuscript and is highlighted in yellow.

Comment 5) Format of tables clearly requires to be improved. Table 1 has too big interlining space for the usual standards in Molecules. So it can be reduced, Table can occupy all the width of the page and the good values could be highlighted using bold or italics. This table and its legend and footnotes should fit in one page, to avoid being splitted between two pages. Interlining space of table footnote should be reduced also.

Authors answer: We thank the reviewer for the comments and suggestions that help to improve the observation of the Tables. We changed the manuscript with the suggestions of the reviewer, namely the reduction of the interlining space in the Table, the good values were highlighted using bold. In addition, the Table and its legend and footnotes are fitted in one page.

Comment 6) Similar changes need to be applied to the remaining tables 2 and 3. In table 2, the good activity values (that justify the MIC determination for selected extracts) could be highlighted (bold or italics).

Authors answer: We thank the reviewer for the comments and suggestions. We changed the tables 2 and 3 with the suggestions of the reviewer, namely the reduction of the interlining space in the Table and the good values were highlighted using bold.

Comment 7) Paragraph in lines 243-251 (just after Table 1) needs to be rewritten. Methanolic extracts do not show higher activity than ascorbic acid, it should be stated something like that the methanolic extract show more antioxidant activity than the extracts obtaining using the remaining solvents, but that the antioxidant activity of the methanolic extracts is slightly lower than the one of the ascorbic acid.

Authors answer: We thank the reviewer for the comments and suggestions. We changed the sentence as:

“Antioxidant activity was higher in the methanol extracts (Table 1) in comparison with the extracts obtained using the remaining solvents, but the antioxidant activity of the methanolic extracts is slightly lower than the one of the ascorbic acid, used as positive control in the DPPH assay. Moreover, the most active were the ones obtained from P. madagascariensis (E6 and E7), P. neochilus (E15 and E16) and P. porcatus (E23 and E24).”

Comment 8) It is not clear which positive control is used in Table 3 for each bacterial strain. Three positive controls are used (vancomycin, rifampicin and norfloxacin). Please denote each one somehow (as example, a, b, c as superscripts) and in the MIC value, denote the respective superscript in function of the positive control used.

Authors answer: We thank the reviewer for the comments and suggestions, which help us to improve the observation of Table 3. It was included the positive control used in each case, and also in the footnote as shown below:

Table 3 MIC values (mg/mL) for the most active Plectranthus spp. extracts

^a) VAN - vancomycin (Gram-positive), RIF - rifampicin (mycobacteria) or NOR - norfloxacin (Gram-negative); well diameter - 5 mm

Comment 9) Format of table 4 clearly needs to be improved. Please center left column and upper rows, and provide a more standardized scientific format, with less lines between cells.

Authors answer: We thank the reviewer for the comments and suggestions. For Table 4 we center the first 2 rows, we standardized with less lines between cells and fit it in one page.

Comment 10) Last paragraph of the body of the article (lines 352-368; "In summary..." could be put as Conclusion, as this section is missed.

 Authors answer: We thank the reviewer for the suggestions, which improved the manuscript.

Reviewer 2 Report

It is interesrting the comparison between extract plants but it could be innovative to try to identify new compounds if they occur instead of confirmig the activity of known standards. 

Author Response

Manuscript ID: biomolecules-489425 entitled “Comparison study of different extracts of Plectranthus madagascariensis, P. neochilus and the rare P. porcatus (Lamiaceae): Chemical characterization and cytotoxic activity”

Dear Reviewer 2:

We appreciate the reviewer2 comments, which have helped us to improve the manuscript. We have carefully considered the suggestions, addressing and incorporating them in the manuscript as detailed below. The modifications in the corrected paper have a yellow background.

Reviewer #2 Comment: It is interesting the comparison between extract plants but it could be innovative to try to identify new compounds if they occur instead of confirming the activity of known standards.

Authors answer: We thank the reviewer for the comment. Indeed the reviewer is correct when states that the identification of new compounds is very important which we actually try to identify. However, in these plants case, no new compounds were identified, only known bioactive molecules. Thus, we improved the work with its new chemical characterization, antioxidant, antimicrobial and cytotoxic activities.

Reviewer 3 Report

Manuscript by Rijo and co-workers describes extraction, characterization, and some bioassay of Plectranthus genus. Basically, the paper is well-presented and summarized. Obtained results would be interested for the reader of this journal. I do not have any points which should be revised in the manuscript. Thus, I think this may be accepted for publication in biomolecules.

Author Response

Manuscript ID: biomolecules-489425 entitled “Comparison study of different extracts of Plectranthus madagascariensis, P. neochilus and the rare P. porcatus (Lamiaceae): Chemical characterization and cytotoxic activity”

Dear Reviewer3:

We appreciate the reviewer3 comments, which have helped us to improve the manuscript. We have carefully considered the suggestions, addressing and incorporating them in the manuscript as detailed below. The modifications in the corrected paper have a yellow background.

Reviewer #3 Comment: Manuscript by Rijo and co-workers describes extraction, characterization, and some bioassay of Plectranthus genus. Basically, the paper is well-presented and summarized. Obtained results would be interested for the reader of this journal. I do not have any points which should be revised in the manuscript. Thus, I think this may be accepted for publication in biomolecules.

Authors answer: We thank the reviewer for the comment.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round  1

Reviewer 1 Report

The manuscript describes a comparison study of different extracts of three species of genus Plectranthus; two of them were already studied in a similar way and this work only contributes the comparative study. However, the work is well done and the biological evaluation is worth to publish after major considerations.

Page 2, line 54: the sentence starting “Different extraction…..technique” is hard to understand, please rewrite.

Where is the Fig. 1 mentioned in line 100 of page 3?

Table 1: The amounts obtained in each extract are really tiny, even the authors are able to weigh thousandths of mg, is that possible? I guess those are the results of the extraction of 10 g of plant with 100 mL of solvent, sometimes with 200 mL, others times the extracts are done with 30 g of plant, is that considered in table 4? Some data about the conventional microwave oven are necessary.

In lines 119 and 120 of page 4, there are data of RSA for some extracts that should also be included in the table, why are not?

If the authors compare different extracts attending the traditional use of the plants, It is not clear why they perform the ultrasound-assisted and supercritical CO2 extractions.

All the identified compounds are already known, but in the Fig 2 there are peaks in the HPLC chromatogram than seem to be in enough quantity to be identified with Rt over 15 min, as in E4, E7, E17, etc. Are they already known? In this sense, Fig 2 and Table 4 should be together, to identify quickly each compound with its Rt.

I miss a section with the conclusions, the authors present the results but no conclusions. 

Reviewer 2 Report

The quite big part of data has been already published in several works (even by the same authors) - as the authors themselves have cited in this manuscript: lines 121, 132, 150, 192. 

The antioxidant activity (table 1) is not completely presented. As a reader, I can not conclude if the other extracts (water, acetone, scCO2 and R-SCFE acetone) have been analysed? In the main text (line 118-121) the aqueous extracts are mentioned in the sense of ''relevant antioxidant capacity'' but there are no such data in the table. Although aqueous extracts data for P. neochilus and P. porcatus have been already published (line 120-121) they should be presented here as well – so, the whole comparative analysis will be completely presented. Thus, this part of analysis (experimentally or only in the sense of results presenting – not clear enough – should be reconsider/rewritten).

The title is incomplete: why the antibacterial and antioxidant activities data are missing (only chemistry and cytotoxicity are included)?

Regarding antibacterial activity: table 2 presents only part of the data. Although it is written that ''extracts not shown were inactive against all the microbial strains tested'', there are still incomplete data for the E6 and E15 extracts against S. epidermidis. The symbol ''-'' in the table can mean ''not tested'' (which is more logic here), or ''not active'' (usually is it n.a.). If it was not tested, it should be. Because, if the authors present comparison study, the data should be comparable meaning complete and certainly not being compared at random.

Quantification of the compounds registered in each extract sample is important and seems to be necessary in this kind of work (since the authors use the chemistry to explain biological activities of the samples).

In general, the manuscript is well-conceived as an idea, still missing some crucial data that should be added. 

Reviewer 3 Report

This manuscript describes the screening results of a number of extracts obtained for three related plant species. The H{LC profiles of some typical extracts are reported, and simple in vitro biological screening for antioxidant, antimicrobial and cytotoxic activities are presented. As such, the data are considered preliminary and they have little relevance to the scope of the Journal.