Bioactive Potential of Pogostemon benghalensis (Burm.f.) Kuntze: Antibacterial, Antioxidant, and Xanthine Oxidase Inhibitory Activities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article investigated the active components of inflorescence and stem bark of Pogostemon benghalensis extracted by three solvents (water, methanol and ethyl acetate). The total phenol content and total flavonoid content were determined. The antioxidant, xanthine oxidase inhibitory and bacteriostatic effects of the extract were studied. This study is an interesting and has some useful information for the application of P. benghalensis stem bark and inflorescence. However, there are some questions need to address:

1. Line 207, The apparatus of US spectrophotometer should be given relevant information, such as instrument model, manufacturer, city and nation.  

2. Line 208: why did the total flavonoid content in the extracts is determined at 415 nm? please provide references.

3. The unit of total phenol content and total flavonoid content in Table 2 should be provided.

4. Please add the error bars in Figure 2 and Figure 4.

5. Line 647: “at” should be deleted.

6. The format of table 2-6 should be changed to a three-line table.

7. The references styling should be revised according to the requirements of journal guidelines.

8. In section 2.5 and Table 1, what is the extraction rate of substance? Not clearly described.

9. The English of this manuscript should be improved.

Comments on the Quality of English Language

The English of this manuscript should be improved.

Author Response

Dear reviewer. We are very much grateful for giving your time to uplift the quality of our manuscript and making more scientific. We have addressed every suggestion and concern and revised the manuscript. Change has been yellow highlighted.

Comment-1-Line 207, The apparatus of US spectrophotometer should be given relevant information, such as instrument model, manufacturer, city, and nation.  

Response 1- We are thankful to reviewer for relevant suggestion. Now we have done suggested addition (Line 201-202).

Comment-2. Line 208: why did the total flavonoid content in the extracts is determined at 415 nm? please provide references.

Response-2- We are thankful to reviewer for critically reviewing our manuscript to screen mistakes microscopically. We would like to apologize for our typo, as it should be 510 nm. Now we have corrected the error. (Line 202)

Comment-3-The unit of total phenol content and total flavonoid content in Table 2 should be provided.

Response-3- As per the suggestion of reviewer we have done necessary addition in the manuscript. (Table 2; Line 439)

Comment-4- Please add the error bars in Figure 2 and Figure 4.

Response-4- We completely agree with reviewers’ recommendation. As per the suggestion of reviewer we have done necessary addition in the manuscript. (Page 12; Line 474 and Page 14; Line 509)

 

Comment-5- Line 647: “at” should be deleted.

Response-5- Thank you so much for rectifying this grammatical mistake. We have corrected now (Page 18; Line 662)

Comment-6- The format of table 2-6 should be changed to a three-line table.

Response-6- As per reviewers’ suggestion we have made three-line table throughout the manuscript.

 

Comment-7- The references styling should be revised according to the requirements of journal guidelines.

Response-7- We have taken the suggestion of reviewer seriously and converted whole reference section as per journal format.

Comment-8- In section 2.5 and Table 1, what is the extraction rate of substance? Not clearly described.

After reading the suggestion of reviewer, we studied literature well and have added small scientific discussion to emphasize on importance of extraction ratio to optimize suitable solvent for extracting wide variety of bioactive constituents. (Page 9; Line 401 to 412).

Comment-9- The English of this manuscript should be improved

After completing the whole review of this manuscript, it has been reviewed by one of co-author (Dr. Hari Prasad Devkota, Assistant Professor at Kumamoto University Japan) to make it correct. Also, we used premium Grammarly software to reduce grammatical mistakes. We have rewritten several sections to improve the quality of English.

Reviewer 2 Report

Comments and Suggestions for Authors

I find this paper interesting. However it lacks some important discussions in regard to previously published papers, e.g. https://link.springer.com/article/10.1007/s42535-022-00557-2

Author Response

Dear Reviewer, we are very thankful for your feedback to improve the quality of this manuscript. The change made has been yellow-highlighted in the manuscript.

Comment- 1- I find this paper interesting. However, it lacks some important discussions in regard to previously published papers, e.g. https://link.springer.com/article/10.1007/s42535-022-00557-2.

 

Response-1-We are very much grateful to the reviewer for the help to make our discussion more scientific and impactful. As per the suggestion, we have discussed the scientific findings of the above-mentioned manuscript and added to our discussion section.  (Page 18 and 19, Line 641-648)

Reviewer 3 Report

Comments and Suggestions for Authors

The paper has an interesting subject, but I have some doubts and questions:

1.     The botanical name of the plant examined in the title should be completed.

2.     It needs to be explained in abstract what ZOI means.

3.     Why the value of MIC and MBC are not presented in abstract?

4.     The introduction is too long and should be shortened.

5.     Line 155: Why were the dried inflorescences crushed by hand rather than ground?

6.     The antimicrobial activity of the extract can hardly be described as strong. What was the criterion used to assess this activity? The extracts show rather weak antibacterial activity against gram+ bacteria and almost no activity against gram- bacteria.

7.     It is worth to present MIC/MBC ratio.

8.     The discussion part is extremely long and contains a lot of repaetion e.g. about several hydroxyl group in polyphenol and their connection with antioxidant activity. It is difficult to read.

9.     The discussion section is extremely long and contains a lot of repetition, e.g. about different hydroxyl groups in polyphenols and their connection with antioxidant activity. It is difficult to read.

10.  Lines 613-614 are trivial and repeat the results. Of course, the extracts were more active against gram-positive bacteria because there is no activity against gram-negative bacteria.

11.  Line 667-671 is very speculative  and actually not true.

12.  However, the biggest drawback of the work is the lack of any analysis of the extracts tested. Not even a qualitative HPLC analysis, let alone an LC-MS spectral analysis. Determining the sum of polyphenolic compounds or flavonoids is not phytochemical analysis.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear reviewer, We are very much grateful for taking the time to uplift the quality of our manuscript and making more scientific. We have addressed every suggestion and concern and revised the manuscript. All the change have been yellow highlighted in the manuscript

Comment-1- The botanical name of the plant examined in the title should be completed.

Response-1- We are thankful to reviewer for great suggestion. Now, we have added complete botanical name of plant in the title. (Line 2).

Comment-2- It needs to be explained in abstract what ZOI means.

Response-2- Now we have added the full form of ZOI in line 23.

Comment-3- Why the value of MIC and MBC are not presented in abstract?

Response-3- Thank you very much for microscopic review of the manuscript. Now we have added the full form of MIC and MBC in the abstract (Line 24-25)

Comment-4-The introduction is too long and should be shortened.

Response-4- We really appreciate the suggestion of reviewer. We have entirely rewritten the introduction section to make short and scientific. (Line 38-52; Line-86--102).

Comment-5- Line 155: Why were the dried inflorescences crushed by hand rather than ground?

Response-5- When we shade dried inflorescence of the P. benghalensis, sample was very crunchy and it grinded to very fine powder after grinding and was very challenging to filter as fine powder got clogged in the filter paper. So, we decided to crus the powder manually and powder size was fine.

Comment-6-   It is worth to present MIC/MBC ratio.  

Response-6- 7As per the reviewer’s suggestion we have added MIC/MBC ration in Table (page 17; Line 564)

Comment-7- The discussion part is extremely long and contains a lot of repetition e.g. about several hydroxyl group in polyphenol and their connection with antioxidant activity. It is difficult to read.

Response-7-After reading the comment of reviewer, we realized that discussion should be trimmed and revised. Thus, we have revised our discussion section by removing repetitions and focusing the mechanism behind our result. The complex discussion about polyphenol, flavonoid, and antioxidant have been deleted and rewritten now. (Page 17 and 18; Line 577-606).

Comment-8.  Lines 613-614 are trivial and repeat the results. Of course, the extracts were more active against gram-positive bacteria because there is no activity against gram-negative bacteria.

Response-8- Now we have deleted that sentence accordingly.

Comment-9-Line 667-671 is very speculative and not true.

Response-9- Appreciating the suggestion given by reviewer, we have deleted that paragraph and rewritten a new logical scientific paragraph in revised draft. (Page 20; Line 698-710)

Comment 10-.  However, the biggest drawback of the work is the lack of any analysis of the extracts tested. Not even a qualitative HPLC analysis, let alone an LC-MS spectral analysis. Determining the sum of polyphenolic compounds or flavonoids is not phytochemical analysis.

Response 10- Thank you for your valuable feedback. We acknowledge the limitations regarding phytochemical analysis. Due to limited access to advanced instruments (e.g., HPLC, LC-MS) in Nepal, we focused on evaluating antibacterial, antioxidant, and xanthine oxidase activities, along with total phenolic and flavonoid contents, as an accessible approach to characterize bioactivity. The primary goal was to motivate undergraduate students using available resources. We are committed to incorporating advanced analyses and animal studies in future research to enhance our findings. We appreciate your suggestions and look forward to applying them as we expand our research capabilities. We have added a separate subheading to highlight the limitations of this study (Page 19; line 673-686

Reviewer 4 Report

Comments and Suggestions for Authors

This article addresses a subject of interest, assessing plant extracts using modern techniques. Various botanical species that have been known for centuries in traditional medicine are still studied to map their composition and discover the exact mechanism of action that explains the therapeutic effects. This study contributes to expanding the knowledge on the antioxidant, antimicrobial, and anti-gout potential of Pogostemon benghalensis inflorescence and stem bark extracts.

The article deserves to be published in Bacteria, however, there are several points in which improvements should be made to strengthen the manuscript.

Although the title is consistent with the presented evidence and arguments, I would suggest shortening it. The last part seems excessive.  

The introduction presents the background of the study. Several traditional effects are mentioned. It would be beneficial for the reader to have a short paragraph regarding the composition of various extracts.

Figure 1 - I would suggest adding the measurement unit directly to the image and removing the plastic ruler.

For all figures and tables that contain abbreviations, please mention them in the legend, as you did for Table 1.

My main concern is related to several numeric values that differ in the text in comparison to the table or figure where they are also presented. Please clarify:

·       Line 440 – 93.36% at a concentration of 500 μg/mL, while in Table 3, in the column of the same concentration and the same methanol bark extract the value is 97.53±1.75.

·       Line 480 - IC50-6.8 μg/mL, while in Figure 4 the value is 12.65.

The discussion paragraphs dedicated to polyphenols and flavonoids (lines 525 – 564) are created containing the same thought process and the same words, and even present the same references. Please consider rephrasing or having only one paragraph that presents both.

It would be beneficial for the reader to have a separate paragraph discussing the limitations of the study.

The references are appropriate. There are self-citations, but they are accompanied by a variety of relevant references from different research groups.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear reviewer. We are very much grateful for giving your time to uplift the quality of our manuscript and making more scientific. We have addressed every suggestion and concern and revised the manuscript. All the change has been yellow highlighted in the revised manuscript.

Comment-1- Although the title is consistent with the presented evidence and arguments, I would suggest shortening it. The last part seems excessive.  

Response-1- We completely agree with the reviewers opinion and hence shortened the title of this article. (Page 1, Line 2-4)

Comment-2- The introduction presents the background of the study. Several traditional effects are mentioned. It would be beneficial for the reader to have a short paragraph regarding the composition of various extracts.

Response-2- As per the genuine suggestion of reviewer, we have added short paragraph in the introduction to provide relevant information on chemical composition of P. benghalensis extracts. (Page 2 and 3; Line 86-91).

 

Comment-3- Figure 1 - I would suggest adding the measurement unit directly to the image and removing the plastic ruler.

Response-3- Thank you very much for close observation to improve the quality of our manuscript. We have done necessary correction in the figure now. (Page 4; Line 137).

Comment-4- For all figures and tables that contain abbreviations, please mention them in the legend, as you did for Table 1.

Response-4- As per the reviewer’s suggestion we have added legend in all the tables and figures. (Figure 2-4 and Table 2-6). All the added abbreviations has been yellow highlighted.

Comment-5- My main concern is related to several numeric values that differ in the text in comparison to the table or figure where they are also presented. Please clarify.

Response-5- We are very much grateful to reviewer for detecting blunder mistakes microscopically. Those were typo errors and we have corrected them carefully.

93. 36% at a concentration of 500 μg/mL, while in Table 3, in the column of the same concentration and the same methanol bark extract the value is 97.53±1.75.

Response- At the concentration of 500 mg/mL, 93.6% inhibition was for the PBFM (P. benghalensis inflorescence methanol extract) whereas 97.53 % was for PBBM (P. benghalensis bark methanol extract) (Line 498)

6. Line 480 - IC50-6.8 μg/mL, while in Figure 4 the value is 12.65.

Response- We really appreciate reviewer for catching these errors. That was typo and it should ne 12.65. Hence, we have corrected accordingly. (Line 407 and 408)

Comment-8- The discussion paragraphs dedicated to polyphenols and flavonoids (lines 525 – 564) are created containing the same thought process and the same words, and even present the same references. Please consider rephrasing or having only one paragraph that presents both.

Response-8- We agree with the logic of reviewers. Now, we have merged two paragraphs as a single by removing repetition. (Page 17; Line 571-591).

Comment-9- It would be beneficial for the reader to have a separate paragraph discussing the limitations of the study.

Response-9- We agree with the suggestion of reviewers. Thus, we have added separate section “Limitation of the study” (Page 19; Line 673-686)

Comment-10- The references are appropriate. There are self-citations, but they are accompanied by a variety of relevant references from different research groups.

Reference -10-Previously there were 4 references cited from my previous publication. Now we have kept only two references as we have adopted major methodology from these articles. (Line 871-875 reference 42 and 43)