Optimization of Extraction Process of Total Alkaloids from Thalictrum delavayi Franch. and Their Therapeutic Potential on Pulmonary Infection Caused by Klebsiella pneumoniae and Escherichia coli

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Therapeutic potential of total alkaloids from Thalictrum delavayi Franch. on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli” is quite interesting, but I have a few suggestions and questions that I would like to share:

1. In the introduction, I suggest providing a more detailed characterization of the antibiotic resistance mechanisms in E. coli and K. pneumoniae.

2. In line 159, I suggest verifying whether the concentration is indeed in mg/mL, as these would be very high concentrations for in vitro work. If the mixture of alkaloids was actually tested at mg/mL, why were they tested at this order of magnitude and not at microg/mL?

3. In the "In Vitro Antibacterial Assay" subsection, I suggest describing how the development was carried out to determine the presence or absence of bacteria for MIC determination. Was levofloxacin used as a standard drug? If so, I suggest citing or stating which standard drug was used. In addition, I suggest citing the vehicle used to prepare the stock solutions of the alkaloids and whether the vehicle was tested as a control. I also have a question: why was the determination of the minimum bactericidal concentration not performed?

4. In the subsection on the determination of Acute Toxicity and Accumulative Toxicity, please cite which organs were analyzed histologically. In addition to the survival rate and weight changes in the animals, were any other changes observed in the mice?

5. I suggest presenting the results of antibacterial activity in the form of graphs or tables to compare the results of the tested alkaloid mixtures with the controls (vehicle and standard drugs). In the text on the results, I suggest that the results of the antibacterial effect of the tested alkaloid mixture be compared with the drugs used as standards. In addition, the effect of the vehicle used on bacterial growth should be commented on.

6. Still regarding the results of antibacterial activity. The text states that the mixture of alkaloids had good activity. What parameters were used to determine that it is indeed a good antibacterial activity, since the MIC was at a concentration in the order of mg/mL (high concentration, since the assay is in vitro)?

7. Regarding the results of the in vivo assay, I suggest standardizing the terminology in relation to the black/control group. In the text of the results, the authors refer to it as the black group, while in the figures it is referred to as the control group, which may confuse the reader.

8. In Table 6, identify the statistical analysis for the positive group.

Author Response

Dear reviewer,

  First of all, the authors would like to express our appreciation to the editor and the anonymous reviewers for your in-depth, rigorous, valuable, and helpful comments, corrections, and suggestions on our manuscript titled “Therapeutic potential of total alkaloids from Thalictrum delavayi Franch. on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli”.

According to the editor and reviewers’ comments, we dealt fully with the criticisms in the revised manuscript, and the changes to the manuscript have been highlighted in red. The revised manuscript has been uploaded and submitted through the “Author Center” online. We also responded point by point to the editor and reviewer’s comments as listed below, along with a clear indication of the location of the revision. We hope that this revision is interesting and novel enough for general readers of Separations.

And the responses to the reviewers’ comments point by point were listed on the next pages.

 

Best regards,

 

 

Responses to the reviewers’ comments point by point were listed as follows:

 

Summary

The manuscript “Therapeutic potential of total alkaloids from Thalictrum delavayi Franch. on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli” is quite interesting, but I have a few suggestions and questions that I would like to share:

 

Comments 1: In the introduction, I suggest providing a more detailed characterization of the antibiotic resistance mechanisms in E. coli and K. pneumoniae.

Response: Thanks for your valuable comments. A more detailed characterization of the antibiotic resistance mechanisms in E. coli and K. pneumoniae has been added in the revised manuscript.

 

Comments 2: In line 159, I suggest verifying whether the concentration is indeed in mg/mL, as these would be very high concentrations for in vitro work. If the mixture of alkaloids was actually tested at mg/mL, why were they tested at this order of magnitude and not at microg/mL?

Response: Thanks for your valuable and rigorous comments. The concentration of Thalictrum delavayi Franch. alkaloids were tested at mg/mL. The reason why they were tested at the order of magnitude (0.39 ~100.0 mg/mL) and not at microg/mL is that the water solubility and in vitro antibacterial assay were carried out by two-fold broth microdilution method for determination of MICs.

 

Comments 3: In the "In Vitro Antibacterial Assay" subsection, I suggest describing how the development was carried out to determine the presence or absence of bacteria for MIC determination. Was levofloxacin used as a standard drug? If so, I suggest citing or stating which standard drug was used. In addition, I suggest citing the vehicle used to prepare the stock solutions of the alkaloids and whether the vehicle was tested as a control. I also have a question: why was the determination of the minimum bactericidal concentration not performed?

Response: Thanks for your valuable comments. The manuscript has been revised carefully. During the antibacterial assay, levofloxacin was used for QC (quality control) range as a reference drug based on its antibacterial effects against E. coli and K. pneumoniae. As your valuable suggestion, we have tested the MBCs against E. coli and K. pneumoniae and supplemented them in the revised manuscript.

 

Comments 4: In the subsection on the determination of Acute Toxicity and Accumulative Toxicity, please cite which organs were analyzed histologically. In addition to the survival rate and weight changes in the animals, were any other changes observed in the mice?

Response: Thanks for your valuable and rigorous comments. “Histopathology observations of heart, liver, spleen, lung and kidney were evaluated” has been added in the revised manuscript.

 

Comments 5: I suggest presenting the results of antibacterial activity in the form of graphs or tables to compare the results of the tested alkaloid mixtures with the controls (vehicle and standard drugs). In the text on the results, I suggest that the results of the antibacterial effect of the tested alkaloid mixture be compared with the drugs used as standards. In addition, the effect of the vehicle used on bacterial growth should be commented on.

Response: Thanks for your valuable and rigorous comments. In the revised manuscript, we have added the results of the antibacterial activity and bactericidal activity of the tested alkaloid mixtures with the controls (vehicle and standard drugs) in Table 6 to compare the results.

 

Comments6: Still regarding the results of antibacterial activity. The text states that the mixture of alkaloids had good activity. What parameters were used to determine that it is indeed a good antibacterial activity, since the MIC was at a concentration in the order of mg/mL (high concentration, since the assay is in vitro)?

Response: Thanks for your valuable and rigorous comments. In the revised manuscript, we have supplemented the results in Table 6 to verify the good antibacterial activity of the tested alkaloid mixtures.

 

Comments 7: Regarding the results of the in vivo assay, I suggest standardizing the terminology the blank/control group. In the text of the results, the authors refer to it as the blank group, while in the figures it is referred to as the control group, which may confuse the reader.

Response: Thanks for your valuable and careful comments. We have verified the terminology as the control group in the revised manuscript.

 

Comments 8: In Table 6, identify the statistical analysis for the positive group.

Response: Thanks for your valuable and careful comments. We are so sorry to miss the description of the statistical analysis for the positive group in the revised manuscript listed in Table 7.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The present work reports the extraction process of total alkaloids from Thalictrum delavayi which was optimized for separations and the therapeutic potential of pulmonary infection caused by Klebsiella pneumoniae  and Escherichia coli.

L 14: gram-negative bacteria>  Gram-negative bacteria! please correct throughout

L 16...Thalictrum delavayi Franch. (T.delavayi) > T. delavayi is a natural and logical abreviation which does not to be mentioned!!! also mind to have a space not T.delavayi throughout!

L19...from T.delavayi... which part? total plant? the utilized part MUST be written!

L20 ..Klebsiella pneumoniae (K.pneumoniae) and Escherichia coli (E.coli)> Klebsiella pneumoniae and Escherichia coli ! there is no need !!!!

L24-25... The total alkaloids from T.delavayi... the sentence MUST be removed as it is speculative... "could alleviate the pathological changes" .. is scientifically vague! please avoid such sentences throughout!

L25...The treatment groups.. are only in drugs! so avoid such definitions as the alkaloids used are NO drugs! just in vitro experimental... thus the authors may state experimental groups etc..

L29-30.... Our studies proved that total alkaloids from T. delavayi had therapeutic effects on mice with K. pneumoniae and E. coli ...> the prelimary experimental data of the tested alkaloids showed K. pneumoniae and E. coli inhibitory activity when compared with....

as there are no tox data available esp. on humans! please avoid drug like claims as the work is just an experimental preliminary work.

The authors should also consult pharmacology experts from pharmaceutical sciences!

L68 ...The voucher specimen number is missing??

L70.. the whole plant ? dryness?? or moisture content? 

L100...remove "simple" just give proper primary source reference!

the in vitro assays need to report also positive and negative controls in the results tables for proper comparison.

The alkaloids incl. berberine cannot be defined as drugs unless they are pharma grade! 

L457 5. Conclusions 

is stated as another abstract, the authors need to state what consequences from the results should be withdrawn? no need to abstract the work again.

Overall, although the work is interesting and the data represented is new, the authors MUST improve the work to be considered for acceptance.

 

 

Author Response

Dear reviewer,

First of all, the authors would like to express our appreciation to the editor and the anonymous reviewers for your in-depth, rigorous, valuable, and helpful comments, corrections, and suggestions on our manuscript titled “Therapeutic potential of total alkaloids from Thalictrum delavayi Franch. on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli”.

According to the editor and reviewers’ comments, we dealt fully with the criticisms in the revised manuscript, and the changes to the manuscript have been highlighted in red. The revised manuscript has been uploaded and submitted through the “Author Center” online. We also responded point by point to the editor and reviewer’s comments as listed below, along with a clear indication of the location of the revision. We hope that this revision is interesting and novel enough for general readers of Separations.

And the responses to the reviewers’ comments point by point were listed on the next pages.

Best regards,

 

 

Responses to the reviewers’ comments point by point were listed as follows:

 

Summary

The present work reports the extraction process of total alkaloids from Thalictrum delavayi which was optimized for separations and the therapeutic potential of pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli.

 

Comments 1: L14: gram-negative bacteria> Gram-negative bacteria! please correct throughout

Response: Thanks for your valuable comments. We have corrected the term “Gram-” throughout the revised manuscript carefully.

 

Comments 2: L 16...Thalictrum delavayi Franch. (T.delavayi) > T. delavayi is a natural and logical abbreviation which does not to be mentioned!!! also mind to have a space not T.delavayi throughout!

Response: Thanks for your valuable comments. We have mentioned the logical abbreviation of Thalictrum delavayi Franch. (T. delavayi) in Line 16 and corrected the term “T. delavayi” throughout the revised manuscript carefully. 

 

Comments 3: L19...from T.delavayi... which part? total plant? the utilized part MUST be written!

Response: Thanks for your valuable and rigorous comments. In this study, the extraction process of total alkaloids from the whole plant of T. delavayi was optimized.

 

 

Comments 4: L20 ...Klebsiella pneumoniae (K.pneumoniae) and Escherichia coli (E.coli)> Klebsiella pneumoniae and Escherichia coli ! there is no need !!!!

Response: Thanks for your valuable and rigorous comments. The revised manuscript has been revised carefully according to the reviewers’ suggestions.

 

Comments 5: L24-25... The total alkaloids from T.delavayi... the sentence MUST be removed as it is speculative... "could alleviate the pathological changes" .. is scientifically vague! please avoid such sentences throughout!

Response: Thanks for your valuable and rigorous comments. The sentence has been removed in the revised manuscript.

 

Comments6: L25...The treatment groups.. are only in drugs! so avoid such definitions as the alkaloids used are NO drugs! just in vitro experimental... thus the authors may state experimental groups etc..

Response: Thanks for your valuable and rigorous comments. In the revised manuscript, the terms such as “treat groups” and “drug …” have been revised carefully because the alkaloids used are NO drugs.

 

Comments 7: L29-30.... Our studies proved that total alkaloids from T. delavayi had therapeutic effects on mice with K. pneumoniae and E. coli ...> the prelimary experimental data of the tested alkaloids showed K. pneumoniae and E. coli inhibitory activity when compared with....

as there are no tox data available esp. on humans! please avoid drug like claims as the work is just an experimental preliminary work.

The authors should also consult pharmacology experts from pharmaceutical sciences!

Response: Thanks for your valuable and rigorous comments. We have consulted pharmacology experts and the manuscript has been revised carefully. The sentences such as “Our studies proved that total alkaloids from T. delavayi had therapeutic effects on mice with K. pneumoniae and E. coli” have been carefully corrected.

 

Comments 8: L68 ...The voucher specimen number is missing??

Response: Thanks for your valuable and rigorous comments. The voucher specimen number has been added to the revised manuscript.

 

Comments 9: L70.. the whole plant? dryness?? or moisture content?

Response: Thanks for your valuable and rigorous comments. The whole dry plant was used and the manuscript has been revised carefully.

 

 

Comments 10: L100...remove "simple" just give proper primary source reference!

the in vitro assays need to report also positive and negative controls in the results tables for proper comparison.

The alkaloids incl. berberine cannot be defined as drugs unless they are pharma grade!

Response: Thanks for your valuable and rigorous comments. In the revised manuscript, term such as “drug …” have been revised carefully because the alkaloids incl. berberine used are NO drugs. Additionally, in vitro assays of the positive and negative controls in the results tables for proper comparison were carried out in Table 7.

 

Comments 11: L457 5. Conclusions are stated as another abstract, the authors need to state what consequences from the results should be withdrawn. no need to abstract the work again.

Overall, although the work is interesting and the data represented is new, the authors MUST improve the work to be considered for acceptance.

Response: Thanks for your valuable and rigorous comments. The manuscript has been revised carefully and the conclusions section has been corrected carefully.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Muy buen trabajo para el control de las bacterias en estudio, que han reportado resistencia a los fármacos. Muestra un precedente sobre el potencial y la eficacia de las plantas.

Author Response

Dear Reviewer,

 

Thank you very much for your positive feedback and encouraging comments on our manuscript. We are delighted to hear that you found our work valuable and that you have no further suggestions for improvement. Your approval is immensely gratifying and motivates us to continue our research with the same rigor and enthusiasm.

We greatly appreciate your time and effort in reviewing our submission.

 

Best regards,

Reviewer 4 Report

Comments and Suggestions for Authors

In their study the authors have performed an optimization of T. delavayi alkaloid extraction and examined their therapeutic potential on mixed bacterial pulmonary infection. The study design is appropriate, methodology and the results clearly presented. 

My comments and suggestion for manuscript improvement are listed here: 

Introduction 

- The description of Thalictrum delavayi alkaloids given in the introduction is somewhat unspecific. Could you specify some of the most typical alkaloid constituents that were reported for T. delavayi? 

- 'Notably, alkaloids have good bacteriostatic activity and the bacteriostatic effect is similar to that of commonly used antimicrobial agents, having the  potential to replace antibiotics presenting a great potential for discovery of novel  anti-inflammatory drugs'. Needs rephrasing (repetitive/unclear) 

- Lines 62-69: the sentence is too long - should be split in two for clarity 

Materials and Methods 

- Lines 152-156 and Table 2. are repetitive/providing the very same information, so author may consider using either text or Table 2. for gradient elution procedure. 

- Line 173: for clarity consider adding 'standard' i.e. 'according to the GB 15193.3-2014 standard' 

- '2.6 Acute Toxicity and Accumulative Toxicity': please, explain organ coefficient here; 'Accumulative Toxicity' should be changed to 'Cumulative Toxicity'? (here are elsewhere) 

Results 

- Lines 325 and 336: please change 'non-acute toxic' to 'not acutely toxic'; 'non-accumulative toxicity' to 'not cumulatively toxic' 

- Paragraph 3.5.1 - please add comment regarding results for positive control shown in Fig. 8 and Fig. 9. Similar pattern seems to be observed for model as well as treated groups (increase in body weight after 5 days of treatment) 

- Figure 9. please rearange the graphics so that the Control is shown first to the left. This was the Figure 9. will be aligned with the order of comments in the text of the respective paragraph and Figure 8. (therefore, easier to the reader to follow). Correct the typo in 'Medium' 

- Table 6. Line 378: please add 'positive' - 'compared with the positive control group' 

- Line 395: add 'compared to the model group' in 'there was no significant difference between the medium-dose and low-dose groups compared to the model group' 

Discussion 

Discussion would be largely improved by (i) discussing/commenting the alkaloid composition in extracts used in the study, (ii) note on toxicity testing, (iii) re-considering text in Lines 449-452 (disrupts the 'flow' of the discussion and maybe not needed here), re-checking Lines 462-463 (unclear, incomplete, repetitive?). 

- Line 467: change 'organ' to 'lung' 

Additional comments 

Cell count should preferably be changed into white blood cell count throughout the paper, but especially in the Abstract.

Thalicarpine, Jatrorrhizine, Tetrandrine, and Berberine - I assume the first letter in alkaloids are not to be written in capitals

The authors are encouraged to re-consider the title of the manuscript since the optimization of alkaloid extraction represents a significant part of it which is not reflected in the title.

Comments on the Quality of English Language

There is a need for minor English language editing (grammar, typos etc.)

Author Response

Response to Reviewer 4 Comments

 

Dear reviewer,

First of all, the authors would like to express our appreciation to the editor and the anonymous reviewers for your in-depth, rigorous, valuable, and helpful comments, corrections, and suggestions on our revised manuscript titled “Optimization of extraction process of total alkaloids from Thalictrum delavayi Franch. and their therapeutic potential on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli.

According to the editor and reviewers’ comments, we dealt fully with the criticisms in the revised manuscript, and the changes to the manuscript have been highlighted in red. The revised manuscript has been uploaded and submitted through the “Author Center” online. We also responded point by point to the editor and reviewer’s comments as listed below, along with a clear indication of the location of the revision. We hope this revision is interesting and novel enough for general readers of Separations.

And the responses to the reviewers’ comments point by point were listed on the next pages.

Best regards,

 

 

Responses to the reviewers’ comments point by point were listed as follows:

 

Comments 1: The description of Thalictrum delavayi alkaloids given in the introduction is somewhat unspecific. Could you specify some of the most typical alkaloid constituents that were reported for T. delavayi? 

Response: Thanks for your valuable and rigorous comments. Typical alkaloid constituents such as dimeric benzylisoquinoline alkaloids (thalidelavines A-E) and isoquinoline alkaloids (2,3,9,10-dimethylenedioxy-8-oxoprotoberberine and 2,3,9,10-dimethylenedioxy-1,8- dihydroxyprotoberberine) were reported for T. delavayi were cited in the introduction section. 

 

Comments 2: - 'Notably, alkaloids have good bacteriostatic activity and the bacteriostatic effect is similar to that of commonly used antimicrobial agents, having the potential to replace antibiotics presenting a great potential for discovery of novel anti-inflammatory drugs'. Needs rephrasing (repetitive/unclear) 

Response: Thanks for your valuable comments. The sentence has been modified in the revised manuscript.

 

Comments 3: Lines 62-69: the sentence is too long - should be split in two for clarity.

Response: Thanks for your valuable and rigorous comments. The sentence in Lines 62-69 has been modified in the revised manuscript.

 

 

Comments 4: Lines 152-156 and Table 2. are repetitive/providing the very same information, so author may consider using either text or Table 2. for gradient elution procedure. 

Response: Thanks for your valuable comments. The gradient elution procedure described in the text in Lines 152-156 has been deleted. 

 

Comments 5: Line 173: for clarity consider adding 'standard' i.e. 'according to the GB 15193.3-2014 standard' .

Response: Thanks for your valuable and rigorous comments. The word ”standard” has been added in Line 173.

 

Comments6: Acute Toxicity and Accumulative Toxicity': please, explain the organ coefficient here; 'Accumulative Toxicity' should be changed to 'Cumulative Toxicity'? (here are elsewhere) 

Response: Thanks for your valuable and careful comments. The organ coefficient has been explained in the section “Acute Toxicity and cumulative Toxicity”; and 'Accumulative Toxicity' has been changed to 'Cumulative Toxicity' in the revised manuscript.

 

Comments 7: Lines 325 and 336: Please change 'non-acute toxic' to 'not acutely toxic'; 'non-accumulative toxicity' to 'not cumulatively toxic' 

Response: Thanks for your valuable and careful comments. The phrase 'non-acute toxic' has been changed to 'not acutely toxic' and 'non-accumulative toxicity' to 'not cumulatively toxic' in Lines 325 and 336.

 

Comments 8: Paragraph 3.5.1 - please add a comment regarding results for positive control shown in Fig. 8 and Fig. 9. Similar pattern seems to be observed for the model as well as treated groups (increase in body weight after 5 days of treatment) 

Response: Thanks for your valuable and suggestive comments. Paragraph 3.5.1 has been carefully checked and improved in the revised manuscript.

 

 

Comments 9: Figure 9. Please rearrange the graphics so that the Control is shown first to the left. This was Figure 9. will be aligned with the order of comments in the text of the respective paragraph and Figure 8. (therefore, easier for the reader to follow). Correct the typo in 'Medium' 

Response: Thanks for your valuable, suggestive and careful comments. Figure 9Please has been rearranged and the typo in 'Medium' has been corrected in the revised manuscript.

 

Comments 10: Table 6. Line 378: Please add 'positive' - 'compared with the positive control group' 

Response: Thanks for your valuable comments. The term 'positive' has been added in Table 6. Line 378.

 

Comments 11:  Line 395: add 'compared to the model group' in 'there was no significant difference between the medium-dose and low-dose groups compared to the model group' 

Response: Thanks for your valuable and rigorous comments. The phrase 'compared to the model group' in 'there was no significant difference between the medium-dose and low-dose groups compared to the model group' in Line 395.

 

Comments 12: Discussion would be largely improved by (i) discussing/commenting the alkaloid composition in extracts used in the study, (ii) note on toxicity testing, (iii) re-considering text in Lines 449-452 (disrupts the 'flow' of the discussion and maybe not needed here), re-checking Lines 462-463 (unclear, incomplete, repetitive?). 

Response: Thanks for your valuable and rigorous comments. The discussion has been carefully checked and improved in the revised manuscript.

 

Comments 13: - Line 467: change 'organ' to 'lung' 

Response: Thanks for your valuable and rigorous comments. In Line 467, 'organ' has been changed to 'lung'.

 

Comments 14: Cell count should preferably be changed into white blood cell count throughout the paper, especially in the Abstract.

Response: Thanks for your valuable and rigorous comments. Cell count has been changed into white blood cell count throughout the whole paper.

 

Comments 15: Thalicarpine, Jatrorrhizine, Tetrandrine, and Berberine - I assume the first letter in alkaloids is not to be written in capitals.

Response: Thanks for your valuable comments. We have changed the first letter to “Thalicarpine, Jatrorrhizine, Tetrandrine, and Berberine” and written in lowercase.

 

Comments 16: The authors are encouraged to re-consider the title of the manuscript since the optimization of alkaloid extraction represents a significant part of it which is not reflected in the title.

Response: Thanks for your valuable comments. We have changed the title to reflect the whole manuscript, and the new title is “Optimization of extraction process of total alkaloids from Thalictrum delavayi Franch. and their therapeutic potential on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli”.

 

Comments 17: There is a need for minor English language editing (grammar, typos etc.)

Response: Thanks for your valuable comments. We have checked the manuscript and edited the manuscript, especially English language editing.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript "Therapeutic potential of total alkaloids from Thalictrum delavayi Franch. on pulmonary infection caused by Klebsiella pneumoniae and Escherichia coli" and I am pleased to recommend it for publication in Separations. The authors have addressed all of the reviewers' concerns thoughtfully and in detail, and the manuscript is now in excellent shape.

I was particularly impressed with the authors' willingness to make significant changes to their work in response to the reviewers' feedback. This demonstrates a commitment to rigor and a willingness to improve the quality of their research.

The manuscript makes a significant contribution to the field of evaluation of the antimicrobial activity of natural products. It is well-written and clearly argued, and it is sure to be of interest to a wide range of readers.

Author Response

Dear Reviewer,

 

Thank you very much for your positive feedback and encouraging comments on our manuscript. We are delighted to hear that you found our work valuable and that you have no further suggestions for improvement. Your approval is immensely gratifying and motivates us to continue our research with the same rigor and enthusiasm.

We greatly appreciate your time and effort in reviewing our submission.

 

Best regards,