Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: In vitro and in vivo experiments

Prapaporn Chaniad; Arisara Phuwajaroanpong; Walaiporn Plirat; Atthaphon Konyanee; Abdi Wira Septama; Chuchard Punsawad

doi:10.1371/journal.pone.0296756

Peer Review History

Original SubmissionJuly 2, 2023
9 Nov 2023 Decision Letter - Branislav T. Šiler, Editor PONE-D-23-19212Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: in vitro and in vivo experimentsPLOS ONE Dear Dr. Punsawad, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Histopathological photographs should be made in a higher magnification, as suggested by Reviewer #2. Several clarifications in Material and Methods, required by both reviewers, are needed as well. Conclusion seems to fail to be sustained by results provided in Table 9. Please provide species authorities for the species listed in L100-102 as well as for Artemisia vulgaris in Table 1. In L323, it is Rane's test instead of Rane test. ============================== Please submit your revised manuscript by Dec 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://aje.com/go/plos) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: The name of the colleague or the details of the professional service that edited your manuscript A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a supporting information file) A clean copy of the edited manuscript (uploaded as the new manuscript file)” 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: General comment: This study assessed the antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment both in vitro and in vivo. The paper is well written and the results well presented. The need for scientific appraisal of antimalarial formulations cannot be overemphasized, hence the study is very relevant. Specific comments to be addressed: The author must however revise line 274 "eliminating waste from the cages daily" is not the standard practice. Authors must also cite the international guideline(s) being referred to in line 276. It is also worthy to note that though the mean parasitaemia level was lower than 90% (which is an indication that both formulations were active as antimalarials) in the invivo study, with the % suppression of both CTLL and PSCD being less than 50 (Table 9) the formulations cannot be said to have showed a good antimalarial (curative) activity, hence conclusion is not well justified. Authors must revise the conclusion. Reviewer #2: • The authors mentioned that “Each plant and formulation 165 were extracted using ethanol and distilled water.”. What was the percentage of the used ethanol? • Please mention the exact temperature where room temperature varies from country to country “at room temperature for 72 h…..”. • Please mention the source of Plasmodium falciparum. • For parasitemia determination “The percentage of parasitemia was determined daily using a light microscope”: I suggest adding a reference (cite) and calculate the percentage of suppression from The antiplasmodial and spleen protective role of crude Indigofera oblongifolia leaf extract traditionally used in the treatment of malaria in Saudi Arabia. Drug Des Devel Ther. 2015 Nov 25;9:6235-46. doi: 10.2147/DDDT.S94673. • If the used mice were adult (6-8 weeks) add adult before mice. • Mention how you have calculated 1× 107 P. berghei-parasitized RBCs to be the used dose. Mention the used chamber. • Did you use the whole blood or you separated the parasitized erythrocytes to be only injected into experimental mice? • In table 10: “Table 10. Percent suppression of the ethanolic extract of the CTLL and PSCD formulations in prophylactic test” mention the day of blood collection. Why this day. • In the histopathology: I can not see any histopathological change with this magnification. I found no figure caption describing the groups and the histopathological change. I can not see the scale bar. I also suggest in addition to add a higher magnification to add a histology score. • I suggest comparing your results with authors used any of the used extracts or mention the activity of other extracts and write on the importants of using medithinal plants against malaria. You can use “ Medicinal plants as a fight against murine blood-stage malaria. Saudi J Biol Sci. 2021 Mar;28(3):1723-1738. doi: 10.1016/j.sjbs.2020.12.014. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Samuel Adjei Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0296756.r001
Revision 1
25 Nov 2023 Author Response POINT-BY-POINT RESPONSES TO THE REVIEWERS’ COMMENTS Editor Histopathological photographs should be made in a higher magnification, as suggested by Reviewer #2. Several clarifications in Material and Methods, required by both reviewers, are needed as well. Conclusion seems to fail to be sustained by results provided in Table 9. Please provide species authorities for the species listed in L100-102 as well as for Artemisia vulgaris in Table 1. In L323, it is Rane's test instead of Rane test. Response: The manuscript has been revised as suggested. Reviewer #1: General comment: This study assessed the antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment both in vitro and in vivo. The paper is well written and the results well presented. The need for scientific appraisal of antimalarial formulations cannot be overemphasized, hence the study is very relevant. Response: Thank you. Specific comments to be addressed: The author must however revise line 274 "eliminating waste from the cages daily" is not the standard practice. Authors must also cite the international guideline(s) being referred to in line 276. Response: The sentence has been revised. Please see page 13, line 275. It is also worthy to note that though the mean parasitaemia level was lower than 90% (which is an indication that both formulations were active as antimalarials) in the in vivo study, with the % suppression of both CTLL and PSCD being less than 50 (Table 9) the formulations cannot be said to have showed a good antimalarial (curative) activity, hence conclusion is not well justified. Authors must revise the conclusion. Response: The conclusion has been revised. Please see page 43, line 857-859 and page 3, lines 49-51. Reviewer #2: • The authors mentioned that “Each plant and formulation 165 were extracted using ethanol and distilled water.”. What was the percentage of the used ethanol? Response: The percentage of ethanol at 95% has been added to the manuscript. Please see page 8, line 165. • Please mention the exact temperature where room temperature varies from country to country “at room temperature for 72 h…..”. Response: Room temperature at 25 °C has been added to the manuscript. Please see page 9, line 169. • Please mention the source of Plasmodium falciparum. Response: The sentence about the source of Plasmodium falciparum has been added to the manuscript. Please see page 9, lines 182-184. • For parasitemia determination “The percentage of parasitemia was determined daily using a light microscope”: I suggest adding a reference (cite) and calculate the percentage of suppression from The antiplasmodial and spleen protective role of crude Indigofera oblongifolia leaf extract traditionally used in the treatment of malaria in Saudi Arabia. Drug Des Devel Ther. 2015 Nov 25;9:6235-46. doi: 10.2147/DDDT.S94673. Response: A suggested reference (cite) has been added. Please see page 9, line 190. • If the used mice were adult (6-8 weeks) add adult before mice. Response: The word “adult” has been added to the manuscript. Please see page 13, line 268. • Mention how you have calculated 1× 107 P. berghei-parasitized RBCs to be the used dose. Mention the used chamber. Response: For the calculation of 1× 107 P. berghei-parasitized RBCs to be the used dose, we calculated from the level of parasitemia in donor mice. When the level of parasitemia in donor mice reached 20–30%, the donor mouse was subsequently sacrificed and blood collected (via cardiac puncture) into a heparinized tube. Based on the parasitemia level of the donor mouse and the red blood cell (RBC) count of a normal mouse, the collected blood was diluted with normal saline (0.9%), so that 1 ml of blood contains 5 x 107 infected RBCs. Each mouse was injected with 0.2 ml of blood containing 1 x 107 P. berghei-infected RBCs via the intraperitoneal route. Ref: Belay, W. Y., Endale Gurmu, A., & Wubneh, Z. B. Antimalarial activity of stem bark of Periploca linearifolia during early and established Plasmodium infection in mice. J Evid Based Complementary Altern Med.2018; 4169397. https://doi.org/10.1155/2018/4169397 • Did you use the whole blood or you separated the parasitized erythrocytes to be only injected into experimental mice? Response: For the infection of mice, we used whole blood. Briefly, blood from donor mice was collected and then diluted with 0.9% physiological isotonic saline before injected into mice in the experiment. • In table 10: “Table 10. Percent suppression of the ethanolic extract of the CTLL and PSCD formulations in prophylactic test” mention the day of blood collection. Why this day. Response: • The word “Day 6” has been added to the table name. Please see page 30, line 563. • We monitored parasitemia on day 6 (day 6 after mice received the extract or day 3 post-infection) because the parasitemia level increased steadily beginning on day 3 post-infection. The reason could be that only approximately one-third of infected red blood cells are able to circulate freely in the blood circulation within 48 hours, whereas the remaining parasites stick to the venules and capillaries. Therefore, blood collection at day 3 post-infection would provide a precise assessment of the parasitemia. Ref: Nwonuma CO, Balogun EA, Gyebi GA. Evaluation of antimalarial activity of ethanolic extract of Annona muricata L.: An in vivo and an in-silico approach. Journal of Evidence-Based Integrative Medicine. 2023;28. • In the histopathology: I cannot see any histopathological change with this magnification. I found no figure caption describing the groups and the histopathological change. I cannot see the scale bar. I also suggest in addition to add a higher magnification to add a histology score. Response: Thank you for your suggestion. A new figure of histopathological change with a higher magnification has been added. • I suggest comparing your results with authors used any of the used extracts or mention the activity of other extracts and write on the important of using medithinal plants against malaria. You can use “Medicinal plants as a fight against murine blood-stage malaria. Saudi J Biol Sci. 2021 Mar;28(3):1723-1738. doi: 10.1016/j.sjbs.2020.12.014. Response: Thank you for your valuable comment. I have already compared results with authors who used any of the used extracts or mentioned the activity of other extracts (please see pages 37–38, lines 720–723) and wrote about the importance of using medicinal plants against malaria (please see pages 34, lines 63–63). Attachments Attachment Submitted filename: POINT-BY-POINT.pdf https://doi.org/10.1371/journal.pone.0296756.r002
7 Dec 2023 Decision Letter - Branislav T. Šiler, Editor PONE-D-23-19212R1Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: in vitro and in vivo experimentsPLOS ONE Dear Dr. Punsawad, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Authors have overseen the Editor's request provided in the previous review round "Please provide species authorities for the species listed in L100-102 as well as for Artemisia vulgaris in Table 1." You may use Kew Garden's Plants of the World Online (https://powo.science.kew.org/). ============================== Please submit your revised manuscript by Jan 21 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0296756.r003
Revision 2
10 Dec 2023 Author Response The Editor's request: "Please provide species authorities for the species listed in L100-102 as well as for Artemisia vulgaris in Table 1." You may use Kew Garden's Plants of the World Online (https://powo.science.kew.org/). Response: We have checked and revised as suggested. Journal requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Response: We have checked and revised as suggested. Attachments Attachment Submitted filename: POINT-BY-POINT.pdf https://doi.org/10.1371/journal.pone.0296756.r004
18 Dec 2023 Decision Letter - Branislav T. Šiler, Editor Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: in vitro and in vivo experiments PONE-D-23-19212R2 Dear Dr. Punsawad, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0296756.r005
Formally Accepted
3 Jan 2024 Acceptance Letter - Branislav T. Šiler, Editor PONE-D-23-19212R2 PLOS ONE Dear Dr. Punsawad, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Branislav T. Šiler Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0296756.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .