Analysis of the chemical constituents and their metabolites in Orthosiphon stamineus Benth. via UHPLC-Q exactive orbitrap-HRMS and AFADESI-MSI techniques

Jianting Ouyang; Danyao Lin; Xuesheng Chen; Yimeng Li; Qin Liu; Delun Li; Haohao Quan; Xinwen Fu; Qiaoru Wu; Xiaowan Wang; Shouhai Wu; Chuang Li; Yi Feng; Wei Mao

doi:10.1371/journal.pone.0304852

Peer Review History

Original SubmissionJanuary 23, 2024
29 Feb 2024 Decision Letter - Andrea Mastinu, Editor PONE-D-24-03002Analysis of the chemical constituents and their metabolites in Orthosiphon stamineus Benth. via UHPLC-Q Exactive Orbitrap-HRMS and AFADESI-MSI techniquesPLOS ONE Dear Dr. Mao, 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. Please submit your revised manuscript by Apr 14 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. 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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: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: Dear Authors The study deals with a meaningful and important question, so the topic interests certain readers in the Plos One. However, the following comments should be addressed because some parts should be revised and improved. 1. In your title, are there any differences between the terms chemical constituent and metabolite? 2. Did you use a QC sample in your LC-MS/MS analysis? If not, please explain it 3. In tables 1, 2, and 3 please write the molecular formula correctly like this C9H10O5 4. For your annotation of metabolite, add information about error mass to make sure your tentative identification is precise and accurate Reviewer #2: This manuscript presents the results of research on the analysis of the chemical compounds content of Orthosiphon stamineus Benth (OS) using UHPLC-Q Exactive Orbitrap- 4 HRMS and AFADESI-MSI techniques. The research is a preliminary stage in utilizing OS as a source of medicine for kidney disease, so that the manuscript compiled is only limited to reporting a list of chemical compounds in OS and metabolites in serum from rats treated with OS extract. In this manuscript there is no in-depth analysis of the data obtained and no detailed discussion of the results of data analysis. Some notes on this manuscript are as follows: 1. The significance of this study only emphasizes the use of UHPLC-Q Exactive Orbitrap-4 HRMS for the analysis of metabolite content of OS and the use of AFADESI-MSI technique to visualize the spatial distribution of compounds in tissues. 2. The method did not describe which part of OS was used and how many biological replicates and technical replicates were conducted in this study. 3. The manuscript does not show data on the content of chemical compounds and metabolites in serum and kidney from rats that did not receive OS extract treatment (control group), so it cannot be compared between the content of chemical compounds and metabolites in the two treatments. 4. The discussion is only limited to page 25, lines 557 - 580. Even then there are confusing parts (lines 565-571). The author illustrates compound M21 at the beginning of the paragraph, but then suddenly compound M21 is explained with M18 with precursor found at m/z342.09508, which belongs to M21. In explaining the mechanism of change of chemical compounds, the authors did not use literatures as a reference. 5. Appendix B table starts from No. 93, instead of 1. There seems to be something missing. 6. Authors must be able to prove that the chemical analysis in OS was carried out from at least 3 biological replicates and at least 2 technical replicates. 7. Authors should add more detail discussion regarding the mechanism of metabolite changes in serum and kidney and compare between control and treatment groups. ****** 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: No 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". 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Revision 1
14 Apr 2024 Author Response Reviewer #1: Dear Authors The study deals with a meaningful and important question, so the topic interests certain readers in the Plos One. However, the following comments should be addressed because some parts should be revised and improved. 1.In your title, are there any differences between the terms chemical constituent and metabolite? Yes, chemical composition refers to the prototype of a compound measured in vitro such as in aqueous decoctions or powdered preparations, which exists as a prototype in the blood or tissues after administration, and metabolites, which are products resulting from metabolic reactions of the prototypical ingredient in the blood(1, 2). This metabolic operation consists of three distinct phases. Phase I metabolism encompasses functionalization reactions while Phase II involves a series of conjugation reactions. Phase III is denoted by transporter-mediated elimination of drugs and/or metabolites from the body, typically executed by the liver, gut, kidney, or lungs. The metabolites produced by prototypes in the metabolic reactions of phase I can be directly excreted or re-excreted after going through the metabolic reactions of phase II, while also excreted through the metabolic reactions of phase II directly.(3) 2.Did you use a QC sample in your LC-MS/MS analysis? If not, please explain it. I did not use QC samples, in order to ensure the stability of the instrument and the reproducibility of the data, I prepared and tested the samples separately at different times, and observed their TIC plots, the overall profile and retention time did not find any significant changes, and combined with the comparison of the BLANK samples, the baseline is smooth, so it ensures that there are no residuals and the data can be reproduced. (S1 Fig). 3.In tables 1, 2, and 3 please write the molecular formula correctly like this C9H10O5 Thank you for your suggestion, I have changed the molecular formulae in Tables 1, 2 and 3 and in the appendix to the correct expressions 4. For your annotation of metabolite, add information about error mass to make sure your tentative identification is precise and accurate Thank you for your suggestion, the Error (ppm) column in the Table 3 is the error value for the molecular mass of the metabolite, all within ±5. Reviewer #2: This manuscript presents the results of research on the analysis of the chemical compounds content of Orthosiphon stamineus Benth (OS) using UHPLC-Q Exactive Orbitrap- 4 HRMS and AFADESI-MSI techniques. The research is a preliminary stage in utilizing OS as a source of medicine for kidney disease, so that the manuscript compiled is only limited to reporting a list of chemical compounds in OS and metabolites in serum from rats treated with OS extract. In this manuscript there is no in-depth analysis of the data obtained and no detailed discussion of the results of data analysis. Some notes on this manuscript are as follows: 1. The significance of this study only emphasizes the use of UHPLC-Q Exactive Orbitrap-4 HRMS for the analysis of metabolite content of OS and the use of AFADESI-MSI technique to visualize the spatial distribution of compounds in tissues. Thanks to your suggestion, this paper also compares the compounds contained in the aqueous decoction of OS with those contained in the powder of OS and finds the necessity of decoctions of traditional Chinese medicines to co-analyse the chemical characterisation of OS not only in vivo and in vitro, but also at the spatial level to characterise the distribution of OS. The UHPLC-Q Exactive Orbitrap-HRMS technique was developed for the first time for the characterisation of kidney tea with good reproducibility and stability, and maximised the extraction of the compounds contained in OS. 2. The method did not describe which part of OS was used and how many biological replicates and technical replicates were conducted in this study. The dry ground portion of the OS was used and three samples were prepared in parallel for each assay and a total of three replications were performed at different times to clarify the stability as well as the accuracy of the method. (S1 Fig.) 3. The manuscript does not show data on the content of chemical compounds and metabolites in serum and kidney from rats that did not receive OS extract treatment (control group), so it cannot be compared between the content of chemical compounds and metabolites in the two treatments. The metabolic content (i.e. peak intensity or peak area) has been demonstrated in Table 3 and Appendix F, showing that the control serum and kidney tissue homogenate samples had low levels of the prototypical substance, flush with baseline and using the extracted ion-pairing approach as a complete peak that could appear, but was present in the drug-containing serum and kidney tissue. In contrast, the peak areas of the compound metabolites were all zero in the control group, indicating their specificity. 4. The discussion is only limited to page 25, lines 557 - 580. Even then there are confusing parts (lines 565-571). The author illustrates compound M21 at the beginning of the paragraph, but then suddenly compound M21 is explained with M18 with precursor found at m/z342.09508, which belongs to M21. In explaining the mechanism of change of chemical compounds, the authors did not use literatures as a reference. Sorry for the confusion due to my mistake, the paragraph has always been about M21, as the numbering was changed during the writing process, here is my mistake. References are cited by accession. 5. Appendix B table starts from No. 93, instead of 1. There seems to be something missing. There is no loss of data, I am sorry for the misunderstanding because of my numbering, the first 92 numbers are compounds present in the OS, because of the display and labelling of the standard's graphs, i.e. Figures 3 and 4, if the numbering starts from 1 it is easy to confuse, so the compounds present in the OS are numbered together with compounds that are not present. 6. Authors must be able to prove that the chemical analysis in OS was carried out from at least 3 biological replicates and at least 2 technical replicates. Thank you for the reminder, the duplicate experimental plots have been added to the attachment. (S1-4 Fig) 7. Authors should add more detail discussion regarding the mechanism of metabolite changes in serum and kidney and compare between control and treatment groups. Thank you for your suggestion, which has been added to the discussion section. Complemented the way of rutin produces metabolites in blood. It has been stated in the literature that the main metabolic pathways involved in rutin in vivo are methylation, glucuronidation, sulphate esterification and their complex reactions(4). M26 and M29 are metabolites produced by different metabolic reactions of rutin. The precursor ion [M+K]+ of M26 occurs at m/z 393.07917，It is possible that a brassinose group (C12H20O9) was first lost, and the reaction was followed by oxidation, methylation and continued loss of CH2O2. The precursor ion [M-H]+ of M29 occurs at m/z 541.02960，The prototypical rutin first binds a glucuronic acid and oxidation reaction at m/z 477 based on the presence of a sulphuric acid and a methyl group at m/z 542 later, in accordance with the literature(4). References: 1. Liu YN, Hu MT, Qian J, Wang Y, Wang SF. Characterization of the chemical constituents of Jie-Geng-Tang and the metabolites in the serums and lungs of mice after oral administration by LC-Q-TOF-MS. Chin J Nat Med. 2021;19(4):284-94. http://doi.org/10.1016/S1875-5364(21)60028-6 2. Xu T, Li S, Sun Y, Pi Z, Liu S, Song F, et al. Systematically characterize the absorbed effective substances of Wutou Decoction and their metabolic pathways in rat plasma using UHPLC-Q-TOF-MS combined with a target network pharmacological analysis. J Pharm Biomed Anal. 2017;141:95-107. http://doi.org/10.1016/j.jpba.2017.04.012 3. Almazroo OA, Miah MK, Venkataramanan R. Drug Metabolism in the Liver. Clin Liver Dis. 2017;21(1):1-20. http://doi.org/10.1016/j.cld.2016.08.001 4. Goyal J, Verma PK. An Overview of Biosynthetic Pathway and Therapeutic Potential of Rutin. Mini Rev Med Chem. 2023;23(14):1451-60. http://doi.org/10.2174/1389557523666230125104101 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0304852.r002
17 May 2024 Decision Letter - Andrea Mastinu, Editor Analysis of the chemical constituents and their metabolites in Orthosiphon stamineus Benth. via UHPLC-Q Exactive Orbitrap-HRMS and AFADESI-MSI techniques PONE-D-24-03002R1 Dear Dr. Mao, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Andrea Mastinu Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. 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 #2: Yes ****** 5. 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 #2: Yes ****** 6. 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 #2: (No Response) ****** 7. 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 #2: No ******** https://doi.org/10.1371/journal.pone.0304852.r003
Formally Accepted
13 Jun 2024 Acceptance Letter - Andrea Mastinu, Editor PONE-D-24-03002R1 PLOS ONE Dear Dr. Mao, 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. Andrea Mastinu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0304852.r004

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