Modulatory effects of rutin and vitamin A on hyperglycemia induced glycation, oxidative stress and inflammation in high-fat-fructose diet animal model

Aqsa Iqbal; Sairah Hafeez Kamran; Farhan Siddique; Saiqa Ishtiaq; Misbah Hameed; Mobina Manzoor

doi:10.1371/journal.pone.0303060

Peer Review History

Original SubmissionDecember 23, 2023
12 Feb 2024 Decision Letter - Pankaj Singh, Editor PONE-D-23-43396Modulatory effects of Rutin and Vitamin A on Hyperglycemia induced Glycation, Oxidative stress and Inflammation in High-Fat-Fructose Diet Animal ModelPLOS ONE Dear Dr. Hafeez Kamran, 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. ============================== In this manuscript, authors have studied entitled “Modulatory effects of Rutin and Vitamin A on Hyperglycemia induced Glycation, Oxidative stress and Inflammation in High-Fat-Fructose Diet Animal Model” and have reported that combined effects of rutin and vitamin A in treated rats significantly reduces the level of HbA1c, hexokinase-2, and D-lactate levels and decreases the level of MDA, CAT, and GPx; and expression of inflammatory cytokines such as IL-6, IL-8, and NF-κB in treated rats. The research findings also indicate that rutin had a strong affinity for several important biomolecules, including as NF-κB, Catalase, MDA, IL-6, hexokinase-2, and GPx which can be hope for the treatment of AGE-related disorders, such as diabetes, autism, Alzheimer’s, atherosclerosis etc. Overall, the manuscript in its current form is not acceptable for publication in the esteemed “Plos One” journal and requires major revision in the manuscript. Author should revise the manuscript as per reviewer’s comments and can resubmit for the publication in Plos One journal. ============================== Please submit your revised manuscript by Mar 28 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, Pankaj Singh, 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. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 4. Please amend the manuscript submission data (via Edit Submission) to include author Mobina Manzoor. 5. Please amend your authorship list in your manuscript file to include author Mobeena Manzoor. 6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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: Authors did a good and timely work and represented in very lucid and scientific languages. They represented all the details. Introduction, Materials methods, result and discussion sections are properly represented. However a few mistakes that I observed, that need to be corrected. 1. Line no 59, 60, 62: objectives need to be corrected. sentence formation. 2. Line no: 92: 3 deoxyglucocone should be 3 deoxyglucosone 3,Line 111-120: References are missing 4. Line 214: Slight modification in method: need to be mention. Please do the correction and submit. Reviewer #2: This article examines the impact of a combination of rutin and Vitamin D on glycation resulting from hyperglycemia. Primarily, in the present analysis of studies on a mouse model of hyperglycemia involving 30 animals divided into six groups (N=5), endpoints including glycation, oxidative stress, inflammatory response, and cellular integrity were evaluated. The results support that the combination therapy has beneficial effects which are additive and potentially synergistic. An in-silico study showing the comparative binding affinities of the two drugs to identical ligands is also provided and provides further support for the observed experimental outcomes. This is a well-designed study; the results are presented well and produce a good story. While it the authors may claim that the combination of the drugs has enhanced effects, it may be too early, without further evidence, to claim synergism. The authors are encouraged to be cautious about this claim (synergism). General comments Abstract: Line 71 – 72: Authors must note that NF kappa B is not a cytokine. Article: • Lines 197-200 (page 8) should be broken down as the description of the procedure at this point is somewhat ambiguous. • Line 298 – the figure reference is incomplete. • Lines 314 – 317 may be broken down as follows: The pancreas of the rats receiving a combination of rutin and vitamin A showed normal morphology of the endocrine pancreas (green arrow), thereby suggesting a synergistic effect of the combination. This is also supported by our biochemical and PCR results. Reviewer #3: The manuscript submitted for the publication in “Plos One” Journal is not acceptable in its current form and require major revision. Please refer following comments to improve the manuscript. Comments: 1. Page 12, Lines 114-118, authors have written that Rutin (quercetin-3-O-rutinoside) is referred as vitamin P, but vitamin P represents Flavonoids or bioflavonoids, a vast group of polyphenolic compounds. 2. In Daily administration of HFFD (30% fat (5% sunflower oil+ 10% cottonseed oil+ 15% saturated fatty acids), why authors have selected combination of different oil instead of pure single oil. What was the basis of their % selection and combination of different oil? 3. Page 15, Lines 187-188, authors should mention that to which groups of animal 10% fructose in drinking water was also given daily for 42 days. 4. Please correct the title of Table 2. Authors mentioned only Binding affinity only for Rutin with target proteins but table has both Rutin and Vitamin A. 5. In methodology section, authors can divide experimental parameters under In vitro, in vivo, and in silico experiments subheads for better understanding of experimental design. 6. In methodology section, In-vitro Fructosamine Assay experimental procedure is incomplete. Authors should also mention the process of Glycation of Bovine Serum Albumin (BSA) weather they have followed the same procedure for Glycation of BSA as in cited reference 23. 7. Page 21, Lines 340, What is MG? 8. Please shift the levelling the Figure 3 (A, B, C……) outside the area of Figure. 9. Page 32, Line 621, Please check the statement : p < 0.001 (when compare with HFFD) but HFFD is not showed in Figure. 10. Authors have mentioned in many experimental results that Rutin or Vitamin A or combination of Rutin or Vitamin A showed better or similar protective effects that the synthetic Metformin. Rutin or Vitamin A are well known compounds from long years then why we are not using natural Rutin or Vitamin A for diabetic patients or to cure other diseases instead of synthetic Metformin still Metformin has many side effects. 11. Authors can improve the image quality of Figure 3 as significant level should be more visible and clear. 12. In Molecular docking study, Authors have discussed the results of Rutin more deeply whereas results of Vitamin A are not discussed very effectively. 13. In Figure’s caption and Table’s title, change case should be uniform. 14. Please remove the word discussion from Results and Discussion heading as authors have given separate heading for Discussion section. 15. Authors should strictly follow Instructions for Authors of Plos One’s Journal. ******* 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: SIRSHENDU CHATTERJEE Reviewer #2: Yes: Monde Ntwasa Reviewer #3: 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.0303060.r001
Revision 1
20 Mar 2024 Author Response Dear Editors and Reviewers, We appreciate you giving us the chance to revise our manuscript, "Modulatory effects of Rutin and Vitamin A on Hyperglycemia induced Glycation, Oxidative stress, and Inflammation in High-Fat-High-Fructose Diet Animal Model." Your insightful and beneficial comments have helped us to improve our manuscript. Every comment has been thoroughly reviewed, and the appropriate modifications have been made. We trust that all the modifications have been carried out correctly, and we hope to receive approval. All the corrections and responses to reviewer comments are as follows. Response to 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 Response: Thank you for your valuable suggestion. We have carefully revised the manuscript according to the instructions provided. 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. Response: We have applied codes that follow the best practice and facilitates reproducibility and reuse. 3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Response: Thank you for your valuable suggestion. We have included the method of sacrifice and method of anaesthesia in the study design section. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. Response: Thank you for your valuable suggestion. We have included all data in the manuscript and remaining data in the Supplementary file attached. 5. Please amend the manuscript submission data (via Edit Submission) to include author Mobina Manzoor. Response: Thank you for your valuable suggestion. We have amended the name Mobina Manzoor. 6. Please amend your authorship list in your manuscript file to include author Mobeena Manzoor. Response: Thank you for your valuable suggestion. We have amended the name Mobeena Manzoor to Mobina Manzoor in the authorship list. 7. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Response: Thank you for your valuable suggestion. We have deleted the Ethical statement from the declaration section and moved to the methods section. Response to Reviewers Reviewer #1 Authors did a good and timely work and represented in very lucid and scientific languages. They represented all the details. Introduction, Materials methods, result and discussion sections are properly represented. However a few mistakes that I observed, that need to be corrected. Response: We are grateful for your encouraging remarks. The responses of the questions are provided. Line no 59, 60, 62: objectives need to be corrected. sentence formation. Response: We have reformed the sentence in Line 59-62 In the current study we investigated the impact of combination of rutin and vitamin A on glycated products, the glyoxalase system, oxidative markers, and inflammation in animals fed a high-fat high-fructose (HFFD) diet. Line no: 92: 3 deoxyglucocone should be 3 deoxyglucosone Response: Thank you for your insightful suggestion. We have rectified to 3-deoxyglucosone. Line 111-120: References are missing Response: Thank you for your valuable suggestion. We have added reference in line 117. Line 214: Slight modification in method: need to be mention. Response: Thank you for helpful comment. We have added the methods. Reviewer #2 This article examines the impact of a combination of rutin and Vitamin D on glycation resulting from hyperglycemia. Primarily, in the present analysis of studies on a mouse model of hyperglycemia involving 30 animals divided into six groups (N=5), endpoints including glycation, oxidative stress, inflammatory response, and cellular integrity were evaluated. The results support that the combination therapy has beneficial effects which are additive and potentially synergistic. An in-silico study showing the comparative binding affinities of the two drugs to identical ligands is also provided and provides further support for the observed experimental outcomes. Response: We appreciate your supportive remarks. This is a well-designed study; the results are presented well and produce a good story. While it the authors may claim that the combination of the drugs has enhanced effects, it may be too early, without further evidence, to claim synergism. The authors are encouraged to be cautious about this claim (synergism). Response: Thank you for your valuable suggestions. We have replaced the word “synergism” with “beneficial’. Abstract: Line 71 – 72: Authors must note that NF kappa B is not a cytokine. Response: Thank you for your valuable suggestion. We have corrected the statement. Article • Lines 197-200 (page 8) should be broken down as the description of the procedure at this point is somewhat ambiguous. • Line 298 – the figure reference is incomplete. • Lines 314 – 317 may be broken down as follows: The pancreas of the rats receiving a combination of rutin and vitamin A showed normal morphology of the endocrine pancreas (green arrow), thereby suggesting a synergistic effect of the combination. This is also supported by our biochemical and PCR results. Response: We have broken down the statements to decrease the ambiguity. Line 298: We have corrected the figure reference. Line 314-317: Thank you for your valuable suggestion. We have broken down the statement in Line 314-317. Reviewer #3: The manuscript submitted for the publication in “Plos One” Journal is not acceptable in its current form and require major revision. Please refer following comments to improve the manuscript. Response: We have made every effort to amend the content in accordance with the helpful recommendations given. Page 12, Lines 114-118, authors have written that Rutin (quercetin-3-O-rutinoside) is referred as vitamin P, but vitamin P represents Flavonoids or bioflavonoids, a vast group of polyphenolic compounds. Response: Thank you for your valuable suggestion. We have corrected the statement. We found rutin referred to as vitamin P in an article (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355559/. But Vitamin P broadly represents more than one flavonoid. We have removed the word Vitamin P from page 12. In Daily administration of HFFD (30% fat (5% sunflower oil+ 10% cottonseed oil+ 15% saturated fatty acids), why authors have selected combination of different oil instead of pure single oil. What was the basis of their % selection and combination of different oil? Response: The HFFD utilized in this research was specifically formulated to induce hyperglycemia, and the cited reference was studied. Various oils were utilized to create a blend of unsaturated and saturated fatty acids. Sunflower and cotton seed oils both include a combination of unsaturated and saturated fatty acids. 15% saturated fatty acids were introduced separately to induce metabolic imbalance and obesity. Page 15, Lines 187-188, authors should mention that to which groups of animal 10% fructose in drinking water was also given daily for 42 days. Response: Thank you for your valuable suggestion. We have added the statement in the study design. Please correct the title of Table 2. Authors mentioned only Binding affinity only for Rutin with target proteins but table has both Rutin and Vitamin A. Response: Thank you for your valuable suggestion. We have corrected the title of the table 2. In methodology section, authors can divide experimental parameters under In vitro, in vivo, and in silico experiments subheads for better understanding of experimental design. Response: We have divided the sections as mentioned in the comment. In methodology section, In-vitro Fructosamine Assay experimental procedure is incomplete. Authors should also mention the process of Glycation of Bovine Serum Albumin (BSA) weather they have followed the same procedure for Glycation of BSA as in cited reference 23. Response: Thank you for your valuable comment. We have added the method of glycation of bovine serum albumin in the in-vitro fructosamine assay. Page 21, Lines 340, What is MG? Response: We have added the abbreviation of methylglyoxal (MG) in the abbreviation list and full name along with abbreviation is mentioned in Line 95 in Introduction Please shift the levelling the Figure 3 (A, B, C……) outside the area of Figure. Response: Thank you for the helpful comment. We have shifted the levelling of the figure 3 outside the figure. Page 32, Line 621, Please check the statement **: p < 0.001 (when compare with HFFD) but HFFD is not showed in Figure. Response: Thank you for your helpful comment. We have corrected the statement below the Fig. 2. Authors have mentioned in many experimental results that Rutin or Vitamin A or combination of Rutin or Vitamin A showed better or similar protective effects that the synthetic Metformin. Rutin or Vitamin A are well known compounds from long years then why we are not using natural Rutin or Vitamin A for diabetic patients or to cure other diseases instead of synthetic Metformin still Metformin has many side effects. Response: Thank you for your valuable comment. In the current investigation, the combination of rutin and vitamin A demonstrated benefits that were comparable to those of metformin. The evidence for substituting rutin and vitamin A combination for metformin is insufficient. Although the purpose of this study was to support the previously published findings, we are still unable to assert that a combination of rutin and vitamin A may replace metformin. We made the necessary corrections to the conclusion section and suggested that more research be done on the combination of rutin and vitamin A for the possible development of therapeutic approaches for conditions associated with advanced glycation end products (AGE). Authors can improve the image quality of Figure 3 as significant level should be more visible and clear. Response: Thank you for your valuable suggestion. We have improved the quality of images. In Molecular docking study, Authors have discussed the results of Rutin more deeply whereas results of Vitamin A are not discussed very effectively. Response: We have added the results of vitamin A in the results and discussion section In Figure’s caption and Table’s title, change case should be uniform. Response: We are grateful for your helpful suggestion. The change case has been uniformed in the titles of figures and tables. Please remove the word discussion from Results and Discussion heading as authors have given separate heading for Discussion section. Response: Thank you. We have removed the word from the heading. Authors should strictly follow Instructions for Authors of Plos One’s Journal. Response: Thank you for your valuable suggestion. We have tried to strictly follow the instructions for authors of Plos One. Attachments Attachment Submitted filename: Response to Reviewers Plos.docx* https://doi.org/10.1371/journal.pone.0303060.r002
27 Mar 2024 Decision Letter - Pankaj Singh, Editor PONE-D-23-43396R1Modulatory effects of Rutin and Vitamin A on hyperglycemia induced glycation, oxidative stress and inflammation in high-fat-fructose diet animal modelPLOS ONE Dear Dr. Hafeez Kamran, 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. ============================== The revised manuscript can be accepted for publication in esteemed "Plos One” Journal with few minor corrections. Authors have addressed each queries very well raised by reviewer and have critically modify the manuscript as per requirement. Following corrections still need to improve the submitted manuscript. Comments: 1. Table 3, Unit should be uniform throughout Table. 2. Please improve the quality of Fig 1, 2 and 5 as words are not visible to read. It is requested that authors should submit the revised manuscript with improved figures. 3. Please correct the order of Figures in submitted manuscript. ============================== Please submit your revised manuscript by May 11 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, Pankaj Singh, 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.0303060.r003
Revision 2
16 Apr 2024 Author Response Dear Editors and Reviewers, We appreciate you giving us the chance to revise our manuscript, "Modulatory effects of Rutin and Vitamin A on Hyperglycemia induced Glycation, Oxidative stress, and Inflammation in High-Fat-High-Fructose Diet Animal Model." Your insightful and beneficial comments have helped us to improve our manuscript. Every comment has been thoroughly reviewed, and the appropriate modifications have been made. We trust that all the modifications have been carried out correctly, and we hope to receive approval. All the corrections and responses to reviewer comments are as follows. Response to Academic Editor Comments Table 3, Unit should be uniform throughout Table. Response: The Table has been corrected. Please improve the quality of Fig 1, 2 and 5 as words are not visible to read. It is requested that authors should submit the revised manuscript with improved figures. Response: The quality of the figures 1, 2 and 5 have been improved. We have uploaded the figures on Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool and ensured that figures meet Plos requirements. Please correct the order of Figures in submitted manuscript. The order of the figures has been corrected. Response to 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 Response: Thank you for your valuable suggestion. We have carefully revised the manuscript according to the instructions provided. 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. Response: We have applied codes that follow the best practice and facilitates reproducibility and reuse. 3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Response: Thank you for your valuable suggestion. We have included the method of sacrifice and method of anaesthesia in the study design section. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. Response: Thank you for your valuable suggestion. We have included all data in the manuscript and remaining data in the Supplementary file attached. 5. Please amend the manuscript submission data (via Edit Submission) to include author Mobina Manzoor. Response: Thank you for your valuable suggestion. We have amended the name Mobina Manzoor. 6. Please amend your authorship list in your manuscript file to include author Mobeena Manzoor. Response: Thank you for your valuable suggestion. We have amended the name Mobeena Manzoor to Mobina Manzoor in the authorship list. 7. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Response: Thank you for your valuable suggestion. We have deleted the Ethical statement from the declaration section and moved to the methods section. Response to Reviewers Reviewer #1 Authors did a good and timely work and represented in very lucid and scientific languages. They represented all the details. Introduction, Materials methods, result and discussion sections are properly represented. However a few mistakes that I observed, that need to be corrected. Response: We are grateful for your encouraging remarks. The responses of the questions are provided. Line no 59, 60, 62: objectives need to be corrected. sentence formation. Response: We have reformed the sentence in Line 59-62 In the current study we investigated the impact of combination of rutin and vitamin A on glycated products, the glyoxalase system, oxidative markers, and inflammation in animals fed a high-fat high-fructose (HFFD) diet. Line no: 92: 3 deoxyglucocone should be 3 deoxyglucosone Response: Thank you for your insightful suggestion. We have rectified to 3-deoxyglucosone. Line 111-120: References are missing Response: Thank you for your valuable suggestion. We have added reference in line 117. Line 214: Slight modification in method: need to be mention. Response: Thank you for helpful comment. We have added the methods. Reviewer #2 This article examines the impact of a combination of rutin and Vitamin D on glycation resulting from hyperglycemia. Primarily, in the present analysis of studies on a mouse model of hyperglycemia involving 30 animals divided into six groups (N=5), endpoints including glycation, oxidative stress, inflammatory response, and cellular integrity were evaluated. The results support that the combination therapy has beneficial effects which are additive and potentially synergistic. An in-silico study showing the comparative binding affinities of the two drugs to identical ligands is also provided and provides further support for the observed experimental outcomes. Response: We appreciate your supportive remarks. This is a well-designed study; the results are presented well and produce a good story. While it the authors may claim that the combination of the drugs has enhanced effects, it may be too early, without further evidence, to claim synergism. The authors are encouraged to be cautious about this claim (synergism). Response: Thank you for your valuable suggestions. We have replaced the word “synergism” with “beneficial’. Abstract: Line 71 – 72: Authors must note that NF kappa B is not a cytokine. Response: Thank you for your valuable suggestion. We have corrected the statement. Article • Lines 197-200 (page 8) should be broken down as the description of the procedure at this point is somewhat ambiguous. • Line 298 – the figure reference is incomplete. • Lines 314 – 317 may be broken down as follows: The pancreas of the rats receiving a combination of rutin and vitamin A showed normal morphology of the endocrine pancreas (green arrow), thereby suggesting a synergistic effect of the combination. This is also supported by our biochemical and PCR results. Response: We have broken down the statements to decrease the ambiguity. Line 298: We have corrected the figure reference. Line 314-317: Thank you for your valuable suggestion. We have broken down the statement in Line 314-317. Reviewer #3: The manuscript submitted for the publication in “Plos One” Journal is not acceptable in its current form and require major revision. Please refer following comments to improve the manuscript. Response: We have made every effort to amend the content in accordance with the helpful recommendations given. Page 12, Lines 114-118, authors have written that Rutin (quercetin-3-O-rutinoside) is referred as vitamin P, but vitamin P represents Flavonoids or bioflavonoids, a vast group of polyphenolic compounds. Response: Thank you for your valuable suggestion. We have corrected the statement. We found rutin referred to as vitamin P in an article (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355559/. But Vitamin P broadly represents more than one flavonoid. We have removed the word Vitamin P from page 12. In Daily administration of HFFD (30% fat (5% sunflower oil+ 10% cottonseed oil+ 15% saturated fatty acids), why authors have selected combination of different oil instead of pure single oil. What was the basis of their % selection and combination of different oil? Response: The HFFD utilized in this research was specifically formulated to induce hyperglycemia, and the cited reference was studied. Various oils were utilized to create a blend of unsaturated and saturated fatty acids. Sunflower and cotton seed oils both include a combination of unsaturated and saturated fatty acids. 15% saturated fatty acids were introduced separately to induce metabolic imbalance and obesity. Page 15, Lines 187-188, authors should mention that to which groups of animal 10% fructose in drinking water was also given daily for 42 days. Response: Thank you for your valuable suggestion. We have added the statement in the study design. Please correct the title of Table 2. Authors mentioned only Binding affinity only for Rutin with target proteins but table has both Rutin and Vitamin A. Response: Thank you for your valuable suggestion. We have corrected the title of the table 2. In methodology section, authors can divide experimental parameters under In vitro, in vivo, and in silico experiments subheads for better understanding of experimental design. Response: We have divided the sections as mentioned in the comment. In methodology section, In-vitro Fructosamine Assay experimental procedure is incomplete. Authors should also mention the process of Glycation of Bovine Serum Albumin (BSA) weather they have followed the same procedure for Glycation of BSA as in cited reference 23. Response: Thank you for your valuable comment. We have added the method of glycation of bovine serum albumin in the in-vitro fructosamine assay. Page 21, Lines 340, What is MG? Response: We have added the abbreviation of methylglyoxal (MG) in the abbreviation list and full name along with abbreviation is mentioned in Line 95 in Introduction Please shift the levelling the Figure 3 (A, B, C……) outside the area of Figure. Response: Thank you for the helpful comment. We have shifted the levelling of the figure 3 outside the figure. Page 32, Line 621, Please check the statement **: p < 0.001 (when compare with HFFD) but HFFD is not showed in Figure. Response: Thank you for your helpful comment. We have corrected the statement below the Fig. 2. Authors have mentioned in many experimental results that Rutin or Vitamin A or combination of Rutin or Vitamin A showed better or similar protective effects that the synthetic Metformin. Rutin or Vitamin A are well known compounds from long years then why we are not using natural Rutin or Vitamin A for diabetic patients or to cure other diseases instead of synthetic Metformin still Metformin has many side effects. Response: Thank you for your valuable comment. In the current investigation, the combination of rutin and vitamin A demonstrated benefits that were comparable to those of metformin. The evidence for substituting rutin and vitamin A combination for metformin is insufficient. Although the purpose of this study was to support the previously published findings, we are still unable to assert that a combination of rutin and vitamin A may replace metformin. We made the necessary corrections to the conclusion section and suggested that more research be done on the combination of rutin and vitamin A for the possible development of therapeutic approaches for conditions associated with advanced glycation end products (AGE). Authors can improve the image quality of Figure 3 as significant level should be more visible and clear. Response: Thank you for your valuable suggestion. We have improved the quality of images. In Molecular docking study, Authors have discussed the results of Rutin more deeply whereas results of Vitamin A are not discussed very effectively. Response: We have added the results and discussion of vitamin A in the results and discussion section In Figure’s caption and Table’s title, change case should be uniform. Response: We are grateful for your helpful suggestion. The change case has been uniformed in the titles of figures and tables. Please remove the word discussion from Results and Discussion heading as authors have given separate heading for Discussion section. Response: Thank you. We have removed the word from the heading. Authors should strictly follow Instructions for Authors of Plos One’s Journal. Response: Thank you for your valuable suggestion. We have tried to strictly follow the instructions for authors of Plos One. Attachments Attachment Submitted filename: Response to Reviewers Plos.docx* https://doi.org/10.1371/journal.pone.0303060.r004
19 Apr 2024 Decision Letter - Pankaj Singh, Editor Modulatory effects of Rutin and Vitamin A on hyperglycemia induced glycation, oxidative stress and inflammation in high-fat-fructose diet animal model PONE-D-23-43396R2 Dear Dr. Hafeez Kamran, 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, Pankaj Singh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0303060.r005
Formally Accepted
29 Apr 2024 Acceptance Letter - Pankaj Singh, Editor PONE-D-23-43396R2 PLOS ONE Dear Dr. Hafeez Kamran, 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. Pankaj Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303060.r006

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