A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells

Swaha Satpathy; Arjun Patra; Muhammad Delwar Hussain; Mohsin Kazi; Mohammed S. Aldughaim; Bharti Ahirwar

doi:10.1371/journal.pone.0240068

Peer Review History

Original SubmissionAugust 20, 2020
14 Oct 2020 Decision Letter - Branislav T. Šiler, Editor PONE-D-20-26098 Antioxidant enriched fraction from Pueraria tuberosa alleviates ovariectomized-induced osteoporosis in rats, and inhibits growth of breast and ovarian cancer cell lines in vitro PLOS ONE Dear Dr. Satpathy, 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. ============================== Reviewer #1 found the manuscript deficient in several values which should be determined in additional measurements/experiments in order to provide a solid scientific backgound for the recorded observations and concluded claims. Statistical testing should be presented in a more profound manner, while the language is to be considerably polished throughout the text. Please, be aware of the attachment with additional comments provided by Reviewer #2. ============================== Please submit your revised manuscript by Nov 28 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-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) PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 3) Please include a copy of Table 3 which you refer to in your text on page 24. 4) Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 5) We noticed there is a bit of text overlap with this previously published paper of yours: https://doi.org/10.1016/j.sajb.2018.05.033, which will need to be addressed. In your revision ensure you cite all your sources (including your own work), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. [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: Partly ******** 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: No Reviewer #2: No ****** 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: The manuscript is a good research area. However, there are few grammatical errors that must be check in the manuscript. All comments raised have been uploaded for authors. The basis for the dosages used in the methodology must be provided by authors. Reviewer #2: The present submission claimed that AEF may alleviate bone loss through its anti-oxidant activity. However, I have a few concerns about the submission. (1) Docking study demonstrated AEF may bind to ERa and ERb. Therefore, ER expression should be examined in the bone. (2) The uterus weight relative to body weight should be determined. (3) The authors claimed that AEF may inhibit hyperlipidemia. However, the histological staings in the provided figure appeared that there is less fat disposition in the femur. In addition, oil red O staings should be evaluated in the bone. Moreover, what is the alterations of serum HDL and LDL? (4) The redox status should be tested in the bone. (5) If possible, the BMD and bone microstructure should be evaluated. (6) The P value should be followed when the authors mentioned the result is significant. (7) Please provide the batch number for the kits and chemicals. (8) Grammatical mistakes have been found in the MS. (9) Please eliminate the description of the anticancer. The current data did not support the assertion. ****** 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". 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. Attachments Attachment Submitted filename: PONE-D-20-26098_reviewer comments.pdf https://doi.org/10.1371/journal.pone.0240068.r001
Revision 1
9 Dec 2020 Author Response Below we present our point by point responses to the reviewer/editorial comments: Reviewer/Editorial Comments Response "Main title: "Antioxidant enriched fraction" has no meaning. Instead "Antioxidant" it can stand "Antioxidant compounds". Moreover, "enriched" sounds like the authors somehow added antioxidant compounds to the fraction they have investigated. Instead of "enriched", "rich"(if they are clearly reach) or "containing" should read (throughout the text). And finally, the authors did not investigate a fraction of P. tuberosa, but a fraction of its extract." We have corrected "Antioxidant enriched fraction" to "Antioxidant-rich fraction" throughout the manuscript. The title is changed as: Antioxidant-rich fraction from Pueraria tuberosa alleviates ovariectomized-induced osteoporosis in rats, and inhibits growth of breast and ovarian cancer cells 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 Corrected as per the PLOS ONE style templates. 2) PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. ORCID iD information is added. 3) Please include a copy of Table 3 which you refer to in your text on page 24. Table 3 was mentioned in the MS by mistake and now it has been corrected as the supplementary material S1. 4) Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Captions are included and updated the in-text citations 5) We noticed there is a bit of text overlap with this previously published paper of yours: https://doi.org/10.1016/j.sajb.2018.05.033, which will need to be addressed. In your revision ensure you cite all your sources (including your own work), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. We have extensively edited, rephrased the revised manuscript for any text overlap with the previous paper. Reviewer's Responses to Questions 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: Partly Changes and corrections have been made in the manuscript to address the comment for Reviewer #2 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? 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: No Reviewer #2: No The manuscript is extensively edited to improve the language. The typographical or grammatical errors have also been corrected. 5. Review Comments to the Author Reviewer #1: The manuscript is a good research area. However, there are few grammatical errors that must be check in the manuscript. All comments raised have been uploaded for authors. The basis for the dosages used in the methodology must be provided by authors. We thank the reviewer for making inspiring comments. We have gone through the manuscript very carefully and corrected grammatical, and spelling mistakes as per the suggestions. The basis for the dose use is provided below. 1) Why and what is the basis for this dose selection? No mortality or any signs of moribund status were found at this dose (2000 mg/kg). Therefore, the LD50 cut-off is 5000 mg/kg (category 5 in the Globally Harmonized Classification System). Usually, 1/5th and 1/10th of the LD50 value can be used for animal experimentation and the dose selected (very small dose compared to LD50 cut-off, 5000 mg/kg) for screening antiosteoporotic activity, where the treatment was for 90 days. We have not observed and mortality of signs of toxicity during this 90 days period also. This information is now provided in the manuscript (page #, lines #-#) 2) In the molecular docking methodology, it was necessary to detail more, for example, which values of the grid box (x, y and z). 1x7R: 15.587, 32.224, 22.304 1x76: 29.554, 37.747, 38.951 This information is now provided in the manuscript (page 14, lines 284-285) No proper methodology in ligand preparation. Provide detailed methodology. 3D conformer (.sdf) of daidzein and genistein were downloaded from PubChem website and used as ligand. FlexXLeadIT 2.1.8 of BiosolveIT uses only 3D conformer of ligands. This information is now provided in the manuscript (page 14, lines 292-293) Genistein: https://pubchem.ncbi.nlm.nih.gov/compound/5280961 Daidzein: https://pubchem.ncbi.nlm.nih.gov/compound/5281708 The information is added in the method section (page#, lines #-#) Why was a reference drug not used to compare with the two compounds used for docking studies? 5-hydroxy-2-(4-hydroxyphenyl)-1-benzofuran-7-carbonitrile was used as internal ligand in 1X76 (ER-α). Genistein was the internal ligand of 1X7R (ER-β), Figure 10 mentioned in page 22-23. Further, As per earlier literature, estradiol (a reference drug) has docking score of -18 and -17 into estrogen receptor α and β active site, respectively, which is mentioned in the discussion section (Page 27, line 548-549). Also, authors should provide the ADMET profile for these compounds as well as the Drug likeness prediction The ADMET profile of the two compounds are now provided in the manuscript (materials section page 14-15, and results section page 23-24) Reviewer #2: The present submission claimed that AEF may alleviate bone loss through its anti-oxidant activity. However, I have a few concerns about the submission. (1) Docking study demonstrated AEF may bind to ERa and ERb. Therefore, ER expression should be examined in the bone. The docking study was done to support the data obtained in animal study and we found good interaction of daidzein and genistein with both ER-α and ER-β. However, ER expression could be carried out in future. We have mentioned it in the revised manuscript (page 22, line 449). (2) The uterus weight relative to body weight should be determined. The weight ratios are: Gr-I (0.1896%); Gr-II (0.0457%); Gr-III (0.1124%); Gr-IV (0.0734%); Gr-V (0.0918%). We have now included this numbers in the revised manuscript (page 20, line 405-407) (3) The authors claimed that AEF may inhibit hyperlipidemia. However, the histological staings in the provided figure appeared that there is less fat disposition in the femur. In addition, oil red O staings should be evaluated in the bone. Moreover, what is the alterations of serum HDL and LDL? We thank the reviewer for pointing this out. We made the statement based on the ability of AEF to reduce the OVX-induced increased level of triglycerides and total cholesterol. We have not measured the serum HDL and LDL levels at the time of animal experiment. (4) The redox status should be tested in the bone. Thanks for the suggestion. As we have not measured the balance between oxidants and antioxidants during the experiment. (5) If possible, the BMD and bone microstructure should be evaluated. We thank the reviewer for the great suggestion. But we could not evaluate the BMD and bone microstructure due to lack of facility at the time of the animal experiment. (6) The P value should be followed when the authors mentioned the result is significant. We thank the reviewer for the excellent suggestion. We have now provided the information in the manuscript (results section in pages 17-20). (7) Please provide the batch number for the kits and chemicals. Thanks to the reviewer for the suggestion. We have now provided the information here, but not mentioned in the MS. Currently, we do not have the batch number of some of the reagents/chemicals as they were over and not provided here. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT): lot #11206BE Dimethyl sulfoxide (DMSO): Lot # S0154 Phosphate Buffered Saline (PBS): Lot # 11419005 Xylazine: FFK8002 Ketamine: 382053 Gentamicin: AHF0051 Calcium kit: CAO-020 Phosphorus kit: PHO-L-B1002 Urea kit: URE-L-B1010 Alkaline phosphatase kit: ALP-L-B1086 Cholesterol kit: HDL-L-B1046 Triglyceride kit: TGR-L-B1106 (8) Grammatical mistakes have been found in the MS. We have searched and corrected the grammatical mistakes Throughout the manuscript (9) Please eliminate the description of the anticancer. The current data did not support the assertion. The Antioxidant-Rich Fraction (ARF) has the potential for treatment of menopausal osteoporosis. In addition, it has anticancer effect in cancer cells. This is advantageous as treatment with estrogens have the side effect including possibility of developing cancer.To make this clear,we have mentioned this in the Introduction section (page 4, paragraph 2), discussion (page 27, lines 554-559), conclusion (page 30, lines 567-568), and abstract (page 2, lines 44) Dr. Swaha Satpathy Attachments Attachment Submitted filename: Responses to reviewers.doc https://doi.org/10.1371/journal.pone.0240068.r002
11 Dec 2020 Decision Letter - Branislav T. Šiler, Editor PONE-D-20-26098R1 Antioxidant-rich fraction from Pueraria tuberosa alleviates ovariectomized-induced osteoporosis in rats, and inhibits growth of breast and ovarian cancer cells PLOS ONE Dear Dr. Satpathy, 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 authors did not properly respond to the comments provided by the Reviewer #2. Two major issues are stated in the reviewers' reports below. In addition, please take notice of the following comments: The authors did not grasp the Editor's comments related to the proper usage of scientific terminology introduced in the previous review round. Let me try to explain in a semantic manner: "Antioxidant" is an adjective and cannot present an object. Therefore, a subject cannot be rich in an attribute, but in some item (noun). The expression "Antioxidant compounds" sound like a logical choice as suggested in the previous review round. The phrase "fraction from Pueraria tuberosa" is unaccomplished, vague. Can authors define how would they describe a fraction of a plant? Instead of that, a fraction of a plant extract would be more appropriate. Considering above mentioned, I would suggest transforming the main title into: "A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells". The same terminology has to be applied throughout the text. L23: No need to introduce the abbreviation of the genus name, i.e. please delete the bracket. Instead of that, the species authority should be introduced as: Pueraria tuberosa (Roxb. ex Willd.) DC. here and also in the L90. In L89: all the Latin names must stand in italics as well as elsewhere in the text. Compound names are randomly capitalized. Please do not write compound names with capital letters unless they present trademarks. ARF (antioxidant-rich fraction) cannot stand from the reasons stated as above. "Fraction rich in antioxidant compounds" and a matching abbreviation can read. Figures 8 and 9 are vertically stretched out. Can you revert them to the original ratio? ============================== Please submit your revised manuscript by Jan 25 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #1: All comments have been addressed Reviewer #2: (No Response) ******** 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes 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 #1: All comments have been addressed. Research ethics was followed as ethical approval was obtained for this study. Reviewer #2: The authors partly responsed my comments. However, (1) The redox status should be tested in the bone. (2) The author should explain why did not test HDL and LDL. ****** 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 #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". 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.0240068.r003
Revision 2
22 Dec 2020 Author Response 1. "Antioxidant" is an adjective and cannot present an object. Therefore, a subject cannot be rich in an attribute, but in some item (noun). The expression "Antioxidant compounds" sound like a logical choice as suggested in the previous review round. The phrase "fraction from Pueraria tuberosa" is unaccomplished, vague. Can authors define how would they describe a fraction of a plant? Instead of that, a fraction of a plant extract would be more appropriate. Considering above mentioned, I would suggest transforming the main title into: "A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells". The same terminology has to be applied throughout the text. Response: The title is changed as: A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells 2. L23: No need to introduce the abbreviation of the genus name, i.e. please delete the bracket. Instead of that, the species authority should be introduced as: Pueraria tuberosa (Roxb. ex Willd.) DC. here and also in the L90. In L89: all the Latin names must stand in italics as well as elsewhere in the text. Response: Corrections have been made throughout the manuscript (Page: 2, 3, 5, 14, 16-22, 25, 27 & 28). 3. Compound names are randomly capitalized. Please do not write compound names with capital letters unless they present trademarks. Response: Changes have been made in the manuscript (page: 5 and supplementary material). 4. ARF (antioxidant-rich fraction) cannot stand from the reasons stated as above. "Fraction rich in antioxidant compounds" and a matching abbreviation can read. Response: ARF (antioxidant-rich fraction) has been stated as "Fraction rich in antioxidant compounds" FRAC throughout the manuscript (Page: 2, 3, 5-7, 9-11, 13, 14, 16-22, 25-28). 5. Figures 8 and 9 are vertically stretched out. Can you revert them to the original ratio? Response: The figures are now in the original ratio Reviewer's Responses to Questions 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. Response: Reviewer #1: All comments have been addressed Reviewer #2: (No Response) Reviewer#2 suggested to report some parameters related to animal experiment. We thank the reviewer and appreciate for the suggestions. We will definitely consider them in our future studies. At this time, it is difficult to conduct the whole animal experiments for only these parameters (redox status in bone and HDL & LDL). We have acknowledged and mentioned them for future studies in the conclusion section. 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 #1: Yes Reviewer #2: Yes 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes Reviewer #2: Yes Review Comments to the Author Reviewer #1: All comments have been addressed. Research ethics was followed as ethical approval was obtained for this study. We thank the reviewer for encouraging comments. Reviewer #2: The authors partly responsed my comments. However, (1) The redox status should be tested in the bone. (2) The author should explain why did not test HDL and LDL. Response: We thank the reviewer and appreciate for the suggestions. We will definitely consider them in our future studies. At this time, it is difficult to conduct the whole animal experiments for only these parameters (redox status in bone and HDL & LDL). We have acknowledged and mentioned them for future studies in the conclusion section. Attachments Attachment Submitted filename: Responses to Reviewers.doc https://doi.org/10.1371/journal.pone.0240068.r004
26 Dec 2020 Decision Letter - Branislav T. Šiler, Editor A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells PONE-D-20-26098R2 Dear Dr. Satpathy, 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.0240068.r005
Formally Accepted
4 Jan 2021 Acceptance Letter - Branislav T. Šiler, Editor PONE-D-20-26098R2 A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells Dear Dr. Satpathy: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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.0240068.r006

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