Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients

Woraanong Prugsakij; Sukanya Numsawat; Ponsawan Netchareonsirisuk; Parkpoom Tengamnuay; Wanchai De-Eknamkul

doi:10.1371/journal.pone.0284853

Peer Review History

Original SubmissionSeptember 4, 2022
5 Jan 2023 Decision Letter - Cheorl-Ho Kim, Editor PONE-D-22-22814 Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients PLOS ONE Dear Dr. De-Eknamkul, 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. ============================== Only a minor comments have been raised and please respond as soon as possible. Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear Authors I have a minor comment as it is interesting in the field. It can be further considered when the minor brevision is made. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: 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: This study is meaningful as a basic study for the development of a hair growth promoting mechanism and androgenic alopecia treatment of Avicennia marina extract and its active ingredient (avicequinone C). We recommended that the following corrections are necessary. 1. There are no results regarding the cytotoxicity of AM and AC. It is necessary to write a separate Fig. In addition, it is necessary to description in detail why the experiment was performed only on low concentrations of the sample with AM (0-40 µg/mL) and AC (0-40 µM), and why the time was measured as 48 hours. 2. When measuring the protein expression of hair growth factor, the AM treatment concentration was effective at 10 µg/mL. When translocation from the cytoplasm to the nucleus was confirmed, the AM treatment concentration was increased by 2 times (20 µg/mL). What is reason? 3. In the cell cycle experiment, are there any results for each AM and AC treatment time period and concentration? What is the rationale for setting AM (10 µg/mL) and AC (10 µM) concentrations? And, what is the rationale for setting 48 hours? 4. Line 438: This may reflect the synergistic effects of the constituents present in the AM extract. → It is necessary to supplement the consideration of other active ingredients present in Avicennia marina. 5. Line 240: 366 font size Line 248: Unit correction required (mM → µM) Line 478: T?? DHT?? Line 528: in vitro, in vivo in italics Line 638: 5 font size Figure 3 (a) 3 Lane, unit correction required mg/mL → µg/mL Figures 1, 5-8 Numbers and symbols overall poor readability. Needs correction ****** 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 ******** [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: Reviewer Note.docx https://doi.org/10.1371/journal.pone.0284853.r001
Revision 1
10 Mar 2023 Author Response Response to Reviewers’ Comments Manuscript: [PONE-D-22-22814] Title: Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients Reviewers’ Comments 1. There are no results regarding the cytotoxicity of AM and AC. It is necessary to write a separate Fig. In addition, it is necessary to description in detail why the experiment was performed only on low concentrations of the sample with AM (0-40 µg/mL) and AC (0-40 µM), and why the time was measured as 48 hours. Response: We have included the results of cytotoxicity of AM and AC as Fig. 2 in the main manuscript with describing the detail of the results and adding figure legend. For the question on the measuring time of 48 hours, this was obtained from the time-course study of 5α-R enzyme activity assay (0, 6, 12, 24, 36 and 48 h) using the primary DPCs (S6 Fig). The results showed that the activity of 5α-R enzyme was significantly increased during. Therefore, the cytotoxicity of AM and AC treatment was reasonably detected at 48h for cell viability. Revision: Line 403-416: “In order to observe the effects of AM and AC on the cell cycle of the isolated DPCs, the primary cells were first determined for the cytotoxicity of both the extract and active compound by using MTT assay for 48 hr. This incubation time was optimized based on the time-course study of 5α-R enzyme activity assay (0, 6, 12, 24, 36 and 48 h) using the primary DPCs (S6 Fig). The results showed that the treatment of AM at the concentration range of 2.5-20 µg/mL and AC at 2.5-20 µM did not cause significant cytotoxicity on DPCs, compared with the non-treated cells (Fig 2). At the maximum non-toxic concentrations of 20 µg/mL AM and 20 µM AC, the cell viability was observed to be in the range of 80-85% which is lower than the acceptable 90% cell viability. Therefore, we selected the concentrations of 10 µg/mL AM and 10 µM AC for cell cycle analysis and further experiments.” Line 428-431: “Fig 2. Cytotoxicity assay of AM and AC on DPCs. DPCs were treated with various concentrations of (a) AM (0-40 µg/mL) and (b) AC (0-40 µM) for 48 h. Cell viability was evaluated by PrestoBlue® assay. The data is presented as mean + SD (n=3). * p < 0.05 versus non-treated cells.” 2. When measuring the protein expression of hair growth factor, the AM treatment concentration was effective at 10 µg/mL. When translocation from the cytoplasm to the nucleus was confirmed, the AM treatment concentration was increased by 2 times (20 µg/mL). What is the reason? Response and Revision: It was a typo error. We are sorry for the mistake. In fact, the protein expression analysis of hair growth factors and translocation were all treated with AM at 10 µg/mL. We have corrected the concentration from 20 µg/mL to 10 µg/mL in the Line 324 and 513. 3. In the cell cycle experiment, are there any results for each AM and AC treatment time period and concentration? What is the rationale for setting AM (10 µg/mL) and AC (10 µM) concentrations? And, what is the rationale for setting 48 hours? Response: For the cell cycle experiment, the concentration and time of AM and AC treatment were decided to set at 10 µg/mL and AC 10 µM which were based on the cytotoxicity result in Fig2. AM (10 µg/mL) and AC (10 µM) showed acceptable cell viability more than 90%. For the question on the measuring time of 48 hours, this was obtained from the time-course study of 5α-R enzyme activity assay (0, 6, 12, 24, 36 and 48 h) using the primary DPCs (S6 Fig). Line 736-737: S6 Fig. A TLC plate shows the optimization of 5α-R activity in various time points. DP primary cells were treated with substrate testosterone to detect the optimum time of dihydrotestosterone formation at 0-48h (lane 1-6). DP primary cells without testosterone (lane 7). Testosterone (lane8) and dihydrotestosterone (lane9). Revision: Line 403-416: “In order to observe the effects of AM and AC on the cell cycle of the isolated DPCs, the primary cells were first determined for the cytotoxicity of both the extract and active compound by using MTT assay for 48 hr. This incubation time was optimized based on the time-course study of 5α-R enzyme activity assay (0, 6, 12, 24, 36 and 48 h) using the primary DPCs (S6 Fig). The results showed that the treatment of AM at the concentration range of 2.5-20 µg/mL and AC at 2.5-20 µM did not cause significant cytotoxicity on DPCs, compared with the non-treated cells (Fig 2). At the maximum non-toxic concentrations of 20 µg/mL AM and 20 µM AC, the cell viability was observed to be in the range of 80-85% which is lower than the acceptable 90% cell viability. Therefore, we selected the concentrations of 10 µg/mL AM and 10 µM AC for cell cycle analysis and further experiments.” 4. Line 438: This may reflect the synergistic effects of the constituents present in the AM extract. → It is necessary to supplement the consideration of other active ingredients present in Avicennia marina. Response This sentence intended to explain the observed results obtained from the AM extract. However, we agree that an additional sentence should be added to make it clearer. Revision: Line 453-456: “This may reflect the synergistic effects of the constituents present in the AM extract [40]. To prove this, however, it is necessary to find other active constituents in AM, followed by observing the 5α-R inhibitory activity of their combinations with AC.” 5. Line 240: 366 font size Revision: We have revised 366 font size. Line 248: Unit correction required (mM → µM) Revision: We have corrected the unit from mM to µM Line 478: T?? DHT?? Revision: We have revised line 496-497 “These results suggest that the AM extract and the AC were able to inhibit both DHT-AR and T-AR complex formation. Line 528: in vitro, in vivo in italics Revision: We have revised italics font in line 545 Line 638: 5 font size Revision: We have revised in line 655. Figure 3 (a) 3 Lane, unit correction required mg/mL → µg/mL Revision: The unit of mg/mL has been changed to µg/mL in Fig. 4 Figures 1, 5-8 Numbers and symbols overall poor readability. Needs correction. Revision: The numbers and symbols were all corrected. Attachments Attachment Submitted filename: Response to reviewers comments.docx https://doi.org/10.1371/journal.pone.0284853.r002
11 Apr 2023 Decision Letter - Cheorl-Ho Kim, Editor Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients PONE-D-22-22814R1 Dear Dr. De-Eknamkul, 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, Cheorl-Ho Kim, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr Wanchai I have carefully read your revision and found it appropriately responded and revised, as you did. I think that your study is interesting in the acting compound, avicequinone C, which was isolated from the Avicennia marina. As the dermal papilla cells isolated from androgenic alopecia patients has been examined for hair growth promotion, the study will enahance our journal Plos One's impact. I am very happy to decide editorial ACCEPT for publication in Plos One. Thank you for your chosing the Plos One, once again, Sincerely Cheorl-Ho Kim PhD, Editor, Plos One Professor SKKU, Biological Science Professor, SHAIST. Reviewers' comments: https://doi.org/10.1371/journal.pone.0284853.r003
Formally Accepted
14 Apr 2023 Acceptance Letter - Cheorl-Ho Kim, Editor PONE-D-22-22814R1 Mechanistic synergy of hair growth promotion by the Avicennia marina extract and its active constituent (avicequinone C) in dermal papilla cells isolated from androgenic alopecia patients Dear Dr. De-Eknamkul: 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 Professor Cheorl-Ho Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284853.r004

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