PONE-D-22-35294

Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer’s disease

PLOS ONE

Dear Dr. Hammad Ismail,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Milkyas Endale, PhD

Academic Editor

PLOS ONE

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2. Please ensure that the specific geographical locations for the collection of the Berberis lycium are specified.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This research was supported by the Committee on Scientific and Technological Cooperation of the Organization of Islamic Conference, Islamabad, Pakistan and the International Foundation for Science, Stockholm, Sweden, through a grant to Dr. Hammad Ismail, Assistant Professor University of Gujrat.”

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“Dr. Hammad Ismail received the funding for research by International Foundation of Science (IFS) under grant no. I-1-F-6453-1 (https://www.ifs.se/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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Additional Editor Comments:

�Title should reflect the in silico study done. Possible the tiles can be revised as "Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer’s disease combined with insilico molecular docking"

�Abstract: the binding affinity values of berberine and rivastigmine to beta secretase should be included for comparison to standard drug.

�EAM37 is a fraction collected from solvent mixture ethyl acetate and methanol (3:7) of silica gel CC separation of methanol roots extract of Berberis lycium (check line 141). Line 350-358, this fraction was analytically analyzed by TLC and HPLC using available laboratory standards of alkaloids, including morphine, quinine, scopolamine, caffeine, and berberine. EAM37 and selected alkaloid standards were compared by their TLC Rf values (Figure 2A-C). On the other hand, HPLC analysis of the EAM37 fraction was also compared with the standard (Figure 2E) berberine and the authors confirmed that the yellow precipitate from EAM37 is berberine from TLC and HPLC. Here we suggest Mass Spectrometry analysis of TOF-MS analysis of berberine and TOF-MS analysis of methanolic root extract of Berberis lycium. We expect the authors fully characterize the mass spectra and compare also with the standard berberine.

�We suggest complete silica gel column chromatographic or preparative RP-HPLC purification of EAM37 fraction, get pure berberine and conduct a comprehensive NMR spectroscopic characterization of the pure berberine. This will help for complete characterization and identification of berberine, not close isomers, instead of relying on TLC Rf values and analytical RP-HPLC retention time values across the manuscript.

�Line 530: It is stated that "Previously, researchers docked the berberine with four enzymes including acetylcholinesterase and found that binding affinities of berberine were very close to the our experimental values representing strong interaction of berberine with the enzyme". Here, we suggest the comparison should be done indicating the respective binding affinity values against respective protein targets and then only one can be convinced of the discussion included.

�Binding interaction profile for berberine both polar and non-polar inercations obtained after molecular docking should be presented in Table and different types of interactions shown in different colours should be presented in figure.

�ADMET properties calculated for Berberine and derivatives from SWISSADME online server should be included.

�We also suggest including molecular dynamics simulations of beta-secretase-berberine complex acquired through molecular docking. The authors can use YASARA suite software or other alternative software's to conduct this simulation.

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This study on bioassays guided isolation of berberine from Berberis lycium and its bioactivity role was done following good scientific procedure. The manuscript can be accepted for publication considering few comments listed below.

-The structure of berberine is not drawn in the manuscript. Its structure should be included in the manuscript.

-The authors identified berberine using TLC and HPLC using standards. Why NMR and mass spectroscopy not used? Those method confirms the purity of the compound. It is better to include NMR data.

-In the abstract the code EAM37 should be replaced by ethyl acetate and methanol (3:7) fraction or other term. EAM37 is a technical code assigned by the authors during experiment and should be avoided from the abstract.

Reviewer #2: The title may be modified as Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer’s disease combined with insilico molecular docking. Accept with minor revision and I attached the comment on pdf.

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Reviewer #1: No

Reviewer #2: Yes: Muhdin Aliye

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Attachments

Attachment

Submitted filename: Comments for Manuscript Number PONE.docx

Attachment

Submitted filename: PONE-D-22-35294.pdf

PONE-D-22-35294R1Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer’s disease combined with insilico molecular dockingPLOS ONE

Dear Dr. Ismail,

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.

Be sure to include:

• Detailed experimental conditions and methods followed in isolation of berberine using preparative HPLC (not analytical HPLC) included in Materials and Methods section
• Line 462-468, please remove the spectral data presented in paragraph form and I strongly recommend presenting the NMR data of berberin in Table form along with literature (reference 46) spectral data within the table for comparison.

Please submit your revised manuscript by May 27 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Milkyas Endale, PhD

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.

Additional Editor Comments:

Improvements made

Title revised as per editors comment

Binding affinity values of berberine and rivastigmine to beta secretase are now included in the abstract.

Berberine obtained from fraction EAM37 is now fully characterized by FT-IR and NMR (Page 21 and 22).

Molecular docking study of berberine over β-secretase was included.

ADMET analysis for berberine and Rivastigmine is now included (authors used SWISSADME online server).

Molecular dynamic simulation was included using Gromacs under Linux system where CHARMM36 force field Jul2021 version from MacKerell Lab was used.

Major revisions required

Page 10, line 215-226, the detection of fraction EAM37 to be similar as berberine was checked by using analytical HPLC (comparing Rt with the reference sample). It is good that the authors compared EAM37 with a reference sample. However, I suggest detailed experimental conditions and methods followed in isolation of berberine using preparative HPLC (not analytical HPLC) should be included here. It is a must to include the quantitative isolation of the compound in materials and methods. I also recommend including the subtitle "HPLC isolation of Berberine" in the methods section.

Page 16: line 361: subtitle" Fraction EAM37 was isolated as yellow precipitates" should be revised as "Isolation of berberine from roots of Berberis lycium”.

Line 462-468, please remove the spectral data presented in paragraph form and I strongly recommend presenting the NMR data of berberin in Table form. Within the same table, literature (reference 46) spectral data of the same compound should also be included and compared.

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[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.

Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer’s disease combined with insilico molecular docking

PONE-D-22-35294R2

Dear Dr. Ismail,

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.

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Kind regards,

Milkyas Endale, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors did several improvments given during the second revision. The major improvments include:

1. Detailed experimental conditions and methods followed in isolation of berberine using preparative HPLC included.

2. Page 16: line 361: subtitle"Fraction EAM37 was isolated as yellow precipitates" is now revised as "Isolation of berberine from roots of Berberis lycium”.

3. Line 462-468, spectral data is presented earlier in paragraph form is now presented within Table and chemical shifts are compared with literature data.

However, within Table 3, please replace the word "reported" by "reference 46 or [46]".

Reviewers' comments: