PONE-D-24-04375Zeaxanthin dipalmitate-enriched wolfberry extract improves vision in a mouse model of photoreceptor degenerationPLOS ONE

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

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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

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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 goal of the authors of the manuscript is to describe a new way to produce extract from Wolfberry containing higher amount of Zeaxanthin dipalmitate (ZD) and to test the effects of this extract (ZDE) in an in vivo model of retinal degeneration induced by intraperitoneaal injection of N-methyl-N-nitrosourea in C57BL6 mice.

first the =y evaluate the quantity and purity of ZD in their extract produced by this new technique and then they evaluate the effect of oraal administration fro 14 days of ZDE on retinal structure, photoreceptor survival, ERG response of the retina submited to several type of stimuli (at various frequencies and/or intensities) and then the evaluate the visulaa function in the whole animal using two different tests in addition they provide certain proofs of context showing the sfatey of the doses of the ZDE used in the supplemental figures.

in general although simple the paper is clear, the methods used classic but pertinent to answer the questions raised and the data tends to support the claims.

the methods used and the statistical analysis seems accurate as well.

i have only minor comments to improve the manuscript:

1 figure Suplementary S2 the file is Corrrupted and cannot be read appropriately and need to be replaced.

the abstract and the intro needs to be checked for english as some sentence may be awkward:

line 35 this sentence needs to rephrased :The adult C57BL/6 mice were treated with ZDE or solvent by daily gavage for 2 weeks, during the middle time (?) the animals were intraperitoneally injected with N-methyl-N-nitrosourea to induce photoreceptor degeneration.

intro line 58: While these products ... needs to be replaced by while this strategy using gene therapy...

In the methods line 106 to 108 the sentence needs to be reformulated for more clarity

line 141 give the figures for the 9mg/kg body weight optimal treatment concentration

in the discussion line 326 : add imporved the survival (of photoreceptors)

in the discussion they say that LgBP is recognized as the main antioxidant molecule in wolfberry. What is the concentration of LbGP in their ZDE ? the authors needs to discuss at the least the others compound present or potentially present in their extract. any HPLC data on this?

i wanted them to discuss why they have no effect on their dark and light bi=ox test evaluating the ability of mice to see difference of luminance while their experiments show an effect on scotopic vision.

Apparently this test is not sensitive enough and does not allow to see a difference between normal mice an mice with retinal degenration. therefore it must be discussed and if it is the case this test should be discarded from the article.

Reviewer #2: Chen et al examined the impact of zeaxanthin dipalmitate (ZD)-enriched wolfberry extract on photoreceptor health in mice with N-methyl-N-nitrosourea-induced retinal degeneration. This is a continuous study from their previous work which has shown one shot of intravitreal ZD (95% purity) rescued photoreceptors in rd10 mice. Overall, the current work is well-designed and the manuscript is well-written. There are only several minor comments to address.

1. Fig 5B: statistics analysis shall be shown and “not significant” shall be labeled on the graph if no statistical significance was found. The statement at line 306-308 shall be toned down if there was no significance.

2. Supplement Figure 2 was too small to see. The authors please correct. The graph shall be exported in JPEG or Tiff. Same as S. Figure 1.

3. Figure legends were embedded in the main text. It shall be moved after references.

4. Line 336, please create the reference including authors, title, journal and publication date for the article under press.

Reviewer #3: Chen et. al. used the their new developed method enriched ZD from wolfberry and test the efficacy of ZDE in MNU-injured retina. The enriched ZDE showed 9 times higher conc. than that in wolfberry. And authors conducted dose response to optimize the dose selection. By daily pretreating mice before MNU injury and until the end of study with ZDE, they demonstrated the neuroprotective effects on MNU-injured retina. It is well-designed study. Below please find my TWO additional comments, to make it better.

1. In the referred previous study by authors' group on ZD, they performed intravitreal injection but not oral treatment. It is understandable that oral given will need high dose, but the metabolic pathway is different. The active pathway thereby could be different with previous study. Can authors add one paragraph in the discussion part?Also, did authors test if ZD reached to the retina post oral treatment? And how much? The molecular weight of ZD is over 1000. Would it affect ZD penetrate the BRB if give orally?

2. How about the purity of ZDE? There are some small peaks in Fig.2C.

3. In supplementary data, authors presented that oral given ZDE has no influence on retinal functions. But it is a short-term toxicity test. In clinic, RP has chronic progression, which is different with RD10 or MNU-injured model. It may need long-term drug given. Have the authors tested the long-term toxicity or side effects by daily oral given?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Zeaxanthin dipalmitate-enriched wolfberry extract improves vision in a mouse model of photoreceptor degeneration

PONE-D-24-04375R1

Dear Dr. Xu,

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.

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Kind regards,

Kin-Sang Cho, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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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

Reviewer #3: 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: No specific comments the authors responded satifyingly to the comments of all three reviewers comments.

Reviewer #2: The authors have addressed my concerns in the current revised manuscript. I have no additional comments.

Reviewer #3: Authors have addressed my concerns and discussed in the discussion part. I would suggest to accept.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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