PONE-D-22-33541Extraction, phytochemical characterization and anti-cancer mechanism of Haritaki Churna: An ayurvedic formulation.PLOS ONE

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PLOS 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: Partly

Reviewer #4: Yes

Reviewer #5: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: 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: No

Reviewer #4: Yes

Reviewer #5: 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: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

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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 authors could improve the overall language of this manuscript to increase readability.

>Introduction: The introduction section of this study fails to relate with the previous study accurately. Rewrite this section and relate why this study is novel and its importance.

>Conclusion: This section is not properly written. You are suggested to write the major findings and its significance in few sentences.

>Some figures are blurry. Include figures with correct resolution.

Reviewer #2: I read with interest the paper and I believe that authors have taken into account a topic worthy to be investigated.

A good match between in vitro investigation and chemical characterization is clearly highlighted.

In my review I want to underline the strenghts, but I need to raise some fundamental limits of the work.

Strenghts:

I appreciate how authors planned the work, aiming first to provide with a simple but well performed protocol that was able to obtain some practical (and thus sounding) preclinical results.

Indeed the fact that authors studied the actual preparation used in ayurveda (i.e. maybe water is not the best solvent to extract Terminalia, but it is the solvent that practioners use), they considered the gastric stability, the effectiveness of phytocomplex, fractions and single compounds and tested samples in cancer and normal cell lines are all together basic good experimental practices in phytotherapy, too often underrated.

Limits:

I have to admit that this work opened many different interesting scientific questions, but many things remain unsolved.

The chemical characterization has been done but, due to the lack of reference standards, the quantification of ellagic acid as well other effective compounds is missing.

Authors should have completed the gastrointestonal stability simulation, by adding the simulation of the activity of bile salts and pancreatin at pH 6.8-7.2.

Finally, molecular mechanism were not investigated.

Please check carefully spelling errors.

Reviewer #3: The pictures in the article are not clear and the pixels do not meet the publication requirements.

The purity of the compound is not high.

Neither the fraction nor the compound activity is very strong.

Reviewer #4: This manuscript reports the extraction, phytochemical characterization, and anti-cancer mechanism of Haritaki Churna. The anti-cancer activity, safety, toxicity, stability analysis of Haritaki churna was tested, and the active ingredient significantly contributed into anti-cancer effect of HCAE was isolated and improved to played activity by degrading the genomic DNA and inducing the cellular viability,The overall logic of the paper is clear.Personally, the manuscript might require little revision prior to be considered to be acceptable or not due to that:

1. In figure 4, the retention time of B, and D need to be increased for easy observation about all compounds in the condition.

2. In figure 5, the structure of the compound is recommended to be beautified in the same format.

3. The accuracy of the mass spectrometry data in the article needs to be improved to four decimal places, and needs to be consistent, such as line 338 and 341.

4. In 3.5, please add the amount of each fraction separated.

Reviewer #5: The current study reported that the ayurvedic formulation, Haritaki churna (HC), has anti-cancer activity in different cancer cell lines and potential mechanisms. The authors checked the bioavailability of HC and it’s activity against different cancer cell lines. They further explored the active pharmaceutical ingredient in HC, tested the contributions of those phytochemicals, and found that Ellagic acid significantly contributes to the anti-cancer effect of HCAE. However, the present form can’t be accepted for publication.

1. The manuscript needs to be polished the English grammar or spelling by a native speaker.

Inconsistent hyphenation throughout the manuscript. Like anti-cancer vs anticancer, gastro-intestinal vs gastrointestinal, etc. Both ways are acceptable, but you have to keep consistent.

American English and British English are mixed used.

Grammar and punctuation mistakes everywhere

2. The last decade has seen remarkable progress in cancer research, with a significant update to many theories. But most of the citations here have been published for more than ten years.

The format of references is seriously inconsistent. For instance, ref 37 is in a different form from ref 36.

3. All experiment data need to have supported tables or figures, for section 3.1, no tables or figures are cited.

4. Section 3.3, the authors stated, “HC aqueous extract had not affected the cellular viability of HEK-293 cells up to concentration of 300 µg/ml. In addition, it is not affecting the cellular morphology as well (Figure 2B).”.

However, we can see significant changes in morphology in HEK293 in Figure 2B. And there is no discussion for these changes.

5. Figure 5, since there are different subfigures, please cite them separately according to the results.

6. Section 3.9, “This increase in anticancer activity may be attributed to their antagonistic action. Their antagonistic action may be (a) because of these phytochemicals competing for the same protein which is crucial in pathway of killing cancer cells or (b) may be because of formation of a complex polyphenol mixture which are unable to perform their individual activities. “

References to support this discussion are missing.

7. Section 3.10. Three independent repeated experiments are needed to get the mean ± SD/ SEM and reliable results. While the results here seem only come from a one-off experiment.

8. Current data is not strong enough to make the conclusion that “ellagic acid is capable of arresting cell cycle progression in S-phase and induction of apoptosis in colorectal cancer cells”

More experiment such as Western Bloting to check the cell cyle stage-dependant makers is needed, such as Cyclin A, Cyclin B…

The same for apoptosis, more experimental evidence is needed.

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Reviewer #1: No

Reviewer #2: Yes: Marco Biagi

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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Extraction, phytochemical characterization and anti-cancer mechanism of Haritaki Churna: An ayurvedic formulation.

PONE-D-22-33541R1

Dear Dr. Trivedi,

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,

Chun-Hua Wang

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 #5: 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 #5: Yes

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

Reviewer #1: Yes

Reviewer #5: Yes

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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 #5: 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 #5: Yes

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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 Response)

Reviewer #5: (No Response)

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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 #5: No

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