Biotechnological Intervention and Withanolide Production in Withania coagulans

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Dear authors,

the review is quite interesting but there are many problems related to English, careless mistakes, repetitions. Please review following the comments below.

Overall, sentences and comments that seem more suitable for discussion and conclusion are spread in other sections like introduction, ethnobotany, results etc. Please revise

Write in vitro in italics, check throughout the text.

 

Abstract

- “The plant is a subshrub that is indigenous to the Mediterranean and some part of South West Asia”. It is not a Mediterranean species, as also indicated in the Figure. Also change it in the Introduction.

-“Its biological ingredients, which are connected to human health are what give it its therapeutic effects and used in conventional medicine system to treat different disease including neurological problems, diabetes,asthma”. Please change this sentence in a clearer and linear one

-Invert the sentence starting with “Multi-omics…” with the last one

Introduction

-When citing again the name of a species and relatives, use the punctate version of the genus (in the body of the text, not at the start of a sentence or paragraph). Therefore, in example: “These two species are known as Withania coagulans, and W. somnifera….”

- “Both species contain valuable metabolites known as "withanolides (WTDs)" whose therapeutic potential has been extensively researched”. Remove the quotes from withanolides, and add the references (since as you said they are extensively present).

-“As the demand for natural remedies increases, it is possible that additional Withania species with medicinal properties will be discovered.” This sentence could be suitable for discussion or conclusion. By the way, at the end of this sentence please use the following one as a start for a new paragraph

-“These techniques can also increase the production of secondary metabolites that have medicinal properties, which can meet the increasing demand for natural remedies.” This sentence repeats the previous ones

-“The proposed review offers novel aspects that make it essential reading for scientists in the field.” Hopefully, but keeping a humbler tone is way better. Please edit.

-Lines 90-97: as above. Please revise.

 

Ethnobotanical and phytochemical profile

-line 99: Use “Withania coagulans” (full version at the beginning of a sentence or a paragraph)

-lines 100-101. 1) subject is missing 2) 150cm wide means that the leaves are larger than the height of the plant. Please revise and standardize units of measure by converting to proportion, not just changing “mm” to “cm”. The same for the flowers.

-Lines 108-111. Please remove this sentence from here, it is more suitable for discussion or conclusion.

 

Table 1.

-If you're writing about ethnobotanical uses, it's different than citing molecules. So document 13 is reported improperly (maybe more than once); the same seems to be for paper 17, and for withaferin (final row)

-Lines 114-117: change in “Due to the widespread usage of W. coagulans in classical medicine and the subsequent commercialization in modern medicine, numerous studies have been conducted to identify, isolate and characterize various phytochemicals from different parts of the plant: among these, steroid lactones, tannins, flavonoids, terpenoids, iridoids, alkaloids and others have been found [22, 24, 25, 26]”

Figure 2: write correctly “Toxicity” and “Respiratory problems”

3. Biotechnological intervention for the conservation and withanolide production

This section is too long with information which is unessential for the review’s subject itself. Concepts are repeated many times. Please deeply revise this section and try to be more concise, avoiding repeating the same things that have been reported previously.

A resume of the main biotechnological approaches is fine, but in this case is too long and generic: it is not a review on the biotechnological methods, but a review on biotechnological interventions for W. coagulans, right? Therefore, write these parts with more emphasis on W. coagulans related biotechnologies.

For instance: lines 302-321 are fine. Only the final part of the paragraph sounds more appropriate for the conclusion section.

i.e.: delete from line 141 to 146 until “…many parts of the world”.

-Lines 182-205 are fine, but please specify also here (first part) if this has been established on W. coagulans or it is a general consideration.

Tables: please revise unifying the verbs. Sometimes the past is used, sometimes the present. Also, revise the singular and plurals, etc.

-Table 4, Nodes, ref 61: change in “Plants had a 75% survival rate after acclimatization”

-Table 4, shoot tip: change in “In every population, acclimatization and survival were observed”

-Table 4, Nodes ref 56: “After 15 days, 67.3% of microshoots start to root. 95% of cultures harden plantlets, and 90% of acclimated plants make it to the field.” This is written in a confusing way, please revise

5. Thin cell layer (TCL) culture and elicitation, (and following)

This should be edited as a subsection of the previous one, so: “4.1. Thin cell layer (TCL) culture and elicitation”. The same for the following sections 6 and 7

 

6. Cell suspension and root culture

Line 490: change in “….tiny clumps of cells in a liquid medium” Media is plural

Line 533: change in “Most medicinal plants are known for accumulating secondary metabolites in their roots”

Line 538: “A.tumefaciens strain C58C1 (pRiA4) was used to inoculate with the leaf section of W. coagulans leaves [38]. This made the roots change so that they could make withanolide A and withaferin A, which are the two main bioactive parts of Withania species” rewrite this sentence

Section 7. Line 601: “As a result of this metabolic study, located and cloned a full-length cDNA sequence of TDC from aerial tissue of Withania coagulans” please rewrite

Section 8: limit the use of “however”

see above

Author Response

Please see the attachment.

 

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

The manuscript with the title “Biotechnological intervention and withanolide production in Withania coagulans” is a a review that gathers a significant number of papers on the topic and has good potential to become a reference in the field. However, the manuscript could be improved. Below are some comments.


In the Introduction section, the justification of the review cannot be given simply by saying “this review would be useful to the specialists” (e.g. Line 95). Readers will decide if the review is useful. Instead identify your potential target audience and address their challenges: scientists, farmers, biotech enterprises etc.

At the end of the introduction Lines 87-97 cannot be considered an appropriate paragraph expressing the aim and objectives for this review. Please give an Aim and a few Objectives. Then, consider that your Chapters and Conclusions section should mirror the objectives expressed. Otherwise how can we know the authors reached their objectives for this review if they do not express them?

I suggest to avoid repeating ideas in different paragraphs and instead group the information by categories. Once an idea has been exhaustively presented, move on to the next. The authors should not return and retake again and again the same point. It is annoying to the reader.

Introduction shall be a justification of a study, considering target audience and the delimited objectives. The botanic details and description shall have their own paragraph – but currently chapters 1 and 2 present scattered information on botany and species distribution. One chapter shall deal with the importance of the species (right now chapter 2 presents both botany and importance). One chapter could be on phytochemistry. Etc…

Please consider this is an agronomy journal and therefore I advise authors to say something about the cultivation potential and germplasm available for this species. Do not omit to underline that natural populations are important gene pools for the breeding programs, and this is why depletion of wild plants populations is problematic for agronomy. Also, breeding could be targeting enhanced phytochemical profiles. Please link your review better to the agronomy topic. Biodiversity and conservation are good but link them better to agronomy issues also.

Please consider that the main message of each chapter shall be easy to identify and do not bury it under redundant text. This would help readers find quickly what interest them.

Best regards.

Grammar inconsistency – e.g. jumping from general remarks to first person pronouns “Biotechnology can play a significant role … we can ensure the sustainable use of this important medicinal plant…”

Author Response

Please see the attachment

 

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The present work aims to discuss the traditional and therapeutic uses, phytochemistry and biotechnology of an endangered species, Withania coagulans. The species is of global interest due to the presence of remarkable compounds possessing various biological activities, being valuable as primary medicinal source for some countries.

I think the overall purpose of this review is really commendable. In this context, in fact, data relating to its conservation status need to be updated and it is important to consider ex situ conservation strategies to decrease the harvest pressure on wild plants.

However, this manuscript has a large part devoted to traditional uses and phytochemistry/pharmacological activities, which have already been extensively covered in other reviews. Therefore, these sections sound “repetitive” if compared to the available literature. If there is any progress from previous reviews, it is not reflected in the way the work is set up now. Some written parts add so little to the cited articles that they practically look like original sentences spun backwards. Sometimes it is difficult to understand which parts or extracts the authors are talking about.

Overall, the manuscript is written in poor English. Sometimes it seems that papers are improperly cited (see comments below).

After all these considerations, I don’t feel entirely confident in suggesting acceptance of the manuscript even after major revisions. Therefore, I’m indicating the reject-resubmit option. So, I would suggest this: authors try to streamline the first parts, and then especially point to the biotechnology section. The tables could also be left as they are, provided that the authors double-check them (there are various errors, incorrect italics, etc.). A review organized in this way falls more properly within the aims of the journal.

I attach here a series of general comments which I hope will assist the authors in rewriting the manuscript.

-Standardize “in vivo” and “in vitro” (should be written in italics).

-Pay attention to the language, both grammatically and semantically.

- You wrote “the most readily available treatment in this system today is traditional medicine”. I guess this is especially true for some countries, but people from other nations (such as European ones) are more interested in buying medicines directly, due to a general lack of knowledge about plants. Could you consider specifying here some of the countries that use traditional medicine as primary source? This problem is also present in other parts of the text.

-in the Introduction, write “Commonly known as Rishyagandha, Indian cheesemaker or paneer-bandh, this species is distributed from North Africa to South Asia”. Indeed, according to GBIF and POWO, this species is not present in the Mediterranean region (https://www.gbif.org/species/3801167).

-You often write withanoloides, or withanaliodes. Please use “withanolides”, standardizing it throughout the text.

- You wrote “Additionally, the lack of a successful breeding process in the wild has brought them to the brink of complete extinction [1].”. I can’t find the article [1] (Pandey et al.). a) Check if this reference is suitable: if so, please add a doi. However, I feel other papers could be more appropriate in this context. b) Please briefly improve this part, highlighting genetic and reproductive biology problems. This could help your discourse on conservation actions.

- Botanical description: you wrote “The leaves are generally lanceolate-oblong (25-75 mm long and 1500 mm broad) and flowers (7-12 cm)”. Please standardize the use of mm or cm

-To me, the seeds don’t seem dark nor spike like, please improve the description.

- change in “is limited due to its long gestation period [10] and unisexual nature of flower”, because the last part is not mentioned in the reference you are quoting.

- When a species’ name appears at the beginning of a sentence, the full version should be used (W. coagulans --> Withania coagulans)

-Phytochemistry: I think this section should be moved before Pharmacological activity. Pay attention to the numbering of the section titles (the “4” is repeated twice).

- in Safety evaluation, change in“However, there are many reports available that reported no toxic effects of the extract nor observed a significant difference in body weight and body temperature between the control and the treated subject [45,41]”. Please note, if you write “many reports”, more than two references are expected. Furthermore, what extracts and what parts are you writing about?

Overall, according to the comments reported above, consider the suitability of these latter sections.

-The part on W. coagulans’ conservation should be improved.

References: as usual required by MDPI, references must be numbered progressively based on the first appearance in the text.

I hope these comments are helpful. Kind regards.

Author Response

Response to the reviewer#1

-Standardize “in vivo” and “in vitro” (should be written in italics)

Explanation: corrected.

-Pay attention to the language, both grammatically and semantically.

Explanation: The MS has been revised thoroughly by using institutional English and grammar editing services. The changes has been highlighted in yellow color.

- You wrote “the most readily available treatment in this system today is traditional medicine”. I guess this is especially true for some countries, but people from other nations (such as European ones) are more interested in buying medicines directly, due to a general lack of knowledge about plants. Could you consider specifying here some of the countries that use traditional medicine as primary source? This problem is also present in other parts of the text.

Explanation: The sentence has been from the introduction part.

-in the Introduction, write “Commonly known as Rishyagandha, Indian cheesemaker or paneer-bandh, this species is distributed from North Africa to South Asia”. Indeed, according to GBIF and POWO, this species is not present in the Mediterranean region (https://www.gbif.org/species/3801167).

Explanation: The sentence has been corrected as … in the east of Mediterranean region and extending to South west part of Asia …[Figure source GBIF].

-You often write withanoloides, or withanaliodes. Please use “withanolides”, standardizing it throughout the text.

Explanation: It has been standardized as “withanolides”.

- You wrote “Additionally, the lack of a successful breeding process in the wild has brought them to the brink of complete extinction [1].”. I can’t find the article [1] (Pandey et al.). a) Check if this reference is suitable: if so, please add a doi. However, I feel other papers could be more appropriate in this context. b) Please briefly improve this part, highlighting genetic and reproductive biology problems. This could help your discourse on conservation actions.

Explanation: As per the suggestions, new information on genetic and reproductive biology has been added to this part. The doi of the paper Pandey et al. has also been added to the reference list

- Botanical description: you wrote “The leaves are generally lanceolate-oblong (25-75 mm long and 1500 mm broad) and flowers (7-12 cm)”. Please standardize the use of mm or cm

Explanation: standardized in cm.

-To me, the seeds don’t seem dark nor spike like, please improve the description

Explanation: The sentence has been corrected. A figure has been added to confirm then color of seed (not for publication)

- change in “is limited due to its long gestation period [10] and unisexual nature of flower”, because the last part is not mentioned in the reference you are quoting.

Explanation: corrected.

- When a species’ name appears at the beginning of a sentence, the full version should be used (W. coagulans --> Withania coagulans)

Explanation: corrected.

-Phytochemistry: I think this section should be moved before Pharmacological activity. Pay attention to the numbering of the section titles (the “4” is repeated twice).

Explanation: The section has been removed and corrected.

- in Safety evaluation, change in“However, there are many reports available that reported no toxic effects of the extract nor observed a significant difference in body weight and body temperature between the control and the treated subject [45,41]”. Please note, if you write “many reports”, more than two references are expected. Furthermore, what extracts and what parts are you writing about

Explanation: the sentence has been corrected as …However, studies are available that did not report any toxic effects of the extract nor observed a significant difference in body weight and body temperature between the control and the treated subject.

-The part on W. coagulans’ conservation should be improved

Explanation: The section has been revised and new information has been added along with figures.

-References: as usual required by MDPI, references must be numbered progressively based on the first appearance in the text.

Explanation: The reference section has been carefully revised and corrected wherever necessary.

 

Additional changes:

- The manuscript was thoroughly revised to improve the English and grammar.

- The reference section has been revised both in the text and in the reference list.

- New references have been added.

- Figures [1-3] have been added to the appropriate sections with their explanations.

- New information in the section on pharmacological activity and safety assessment has been added.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Journal- Agronomy

Manuscript ID: agronomy-2169776

Title-" Traditional and clinical uses, phytochemistry, pharmacology and conservation of Withania coagulans (Stocks) Dunal – A review"

 

1.     What is a GRAS (Generally recognized as safe) status of W. coagulanse? It is essential, and the authors can include this information in the introduction section.

2.     In section 4 "Pharmacological activities" the authors described the pharmacological activities of  W. coagulanse. In related text and Table 3, the authors discussed the activity reported with extracts prepared from different parts of W. coagulanse. It is established that the high medicinal values of W. coagulanse are due to the presence of specific phytochemicals. Here, the authors need to include the pharmacological activities of major active phytochemicals of W. coagulanse. Authors can see some examples of active phytochemicals in this article DOI: 10.1211/jpp.62.02.0001.

3.     What is the recommended concentration of W. coagulanse for human consumption? The Authors need to mention this concentration as per safety regulatory agencies like US-FDA, FAO/WHO and Council of Europe and the United Kingdom, Food Additive and Contaminants Committee etc.

4.     Instead of section 4, the Phytochemistry section should be at number 5.

5.     In the phytochemistry section, authors can give some chemical structures of major phytochemicals of W. coagulanse responsible for biological activities.

6.     W. coagulanse is a high medicinal value plant and is used as an essential ingredient in many preparations of herbal medicine, botanicals, and dietary supplements. At a safety point, the US Food and Drug Administration (FDA) encourages evaluating herb-drug interaction studies of medicinal or dietary important herbs. In section 5, "Safety evaluation," the authors need to add some lines about "Herb-Drug interaction". For this the following articles https://0-doi-org.brum.beds.ac.uk/10.1016/j.jep.2022.115822; Am Fam Physician. 2017;96(2):101-107;  https://0-doi-org.brum.beds.ac.uk/10.1016/j.heliyon.2020.e05357 should be helpful and can be used as references.

7.     Where are the figures?

Author Response

Response to the reviewer#2

1. -What is a GRAS (Generally recognized as safe) status of  coagulanse? It is essential, and the authors can include this information in the introduction section.

Explanation: To the best of my knowledge, information on the GRAS status of Withania coagulans is not clearly available and that is why it has not been added to the introduction part.

2. In section 4 "Pharmacological activities" the authors described the pharmacological activities of   coagulanse. In related text and Table 3, the authors discussed the activity reported with extracts prepared from different parts of W. coagulanse. It is established that the high medicinal values of W. coagulanseare due to the presence of specific phytochemicals. Here, the authors need to include the pharmacological activities of major active phytochemicals of W. coagulanse. Authors can see some examples of active phytochemicals in this article DOI: 10.1211/jpp.62.02.0001.

Explanation:  Information has been added. In addition, the pharmacological activities of major active phytochemicals has been discussed in table 4.

3. What is the recommended concentration of  coagulanse for human consumption?The Authors need to mention this concentration as per safety regulatory agencies like US-FDA, FAO/WHO and Council of Europe and the United Kingdom, Food Additive and Contaminants Committee etc.

Explanation: A detail information on this topic has been added to the heading section safety evaluation. However, information on the recommended concentration of W. coagulans (whole plant) by US-FDA, FAO/WHO is lacking. The other species of Withania, W. somnifera is more studied comparatively to the W. cogulans and experimental based information is available and we added to the MS. In addition, the available reports [doi: 10.1002/ptr.4854, https://0-doi-org.brum.beds.ac.uk/10.1016/j.tiv.2012.09.004, DOI: 10.1211/jpp.62.02.0001] and US based certified physician blog/websites [Which Ashwagandha Dosage Is Right for You? - Dr. Axe (draxe.com),  What are withanolides, and can they improve your health? | NutriRise], also gives us the information on the phytochemicals specially withanolides.

“Most experts recommend starting with a dose of about 300 to 500 milligrams per day of ashwagandha extract, with withanolides in the range of 5 percent to 10 percent. A full dose of ashwagandha would be between 1,000–1,500 milligrams per day of extract.” 

The above information has been taken from the website [Which Ashwagandha Dosage Is Right for You? - Dr. Axe (draxe.com)], however, the approval from US-FDA, FAO/WHO and Council of Europe and the United Kingdom, Food Additive and Contaminants Committee etc. is a matter of discussion.

Another sources claim that Aswaghandha is not FDA approved or regulated [https://www.trustednutrients.com/blogs/health-tips/your-guide-to-ashwagandha-frequently-asked-questions#:~:text=John's%20wort%2C%20and%20more.,FDA%20approved%2C%20or%20FDA%20regulated.].

Overall, a direct information on approved dose of W. cogulans is lacking.

4. Instead of section 4, the Phytochemistry section should be at number 5.

Explanation: corrected

5. -In the phytochemistry section, authors can give some chemical structures of major phytochemicals of  coagulanseresponsible for biological activities.

Explanation: I have already added the figures as figure number 1 in this section, which contains the chemical structure of the 10 most important phytochemicals.

6. coagulanseis a high medicinal value plant and is used as an essential ingredient in many preparations of herbal medicine, botanicals, and dietary supplements. At a safety point, the US Food and Drug Administration (FDA) encourages evaluating herb-drug interaction studies of medicinal or dietary important herbs. In section 5, "Safety evaluation," the authors need to add some lines about "Herb-Drug interaction". For this the following articles https://0-doi-org.brum.beds.ac.uk/10.1016/j.jep.2022.115822; Am Fam Physician. 2017;96(2):101-107;  https://0-doi-org.brum.beds.ac.uk/10.1016/j.heliyon.2020.e05357 should be helpful and can be used as references.

Explanation: The information on Herb-Drug interaction has been added.

7. -Where are the figures?

Explanation: I had successfully uploaded the figures when I submitted my manuscript. I do not know why the figures were not included in the MS for review. I may have uploaded them as a separate file.

Now I have included them in the revised file MS.

 

Additional changes:

- The manuscript was thoroughly revised to improve the English and grammar.

- The reference section has been revised both in the text and in the reference list.

- New references have been added.

- Figures [1-3] have been added to the appropriate sections with their explanations.

- New information in the section on pharmacological activity and safety assessment has been added.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I have already seen the edited version of the article and appreciate the efforts the authors have made to improve the manuscript. I really think the work is important in some parts.

However, as I already wrote in the first review report, the entire first part (which is substantial) up to the biotechnology section is a sort of repetition of articles that have already been published. This observation of mine has not received any explanation from the authors in the review round. So, for me the problem remains the same.

Author Response

Dear Sir/Madam

Thank you for reviewing my manuscript, your valuable comments help to improve the quality of the manuscript.

Regarding your comment, I must inform you that MS is not a repeat of previously published articles. Much updated information has been included in the section on medicinal uses, phytochemistry, and pharmacological activity. We have presented much of the data in tabular form using unique methods and different from available publications.

On the other hand, I would like to point out that the previous article did not include information about the safety assessment of W. coagulans. Our current article addresses the toxicity and scope of W. coagulans for drug analysis, which again is a new addition to this article.

In addition, the section on conservation of W. coagulans includes almost all developed tissue culture protocols, including recently developed protocols. In addition, we also discussed micropropagation and synthetic seed development protocol (Figure 3) developed by our research group. The photograph is an original work and is not shown anywhere else. Such a type of presentation has not been given in any previously published review paper.

We have provided data on explant sterilization, nutrient composition, and culture conditions for direct and indirect organogenesis in the text and key data in tables. Thus, our report differs from other published reports on W. cogulans.

 

I hope the above will be helpful in deciding on my manuscript. Once again, I thank the reviewers and editors.

Thank you

Zishan

Author Response File: Author Response.docx

Reviewer 2 Report

Journal- Agronomy (ISSN 2073-4395)

Manuscript ID: agronomy-2169776

Title-"Traditional and clinical uses, phytochemistry, pharmacology and conservation of Withania coagulans (Stocks) Dunal-A review"

 

Reviewer comments

Dear Editor,

The authors have fulfilled all the queries/comments as it was asked previously. Now the manuscript is well written. I believe that it is a nice piece of work for being published in the Agronomy. Finally, I recommend that the paper be accepted for publication in its present form.

 

Decision- Accept 

Author Response

Dear Sir/Madam

Thank you for reviewing my manuscript, your valuable comments help to improve the quality of the manuscript.

 

Thank you

Zishan