The Multifaceted Therapeutic Potential of Saffron: An Overview Based on Research and Patents

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have revised the original manuscript based on the comments of two reviewers (of which I was one) and resubmitted a new manuscript. I have read the revised version and taken notice of the rebuttal of the authors to both reviews of the first version.

 

My conclusion is that the changes made are mostly good improvements but that there still is ample room for further improvements. Actually, further adaptations are needed. I hope that my suggestions below are helpful for the authors to bring the manuscript on a higher quality level and I advise the authors to more critically look at their own work.

 

The title has improved. However, in the title there is ‘anti-cancer activities’, in the abstract I read ‘cytotoxic and antitumoral properties’, and in the further text these terms are not always used consistently. In my opinion, anti-tumor and especially anti-cancer, may be too suggestive. This is underpinned by the authors in their response to comment no. 4 of reviewer 2. Please check again carefully and critically, and make this consistent throughout the entire manuscript. I suggest to consequently used ‘cytotoxicity’ for in vitro work and ‘anti-tumor’ for in vivo work. As the authors state, clinical trials are missing so far. This limitation should be made very clear in text; it is an important message and shows the bounderies of current research on the topic. There is still a long way to go before patients with cancer will be treated with saffron (preparations).

 

The abstract overall is really much better that the first version.

 

The introduction starts with a long focus on cancer and cytotoxic properties of saffron. A few sentences (line 50-51) about anti-oxidant and anti-inflammatory properties seems a bit ‘glued’ to the previous paragraphs. The title however starts with these biological activities. I think that this is the place where prevention should be mentioned and elaborated.

 

Line 85-90: The aim can be formulated clearer. Not the importance is underscored (that might be a conclusion), but existing literature is reviewed followed by patents.  

 

If I understand well, the only relevant focus is on stigmas of Crocus sativus. The plant part used should be better and clearer described in the Introduction, Lines 53-65 give information that distracts a bit from the main topic. Reformulate this part. Start with the plant as such, then go to the medicinally interesting part. The chemical composition of the whole plant can be left out. Focus on the stigmas, saffron, and its major constituents.

 

Figure and its legend should be revised. The figure shows compounds like protocrotin, which are not further discussed or mentioned (see also lines 101-102). The figure also contains ‘GeOH’ and ‘GIOH’ of which the meaning remains unclear. Figure has been taken from a reference but there the context was different. If you state (underpinned with references) that zeaxanthin yields safranal, crocetin, etc. (decomposition; what happens), that would we enough because the latter compounds are apparent markers for biological activity of saffran.

 

Resources and Methods: please add an appropriate reference for PRISMA. Which period was used for the literature review? Add exclusion criteria used (see also figure 3).

 

In the text about the biological activities now many values are mentioned. First of all, in most cases a unit lack which is a huge omission. Then, the values as such don’t say that much. It is the comparison like ethanolic versus aqueous extracts (see 3.1). Mentioning the solvents / extraction fluids is essential. Also, the assays should be specified as well as the composition or main compounds of the extracts. Maybe the latter is not given in the references, but mention that limitation. You may use a table.

 

Section 3.2: ‘many different models’ (line 130) is vague. Also here, provide more detailed info in the form of tables and mention limitations. Line 141: What is ‘a larger selective ratio’?

Line 145-146: I do not understand this conclusion. Probably a sentence taken from a paper without further context. 

 

Section 3.3: similar comments and suggestions as above. Line 164-165: a reference is missing.

 

Section 3.4: line 167, what kind of extract? Line 172, another extract. This is too vague. Values presented as such do not say much. A reference or comparison is needed. 

 

Table 1 mentions refs 44-46 but they are not elaborated in the text above the table. Make explicit in the legend of the table that the data concern in vivo work with experimental animals. 

 

Section 4.1: refer to the figure with the PRISMA diagrams.

 

Many patents seem to come from China. Strange that table 2 stops in 2012 while in the latter years many more patents were filled.

 

Line 245: what do you mean with ‘plant or animal origin’? Especially the word ‘animal’ raises questions to me.

 

The presentation and discussion of the patent part should be better structured. Is it chronologically done? Or maybe better combine comparable patents? This part has to be rewritten to give it a better structure and to make its relevance better understandable for the reader.

 

I absolutely miss a critical evaluation of the literature results, a ‘helicopter overview’. The Conclusion part is very positive, while I think there are serious flaws and limitations. Are we still far away from clinical application, or do the authors envisage a bright future for cliicla use of saffron?

 

My conclusion is that the manuscript underwent improvements but that there is still plenty of room for further improvements. This should be done to make the work acceptable for publication. The topic as such is interesting enough. 

Comments on the Quality of English Language

Some minor improvemnets will be needed (but I am not a native English speaker).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I reviewed the article entitled "Antioxidant, Anti-Inflammatory, and Anti-Cancer Activities of Active Phytochemicals of Saffron: An Overview Based on Research and Patents", written by Yahya Ramadan Elfardi, Reda El Boukhari, Ahmed Fatimi, and Latifa Bouissane.

The purpose of the paper is to describe the antioxidant, anti-inflammatory, and anticancer properties of saffron and its main constituents. In addition, the authors provide useful information regarding the evolution of patents related to cytotoxic or anticancer applications of saffron, while also highlighting their limitations.

The scientific collection is very interesting. However, some problems as indicated below should be solved before the paper can be considered for publication in this journal. This version of the manuscript is not complete. Below, I present my objections/suggestions in detail.

 

English and style

·       English language and style are exhaustive.

 

General remarks:

·       I suggest including a table describing studies on the antioxidant, anti-inflammatory, and cytotoxic activity of saffron, as done for anti-cancer activity.

·       In addition to crocin, crocetin, and safranal, saffron possesses other phytochemical compounds (e.g., kaempferol). What is the contribution of these molecules on anti-inflammatory, antioxidant, cytotoxic, and anticancer activity?

 

Abstract:

·       The abstract is well-written and exhaustive in all its parts, I have no suggestions.

 

Introduction:

·       The introduction is very interesting, but it should be implemented.

 

-       Line 51: Regarding scientific studies on antioxidant and anti-inflammatory properties of natural compounds, I suggest the authors add references belonging to other research groups. Therefore, I encourage the authors to include the following references: (doi= 10.1186/s12906-017-2070-z; doi= 10.3389/fphys.2023.1225552; doi= 10.3390/molecules28010026).

-       Line 67: (a) and (b) should be modified in (A) and (B), respectively.

-       Line 80: Could the phytochemical composition of saffron differ depending on where it is grown or the season?

 

Resources and methods:

·       The resources and methods are complete, I have only a suggestion:

 

-       Line 106: (a) and (b) should be modified in (A) and (B), respectively.

 

Review of saffron’s main biological activities:

·       This section is very interesting, but it should be implemented.

 

-       Line 108: The antioxidant activity of saffron is provided by the ability of its components to counteract several reactive oxygen species, such as superoxide anion, hydroxyl radical, peroxyl, and alkoxyl radicals (doi:10.3390/nu14030597). It might be interesting to further investigate this aspect.

-        

Patents on saffron to prevent and treat cancer:

·       In my opinion, this section is very interesting and has a great scientific resonance. I especially appreciated the description of the limitations of the patents provided so far. I have no suggestions.

 

Observations:

·       This section is well-written and exhaustive in all its parts, I have no suggestions.

 

Conclusion:

·       The conclusion is complete, I have no suggestions.

 

References

 

·       The references of this work are quite recent and adequate.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Dear Editor,

The MS seems to be suitable for publication in DDC du to its citation potential, however, it needs English editing. 

Comments on the Quality of English Language

Dear Editor,

The MS seems to be suitable for publication in DDC du to its citation potential, however, it needs English editing. 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors prepared a modified manuscript. They explain a lot in the rebuttal, but the changes made in the manuscript are only minor. For instance, comments 15, 16, 17, 18 did not lead to adaptations.

Furthermore, the words 'cytotoxicity', 'anti-tumor', 'antitumoral' are still used inconsistently.

The connection between antioxidant and anti-inflammatory activity on the one side and cytotoxicity on the other side is still not made in the introduction. This means that the rationale of the work is not properly defined. Please use lines 118-119 for your introduction and work that out.

It remains unclear on most places what the focus is: the whole plant or stigmas. I guess the last, but then the entire list of constituents of the whole plant (lines 66-74) is redundant unless placed in a different context. 

Figure 2 is not explained. It contains a lot of detailed information not given attention to (like GIOH, GeOH) as I mentioned in my previous review.

I don't understand what the statement 'the of IC50 is always given without unit' is based on. They are concentrations and do have a unit (compare with LD50 in text).

A critical view still lacks.

My conclusion is that the current manuscript still needs major modifications and improvements. 

Comments on the Quality of English Language

I am not a native speaker, but the language / grammar needs to be improved on various places.

Author Response

Comment No. 1. The authors prepared a modified manuscript. They explain a lot in the rebuttal, but the changes made in the manuscript are only minor. For instance, comments 15, 16, 17, 18 did not lead to adaptations.

Response 1: Thank you for your feedback and insightful comments. We acknowledge the importance of providing a critical evaluation of the literature results and appreciate your suggestion.

Old Comment No. 1.15. Many patents seem to come from China. Strange that table 2 stops in 2012 while in the latter years many more patents were filled.

New Response 1.15: Thank you. Depending on the jurisdiction, and particularly its publication policy, various types of patent documents are published at different stages throughout the lifecycle of a patent application. For this study on saffron, we focus exclusively on patent applications (depicted in Figure 4, green) and granted patents (depicted in Figure 4, blue). In most patenting authorities, patent applications are first published 18 months after their priority or filing date, regardless of whether they have been granted. However, some jurisdictions only publish granted patents. In such cases, pending applications may not be made public until the grant is published and may remain unknown if the application fails during examination or is withdrawn. In this study, China emerges as the leader in patenting, accounting for approximately 60% of the patents. Through our research in this domain, we identified 271 patent applications and 25 granted patents. Only 6 granted patents were deemed relevant to the traditional Chinese pharmacopeia. Furthermore, we offer an explanation for the cessation of patent publication dates in 2012, attributing it to the matching score utilized in Elastic search. The optimal approach would have been to analyze and summarize the 25 granted patents by 2023. However, incorporating such detailed information into this paper dedicated to research papers and patents would risk overwhelming the reader and diluting the focus. Therefore, a separate future study solely dedicated to patents, taking into consideration the insights from this report, would be advisable for a more coherent and comprehensive examination of patent-related topics. (Lines 481-501)

Old Comment No. 1.16. Line 245: what do you mean with ‘plant or animal origin’? Especially the word ‘animal’ raises questions to me.

New Response 1.16: Thank you. The ‘plant or animal origin’ refers to the origin of the Chinese medicinal materials used for the patented formulation. e.g., phellodendron bark, glossy ganoderma, saffron for the plant origin, and viper or scorpion venom for the animal origin raw materials. To avoid any confusion for readers, the terms ‘plant or animal origin’ have been removed. (Lines 284-288)

Old Comment No. 1.17. The presentation and discussion of the patent part should be better structured. Is it chronologically done? Or maybe better combine comparable patents? This part has to be rewritten to give it a better structure and to make its relevance better understandable for the reader.

New Response 1.17: Thank you. As already described in Sub-Section 4.2, the first group of patents concerns the use of stigmas of saffron as a component of anti-tumor remedies based on traditional Chinese pharmacopeia. The second group of patents studied concerns the use of one or more major’ biomolecules from saffron for the formulation of anti-tumor remedies. Patents inspired by traditional Chinese medicine have some common points. Following your previous suggestion, the proposed structure uses the two methods. Patents based on traditional Chinese know-how were grouped to permit the comparison. While the rest of the patents were presented in chronological order. (Lines 274-429)

Old Comment No. 1.18. I absolutely miss a critical evaluation of the literature results, a ‘helicopter overview’. The Conclusion part is very positive, while I think there are serious flaws and limitations. Are we still far away from clinical application, or do the authors envisage a bright future for cliicla use of saffron?

My conclusion is that the manuscript underwent improvements but that there is still plenty of room for further improvements. This should be done to make the work acceptable for publication. The topic as such is interesting enough. 

New Response 1.18: Thank you. In response to your concerns, we have carefully reviewed our manuscript and incorporated a thorough assessment of the limitations and potential flaws into our conclusion. We recognize that while our study sheds light on certain aspects of saffron from a patent perspective, there are still significant challenges and uncertainties regarding its clinical application. Saffron has been well documented and studied, with numerous research works and reviews published. In the Observations section, we conducted a critical evaluation of the literature results. Additionally, our approach in this manuscript was to examine saffron from a patent point of view, aiming to avoid the repetitive style adopted in existing works and to provide new insights into less-studied aspects of this plant. To address your comments and provide a comprehensive overview, we have added a new section titled "Future Trends and Implications" to the manuscript. This section discusses the current state of saffron research, highlighting both its potential and the obstacles that remain before it can be widely adopted in clinical settings. We hope these additions enhance the manuscript’s critical perspective and provide a balanced view of the future of saffron in medical applications. We agree that while the topic is indeed interesting and holds promise, further improvements and extensive research are necessary to make the work acceptable for publication and to advance our understanding of saffron’s clinical potential. (Lines 452-475)

Comment No. 2. Furthermore, the words ‘cytotoxicity’, ‘anti-tumor’, ‘antitumoral’ are still used inconsistently.

Response 2: Thank you. We have carefully reviewed our manuscript and improved on this point. (Lines 13, 16, 23, etc.)

Comment No. 3. The connection between antioxidant and anti-inflammatory activity on the one side and cytotoxicity on the other side is still not made in the introduction. This means that the rationale of the work is not properly defined. Please use lines 118-119 for your introduction and work that out.

Response 3: Thank you for your feedback and insightful comments. We have carefully reviewed our manuscript and improved on this point. (Lines 41-57)

Comment No. 4. It remains unclear on most places what the focus is: the whole plant or stigmas. I guess the last, but then the entire list of constituents of the whole plant (lines 66-74) is redundant unless placed in a different context. 

Response 4: Thank you for your feedback and insightful comments. We have carefully reviewed our manuscript and improved on this point. (Lines 74, 79, 82, 92, 161, 227, etc.)

Comment No. 5. Figure 2 is not explained. It contains a lot of detailed information not given attention to (like GIOH, GeOH) as I mentioned in my previous review.

Response 5: Thank you. In response to your concerns, we have carefully revised the legend of Figure 2 as well as described all related information in the manuscript. More specifically, we proposed the different biosynthetic pathways for all bioactive compounds, with particular attention to GIOH, GeOH, etc. (Lines 80-94, 106-110)

Comment No. 6. I don’t understand what the statement ‘the of IC50 is always given without unit’ is based on. They are concentrations and do have a unit (compare with LD50 in text).

Response 6: Thank you for your insightful comment. To address your comment and provide a comprehensive overview for this parameter, we have added the unit of IC50 based on reviewed literature (i.e., ref. [15], [49], [61], [64], [65]) (Lines 145-148, 169-173, 211-215, 226-231)

Comment No. 7. A critical view still lacks.

My conclusion is that the current manuscript still needs major modifications and improvements.

Response 7: Thank you for your feedback and insightful comments. We acknowledge the importance of providing a critical evaluation of our manuscript and appreciate your comments and suggestions. In response to your concerns, we have carefully reviewed our manuscript and improved the introduction section. We have also incorporated a thorough assessment of the limitations and potential flaws in our Conclusion section. We recognize that while our overview sheds light on certain aspects of saffron from a review perspective (research and patents), there are still significant challenges and uncertainties regarding its clinical application. Furthermore, to address your comments and provide a comprehensive overview, we have added a new section titled "Future Trends and Implications" to the manuscript. We hope these additions enhance the manuscript’s critical perspective and provide a balanced view of the future of saffron in medical applications. We agree that while the topic is indeed interesting and holds promise, we have conducted a thorough review of the manuscript to improve coherence and overall readability to ensure it meets the high standards expected for publication in DDC and to advance our understanding of saffron’s clinical potential. Finally, it should be noted that this revised version now contains 98 bibliographic references, compared to 82 previously, which represent 16 new added references.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Several steps were needed to transform a poor-quality manuscript to one that merits publication. I want to compliment the authors with the final result.

I have, however, a few minor comments that should be taken into consideration before the paper can be accepted by DDC.

Line 58: Plant family names in Laten are not reflected in italics (species names do, in contrast).

Section 3: Various values from the literature are given in this section: mean and sd. Mentionig an sd is no very informative if n = is unknown. I suggest to confine to mean values only. Furthermore, in 3.2 units are missing. Please add. What is meant with 'latency' (lines 163-164)? What are 'whole extracts'? (line 168).

Section 5 (and maybe also 6): I miss on short discussion on safety aspects of saffron. Probably not much is know on this topic, but with increasing use by humans more insight into the possible occurrence of adverse reactions will be obtained. I suggest to add one of. few sentences to show the reader that tis is a relevant aspect to consider as well. 

 

Comments on the Quality of English Language

Minor editing of the language will be needed, is my feeling (but I am not a native English speaker / writer).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

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

Comments and Suggestions for Authors

This might be an interesting review of the use of saffron to prevent and treat cancer. However, the interesting results promising a use are only purely described in the manuscript. The authors are not chemists. Some chemists must be approached to explain e.g. fig. 5 and other chemical issues in the manuscript. Some additional comments are below:

Line -43- Also, in recent years, natural products have become an important source for developing new drugs. Morphine was isolated in 1805, which is not recent for me. Natural products have always been a source of new drugs.

Line -69- alfa and beta, use Greece letters

Line -111- In most of the discussion activities are reported, but it is not stated at what concentrations/dose these activities are observed. The information is useless without this information. As an example, crocin is reported to inhibit COX-1 with IC50 9.7, which is four times higher than indomethacin. Consequently, is dubious if this compound could be developed into a drug. Such exact data as concentration or dose concerning the different bioassays are missing.

-Line 127- A similar antitumor effect is observed at a dose of 200 mg/kg, which is an excessively high dose. Please, comment on this.

Line -143- 4120–556 mg/kg, why not 556 – 4120

Line -150-154- The LD50 values are close to the doses for treatment of cancer diseases. Please, comment on this.

Line -159- the use of saffron and its derivatives in the fight against cancer, Saffron is a spice. You can not make derivatives of a spice.

Figure 5: The text in the boxes makes no sense. The authors should approach a chemist to explain the chemistry in the patent. Even though it is written in Chinese the formulas make it possible to understand the chemistry in CN101157645B, which is very different from that suggested by the authors.

Line 233-271- Some patents issued on traditional Chinese medicine are listed. However, no critical evaluation of these is given. I would be interesting to see the chemistry and the clinical trials reported in these patents instead of just the patent number.

 

Similarly, the experiments in which sorafenib was co-administered with crocin or crocin derivatives should be elaborated.  Crocin salts are mentioned. I am not sure how to make a salt of crocin since this is not an acid. Figure 8 illustrates this experiment, but the outcome is not clearly explained. 

Comments on the Quality of English Language

no comments.

Reviewer 2 Report

Comments and Suggestions for Authors

With this manuscript the authors try to show the importance of saffron as an herbal medicine and its relevance for further drug development. However, the result is not very convincing and manuscript contains major flaws.

 

The title does not cover the content and is too suggestive. With the title as basis I will show the major flaws of the manuscript.

 

The authors talk about ‘saffron’ in different contexts. Saffron stand for the entire plant, Crocus sativus, as well as for the medicinally used part, the stigmas. This should be made clearer throughout the manuscript.

 

When we talk about cancer prevention and cancer treatment, this usually refers to humans. Most of what is presented in the manuscript comes from preclinical research, however. When discussing the literature, make clear which studies are in vitro and which study results come from work with experimental animals. Antitumor, anticancer refers to in vivo effects, including in humans. In vitro cytotoxicity refers to work done with cell lines. This should be distinguished an made clearer throughout the manuscript. So, start with in vitro cytotoxicity, then discuss in vivo antitumor activity in animals and finally pay attention to anticancer effects in humans (as far as such information is available for saffron or its bioactive active constituents). 

 

Prevention is elaborated poorly in the manuscript. In this context antioxidant and anti-inflammatory effects may be discussed. It is unclear to me why separate sections are dedicated to these effects (3.1 and 3.2) as this is not the focus of the work.

 

Extracts and products used should be described and characterised in more detail and much better. A ‘saffron extract ‘does not say anything, unless you know about the extraction fluid and the main constituents of the extract. Also, which plant part was used? Dried? Fresh? In addition, more concrete data should be given abut cell lines, concentrations, controls, etc. when experiments published in literature are discussed (use tables).

 

The focus of the work, reflected in the first part of the title should be made clearer at the end of the Introduction. Line 76: ‘we present some of the saffron activities’: This is a good examples of being vague and lack of focus.

 

The second part of the title reads ‘an overview based on research and patents’. This is vague and it remains vague when having read the manuscript. The authors reviewed the literature, but how did they do this? Under heading 2 there is some information, but is it a systematic review or a scoping review? What were inclusion and exclusion criteria? Add a PRISMA flow diagram. I looks as if the authors used a certain bias.

 

The abstract is too general. Also, consequently use the specific terms (like saffron, cytotoxicity, antitumor, as I wrote earlier). Line 18: what do you mean with ‘a cross-section of the literature’? How can researchers be inspired? How can the results so far be used for drug development? This is suggested in the abstract, but missing or poorly worked out in the manuscript.

 

Table 2 lacks detailed information. Please give characteristics of extracts used. If details are not given in the literature, it is a point for critical discussion.

 

The ample information on the plant (lines 45-64) should be shortened with reference to literature. There is nothing new here. Also, Figure 1 and Table 1 are redundant, in my opinion.

 

Important is Figure 2 with the main components of saffron (in this case the dried stigmas). How are the main compounds connected to each other? Refer to these compounds when discussing the products. But leave out mentioning irrelevant compounds which are present in low amounts only (line 65-72). Just refer to reference 7, which gives a good overview. 

 

What does the work submitted add to the content of reference 7? I have the feeling that these authors share a lot of information that is partly a repeated in the manuscript by Elfardi et al..

 

Part 4 on patents contains figures (4, 5, 6, 7. 8) that are taken literally from the respective patents. They are amply discussed in the text. I think the latter is more than sufficient. I suggest to leave out the figures.

 

What lacks is a good discussion about the patents. What did they yield so far? What is their clinical relevance? 

 

In general, the information found in literature should be assessed much more critically, with the focus on clinical applications. Only then ‘to prevent and treat cancer’ in the title may be justified. The possible role of cancer prevention by products from saffron stigmas as such is hardly discussed. 

 

In conclusion, this manuscript is unacceptable for publication. The value for the scientific community is very limited in the way the literature data are presented and worked out now.

 

 

Comments on the Quality of English Language

I am not a native English speaker, but in general the English seems quite acceptable. Some minor adaptations will be needed.