Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Reviewer comments and suggestions

 

The authors in this study investigated papain in preventing fibrin(ogen)lytic, anticoagulant, and antithrombotic activities. Hence, they examined how papain influences fibrinogen and the process of blood coagulation. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bβ, and γ-bands and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Hence, the author suggested that papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties that may be used in preventing or treating cardiovascular issues and thrombosis-related diseases

 

Overall, the manuscript is well written. I have listed the concerns and comments that needed to be explained or modified.

 

 

1. Line 51-52 Please explore some studies for common reader of your manuscript
2. Line 77-78 These points needed appropriate references
3. Comments for last paragraph of introduction section “The authors said lacking, and they added references, which I think is not needed here. The authors need to be well introduce there hypothesis based on the previous absence in the literature.”
4. Line 280 “κ-Carrageenan-Induced Rat-Tail Thrombosis Model” The authors need to introduce this model in introduction part
5. First paragraph of discussion section “Need to discuss the novelty of their own finding, not others in the first paragraph.”
6. Line 330-331 You need to discuss it with proper explanations and references
7. Line 381 -383 Did the authors mention the limitations of this study? It would be better to add. All figures should be in the appropriate place.
8. The conclusion should be in another section
9. All references needs to be modified so that they follow the MDPI journal guidelines

Author Response

Response to reviewers 1:

 

Thank you for your valuable feedback on my manuscript. I appreciate your time and effort in reviewing my work. According to your comments, we have finally changed manuscript as follow. In the manuscript, revised portions are shown in red color.

 

Points 1: Line 51-52 Please explore some studies for common reader of your manuscript.

 

-Answer: Thank you for your feedback. We appreciate your input, and we have revised the sentence to enhance clarity. We have included two more references that we feel will make the work more accessible to a wider readership. We hope that the inclusion of these references will address your concern and provide readers with a more comprehensive understanding of the topic.

 

Point 2. Line 77-78 these points needed appropriate references.

-Answer: In response to your comment about needing appropriate references for the points mentioned in lines 77-78, we have carefully selected and included relevant references to support and strengthen the statements made in that section.

 

Point 3. Comments for last paragraph of introduction section “The authors said lacking, and they added references, which I think is not needed here. The authors need to be well introduce there hypothesis based on the previous absence in the literature.

 

-Answer: Thank you for your valuable feedback regarding introduction part. We appreciate the time and effort you have dedicated to providing feedback. Regarding your suggestion to remove references in the last paragraph of the introduction, we have carefully reconsidered our approach. We agree with your emphasis on introducing our hypothesis based on the existing absence in the literature. In response to your recommendation, we have revised the paragraph by eliminating unnecessary references, ensuring a more focused and direct introduction of our hypothesis.

 

Point 4. Line 280 “κ-Carrageenan-Induced Rat-Tail Thrombosis Model” The authors need to introduce this model in introduction part.

 

-Answer: We appreciate your insightful comment regarding the introduction of the “κ-Carrageenan-Induced Rat-Tail Thrombosis Model.” In response to this suggestion, we carefully considered the placement of information related to the thrombosis model. While we understand the traditional approach of introducing models in the introduction section, we made a deliberate decision to provide a comprehensive and focused discussion on the κ-Carrageenan-Induced Rat-Tail Thrombosis Model in the discussion section.

Our intention to ensure that the model's introduction is contextualized within the broader framework of our experimental results and their interpretation. By presenting the model in the discussion section, we aimed to provide readers with a more immediate understanding of its relevance to our specific findings.

 

Point 5. First paragraph of discussion section “Need to discuss the novelty of their own finding, not others in the first paragraph.”

 

-Answer: Thank you for your thoughtful feedback. We appreciate your suggestion to emphasize the novelty of our own findings in the first paragraph of the discussion section. We have carefully considered this advice and restructured the manuscript accordingly.

 

Point 6. Line 330-331 you need to discuss it with proper explanations and references.

 

-Answer: In response to your comment about needing appropriate references for the points mentioned in lines 330-331, we have carefully selected and included relevant references to support and strengthen the statements made in that section.

 

Point 7. Line 381 -383- Did the authors mention the limitations of this study? It would be better to add. All figures should be in the appropriate place.

 

-Answer: Regarding your query about the limitations of the study, we acknowledge the importance of highlighting the constraints in our research. In line 381-383, we have now included a section addressing the limitations of our study. We would like to address the concern you raised regarding the figures. We modified the picture to place it in as appropriate a place as possible.

However, it seems that due to the formatting requirements of IJMS, there might be a discrepancy in the visual presentation.

 

Point 8. The conclusion should be in another section.

 

-Answer: We greatly appreciate your constructive feedback on the manuscript's organization, particularly the placement of the conclusion. We revised the manuscript to include a dedicated conclusion section in response to this valuable suggestion.

 

Point 9. All references needs to be modified so that they follow the MDPI journal guidelines.

 

-Answer: Thank you for the insightful feedback and your attention to detail regarding the references. I appreciate your guidance on adhering to the MDPI journal guidelines. In the revised version of the manuscript, I will carefully review and modify each reference according to the MDPI guidelines.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Authors did not address the concern around the PT and APTT prolongation times suggested by this reviewer. Authors need to consult with a hemostasiologist. They do present interesting findings around papain, but fail to convey this message in a scientific manner that would not be misleading to the audience, but it seems from the manuscript currently that authors are confused themselves about hemostatic processes.   

Author Response

Response to reviewers 2:

Thank you for your valuable feedback on our manuscript. I appreciate your time and effort in reviewing my work. According to your comments, we have changed manuscript as follow. In the manuscript, revised portions are shown in red color.

 

Points 1: Authors did not address the concern around the PT and APTT prolongation times suggested by this reviewer. Authors need to consult with a hemostasiologist. They do present interesting findings around papain, but fail to convey this message in a scientific manner that would not be misleading to the audience, but it seems from the manuscript currently that authors are confused themselves about hemostatic processes.

-Answer: Thank you for your valuable comments and concerns. The anticoagulant activity of papain was evaluated with in vitro study using PT and APTT assays. Both tests are commonly used to determine defects in the extrinsic, intrinsic, and common pathways of the blood coagulation cascade. In addition, we recognize the necessity to research which clotting factors are impacted by papain for anticoagulation. However, our finding demonstrates that papain has anticoagulant activity as evidenced by prolonged induction in both PT and APTT. We present a review paper (http://dx.doi.org/10.1155/2015/876426) for the reviewer's understanding. In this paper, we can see several cases of increased PT and APTT, similar to our results, and it is stated that when the two parameters are increased, it has anticoagulant properties.

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

In this section words written by the authors of this manuscript are in italics.

Lines 48-9

Papain's therapeutic potential is evident in the pharmaceutical sector, where it is utilized for its anti-inflammatory, analgesic, and wound-healing properties [6].

Ref. 6 Ajlia, S. A., Majid, F. A., Suvik, A., Effendy, M. A., & Nouri, H. S. (2010). Efficacy of papain-based wound cleanser in promoting wound regeneration. Pakistan journal of biological sciences: PJBS, 13(12), 596-603

This sentence is misleading. Where the authors write where it is utilized

it would better be stated as where it has been studied ..

Lines 79-81

This has led many researchers to search for safer alternatives from natural sources for the treatment of thrombotic diseases due to their composition of multiple constituents, each with the potential to target multiple sites. 

There are no citations for this statement. The sentence should be cited or removed. The implicit suggestion that natural sources are associated with safety is inappropriate. Also, this sentence, and that in Lines 48-9, is unethical in that the authors ignore the US Food and Drug administration determination that the use of papain has resulted in potentially life-threatening hypersensitivity reactions. According to the FDA report on the topical use of papain, "reactions described include serious cases of anaphylaxis and anaphylactic shock that started within 15 minutes of topical papain use and resulted in hospitalizations, including admissions to the intensive care unit."

It is unethical for the authors to simply ignore this report. If they do not know about this, then their literature search prior to publication is vastly deficient. 

see https://web.archive.org/web/20170301223514/https://www.fda.gov/OHRMS/DOCKETS/98fr/FDA-2008-N-0481-n.pdf

Lines 81, 82, 83

These products can exhibit pleiotropic and synergistic effects, enhancing therapeutic efficacy. 

There is no citation given for this sentence. This sentence is pure speculation. The sentence should be cited or removed. It also ignores the hazards identified by the US FDA.

Line 82

 In traditional medicine, various cysteine proteases preparations have been employed to enhance platelet aggregation and address thrombosis-related diseases [22]. 

ref. 22 (self-citation) Yang, H. R., Hwang, D. H., Prakash, R. L. M., Kim, J. H., Hong, I. H., Kim, S., ... & Kang, C. (2022). Exploring the Fibrin (ogen) olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model. Nutrients, 14(17), 3552. 

The word employed suggests clinical use outside of a research setting. Do the authors have evidence for this? The word employed should be replaced with the word studied. 

Line 107 

Proteolytic activity was evaluated as described by Segers et al. with some slight modifications [26]. 

Segers is not an author of reference 26. Also, it is odd that the authors do not cite papers that measure the activity of papain. The authors use a very complicated assay that involves centrifugation, very much in contrast to the simple literature assays. Why do they do that? Is the complicated assay that they use even reliable?

Line 135, 136 

Following this, drops of either 20 μL papain at varying concentrations (0, 0.0125, 0.025, 0.05, or 0.1 U) or a positive control (plasmin at 1 mg/mL) were applied to the disks.

Papain in this paper is given in units of "U". What is "U"? How does "U" relate to papain's mg/mL concentration or to its molar concentration? There are ways to chemically titrate papain to determine the exact molar concentration, but the authors seem to be unaware of this. 

Line 224

Papain has a molecular weight of about 37 kDa, with a similar result from Nurhayati et al [30] 

and

Lines 226, 227
Papain has a molecular weight exceeding 75 kDa, and its fibrinolytic activity, as illustrated in Figure 1B, displayed its efficacy in degrading fibrin.

and Line 321  ... papain (37-75 kDa)

The literature molar mass of papain is reported in many places to be 23 kDA. Where do the 37 and 75 kDa numbers come from? Why are these deviations from the literature value not explained or addressed? Does this not suggest that the authors are using some other enzyme? Is their preparation contaminated with papaya protease III or chymopapain, or something else? See https://pubmed.ncbi.nlm.nih.gov/3919769/ an article which strangely enough is not cited here. 

Lines 246, 247

Our experimental findings indicate that papain's stability is not confined to the previously established conditions but extends to a pH as high as 11. 

How does this compare with the literature data on this topic, literature that is not cited? Does this not suggest that the authors are not actually observing the activity of papain, but of something else?

Here is a well-characterized and high-quality pH profile for papain. How do the author's results compare to this? 

see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1177120/?page=4

 

Comments on the Quality of English Language

No comment. 

Author Response

Response to reviewers 3:

Thank you for your valuable feedback on my manuscript. I appreciate your time and effort in reviewing my work. According to your comments, we have finally changed manuscript as follow. In the manuscript, revised portions are shown in red color.

Points 1: Lines 48-9

Papain's therapeutic potential is evident in the pharmaceutical sector, where it is utilized for its anti-inflammatory, analgesic, and wound-healing properties [6].

Ref. 6 Ajlia, S. A., Majid, F. A., Suvik, A., Effendy, M. A., & Nouri, H. S. (2010). Efficacy of papain-based wound cleanser in promoting wound regeneration. Pakistan journal of biological sciences: PJBS, 13(12), 596-603

This sentence is misleading. Where the authors write where it is utilized

It would better be stated as where it has been studied.

 

-Answer: We appreciate your feedback and agree that the sentence's wording may cause some confusion. Based on your concern, we recognize that instead of the word used, studied may be more accurately conveyed and changed accordingly.

 

Points 2: Lines 79-81

This has led many researchers to search for safer alternatives from natural sources for the treatment of thrombotic diseases due to their composition of multiple constituents, each with the potential to target multiple sites. 

There are no citations for this statement. The sentence should be cited or removed. The implicit suggestion that natural sources are associated with safety is inappropriate. Also, this sentence, and that in Lines 48-9, is unethical in that the authors ignore the US Food and Drug administration determination that the use of papain has resulted in potentially life-threatening hypersensitivity reactions. According to the FDA report on the topical use of papain, "reactions described include serious cases of anaphylaxis and anaphylactic shock that started within 15 minutes of topical papain use and resulted in hospitalizations, including admissions to the intensive care unit."

It is unethical for the authors to simply ignore this report. If they do not know about this, then their literature search prior to publication is vastly deficient. 

see https://web.archive.org/web/20170301223514/https://www.fda.gov/OHRMS/DOCKETS/98fr/FDA-2008-N-0481-n.pdf.

-Answer: We have carefully considered your feedback and would like to address each of your concerns. We acknowledge the absence of citations for the statements in Lines 79 and 81. In the revised manuscript, we have added appropriate references (please see References 7) to support the relevant statements.

 In response, we have modified the language in the relevant sentences to use the term “therapeutic” instead of “safer”. We take the issue raised about the US FDA determination on papain seriously. To address this concern, we have included a reference to the FDA report on the adverse reactions associated with the topical use of papain. It is essential for us to provide a comprehensive view of the potential risks associated with papain.

 We want to emphasize that our experiments involved the intravenous (IV) administration of papain in rats. We appreciate your diligence in highlighting the FDA's concerns related to topical application. Our data from the IV route indicates no side effects or toxic effects through our using concentrations. Considering that many thrombolytic drugs have known side effects, our study aims to identify potential therapeutic candidates for thrombolysis. We acknowledge the need for further investigation underlying the side effects.

Points 3: Lines 81, 82, 83

These products can exhibit pleiotropic and synergistic effects, enhancing therapeutic efficacy. 

There is no citation given for this sentence. This sentence is pure speculation. The sentence should be cited or removed. It also ignores the hazards identified by the US FDA.

Line 82

 In traditional medicine, various cysteine proteases preparations have been employed to enhance platelet aggregation and address thrombosis-related diseases [22]. 

ref. 22 (self-citation) Yang, H. R., Hwang, D. H., Prakash, R. L. M., Kim, J. H., Hong, I. H., Kim, S., ... & Kang, C. (2022). Exploring the Fibrin (ogen) olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model. Nutrients, 14(17), 3552. 

The word employed suggests clinical use outside of a research setting. Do the authors have evidence for this? The word employed should be replaced with the word studied. 

 

-Answer: We appreciate your diligence in highlighting the need for proper citation and the speculative nature of the sentence regarding pleiotropic and synergistic effects. Upon careful consideration, we agree with your assessment. In our revised manuscript, we have chosen to remove the sentence of line 81 to ensure the accuracy and reliability.

We appreciate your attention and agree with your suggestion to replace the word 'employed' with 'studied' in the manuscript for line 82-83. In the revised manuscript, we make the necessary adjustment by replacing 'employed' with 'studied' to accurately reflect the nature of our work and avoid any potential misinterpretation.

Points 4: Line 107 

Proteolytic activity was evaluated as described by Segers et al. with some slight modifications [26]. 

Segers is not an author of reference 26. Also, it is odd that the authors do not cite papers that measure the activity of papain. The authors use a very complicated assay that involves centrifugation, very much in contrast to the simple literature assays. Why do they do that? Is the complicated assay that they use even reliable?

 

-Answer: We appreciate your diligence in bringing this to our attention. We acknowledge the oversight in referencing Segers et al. and we will promptly rectify this error by accurately citing the appropriate source. We understand your observation about the complexity of the proteolytic assay used in our study compared to simpler literature assays. Our decision to employ a more intricate assay, involving centrifugation, was motivated by the specific requirements of our experimental design. In response to your suggestion, we will include the references specifically related to measuring papain activity in the discussion section where required.

 

Points 5: Line 135, 136 

Following this, drops of either 20 μL papain at varying concentrations (0, 0.0125, 0.025, 0.05, or 0.1 U) or a positive control (plasmin at 1 mg/mL) were applied to the disks.

Papain in this paper is given in units of "U". What is "U"? How does "U" relate to papain's mg/mL concentration or to its molar concentration? There are ways to chemically titrate papain to determine the exact molar concentration, but the authors seem to be unaware of this. 

 

-Answer: Thank you for your insightful comments regarding the use of "U" and its relation to papain's mg/mL concentration or molar concentration. In our manuscript, "U" refers to enzymatic activity units, specifically papain activity. There is a mistake in the presentation for Unit. So, we changed and fixed the material and method sections, as well as Figure 3.

Points 6: Line 224

Papain has a molecular weight of about 37 kDa, with a similar result from Nurhayati et al [30].

And Lines 226, 227
Papain has a molecular weight exceeding 75 kDa, and its fibrinolytic activity, as illustrated in Figure 1B, displayed its efficacy in degrading fibrin.

and Line 321 ... papain (37-75 kDa)

The literature molar mass of papain is reported in many places to be 23 kDA. Where do the 37 and 75 kDa numbers come from? Why are these deviations from the literature value not explained or addressed? Does this not suggest that the authors are using some other enzyme? Is their preparation contaminated with papaya protease III or chymopapain, or something else? See https://pubmed.ncbi.nlm.nih.gov/3919769/ an article which strangely enough is not cited here. 

 

-Answer: We appreciate your assistance in revising our work regarding the molecular mass of papain. We reported the molecular weight of papain as 37-75 kDa, which may seem inconsistent with the commonly cited literature value of 23 kDa. We would like to emphasize that this observed range is a result of conducting our experiments under non-reducing conditions. The choice of non-reducing conditions was intentional and aimed at preserving the native state of papain by avoiding disulfide reduction and active-site regeneration. We utilized non-reducing conditions to ensure that the observed molecular mass represents the enzyme's natural state, which is essential for studying its enzymatic activity. We also added references, where SDS-PAGE and zymography assay were conducted to study the molecular mass of papain for better clarification. We want to assure that our preparation was not contaminated with other enzymes such as papaya protease III or chymopapain. We implemented experimental setup to ensure the purity of the papain preparation. Our experimental design aimed to provide a comprehensive understanding of papain's enzymatic activity under non-reducing conditions.

Points 7: Lines 246, 247

Our experimental findings indicate that papain's stability is not confined to the previously established conditions but extends to a pH as high as 11. 

How does this compare with the literature data on this topic, literature that is not cited? Does this not suggest that the authors are not actually observing the activity of papain, but of something else?

Here is a well-characterized and high-quality pH profile for papain. How do the author's results compare to this? 

see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1177120/?page=4

-Answer: Thank you for your valuable comments and concerns regarding the observed stability of papain at a pH as high as 11 in our experimental findings. We acknowledge the importance of comparing our results with existing literature data. While our findings may appear to deviate from established conditions, it is essential to note that variations in experimental protocols, sample preparation, and assay techniques can contribute to discrepancies. To address this concern, we have included a comprehensive comparison with relevant literature data in the revised manuscript (please see References 35 and 36). These studies, which was not cited in the initial submission, provide additional context to the observed stability of papain at high pH values. Last but not the least we added the references that you suggested.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

This paper again is much improved. There is a remaining problem that the authors ought to be able to address.

This relates to the ability of other researchers to reproduce this work. To do this, the authors must clearly explain how many mg. or micrograms/mL of purified papain correspond to a U/mL of papain. This should be done either by explaining that item clearly or by providing a citation. Best would be both. This is a very simple thing, but the paper should not be published unless this issue is addressed. 

For instance, if someone wanted to repeat this experiment in Line 134

Following this, drops of either 20 μL papain at varying concentrations (0, 0.0125, 0.025, 0.05, or 0.1 U/mL) or a positive control 135 (plasmin at 2 U/mL) were applied to the disks. 

What is the mg or microgram or nanogram per mL concentration of papain in 0.1 U/mL? How the authors define U/mL? What is the citation for U/mL?

Lines 454 to 486 seem to be items that the authors have not addressed. 

 

Comments on the Quality of English Language

In Line 81

In traditional medicine, various cysteine proteases preparations have been studied to enhance platelet aggregation and address thrombosis-related diseases [25].

The word proteases should be protease. The next word, preparations, given as the plural, is correct. 

In Line 71 

Clinical use of drugs includes heparin, warfarin, and their derivatives, primarily employed for inhibiting blood coagulation factors [19]. 

This is perhaps better given as 

Heparin, warfarin, and their derivatives are, in clinical settings, primarily used to inhibit blood coagulation factors [19]. 

Author Response

Response to reviewers 3:

 

Thank you for your valuable feedback on my manuscript. I appreciate your time and effort in reviewing my work. According to your comments, we have finally changed manuscript as follow. In the manuscript, revised portions are shown in blue color.

 

Point 1: Line 135,136

Following this, drops of either 20 μL papain at varying concentrations (0, 0.0125, 0.025, 0.05, or 0.1 U/mL) or a positive control (plasmin at 2 U/mL) were applied to the disks.

What is the mg or microgram or Nano gram per mL concentration of papain in 0.1 U/mL? How the authors define U/mL? What is the citation for U/mL?

-Answer: In response to your query, the concentration of papain in milligrams per milliliter (mg/mL), micrograms per milliliter (μg/mL), or nanograms per milliliter (ng/mL) can be calculated using the specific activity of papain, which is provided in units per milligram of protein (U/mg protein).

The specific activity of papain preparation is 10 U/mg protein. Therefore, to express the concentration of papain in alternative units, one can use the following conversions:

Concentration in (mg/mL):

=Specific Activity ×Concentration (U/mL)

=10 U/mg protein×0.1 U/mL

=10U/mg protein×0.1U/mL=1mg/mL

Therefore, 0.05, 0.025, 0.0125 U/mL of papain contain 0.5, 0.25, 0.125 mg/mL, respectively.

In addition, we present papers for papain (DOI: 10.1007/s11947-019-2245-3), and plasmin (https://doi.org/10.1080/09537100902803635) for the reviewer's understanding.

 

Points 2: Lines 454 to 486 seem to be items that the authors have not addressed. 

 

-Answer: Thank you for your constructive feedback on our manuscript. We addressed it according to author contributions.

 

Points 3: Line 81 In traditional medicine, various cysteine protease preparations have been studied to enhance platelet aggregation and address thrombosis-related diseases [25].

 

The word proteases should be protease. The next word, prepareations, given as the plural, is correct.

 

-Answer: Thank you for your feedback. We have considered your suggestion regarding the use of "proteases" in the manuscript. We changed the word protease instead of proteases. The updated text on line 81 now reads:

"In traditional medicine, various cysteine protease preparations have been studied to enhance platelet aggregation and address thrombosis-related diseases [25]."

 

Point 4. Clinical use of drugs includes heparin, warfarin, and their derivatives, primarily employed for inhibiting blood coagulation factors [19].

This is perhaps better given as

Heparin, warfarin, and their derivatives are, in clinical settings, primarily used to inhibit blood coagulation factors [19]

-Answer: Thank you for your constructive feedback on our manuscript. We appreciate your suggestion for improving the clarity of the sentence. In response to your recommendation, we have revised the sentence as follows:

"Heparin, warfarin, and their derivatives are, in clinical settings, primarily used to inhibit blood coagulation factors [19].

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

Comments and Suggestions for Authors

This is an interesting exploratory paper on the use of papain in the possible treatment of cardiovascular disease. It has problems that the authors can rectify. 

At the beginning of the paper there are statements of fact that need to be referenced. These are on lines 16-19.

In lines 24-27 there are descriptive numbers about papain's molar mass, heat stability, and pH profile. The authors write as if this were new information; they must cite the earlier literature where all of these things are described. 

In line 26, the language about the molar mass is odd. "Papain appears to have a molecular weight of around 37 kDa. Appears is an inappropriate word (unless the authors dispute the prior work on this topic.) The number 37 kDa should have a citation. 

The same topic reappears in line 227

The molecular weight of papain appears to be approximately 37 kDa.

This sentence should simply cite the literature molecular mass of papain. The words "appears" and "approximately" are inappropriate or should be explained with references. 

The same issue reappears in section 3.2. Effect of Temperature and pH on Protease Activity and Stability 233

This section should include references to prior work that describes the effect of temperature and pH on papain activity and stability. The authors should not pretend, as they do by omission, that this kind of research has not already been done. Their results must be compared to the prior literature results. 

On line 88 the authors write that "Furthermore, the constituents of natural products tend to have fewer side effects on the body system [22].

This sentence is a generalization that is not appropriate for a research paper and is not supported by a reading of the reference. This sentence reads as if it were taken from a commercial advertisement. 

On line 272 the authors write that

... papain possesses components that function as anticoagulants. 

This sentence is written in a way to suggest that their preparation of papain contained contaminants and impurities. That is a reasonable conclusion from the words papain possesses components. If that is what the authors mean, then they should acquire a purer preparation of the enzyme that does not have extraneous components. Otherwise, they can simply write that papain functions as an anticoagulant.

In lines 285-287, the authors write that ...

With the rapid advancements in science and technology, traditional medicine has emerged as an appealing subject for researchers. Particularly, there is a growing interest in conducting evidence-based research on natural products, not only to explore their traditional uses but also to potentially develop them into formal medications. 

These sentences suggest, without any citation given, that papain is part of traditional medicine. None of the references indicate that. There are references cited in this paper to materials that contain papain, but I see no citations that have evidence that papain is the active ingredient in any traditional medicines. 

Also, the authors write there is a growing interest in conducting evidence-based research on natural products.

There always has been great interest in using natural products in medicine. This is not new. Many, if not most antibiotics (think of penicillin) are natural products or are derived from them. However, to say that there is a growing interest is not self-evident. To use this sentence, the authors must provide evidence from sociological studies of research and interests among researchers on this topic. Since the sentences here are not cited, they should either be supported by appropriate citations or be removed. 

On line 291 the authors write that

Consequently, it has been employed as a conventional therapeutic approach for diverse medical conditions [31].

A reading of reference 31 does not demonstrate that the enzyme, papain, has been used as a conventional therapeutic. Its use in cooking does not qualify. Absent additional supporting citations that directly use purified papain in therapy, this sentence must be removed. 

On lines 347 and 348, the authors write that

As a consequence, the search for more inexpensive and safer fibrinolytic enzymes and thrombolytic medications derived from natural sources becomes a crucial issue.

This sentence makes no sense at all, since the agents being criticized are also derived from natural sources. Streptokinase, urokinase, pro-urokinase, and Factors FII, V, VII, VIII, IX, XI, and XII all come from natural sources. The words derived from natural sources in this sentence should be deleted, since those words falsely suggest that the other agents are not derived from natural sources. Falsely suggesting, by omission, that the other agents are not derived from natural sources becomes an ethical issue for this manuscript. 

The most serious problem with this paper is that the authors fail to include references to prior work that shows papain to have a broad specificity. Papain resembles the digestive enzyme trypsin in this respect, and does not resemble the restricted specificities of streptokinase, urokinase, Factors FII, V, VII, VIII, IX, XI, and XII. Suggesting that papain could be a treatment in vivo ignores its broad specificity, so broad that many, if not most, perhaps even all human proteins would be degraded by the enzyme. Of course, with such a broad specificity, it would be expected to dissolve blood clots. But it is also expected to damage nearly everything it touches. 

The failure of the authors to deal with or cite the broad specificity of the enzyme that they are studying is not acceptable, since that broad specificity is expected to have a large negative impact on its possible medicinal use.

The authors should add a disclaimer that there is no evidence that papain is safe to use in an intravenous manner. 

 

 

Comments on the Quality of English Language

The quality of the English used in this paper is excellent. However, some of the verbiage used will mislead readers who are not familiar with the field. The main problem is the peculiar use of the word "natural" to describe papain but is not used to describe any other agent mentioned in the paper that derives from natural sources. Yes, papain is natural. But so are other naturally produced enzymes and proteins mentioned in this paper. i.e,. streptokinase , urokinase, pro-urokinase, and Factors FII, V, VII, VIII, IX, XI, and XII. If the authors wish to label papain as natural, then they are also obliged to label these enzymes and proteins as natural.

This also applies to other natural agents used in the paper, such as κ-carrageenan. Of course, recombinant reteplase and alteplase do not need to be labeled as natural, since they are not. 

In line 47, the paper states that Papaya latex contains a significant concentration of papain enzyme, which have the ability to ....

The word "have" is not appropriate. The authors should use the word "has" instead. 

 

Author Response

REVIEWERS COMMENTS FROM IJMS

 

Response to reviewers 1:

 

To reviewer 1: Thank you for your valuable feedback on my manuscript. According to your comments, we have finally changed manuscript as follow. In the manuscript, revised portions are shown in red color.

 

Points 1 & 2:

• At the beginning of the paper there are statements of fact that need to be referenced. These are on lines 16-19.

 

• In lines 24-27 there are descriptive numbers about papain's molar mass, heat stability, and pH profile. The authors write as if this were new information; they must cite the earlier literature where all of these things are described.

 

-Answer: Thank you for the valuable comment. The guidelines of the journal where we submitting our manuscript recommend that references should not be included in the abstract. To address your concern, we have ensured that the necessary references are included in the introduction section, where they support the discussion of the relevant literature and research context. This placement allows readers to access the foundational knowledge they need while maintaining the abstract's clarity and brevity.

 

Point 3. In line 26, the language about the molar mass is odd. "Papain appears to have a molecular weight of around 37 kDa. “Appears” is an inappropriate word (unless the authors dispute the prior work on this topic.) The number 37 kDa should have a citation.

 

-Answer: Thank you for your valuable feedback regarding the language and citation in line 26 of our manuscript. Though references should not be added in the abstract we just changed language which is marked in red color.

Point 4. The same topic reappears in line 227.

The molecular weight of papain appears to be approximately 37 kDa.

This sentence should simply cite the literature molecular mass of papain. The words "appears" and "approximately" are inappropriate or should be explained with references. 

 

-Answer: Thank you for the valuable suggestion. We added references as well as correct sentence you pointed them out and marked within red color.

 

Point 5. The same issue reappears in section 3.2. Effect of Temperature and pH on Protease Activity and Stability 233.

This section should include references to prior work that describes the effect of temperature and pH on papain activity and stability. The authors should not pretend, as they do by omission, that this kind of research has not already been done. Their results must be compared to the prior literature results.

 

Answer: We appreciate your valuable feedback on our manuscript, particularly your suggestion to include references to prior work in Section 3.2 to provide a comprehensive context for our research findings. We recognize the importance of drawing connections between our results and existing literature. In response to your comment, we will incorporate references and compare our results to the prior literature, highlighting the contributions and insights our study brings to the field.

 

Point 6. On line 88 the authors write that "Furthermore, the constituents of natural products tend to have fewer side effects on the body system [22].

This sentence is a generalization that is not appropriate for a research paper and is not supported by a reading of the reference. This sentence reads as if it were taken from a commercial advertisement.

 

Answer: Thank you for your insightful feedback regarding the reference in our manuscript. We agree that the reference may not be suitable in the context of a research paper, and we understand your concern about the generalization it presents. We recognize the importance of maintaining a high standard of academic rigor and avoiding statements that may appear commercial or overly generalized. We appreciate your guidance in improving the quality of our manuscript, and we believe that the removal of this reference will enhance the overall clarity and scientific integrity of our work.

 

Point 7. On line 272 the authors write that

...papain possesses components that function as anticoagulants. 

This sentence is written in a way to suggest that their preparation of papain contained contaminants and impurities. That is a reasonable conclusion from the words papain possesses components. If that is what the authors mean, then they should acquire a purer preparation of the enzyme that does not have extraneous components. Otherwise, they can simply write that papain functions as an anticoagulant.

 

Answer: Thank you for your feedback on our manuscript and your observation regarding the wording on line 272. We would like to confirm that we utilized a high-grade papain preparation sourced from Sigma-Aldrich, a reputable supplier known for providing quality enzymes.in the materials and method part we enclosed papain catalogue number which contain details about impurities and others. Our choice of this specific papain preparation was made with the intention of ensuring the highest purity and quality. Upon reflection, we understand how the phrase "papain possesses components" could be misinterpreted as implying impurities or contaminants. To eliminate any confusion, we will revise the sentence as you suggested to state that "papain functions as an anticoagulant."

 

Point 8. In lines 285-287, the authors write that...

With the rapid advancements in science and technology, traditional medicine has emerged as an appealing subject for researchers. Particularly, there is a growing interest in conducting evidence based research on natural products, not only to explore their traditional uses but also to potentially develop them into formal medications.

These sentences suggest, without any citation given, that papain is part of traditional medicine. None of the references indicate that. There are references cited in this paper to materials that contain papain, but I see no citations that have evidence that papain is the active ingredient in any traditional medicines.

Also, the authors write there is a growing interest in conducting evidence-based research on natural products.

There always has been great interest in using natural products in medicine. This is not new. Many, if not most antibiotics (think of penicillin) are natural products or are derived from them. However, to say that there is a growing interest is not self-evident. To use this sentence, the authors must provide evidence from sociological studies of research and interests among researchers on this topic. Since the sentences here are not cited, they should either be supported by appropriate citations or be removed.

 

Answer: We appreciate your valuable feedback on our manuscript, particularly your suggestion to include references. We acknowledge the importance of referencing any claims made in our manuscript, and upon reviewing your comment, we agree that this specific statement could benefit from more precise language and citation to support the assertion. That’s why we added citations.

The observation regarding the phrase "growing interest" is indeed valid, and we understand your point about the longstanding and continuous interest in using natural products in medicine. Alternatively, we may choose to rephrase the sentence to emphasize the contemporary relevance of such research without implying that it is entirely new. For instance, we could say, "There is a continued interest in conducting evidence-based research on natural products," to better align with the historical context.

 

Point 9. On line 291 the authors write that

Consequently, it has been employed as a conventional therapeutic approach for diverse medical conditions [31].

A reading of reference 31 does not demonstrate that the enzyme, papain, has been used as a conventional therapeutic. Its use in cooking does not qualify. Absent additional supporting citations that directly use purified papain in therapy, this sentence must be removed. 

 

Answer: Upon revisiting the reference (31), we agree that it does not provide sufficient support for the assertion that papain has been used as a conventional therapeutic in the context of our research. To address your concern, we will remove the sentence in question to ensure that our paper aligns with the highest standards of academic rigor. We appreciate your vigilance in this regard and your guidance in maintaining the quality of our work.

 

Point 10. On lines 347 and 348, the authors write that

As a consequence, the search for more inexpensive and safer fibrinolytic enzymes and thrombolytic medications derived from natural sources becomes a crucial issue.

This sentence makes no sense at all, since the agents being criticized are also derived from natural sources. Streptokinase, urokinase, pro-urokinase, and Factors FII, V, VII, VIII, IX, XI, and XII all come from natural sources. The words derived from natural sources in this sentence should be deleted, since those words falsely suggest that the other agents are not derived from natural sources. Falsely suggesting, by omission, that the other agents are not derived from natural sources becomes an ethical issue for this manuscript.

 

Answer: We greatly appreciate your insightful review and the attention you've given to our manuscript. Upon a closer examination of the sentence in question, we agree that the phrase "derived from natural sources" may create confusion and is not accurate in this context. We promptly removed the sentence and ensure that our paper reflects precise and well-supported information.

 

Point 11: The most serious problem with this paper is that the authors fail to include references to prior work that shows papain to have a broad specificity. Papain resembles the digestive enzyme trypsin in this respect, and does not resemble the restricted specificities of streptokinase, urokinase, Factors FII, V, VII, VIII, IX, XI, and XII. Suggesting that papain could be a treatment in vivo ignores its broad specificity, so broad that many, if not most, perhaps even all human proteins would be degraded by the enzyme. Of course, with such a broad specificity, it would be expected to dissolve blood clots. But it is also expected to damage nearly everything it touches.

The failure of the authors to deal with or cite the broad specificity of the enzyme that they are studying is not acceptable, since that broad specificity is expected to have a large negative impact on its possible medicinal use.

The authors should add a disclaimer that there is no evidence that papain is safe to use in an intravenous manner. 

 

Answer: It is true that papain is a proteolytic enzyme with broad substrate specificity, and this characteristic is well-documented in the literature. We acknowledge that our paper did not include references to prior work that illustrates the broad specificity of papain. We apologize for this oversight and will include the relevant references in our revised manuscript to provide a more comprehensive background on papain.

In our manuscript, we have provided detailed information regarding the safety and toxicity of papain when used in animal experiments, specifically in line materials and method section (2.11), where we reported that the animal experiments demonstrated no toxic effects associated with papain administration. This finding suggests that, under the experimental conditions outlined in our study, intravenous use of papain did not produce adverse effects in the animal models.

 

Point 12: In line 47, the paper states that Papaya latex contains a significant concentration of papain enzyme, which have the ability to....

The word "have" is not appropriate. The authors should use the word "has" instead.

 

Answer: Thank you for your thoughtful feedback. We appreciate your close attention to our paper. We agree with your suggestion to replace "have" with "has" in line 47. The correct usage should be "has" since we are referring to the singular noun "papain enzyme." We acknowledge the importance of precision in our writing.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Authors wrote a nice manuscript describing the worm they have performed in regard to papain’s fibrinolytic activity. It is presented in a nice a clear manner. There is however, a point that authors make prematurely. Describing the results of PT and APTT tests, authors prompt to the conclusion that papain affect intrinsic and extrinsic pathway, which does not necessarily have to the case. Since PT and APTT methodology they used is based on fibrin formation, anything that has fibrinolytic activity would affect PT and APTT. So this conclusion cannot be supported from the data authors show.

Comments on the Quality of English Language

There is some English editing required. 

Author Response

REVIEWERS COMMENTS FROM IJMS

Response to reviewers 2:

To reviewer 2: Authors wrote a nice manuscript describing the worm they have performed in regard to papain’s fibrinolytic activity. It is presented in a nice a clear manner. There is however, a point that authors make prematurely. Describing the results of PT and APTT tests, authors prompt to the conclusion that papain affect intrinsic and extrinsic pathway, which does not necessarily have to the case. Since PT and APTT methodology they used is based on fibrin formation, anything that has fibrinolytic activity would affect PT and APTT. So this conclusion cannot be supported from the data authors show.

Answer: Thank you for taking the time to review our manuscript. We revised the relevant section of the manuscript to clarify that the observed changes in PT and APTT results are likely due to papain's fibrinolytic activity rather than direct effects on the intrinsic and extrinsic coagulation pathways. We did modification on the basis of your valuable comment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigate a potential role of papain on Fibrin(ogen)lytic, Anticoagulant, and Antithrombotic. They fail to address that the coagulation-related effects of papain on the conversion of fibrinogen into fibrin are already known for almost a century. Please refer to published papers and discuss them. 10.1085/jgp.20.4.543 Please, also discuss this paper: 10.1039/d2tb02220f

The text in general needs to be revised for plagiarism.

References need to be revised.

Author Response

REVIEWERS COMMENTS FROM IJMS

 

Response to reviewers 3:

To reviewer 3: The authors investigate a potential role of papain on Fibrin(ogen)lytic, Anticoagulant, and Antithrombotic. They fail to address that the coagulation-related effects of papain on the conversion of fibrinogen into fibrin are already known for almost a century. Please refer to published papers and discuss them. 10.1085/jgp.20.4.543 Please, also discuss this paper: 10.1039/d2tb02220f

The text in general needs to be revised for plagiarism.

References need to be revised.

Answer: We appreciate your valuable feedback on our manuscript. In response to your insightful comments, we would like to acknowledge and address the well-established knowledge on the coagulation-related effects of papain in the conversion of fibrinogen into fibrin. This phenomenon has indeed been documented for nearly a century, and we appreciate the reviewers' emphasis on this historical context. In this discussion, we will refer to the cited papers and integrate them into our study's framework with addition of sentences.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Due to the modulation of the intrinsic and extrinsic coagulant pathways, papain act as anticoagulants. Line 281

How do you modulate the coagulation pathways?  This sentence makes little sense. 

This suggests that papain may exert its anti-coagulant effects by targeting blood coagulation pathways while also inhibiting the conversion of fibrinogen into fibrin, thereby impeding the clotting process.    Lines 359-360

The conversion of fibrinogen to fibrin is part of the coagulation pathway. For that reason, this sentence makes no sense to this reviewer. 

 Our experimental utilization of papain as a natural cysteine protease resulted in the creation of a papain composite with thrombin-like properties, Line 310

The words papain composite suggest that the authors are using a modified papain. Is that the case? If so, the structure of this composite should be clearly described. 

Comments on the Quality of English Language

Due to the modulation of the intrinsic and extrinsic coagulant pathways, papain act as anticoagulants. Line 281

The authors could write that  .... papain acts as an anticoagulant.   

When compared with our previous paper about the pH stability and thermostability of papain it shows broader specificity. Line 326

What does the word it refer to here? This is a very confusing sentence.