Chemical Characterization and Antiproliferative Evaluation of Compounds Isolated from White Shrimp (Penaeus vannamei) By-Products

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Good manuscript
   

   Is the phthalate compound belongs to biosynthetic pathway in shrimp?

   Or it is a bi product related to pollution in marine system

   Is there any isolated compounds from vertebra related to Phethalate.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a study on the components of isolate extracts obtained from the white shrimp species Penaeus vannamei, including discussions on the physicochemical characteristics of the extract fractions and their cytotoxicity. Cancer remains a major global health challenge and a leading cause of mortality; consequently, the search for effective therapeutic agents continues to be a relevant and ongoing research focus. The article is well-structured, clearly articulating its objectives and tasks. The authors support their findings with recent literature, the majority of which originates from the last five years. Modern physicochemical analysis techniques, such as ¹H and ¹³C NMR spectroscopy, infrared, and UV-visible spectroscopy, are employed alongside up-to-date biological investigation methodologies. The assessment of cytotoxicity involves cell lines such as HCT-116, A-549, HeLa, MDA-MB-231, 22Rv1, as well as a non-cancerous cell line (ARPE-19), with positive controls including doxorubicin and cisplatin. Additionally, results concerning cell viability of bioactive fractions, as well as data from fluorescence cell staining and microscopy assays, are presented.

A noteworthy point for discussion relates to the use of carotenoids in oncology. The literature presents both supportive and critical evidence regarding their therapeutic potential. For instance, J. Xu et al. (Mol. Cell Probes, 2019, 46, 101419) demonstrated a positive influence of lycopene on the ovarian cancer cell line SKOV3. Conversely, Y. Zhang et al. (Nutrients, 2022, 14, 1284) critically discussed the potential carcinogenic effects of certain carotenoids, including the risks associated with β-carotene supplementation. To strengthen the manuscript, a more detailed discussion of this topic, weighing the pros and cons of carotenoid use in oncological contexts, would be beneficial.

Furthermore, several considerations could be incorporated into the Results and Discussion sections to enhance comprehensiveness. The primary focus of the study is on specific bioactive components – namely, bis(2-ethylhexyl) terephthalate and carotenoids such as violaxanthin and neoxanthin – in extracts from the exoskeleton and cephalothorax of P. vannamei. First, it would be valuable to include data on the cytotoxicity of these compounds evaluated individually. Isolating the main constituents and assessing their biological activity independently could provide more precise insights. If existing literature reports on the cytotoxic effects of bis(2-ethylhexyl) terephthalate, violaxanthin, or neoxanthin, integrating or comparing these findings would allow for more comprehensive conclusions regarding their bioactive roles.

Second, extending the study to include a stepwise extraction from shrimp samples collected across different seasons or catches could elucidate the stability and variability of bioactive compound concentrations. This approach would provide insights into the consistency of the active substances over time and their potential for reliable future applications.

Third, for compounds with established biological activity – especially those derived from marine organisms – assessing residual heavy metal contamination is critical. Such data are essential for evaluating antitumor effects and understanding mechanisms of action.

Finally, including spectra of individual compounds as supplementary material would facilitate verification and support further research.

As a final suggestion, the authors might consider reducing the current self-citation rate (approximately 17%) to improve the objectivity and balance of references.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

The authors present an interesting study investigating bioactive compounds derived from white shrimp by-products and their antiproliferative effects on prostate cancer cells. The experimental design is appropriate, and the study is strengthened by the inclusion of microscopic imaging and cell viability assays, which provide valuable visual and quantitative support for the reported biological effects. However, despite the scientific relevance of the topic and the experimental effort, the manuscript contains numerous grammatical and language errors, as well as colloquial expressions that are not appropriate for a scientific publication. These issues significantly affect the clarity and readability of the text and must be thoroughly corrected. Scientific terminology should be used consistently, and informal or ambiguous wording should be replaced with precise, discipline-appropriate expressions. In addition, the manuscript would benefit from a more comprehensive and up-to-date review of the scientific literature. The authors state that marine-derived compounds, including those obtained from shrimp, have gained increasing attention as important sources of bioactive molecules. Such claims should be adequately supported by citing recent research articles and, where relevant, patents published in the last few years. This applies particularly to the Introduction and Conclusions, where the broader scientific and translational relevance of the study should be clearly justified.

Overall, while the study addresses an important topic and presents promising results, substantial language revision and strengthening of the literature context are required before the manuscript can be considered for publication.

Line 34:Cancer is characterized by the uncontrolled growth and reproduction of cells, and it can arise and spread to any part of the body

• The term “reproduction of cells” is considered colloquial. In scientific writing, “cell proliferation” is the appropriate term.
• specifying invasion and metastasis provides a more proffesional description of cancer progression, as the term “spread” alone is nonspecific.
• “any part of the body” can be refined to more accurately reflect tissue and organ specificity.

For example: Cancer is characterized by uncontrolled cell proliferation and the ability to invade surrounding tissues and metastasize to distant organs.

Line 40: chemopreventives agents- chemopreventive agents

Line 42: compounds that intervene in the process of cell division or promote cell death collectively referred to as antiproliferative compounds - have become the focus of inten sive study.

Apoptosis is not same as antiproliferation. I recommend to correct for: compounds that intervene in the process of cell division or promote cell death collectively referred to as antiproliferative compounds - have become the focus of inten sive study.

(Apoptosis is not same as antiproliferation.)

Line 43: Marine organisms have reacently gained attention as a source of compounds with anticancer properties.  Correct: recently

Add literature source with examples from recent articles : For example: Marine natural products as a source of novel anticancer drugs: an updated review (2019–2023), Hesham R. El-Seedi et al., 2025

 

Line 44: These include molecules directly isolated from marine environments or those serving as models for identifying novel targets in cancer therapy .

Correct grammar: These include molecules directly isolated from marine environments, as well as compounds that serve as lead structures or molecular templates for the development of novel anticancer agents.

LINE 49–50 Previous studies have examined the chemopreventive potential of by-products from white shrimp (P. vannamei), demonstrating antioxidant and antimutagenic properties.

 

The sentence is generally correct; however, the term “chemopreventive potential” is used too broadly, as antioxidant and antimutagenic activities alone do not necessarily equate to chemoprevention in vivo. Clarification of the context is recommended.

Example: Previous studies have examined the potential chemopreventive-related properties of by-products from white shrimp (P. vannamei), demonstrating antioxidant [5] and antimutagenic [6] activities.

LINE 51–52 Additionally, lipidic* compounds extracted from the muscle tissue of both wild shrimp (Penaeus stylirostris) and white shrimp (P. vannamei) have shown antiproliferative effects against various human cancer lines.
The phrase “human cancer lines” is imprecise and should be replaced with “human cancer cell lines”. In addition, specifying in vitro activity would improve scientific accuracy.

For example: Additionally, lipidic compounds extracted from the muscle tissue of both wild shrimp (Penaeus stylirostris) and white shrimp (P. vannamei) have shown antiproliferative effects against various human cancer cell lines in vitro …

*Also, I reccoment clarify the “ lipidic”: lipid-derived compounds, fatty acid–rich lipid fractions or lipophilic compounds …

 

LINE 54–56: Investigating the impact of bioactive compounds – such as carotenoids found in P. vannamei – on cancer cell lines, along with the analysis of morphological features associated with cell death, is crucial for advancing in the understanding of the molecular mechanisms underlying cancer progression and treatment.

The phrase “advancing in the understanding” is grammatically incorrect. Moreover, the sentence is overly long and stylistically heavy, reducing clarity.

Please correct for example like: …Investigating the effects of bioactive compounds, such as carotenoids found in P. vannamei, on cancer cell lines, together with the analysis of morphological features associated with cell death, is crucial for advancing the understanding of molecular mechanisms underlying cancer progression and treatment  

LINE 57–58 These studies reveal how natural compounds interact with specific signaling pathways and modulate key cellular processes, offering opportunities to develop targeted therapies with fewer adverse effects.

but slightly speculative; it would benefit from more cautious wording. I recommend add some literature sources and examples of signalling pathways.

These studies provide insight into how natural compounds interact with specific signaling pathways and modulate key cellular processes, potentially offering opportunities for the development of targeted therapies with reduced adverse effects.

 

LINE 59–61  Moreover, identifying distinct morphological markers of cell death helps in recognizing specific pathways that could be activated or suppressed in pathological contexts, thereby optimizing therapeutic strategies.

The phrase “helps in recognizing” is stylistically weak.
Moreover, identifying distinct morphological markers of cell death facilitates the recognition of specific pathways that may …

 

LINE 63–66 Given these considerations, the search for compounds that prevent or inhibit cancer development has become essential. White shrimp, one of the most widely consumed seafood products worldwide, may contain biologically activity, therefore, the aim of this study was to isolate and identify the compounds from P. vannamei by-products with antiproliferative activity against human cancer cell lines.

• “biologically activity” – biological activity ?
• sentence is overly long and should be split
• the study aim should be stated more clearly and formally

 

LINE 178–180 Inconsistency in variability reporting: standard deviation is used here, whereas figure captions later report standard error. This should be unified throughout the manuscript.

Use either SD or SEM consistently in text, tables, and figure legends (compare lines 178 and 191- standard deviation and standard error).

 

Line 181-185: The conclusion of “selective activity” is overstated. ARPE-19 is a single immortalized cell line and cannot represent healthy cells in general. The wording should be more cautious.

 

Line 186- 188: A dose-response relationship was observed…

The statement is acceptable, however, no statistical model or IC50 calculation is mentioned here. Consider specifying whether the relationship was statistically tested, same in lines 213–214

 

Line 190-192: 22Rv1 viability exposed to exoskeleton hexanic and acetonic extracts…

 “Hexanic” and “acetonic” are non-standard adjectives in scientific English, use hexane and acetone extracts…

 

LINE 205–207 (Figure 2) …surpassing the efficacy of cis-platin and doxorubicin…

 

Direct comparison with chemotherapeutic drugs at identical mass concentrations is potentially misleading due to differences in purity, molecular weight, and mechanism of action.

I reccoment write like …showing a stronger inhibitory effect than cisplatin and doxorubicin under the tested conditions.

 

LINE 199–203 …efficacy of cisplatin and doxorubicin…

 

The claim is strong and should be tempered. “Efficacy” implies clinical relevance, which is not established. Correct whis fragment like :fraction H3 showed the strongest antiproliferative effect among the tested fractions…

 

LINE 233–236 Elucidation of the compounds presents in the fraction H3 was performed…

Grammatical errors …“presents”and lack of clarity.

Pleas correct this sentence for example:

The structural elucidation of compounds present in fraction H3 was performed using ¹H and ¹³C NMR spectroscopy.

 

LINE 237–243 The NMR signal assignment is overly verbose and contains repeated grammatical errors “correspond” -> “corresponds”. Consider condensing and moving detailed assignments to Supplementary Materials.

LINE 265–267 Based on the combined data… dibutyl terephthalate was identified as one of the major constituents.

Ok, but I understand that sentence contradicts earlier statements identifying Bis(2-ethylhexyl) terephthalate as the major compound. Clarification is required.

 

LINE 275–278 These peaks coincide with… neoxanthin and violaxanthin.

UV–Vis spectra alone are insufficient for definitive carotenoid identification. The wording should reflect tentative assignment. maybe some earlier your published article where the reader can check the spectrum assignment?

LINE 296–299 Due to complexity composition…

Grammatical error “complexity composition”. Also, the argument is valid but should be more concise. The comparison with dibutyl phthalate activity in leukemia cells should explicitly state that this does not necessarily translate to prostate cancer models.

LINE 320–324…it is unlikely to be the main contributor… carotenoids are more likely responsible…
The conclusion is reasonable but should acknowledge the mixed composition of H3 and potential synergistic effects.

…suggesting that carotenoids such as violaxanthin and neoxanthin, possibly in combination with other minor constituents, contribute to the observed activity..

 

Comments on the Quality of English Language

The overall quality of the English language in the manuscript is below the standard required for publication in an international scientific journal. The text contains numerous grammatical errors, incorrect verb forms, missing articles, improper prepositions, and inconsistent use of singular and plural forms. In several sections, sentence structure is awkward or unclear, which negatively affects readability and comprehension. In addition, the manuscript frequently employs colloquial or non-scientific expressions, such as imprecise terminology  “reproduction of cells”, “lipidic compounds”, “gained attention” without appropriate context and informal phrasing that should be replaced with established scientific language. Scientific terms are sometimes used inconsistently, and several statements lack the precision expected in academic writing. The manuscript would benefit from substantial language editing by a native English speaker or a professional scientific editing service. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript incorporates significant corrections made by the authors. Additionally, the detailed responses to the reviewer’s comments help clarify the study’s main objectives and the scope of the research conducted.

The changes introduced in the Introduction section strengthen the presentation of the study’s aims and provide a comprehensive overview of the biological activity of compounds derived from various marine organisms. These modifications improve the clarity of the manuscript, facilitate a better understanding of the importance of studying marine organisms and their biochemical products, and highlight the broader scientific relevance of the work within the context of global research efforts in this area.

Revisions related to the discussion of the potential benefits and risks associated with carotenoids add further depth to the manuscript and provide valuable context to this complex, unresolved issue.

The authors’ detailed explanation regarding the possible biological effects of residual heavy metal concentrations offers insight into the practical insolubility of metal salts and complexes in the solvents specified in the study, such as hexane, methanol, and acetone. However, it is well known that many metal-containing compounds, salts, or complexes can exhibit partial or even significant solubility in these solvents. Additionally, residual concentrations of heavy metal ions at the picogram level can sometimes be sufficient to exert biological activity. It is understood that the authors have considered this and may further refine their experimental approach in future studies.

It is somewhat unfortunate that access to the supplementary materials associated with this manuscript was not possible, as such materials would have further supported the interpretation and understanding of the work.

Overall, these comments do not diminish the scientific value of the study. I recommend acceptance of the manuscript for publication.

Author Response

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Author Response File: Author Response.docx