Astaxanthin as a Natural Photoprotective Agent: In Vitro and In Silico Approach to Explore a Multi-Targeted Compound

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Recommendation: Major Revision

The manuscript investigates the photoprotective effects of Astaxanthin against UVB-induced damage in HaCaT keratinocytes. The study employs a comprehensive approach ranging from antioxidant assays and cell functional experiments (migration, adhesion, apoptosis) to in silico molecular docking targeting inflammatory pathways (JAK2/STAT3). Additionally, the authors formulated an Astaxanthin-enriched cream and evaluated its SPF. The topic is relevant to dermatological science and cosmetic applications. However, there are significant concerns regarding the validation of the proposed mechanisms and the presentation of quantitative data that need to be addressed before publication.

Major Comments

1. Lack of Experimental Validation for Molecular Docking Results: The authors performed in silico molecular docking and predicted strong binding affinities between Astaxanthin and key targets such as JAK2 (-9.9 kcal/mol), STAT3, and MMPs. However, the results section does not explicitly mention any wet-lab validation (e.g., Western Blot, qPCR, or immunofluorescence) to confirm these interactions. Computational prediction alone is insufficient to claim a mechanism of action. The authors must provide experimental evidence showing that Astaxanthin treatment actually inhibits the phosphorylation or expression of JAK2, STAT3, and NF-kB in UVB-irradiated cells. The experiments must be performed.
2. Insufficient Quantitative Data in Abstract: The abstract relies heavily on qualitative descriptors (e.g., "mitigated," "reduced," "enhanced," "strong binding") but lacks specific quantitative data. Please include key numerical results in the abstract to demonstrate the magnitude of the effects. Specifically:

• The SPF value of the formulated cream (this is a critical translational outcome).
• The percentage of recovery in cell viability (MTT assay).
• The fold-change in ROS reduction or apoptotic rates.

Pls revise abstract part carefully.

 

3. Rationale for UVB Dosage: The study uses a dose of 30 mJ/cm².

Please justify this specific dosage in the Methods section. Is this a cytotoxic dose (IC50) or a sub-lethal dose intended to induce senescence/inflammation? The rationale for selecting this intensity should be clearly stated.

4. Unacceptable Figure Quality: The quality of the figures is far below the standard required for publication.

• Specific Example: In Figure 3A (Line 269), the formatting is sloppy. The lines (e.g., the underline for "AST") are not straight, and the alignment of labels is poor. This lack of precision raises concerns about the author's basic research standards and attention to detail.
• Requirement: All figures must be professionally re-generated. Do not simply resize low-resolution images. Use professional graphing software (e.g., GraphPad Prism, Origin, or Illustrator) to ensure high resolution (300+ dpi), consistent font sizes, and precise alignment.

5. Careless Errors in Text: There are obvious typographical errors that indicate a lack of proofreading.

• Specific Example: In Line 333, the text reads: "...pendent manner (p < 0.001 (p < 0.001). (Figure6)". This repetition and formatting error is confusing. The entire manuscript must be carefully proofread to eliminate such distinct errors.

6. Lack of Quantitative Description and Statistical Analysis: The Results section describes findings qualitatively (e.g., "increased," "decreased") but fails to provide specific data points or statistical details in the text. Provide quantitative data in the text (e.g., "Cell viability increased from 45% ± 3% to 80% ± 4%"). Clearly state the statistical significance for each comparison (e.g., "p < 0.01 vs. control").

 

Author Response

Reviwer1

Recommendation: major revision

“The manuscript investigates the photoprotective effects of astaxanthin against UVB-induced damage in HaCaT keratinocytes. The study employs a comprehensive approach ranging from antioxidant assays and cell functional experiments (migration, adhesion, apoptosis) to in silico molecular docking targeting inflammatory pathways (JAK2/STAT3). Additionally, the authors formulated an astaxanthin-enriched cream and evaluated its SPF. The topic is relevant to dermatological science and cosmetic applications. However, there are significant concerns regarding the validation of the proposed mechanisms and the presentation of quantitative data that need to be addressed before publication.

Major Comments

1. “Lack of Experimental Validation for Molecular Docking Results: The authors performed in silicomolecular docking and predicted strong binding affinities between Astaxanthin and key targets such as JAK2 (-9.9 kcal/mol), STAT3, and MMPs. However, the results section does not explicitly mention any wet-lab validation (e.g., Western Blot, qPCR, or immunofluorescence) to confirm these interactions. Computational prediction alone is insufficient to claim a mechanism of action. The authors must provide experimental evidence showing that Astaxanthin treatment actually inhibits the phosphorylation or expression of JAK2, STAT3, and NF-kB in UVB-irradiated cells. The experiments must be performed.

Response: We thank the reviewer for this important comment and we are fully agree that molecular docking alone cannot establish a definitive mechanism of action. Our intention was not to claim inhibition of JAK2/STAT3 or NF-κB, but rather to provide predictive in silico insights supporting the multifunctional effects observed in vitro.

We respectfully admit, however, that performing additional protein level validation experiments such as Western blot, qPCR, or immunofluorescence is beyond the scope and resources of the current study, which was designed as an initial exploratory investigation combining cellular assays with computational analysis.

However, to address the reviewer’s concern, we have made the following revisions:

1. We clarified the role of docking in the manuscript

We now explicitly state in the discussion section that docking results are predictive, not confirmatory.

Line 579: “ Although docking simulations predicted strong binding affinities between astaxanthin and JAK2, STAT3, COX-2, NF-κB, MMP2, and MMP9, these results remain computational predictions”.

2. We added a dedicated limitations paragraph

A new paragraph acknowledges that in vitro and in vivo analyses of JAK2/STAT3/NF-κB phosphorylation are required in future work to validate the proposed mechanism.

Line 581: “The current study did not include protein-level analyses such as phosphorylation assays, Western blotting, or immunofluorescence. Therefore, we cannot conclude direct molecular inhibition. Future work will focus on experimentally in vitro and in vivo validating these interactions in UVB-exposed keratinocytes...

3. We removed any language implying established causality

We revised statements that could be interpreted as mechanistic claims and replaced them with wording such as “may contribute,” “suggests a potential interaction,” and “requires further confirmation.”

4. We modified the conclusion

We removed wording that implied direct mechanistic inhibition and clarified that our conclusions pertain to photoprotective cellular outcomes, supported (but not proven) by docking.

6. Conclusions

This study suggests that astaxanthin exerts multifaceted photoprotective effects in HaCaT keratinocytes. Astaxanthin improved cell survival by reducing intracellular ROS and NO levels and by preserving lysosomal stability under UVB-induced stress. The molecular docking analysis provided supportive, hypothesis-generating insights by predicting favorable interactions with several proteins involved in inflammation and extracellular matrix remodeling (JAK2/STAT3, COX-2, NF-κB, MMPs) as well as focal adhesion kinase (FAK). These computational predictions complement the cellular findings. Altogether, the present data indicate that astaxanthin is a promising candidate for mitigating cellular processes associated with UV-induced skin damage, while further mechanistic validation remains necessary”.

2. “Insufficient Quantitative Data in Abstract: The abstract relies heavily on qualitative descriptors (e.g., "mitigated," "reduced," "enhanced," "strong binding") but lacks specific quantitative data. Please include key numerical results in the abstract to demonstrate the magnitude of the effects. Specifically: The SPF value of the formulated cream (this is a critical translational outcome).The percentage of recovery in cell viability (MTT assay).The fold-change in ROS reduction or apoptotic rates. Pls revise abstract part carefully”.

Response:  We thank the reviewer for highlighting the need for quantitative data in the abstract. We have carefully revised the abstract to include key numerical results demonstrating the magnitude of the effects.  Line 12: “Ultraviolet B radiation is a major cause of skin aging, cellular senescence, and inflammaging, mediated by the excessive production of reactive oxygen species (ROS) and induction of apoptosis. This study evaluated the photo-protective effects of astaxanthin, one of natural strongest antioxidants, in UVB-treated keratinocytes. Antioxidant capacity of astaxanthin was evaluated by ABTS, DPPH, NBT/riboflavin/SOD assays. Hacat cells were exposed to 30mJ/cm² of UVB radiation. Photoprotective effects and accumulated ROS were evaluated in UVB-irradiated HaCaT cells by MTT and DCFH-DA assays. Nitric oxide levels were quantified using the Griess reagent. Apoptosis was assessed by dual staining using acridine orange/ethidium bromide, lysosomal integrity by acridine orange uptake, cell migration by scratch. Cell adhesion was assessed on ECM-coated Nunc plates. Finally, we formulated 0.5% enriched-astaxanthin cream. Astaxanthin mitigated UVB-induced damage by reducing intracellular ROS levels by 3.7 fold, decreasing nitric oxide production by 48% at higher concentration, and maintaining lysosomal integrity. The caratenoind Astaxanthin significantly enhanced cell viability, increasing it from 60.64 ± 8.3% in UV-treated cells to 102.1 ± 3.22% at 40 µM. Moreover, treated cells showed a significant reduction (p<0.001) in the apoptotic rate (37.7 ± 3.1 vs 87.7 ± 3.8 in UVB-irradiated cells, as evidenced by reduced chromatin condensation and nuclear fragmentation. Astaxanthin also enhanced tissue repair, as evidenced by increased cell migration and adhesion to several extracellular matrix (ECM) proteins (poly-L-lysine, laminin, fibrinogen, vitronectin and collagen I). In silico molecular docking predicted strong binding affinities between astaxanthin and key cellular targets, including JAK2 (−9.9 kcal/mol, highest affinity), STAT3, FAK, COX2, NF-k-B, MMP2, and MMP9.The formulated cream demonstrated an in vitro SPF of 7.2±2.5. Astaxanthin acts as a multifunctional photoprotective compound, providing a strong rationale for its incorporation into cosmetic and dermatological formulations, as further supported by the successful formulation and in vitro SPF estimation of an astaxanthin-enriched cream”.

3. “Rationale for UVB Dosage: The study uses a dose of 30 mJ/cm². Please justify this specific dosage in the Methods section. Is this a cytotoxic dose (IC50) or a sub-lethal dose intended to induce senescence/inflammation? The rationale for selecting this intensity should be clearly stated”.

Response: We thank the reviewer and we added the clarification in Line 157:  “2.5. UVB Radiation Cells were pretreated with different concentrations of astaxanthin for 2 h before UVB irradiation. The UVB irradiation was conducted at an energy dose of 30 mJ/cm² using a Vilber-Lourmat VL-4LC UV lamp (8W, 230V, 50/60 Hz). The dose of 30 mJ/cm² was selected because it provides a reproducible, sublethal oxidative stress model in HaCaT keratinocytes and is widely used in photoprotection studies [24–26]. Hong et al. (2022) and Ha et al. (2022). Before experiments, the irradiance at the cell surface was measured with a calibrated UV radiometer (VLX-3W, Vilber) at the culture plane. Exposure time was calculated using:

 

 Time (s)

Before each experiment, the irradiance at the cell surface in 75 cm² flasks was measured with a calibrated UV radiometer (VLX-3W, Vilber) at the culture plane. For our experiment, the irradiance at the cell surface was 1.5 mW/cm², corresponding to an exposure time of 20 s for the 30 mJ/cm² dose. All irradiations were performed with the culture medium removed. Following irradiation, fresh medium was added, and cells were incubated at 37°C for the indicated periods. Thereafter, cells were either processed for analysis or maintained in culture as required”.

1. Unacceptable Figure Quality: The quality of the figures is far below the standard required for publication.

• Specific Example: In Figure 3A (Line 269), the formatting is sloppy. The lines (e.g., the underline for "AST") are not straight, and the alignment of labels is poor. This lack of precision raises concerns about the author's basic research standards and attention to detail. Requirement: All figures must be professionally re-generated. Do not simply resize low-resolution images. Use professional graphing software (e.g., GraphPad Prism, Origin, or Illustrator) to ensure high resolution (300+ dpi), consistent font sizes, and precise alignment.

Response: For pointing out the issues with figure quality. We have carefully re-generated all figures, including Figure 3A, using GraphPad Prism 10. All figures are now high-resolution (≥300 dpi), with consistent font sizes, precise alignment, and clear labeling to ensure clarity and publication quality.

1. “Careless Errors in Text: There are obvious typographical errors that indicate a lack of proofreading. Specific Example: In Line 333, the text reads: "...pendent manner (p < 0.001 (p < 0.001). (Figure6)". This repetition and formatting error is confusing. The entire manuscript must be carefully proofread to eliminate such distinct errors”.

• Response: We thank the reviewer for pointing out the typographical and formatting errors. The manuscript has been carefully proofread and revised to correct all such mistakes, including the specific example noted in Line 333 (“...pendent manner (p < 0.001 (p < 0.001). (Figure6)”), which has now been corrected. All figures, legends, and text have been reviewed to ensure clarity, proper formatting, and consistency throughout the manuscript.

1. Lack of Quantitative Description and Statistical Analysis: The Results section describes findings qualitatively (e.g., "increased," "decreased") but fails to provide specific data points or statistical details in the text. Provide quantitative data in the text (e.g., "Cell viability increased from 45% ± 3% to 80% ± 4%"). Clearly state the statistical significance for each comparison (e.g., "p < 0.01 vs. control").

Response: We appreciate the reviewer’s valuable comment regarding the lack of quantitative details in the results section. The manuscript has been revised to include specific quantitative data for all reported findings. For example, statements previously described qualitatively as “increased” or “decreased” now provide precise values along with measures of variability (mean ± SD) and statistical significance. All comparisons now clearly indicate the statistical analysis performed, and the results section has been carefully updated to ensure that the data presentation meets publication standards.

Reviewer 2 Report

Comments and Suggestions for Authors

This research provided valuable information to demonstrate the astaxanthin acted as a multifunctional photoprotective compound. However, there are some points that need to be improved in the manuscript.

Major Comments:

1. In Line 142, the UVB irradiation was conducted at an energy dose of 30 mJ/cm². Why choose the energy dose of 30 mJ/cm², and what considerations or references were made to previous studies? What is the irradiation duration? Please describe these details.
2. Please label the individual panels in Figure 1 as (A), (B), and (C) to clearly indicate which bar graph corresponds to the DPPH and ABTS assays, respectively.
3. The concentration of astaxanthin in the methods section is described as 2.5-80 μ However, the x-axis of Figure 1 uses the unit μg/mL. Please unify the dosage unit of astaxanthin throughout the manuscript. Furthermore, inconsistencies in the units for astaxanthin are also present between the figure titles and the bar graphs in Figures 2 and 5 (as mentioned in Line 249 and Line 310). All units must be consistent.
4. The study provided evidence that ATX pretreatment can significantly restore UVB-impaired cell adhesion across a range of ECM substrates. In the section 3.7, ATX restores adhesion to poly-L-lysine, laminin, fibrinogen, and collagen I, but not to vitronectin. This is a very interesting discovery. It suggests that the protective effect of astaxanthin may be highly specific. But the discussion section did not delve into the signaling pathways potentially regulating the changes in these matrix proteins. This could help to better elucidate the mechanistic targets of astaxanthin.
5. Although the molecular docking analysis suggests potential binding of AST to key proteins like JAK2 and STAT3 etc, this finding lacks experimental validation at the cellular level. It is recommended that the authors perform analyses such as Western Blot to assess the phosphorylation levels (activation status) of JAK2 and STAT3, and utilize qPCR or Western Blot to examine the expression changes of downstream target genes. These data are crucial for establishing a complete chain of evidence, to better elucidate the mechanism of action of astaxanthin.

Minor Comment:

1. The first sentence of the manuscript is missing its initial capital letter.
2. Please ensure that the units for the AST dosage are consistent throughout the manuscript, including both the main text and all figures.
3. The reference list requires standardization. Please ensure that journal abbreviations (or full names) are used consistently across all references.
4. Please provide higher-resolution images with greater clarity for the manuscript, particularly for the molecular docking results figure.

Author Response

Reviwer 2

1. “In Line 142, the UVB irradiation was conducted at an energy dose of 30 mJ/cm². Why choose the energy dose of 30 mJ/cm², and what considerations or references were made to previous studies? What is the irradiation duration? Please describe these details”.

Response:  We thank the reviewer for this request for clarification. We added the requested clarification in Line 158 “ 2.5. UVB Radiation

Cells were pretreated with different concentrations of astaxanthin for 2 h before UVB irradiation. The UVB irradiation was conducted at an energy dose of 30 mJ/cm² using a Vilber-Lourmat VL-4LC UV lamp (8W, 230V, 50/60 Hz). The dose of 30 mJ/cm² was selected because it provides a reproducible, sublethal oxidative stress model in HaCaT keratinocytes and is widely used in photoprotection studies [24–26]. Hong et al. (2022) and Ha et al. (2022). Before experiments, the irradiance at the cell surface was measured with a calibrated UV radiometer (VLX-3W, Vilber) at the culture plane. Exposure time was calculated using:

 

 Time (s)

 

Before each experiment, the irradiance at the cell surface in 75 cm² flasks was measured with a calibrated UV radiometer (VLX-3W, Vilber) at the culture plane. For our experiment, the irradiance at the cell surface was 1.5 mW/cm², corresponding to an exposure time of 20 s for the 30 mJ/cm² dose. All irradiations were performed with the culture medium removed. Following irradiation, fresh medium was added, and cells were incubated at 37°C for the indicated periods. Thereafter, cells were either processed for analysis or maintained in culture as required.”

2. “Please label the individual panels in Figure 1 as (A), (B), and (C) to clearly indicate which bar graph corresponds to the DPPH and ABTS assays, respectively.”

Response: Thank you for this suggestion. We have revised Figure 1 to clearly label the individual panels as (A), (B), and (C), indicating which bar graph corresponds to the DPPH and ABTS assays, respectively. This clarification improves the figure’s readability and ensures that the data are easily interpretable.

3. “The concentration of astaxanthin in the methods section is described as 2.5-80 μ However, the x-axis of Figure 1 uses the unit μg/mL. Please unify the dosage unit of astaxanthin throughout the manuscript. Furthermore, inconsistencies in the units for astaxanthin are also present between the figure titles and the bar graphs in Figures 2 and 5 (as mentioned in Line 249 and Line 310). All units must be consistent”

Response: We thank the reviewer for pointing out this inconsistency. We have carefully reviewed the manuscript and unified the concentration units of astaxanthin throughout the text, figure titles, and axes. All concentrations are now consistently reported as μM in the Methods section, Figures 1, 2, and 5, and corresponding figure legends.

4. The study provided evidence that ATX pretreatment can significantly restore UVB-impaired cell adhesion across a range of ECM substrates. In the section 3.7, ATX restores adhesion to poly-L-lysine, laminin, fibrinogen, and collagen I, but not to vitronectin. This is a very interesting discovery. It suggests that the protective effect of astaxanthin may be highly specific. But the discussion section did not delve into the signaling pathways potentially regulating the changes in these matrix proteins. This could help to better elucidate the mechanistic targets of astaxanthin.

Response” We thank the reviewer for highlighting this important point. Indeed, our results show that astaxanthin (ATX) pretreatment significantly restores UVB-impaired adhesion to poly-L-lysine, laminin, fibrinogen, and collagen I, but not to vitronectin, suggesting a substrate-specific protective effect. We added this discussion of this finding in Line 523: “ However, astaxanthin likely mediates its substrate-specific restoration of UVB-impaired cell adhesion, effective on poly-L-lysine, laminin, fibrinogen, and collagen I but not vitronectin, through inhibition of matrix metalloproteinases, such as MMP 2, MMP 7, and MMP-9, which degrade these ECM components under oxidative stress. This protection involves suppression of the PI3K/AKT/mTOR/NF-κB signaling axis activated by UVB-induced ROS, thereby preserving ECM integrity and integrin-mediated adhesion for β1-integrin substrates (like laminin and collagen I) while sparing αvβ3/β5-dependent vitronectin interactions. The vitronectin selectivity may reflect astaxanthin's limited modulation of RGD-specific pathways or urokinase plasminogen activator systems, highlighting a mechanistic target for future integrin/MMP-focused studies [19,42,43]. »

5. “Although the molecular docking analysis suggests potential binding of AST to key proteins like JAK2 and STAT3 etc, this finding lacks experimental validation at the cellular level. It is recommended that the authors perform analyses such as Western Blot to assess the phosphorylation levels (activation status) of JAK2 and STAT3, and utilize qPCR or Western Blot to examine the expression changes of downstream target genes. These data are crucial for establishing a complete chain of evidence, to better elucidate the mechanism of action of astaxanthin”.

Response:

Response: We thank the reviewer for this important comment and we are fully agree that molecular docking alone cannot establish a definitive mechanism of action. Our intention was not to claim inhibition of JAK2/STAT3 or NF-κB, but rather to provide predictive in silico insights supporting the multifunctional effects observed in vitro.

We respectfully admit, however, that performing additional protein level validation experiments such as Western blot, qPCR, or immunofluorescence is beyond the scope and resources of the current study, which was designed as an initial exploratory investigation combining cellular assays with computational analysis.

However, to address the reviewer’s concern, we have made the following revisions:

1. We clarified the role of docking in the manuscript

We now explicitly state in the discussion section that docking results are predictive, not confirmatory.

Line 579: “ Although docking simulations predicted strong binding affinities between astaxanthin and JAK2, STAT3, COX-2, NF-κB, MMP2, and MMP9, these results remain computational predictions”.

2. We added a dedicated limitations paragraph

A new paragraph acknowledges that in vitro and in vivo analyses of JAK2/STAT3/NF-κB phosphorylation are required in future work to validate the proposed mechanism.

Line 581: “The current study did not include protein-level analyses such as phosphorylation assays, Western blotting, or immunofluorescence. Therefore, we cannot conclude direct molecular inhibition. Future work will focus on experimentally in vitro and in vivo validating these interactions in UVB-exposed keratinocytes...

3. We removed any language implying established causality

We revised statements that could be interpreted as mechanistic claims and replaced them with wording such as “may contribute,” “suggests a potential interaction,” and “requires further confirmation.”

4. We modified the conclusion

We removed wording that implied direct mechanistic inhibition and clarified that our conclusions pertain to photoprotective cellular outcomes, supported (but not proven) by docking.

“6. Conclusions

This study suggests that astaxanthin exerts multifaceted photoprotective effects in HaCaT keratinocytes. Astaxanthin improved cell survival by reducing intracellular ROS and NO levels and by preserving lysosomal stability under UVB-induced stress. The molecular docking analysis provided supportive, hypothesis-generating insights by predicting favorable interactions with several proteins involved in inflammation and extracellular matrix remodeling (JAK2/STAT3, COX-2, NF-κB, MMPs) as well as focal adhesion kinase (FAK). These computational predictions complement the cellular findings. Altogether, the present data indicate that astaxanthin is a promising candidate for mitigating cellular processes associated with UV-induced skin damage, while further mechanistic validation remains necessary”.

 

Minor Comment:

1. The first sentence of the manuscript is missing its initial capital letter.

Response: We corrected accordingly

1. Please ensure that the units for the AST dosage are consistent throughout the manuscript, including both the main text and all figures.

Response: We have corrected accordingly

 

1. The reference list requires standardization. Please ensure that journal abbreviations (or full names) are used consistently across all references.

Response: All references have been accurately checked for style, with ZOTERO. All of them appear now consistently in accordance to Journal’s guidelines.

1. Please provide higher-resolution images with greater clarity for the manuscript, particularly for the molecular docking results figure.

Response: We provided higher-resolution images

Reviewer 3 Report

Comments and Suggestions for Authors

The development of photoprotective agents remains an important goal in pharmaceutical sciences. In the present manuscript, Lahmar et al. report on their research into the photoprotective properties of astaxanthin, employing both in vitro and in silico methods. The compound demonstrated promising results, which makes it potentially interesting for the readers of Scientia Pharmaceutica and aligns well with the journal’s scope. However, several aspects require improvement prior to publication:

1) The introduction does not clearly explain why current treatment methods are inadequate. Additionally, including epidemiological data would be beneficial to contextualise the study’s significance.

2) The authors used the percentage of inhibition to characterise antioxidant activity in the ABTS and DPPH assays. However, this parameter depends on the concentration of radicals in the analysed solutions and may therefore vary across different studies. A more relevant approach would be to use a stoichiometric coefficient, which reflects the number of radicals scavenged by one molecule of the analysed compound [10.1002/kin.21572; 10.3390/ijms26125659].

3) There appears to be an error in the formula presented on line 139.

4) Regarding the molecular docking study. What was the pH of the medium used in the simulations? How was the ionization state of both the ligands and the biological target accounted for in the modelling?

5) To validate the docking model, a redocking experiment should be performed. The results should include data on binding affinity and root-mean-square deviation (RMSD) values to assess the reliability of the protocol.

6) Please provide RMSD data for the current molecular docking results to allow assessment of the structural stability and consistency of the predicted poses.

Thank you for your attention to these details. I look forward to receiving the revised manuscript for re‑evaluation in the near future.

With best regards,

The Reviewer

Author Response

1) The introduction does not clearly explain why current treatment methods are inadequate. Additionally, including epidemiological data would be beneficial to contextualise the study’s significance.

Response “We thank the reviewer for this valuable comment. We agree that contextualizing the burden of UV-related skin damage and the limitations of current therapeutic strategies strengthens the rationale of the study. To address this point, we have revised the introduction by adding this paragraph Line 52 “Current photo-protection strategies, predominantly reliant on topical sunscreens, remain inadequate due to inconsistent full-spectrum coverage [5]. Inorganic filters such as zinc oxide and titanium dioxide provide reliable UVA/UVB blockade but are marred by aesthetic drawbacks like visible white casts. Furthermore, organic filter like avobenzone exhibit photolability under UV exposure, undergoing rapid photodegradation that generates reactive byproducts. This instability has raised environmental concerns, as avobenzone and related organic UV filters like oxybenzone accumulate in marine ecosystems and contribute to coral reef toxicity by promoting bleaching, DNA damage, and impaired larval development[6,7]. Epidemiologically, UVB drives basal and squamous cell carcinomas (5.4 million cases annually in the US), while UVA predominates in melanoma etiology, with global incidence escalating 4-6% yearly and exceeding 325,000 new cases[8].

2) The authors used the percentage of inhibition to characterise antioxidant activity in the ABTS and DPPH assays. However, this parameter depends on the concentration of radicals in the analysed solutions and may therefore vary across different studies. A more relevant approach would be to use a stoichiometric coefficient, which reflects the number of radicals scavenged by one molecule of the analysed compound [10.1002/kin.21572; 10.3390/ijms26125659].

Response: We thank the reviewer for this valuable and pertinent comment. We agree that percentage inhibition may be influenced by the initial radical concentration and therefore does not always allow direct comparison between studies. In response to this suggestion, we have calculated the stoichiometric coefficient, which represents the number of radical species scavenged per molecule of antioxidant. The stoichiometric coefficients for both ABTS•⁺ and DPPH• assays were calculated based on the molar ratio between the consumed radical species and the antioxidant concentration. These results are now presented in the revised manuscript (Figures 1), providing a more robust and concentration-independent evaluation of antioxidant capacity. The corresponding methodology has been added to the Materials and Methods section.

3) There appears to be an error in the formula presented on line 139.

Response: The mistake was amended.

4) Regarding the molecular docking study. What was the pH of the medium used in the simulations? How was the ionization state of both the ligands and the biological target accounted for in the modelling?

Response: Thank you for the comment. The docking was done at physiological pH (~7.4). The ionization of the ligand and proteins was automatically assigned by AutoDockTools at this pH. This clarification has been added to the Molecular Docking Procedure.

5) To validate the docking model, a redocking experiment should be performed. The results should include data on binding affinity and root-mean-square deviation (RMSD) values to assess the reliability of the protocol.

Response: Response: Thank you for the suggestion. The redocking results showed an RMSD value between 1.22-2.25 Å, which falls within the acceptable threshold (< 2.0 Å), confirming that the docking protocol accurately reproduces the experimental binding pose. The binding affinity obtained from the redocking simulation was consistent with the expected range for the native ligand. These results validate the docking procedure and confirm that the protocol is suitable for reliable prediction of ligand–protein interactions.

The method has been added in “Molecular Docking Procedure”:

-Line 240: “To validate the molecular docking protocol, a redocking experiment was performed. The co-crystallized ligand of the target protein was removed and re-docked into its binding site using the same docking parameters as for all tested compounds. The similarity between the experimental and predicted poses was evaluated by calculating the RMSD”.

And the results has been described:

Line 408: “The docking model was validated through a redocking study, yielding an RMSD between 1.22-2.25 ÅÅ, which is within the accepted threshold for reliable pose prediction. The binding affinity obtained from the redocking simulation was consistent with the expected range for the native ligand. These results validate the docking procedure and confirm that the protocol is suitable for reliable prediction of ligand–protein interactions”.

6) Please provide RMSD data for the current molecular docking results to allow assessment of the structural stability and consistency of the predicted poses.

We thank the reviewer for this important comment. RMSD values for the predicted docking poses have now been added in Table1.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

pls carefully check the  Figure 3. Effect of astaxanthin on the migration of HaCat cells. 

the Bright field microsocpy pic were wrong.

Author Response

We thank the reviewer for this comment. The bright-field microscopy images related to the scratch assay have been carefully revised. Representative fields were re-selected from the original raw data to ensure consistency between experimental conditions and time points (T0 and T24). Manual annotations and subjective outlines were removed. The images are now presented solely for illustrative purposes, while the quantitative assessment of wound closure was objectively performed using ImageJ and is reported in the associated quantitative figure. Image clarity and contrast were improved, and a scale bar (100 µm) was added to all panels. These corrections do not affect the quantitative analysis, statistical significance, or conclusions of the study.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for authors' detailed responses. The corresponding additions and revisions in the manuscript addressed the concerns raised. In particular, I appreciate that you have clearly stated in the Discussion that the docking results are predictive rather than confirmatory, which enhances the overall rigor of the study.

Author Response

Thank you for your efforts to correct the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

My comments were taken into account by the authors. The rest were commented on, and I accept the arguments presented. The results indicate that it is worthwhile.

Author Response

We sincerely thank the reviewer for their constructive feedback and for acknowledging the revisions made. We appreciate the reviewer’s comments and are glad that the arguments and changes have satisfactorily addressed the concerns. 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The scale bar in Figure 3 is incorrectly labelled; it should be μm (micrometres) rather than μM. Additionally, please ensure the scale bar is defined in the figure legend.

Author Response

Response: The scale bar in Figure 3 has been corrected from μM to μm, and the scale bar definition has been added to the figure legend accordingly.