Exploring the Neuroprotective Properties of Capsanthin: Antioxidant Defense and Inflammatory Responses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for sharing your manuscript. The study explores the neuroprotective potential of capsanthin in a glutamate-induced SH-SY5Y model, and the experimental flow is generally clear with interesting observations on oxidative stress and inflammatory markers. A key concern is that the work relies entirely on a single in vitro model using a capsanthin concentration that is not physiologically achievable. In addition, the original image files lack identifiable markers, making it difficult to verify the data. These issues limit the strength, reproducibility, and broader relevance of the conclusions, and additional validation would be needed to support the claims.

Major Concerns

• The study depends solely on a single SH-SY5Y in vitro model, which does not reflect physiological CNS conditions, cellular interactions, or BBB characteristics.

• No in vivo or ex vivo validation is provided, leaving the proposed neuroprotective mechanism unsupported at a biological level.

• The capsanthin concentration used is non-physiological and lacks pharmacokinetic justification, limiting translational relevance.

• Original images lack essential identifiers (sample IDs, MW markers, labels), making verification of authenticity and reproducibility impossible.

• Supplementary data are minimally informative and do not provide raw or supporting evidence necessary for transparency.

• Discrepancies between caspase-3 protein and mRNA data remain unexplained, weakening the mechanistic interpretation.

• The glutamate model is narrow in scope and does not incorporate chronic or disease-relevant exposure conditions.

• Neuroinflammation analysis does not consider the limitations of a glia-free neuronal system, where cytokines like IL-6, IL-8, and TNF-α are primarily glia-derived.

• Data transparency is insufficient, and the conclusions extend beyond what the results can reliably support.

Minor Concerns

• Figure labeling, statistical annotations, and sample size indicators should be clarified and standardized.

• Reagent details, catalog numbers, and batch information should be fully reported to ensure reproducibility.

• Additional references may be needed to contextualize capsanthin’s previously reported biological activity.

• Terminology consistency should be improved across sections (e.g., RA-differentiated SH-SY5Y vs. differentiated SH-SY5Y).

• The discussion would benefit from deeper explanation of mitochondrial involvement and alternative interpretations of ATP recovery.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Thank you for sharing your manuscript. The study explores the neuroprotective potential of capsanthin in a glutamate-induced SH-SY5Y model, and the experimental flow is generally clear with interesting observations on oxidative stress and inflammatory markers. A key concern is that the work relies entirely on a single in vitro model using a capsanthin concentration that is not physiologically achievable. In addition, the original image files lack identifiable markers, making it difficult to verify the data. These issues limit the strength, reproducibility, and broader relevance of the conclusions, and additional validation would be needed to support the claims.

Major Concerns

• The study depends solely on a single SH-SY5Y in vitro model, which does not reflect physiological CNS conditions, cellular interactions, or BBB characteristics.

We agree. Our work is based on an RA-differentiated SH-SY5Y neuronal model, which is widely used but cannot fully capture the complexity of the CNS microenvironment (cell–cell interactions, multicellular signaling, or BBB properties). We strengthened the Limitations and future directions section to explicitly state these limitations and to clarify that our conclusions are restricted to this neuronal in vitro system. We also added a clearer statement that further validation in more complex models is necessary.

 

• No in vivo or ex vivo validation is provided, leaving the proposed neuroprotective mechanism unsupported at a biological level.

We acknowledge this limitation. Our study provides in vitro mechanistic evidence for capsanthin’s protective actions under glutamate-induced excitotoxic stress but does not claim biological validation at the organismal level. We revised the Discussion and Conclusions to emphasize that in vivo/ex vivo confirmation remains necessary, and we explicitly propose neuron–glia co-cultures, organotypic slices, and animal models as logical next steps for translation.

 

• The capsanthin concentration used is non-physiological and lacks pharmacokinetic justification, limiting translational relevance.

Thank you for highlighting this. We revised the Discussion to explain why a single protective concentration was selected for mechanistic assays, the highest non-cytotoxic dose to maximize detectability of protective effects, and provide a clear translational framing: we converted the applied concentration into the micromolar range and discussed its relation to reported dietary/plasma levels and the critical limitation imposed by blood–brain barrier permeability. We explicitly state that BBB transport/bioavailability and pharmacokinetics are required before any meaningful in vivo dose extrapolation can be made.

 

• Original images lack essential identifiers (sample IDs, MW markers, labels), making verification of authenticity and reproducibility impossible.

We agree that raw data presentation must allow authenticity and reproducibility checks. The uncropped raw blot images are provided in the Supplementary Materials; however, we recognize that the current presentation can be improved. Therefore, an additional Supplementary Figure S7 was added, showing the uncropped membranes with sample lane IDs and molecular-weight marker information indicated.

In addition, we note that the corresponding processed blot figures shown in the Results already include the sample/group names and the expected molecular weight marker annotation next to each band to support clear interpretation.

The MW ladder was not captured in the chemiluminescent channel because we use a fluorescent marker, but the marker is clearly visible on the membrane (pre-stained blue reference) while the target protein was detected by ECL.

 

• Supplementary data are minimally informative and do not provide raw or supporting evidence necessary for transparency.

We have expanded the Supplementary Materials to increase transparency. In addition to the existing preliminary viability and ROS optimization figures (used to select non-cytotoxic yet ROS-inducing conditions), we will include uncropped blot membranes with lane identifiers and marker information. In addition, following the recommendation of Reviewer 4, the Caspase-3 western blot data have been moved to the Supplementary Figure section (S5), as no statistically significant differences were detected between the experimental groups. This repositioning avoids over-representation within the main figures while still providing transparency and full access to the original protein-level results.

Besides, representative phase-contrast images of undifferentiated and 5-day RA-differentiated SH-SY5Y.

 

• Discrepancies between caspase-3 protein and mRNA data remain unexplained, weakening the mechanistic interpretation.

We apologize that this distinction was not stated clearly. We clarified the text in Results/Methods to specify that the RT-qPCR assay quantified CASP3 mRNA reflecting transcriptional regulation of the pro-caspase-3 gene, whereas the Western blot assessed cleaved caspase-3, the activated executioner form. Thus, an increase in CASP3 transcript does not necessarily imply increased cleavage/activation at the protein level, particularly under conditions of incomplete execution of apoptosis or limited downstream activation. We added this clarification explicitly to ensure mechanistic interpretation is accurate and reproducible. The Discussion section was supplemented with additional explanations, please see from Line 735.

 

• The glutamate model is narrow in scope and does not incorporate chronic or disease-relevant exposure conditions.

Thank you for your comment. Our design focuses on acute and early excitotoxic stress (two glutamate concentrations and two incubation time points). We strengthened the Limitations section to clearly state that chronic low-dose excitotoxicity and long-term capsanthin protection were not assessed, and we outline this as an important direction for future work. Please see from Line 810.

 

• Neuroinflammation analysis does not consider the limitations of a glia-free neuronal system, where cytokines like IL-6, IL-8, and TNF-α are primarily glia-derived.

We agree and revised the Discussion accordingly. We explicitly state that cytokine changes observed here are neuron-derived and therefore represent neuronal-specific inflammatory signaling, not the full neuroinflammatory response that in vivo is dominated by microglia/astrocytes. We also state that confirming the immunomodulatory effects of capsanthin requires models incorporating neuron–glia crosstalk.

 

• Data transparency is insufficient, and the conclusions extend beyond what the results can reliably support.

We appreciate this critique and addressed it in two ways. We improved transparency by expanding Supplementary materials. We refined the Discussion to avoid overgeneralization, explicitly framing capsanthin as a promising in vitro candidate that warrants validation rather than a confirmed therapeutic intervention.

 

Minor Concerns

• Figure labeling, statistical annotations, and sample size indicators should be clarified and standardized.

We apologise and thank you for your comment. We standardized figure labeling and ensured consistent annotation across figures (time-point labeling, panel naming, significance symbols, and explicit n reporting in captions). The figure legends were supplemented. The Statistics of the Materials and Methods contain the p-value (p<0.05) we used in each measurement.  The Statistics of the Materials was supplemented with the number of the independent experiments (n) and the repeats within the experiments.

 

• Reagent details, catalog numbers, and batch information should be fully reported to ensure reproducibility.

We expanded the Materials and Methods section to include detailed reagent information, supplier details, and catalog numbers. Where lot IDs were available from the same experimental series, these were added to improve reproducibility.

 

• Additional references may be needed to contextualize capsanthin’s previously reported biological activity.

We added additional references to better contextualize capsanthin’s reported antioxidant and immunomodulatory actions and to frame the novelty and limitations of our neuronal in vitro observations. Please see references doi:10.12691/jfnr-12-7-2; doi:10.1038/s41420-025-02368-1; doi:10.1093/jn/127.8.1475; doi:10.1079/BJN2003842; doi:10.3390/antiox11101931.

 

• Terminology consistency should be improved across sections (e.g., RA-differentiated SH-SY5Y vs. differentiated SH-SY5Y).

Thank you for your comment. We revised terminology throughout the manuscript and now use a consistent form, RA-differentiated SH-SY5Y cells, to avoid ambiguity.

 

• The discussion would benefit from deeper explanation of mitochondrial involvement and alternative interpretations of ATP recovery.

Thank you for your suggestion. We expanded the mitochondrial-focused discussion to explain better how mitochondrial redox balance, energy status, and partial apoptotic signaling can coexist, and we added alternative interpretations (e.g., sublethal stress response, incomplete caspase cascade activation, metabolic compensation) consistent with the observed ATP preservation under capsanthin. Please see the Discussion from Line 746. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Overall evaluation:

This study aimed to clarify the potential antioxidant and anti-inflammatory effects of capsanthin, both alone and in a model of glutamate-induced neuronal cell damage.  The study design is reasonable, the methods are described correctly, and the results have some application value, but there are some problems in the manuscript that need to be improved.

 

Specific modification suggestions:

1.Line14, the font "Capsanthin" does not need to be bold.

2.In the Abstract part, the research background is introduced too much. In the abstract part, the purpose of the research is briefly summarized in one sentence, and the focus should be on the research results.

3.Line60-66, please explain which part of the structure of "Capsanthin" gives it "high antioxidant capacity" and which part of the structure gives it "orange-red color".

4.In the Introduction part, the content is a little too much, and it is suggested to delete it appropriately.In addition, it is necessary to explain the deficiencies of existing research and the innovation of this research.

5.Line111-112, there are "g" and "rpm" in the unit of centrifugal speed, please unify the unit in the full text.

6.Line159, please explain the method used to determine the "85-98% purity".

7.Figure 2 (F) (G), it is suggested that "GP×24h" in the figure be changed to "GP 24h".

8.It is recommended to supplement the results of cell morphology pictures.

9.It is suggested that the "Conclusions" section be supplemented.

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

Overall evaluation:

This study aimed to clarify the potential antioxidant and anti-inflammatory effects of capsanthin, both alone and in a model of glutamate-induced neuronal cell damage.  The study design is reasonable, the methods are described correctly, and the results have some application value, but there are some problems in the manuscript that need to be improved.

 

Specific modification suggestions:

1.Line14, the font "Capsanthin" does not need to be bold.

We apologise and thank you for noting this formatting issue. We removed the bold formatting from the word “Capsanthin” in the Abstract to ensure consistent typography throughout the manuscript.

 

2.In the Abstract part, the research background is introduced too much. In the abstract part, the purpose of the research is briefly summarized in one sentence, and the focus should be on the research results.

Thank you for your suggestion. The Abstract was streamlined by shortening the background to one sentence, stating the aim in a single clear sentence, and emphasizing the key findings, such as redox/antioxidant, cytokines, mitochondrial/apoptotic outcomes.

 

3.Line60-66, please explain which part of the structure of "Capsanthin" gives it "high antioxidant capacity" and which part of the structure gives it "orange-red color".

We clarified this in the Introduction by explicitly linking the extended conjugated polyene chain to visible-light absorption and thus the orange–red color, and the conjugated double-bond system and terminal oxygen-containing functional groups to radical scavenging and singlet oxygen quenching, supporting its high antioxidant capacity. Please see from Line 85.

 

4.In the Introduction part, the content is a little too much, and it is suggested to delete it appropriately.In addition, it is necessary to explain the deficiencies of existing research and the innovation of this research.

Thank you for your suggestion. We agree and revised the Introduction accordingly. There have not been many published results regarding the biological role of capsanthin, which is a natural compound that could be used. We removed overly general background and strengthened the rationale by explicitly stating the main knowledge gap: limited mechanistic evidence for capsanthin in neuronal stress and cytokine regulation, and the innovation of the present study, the integrated analysis of oxidative stress, antioxidant defense, cytokine profile, and mitochondrial/apoptotic markers in RA-differentiated neurons. This is aligned with our aim statement in the revised text.

 

5.Line111-112, there are "g" and "rpm" in the unit of centrifugal speed, please unify the unit in the full text.

Thank you for your comment. We standardized centrifugation reporting to a single unit throughout the manuscript (relative centrifugal force, × g). In the revised Methods, the step previously reported in rpm was converted and expressed in × g for consistency.

 

6.Line159, please explain the method used to determine the "85-98% purity".

Thank you for your comment. We clarified this in the Materials and Methods. The reported purity range was based on the manufacturer’s certificate of analysis, determined by HPLC-based purity assessment (as provided by the supplier). We added this information where capsanthin is first described.

 

7.Figure 2 (F) (G), it is suggested that "GP×24h" in the figure be changed to "GP 24h".

The label was revised to “GPx 24 h” and the corresponding 48 h label was formatted consistently, improving readability and nomenclature consistency with the text and caption. GPx is the standard abbreviation in the scientific literature for glutathione peroxidase.

 

8.It is recommended to supplement the results of cell morphology pictures.

We added representative phase-contrast images of undifferentiated and 5-day RA-differentiated SH-SY5Y cells are provided in Supplementary Figure S6. Images were acquired using an EVOS XL Core microscope (Invitrogen, Carlsbad, CA, USA) with a Plan PH2 20×/0.40 objective.

 

9.It is suggested that the "Conclusions" section be supplemented.

Thank you for your suggestion. We added the Conclusions to summarize the main findings better. Please see Line 857.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The author's study uses a 5-day RA-differentiated SH-SY5Y cell model to simulate dopaminergic neurons, followed by glutamate treatment to induce neuronal dysfunction. The study focuses on the effects of capsanthin on differentiated neuronal cells and explores its impact on oxidative stress, antioxidant response, inflammation, and apoptosis after glutamate exposure. However, the manuscript has the following issues:

1.The manuscript lacks identification spectra for capsanthin extraction and isolation. At the very least, an LC-MS/MS or other spectra should be provided to facilitate the reader’s understanding.

2.The manuscript lacks images of RA-differentiated SH-SY5Y cells, as well as images showing cell damage and apoptosis. The entire paper consists of histograms, and lacks cell morphology images, which makes it difficult for the readers to understand and is unacceptable.

3.Abstract: It can be further simplified and optimized to improve readability.

4.Results: Some content without statistical results can be removed and discussed instead, which would help emphasize the key points. For example, Figure 5. Apoptotic changes in cells after capsanthin and glutamate treatment. (A-D) The caspase-3 protein levels after 24h (A,C) and 48h (B,D) of capsanthin and glutamate treatment.

Author Response

Reviewer 4.

Comments and Suggestions for Authors

The author's study uses a 5-day RA-differentiated SH-SY5Y cell model to simulate dopaminergic neurons, followed by glutamate treatment to induce neuronal dysfunction. The study focuses on the effects of capsanthin on differentiated neuronal cells and explores its impact on oxidative stress, antioxidant response, inflammation, and apoptosis after glutamate exposure. However, the manuscript has the following issues:

1.The manuscript lacks identification spectra for capsanthin extraction and isolation. At the very least, an LC-MS/MS or other spectra should be provided to facilitate the reader’s understanding.

Thank you for this important suggestion. In the manuscript, following the description of capsanthin, we have indicated that the spectroscopic data were consistent with previously reported values. In our opinion, the identification of the compound in this context is adequately supported by HPLC-based purity assessment, melting point determination, UV-Vis spectrum, and comparison with an authentic reference sample, which are generally accepted criteria for confirmation of identification. In addition, to improve transparency and reproducibility, we have included a detailed description of the HPLC method in the Materials and Methods section. Please see Line 203.

 

2.The manuscript lacks images of RA-differentiated SH-SY5Y cells, as well as images showing cell damage and apoptosis. The entire paper consists of histograms, and lacks cell morphology images, which makes it difficult for the readers to understand and is unacceptable.

We agree that representative morphology images improve interpretability. We have added representative phase-contrast images of undifferentiated and RA-differentiated SH-SY5Y cells to the Supplementary section (Supplementary Figure S6). Images were acquired using an EVOS XL Core microscope (Invitrogen, Carlsbad, CA, USA) with a Plan PH2 20×/0.40 objective.

Regarding apoptosis-specific imaging, our apoptosis assessment in this work focused on molecular endpoints, RT-qPCR for apoptosis regulators, caspase-9 ELISA, and caspase-3 immunoblotting. We acknowledge that additional imaging-based apoptosis assays (e.g., Annexin V/PI, TUNEL, or mitochondrial membrane potential staining) would further strengthen the cellular-level interpretation. However, at present, our department and faculty do not have access to the required instrumentation to perform fluorescent apoptosis marker labeling/imaging. We therefore indicate these imaging-based approaches as an important future direction once appropriate equipment becomes available.

 

3.Abstract: It can be further simplified and optimized to improve readability.

Thank you for your comment. We revised the Abstract to reduce background content, state the aim more concisely, and emphasize the core findings and mechanistic outcomes, improving readability and flow.

 

4.Results: Some content without statistical results can be removed and discussed instead, which would help emphasize the key points. For example, Figure 5. Apoptotic changes in cells after capsanthin and glutamate treatment. (A-D) The caspase-3 protein levels after 24h (A,C) and 48h (B,D) of capsanthin and glutamate treatment.

Thank you for your suggestion. Following this recommendation and consistent with Editor’s request, we removed the caspase-3 Western blot results from the main Results figures. We moved them to the Supplementary Figures (S5), since no statistically significant differences were detected between groups. This change reduces non-significant content in the main Results section and improves focus on the key statistically supported findings, while retaining full transparency by providing the underlying data in the Supplementary Materials.

We thoroughly revised the manuscript for grammar, style, and clarity to improve readability and ensure that the research is communicated more clearly.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The study provides valuable data on the protective effects of capsanthin against glutamate-induced stress. Several additions, however, would help clarify the proposed mechanisms. Since mitochondrial protection is a major conclusion, an assessment of membrane potential (JC-1 or TMRE) would more directly support this point. Likewise, because cleaved caspase-3 and viability remain unchanged, an Annexin V/PI assay would help determine whether apoptosis truly progresses or whether the cells remain in a reversible stress state.

The use of a single capsanthin concentration would be more convincing with a brief dose–response validation. Similarly, the glutamate concentrations employed are higher than those typically used in excitotoxicity models; a simple comparison with a physiologically relevant range (e.g., 300–500 µM) would improve interpretability. Finally, some supplementary figures are difficult to follow due to minimal labeling, and clearer annotation would enhance readability.

Overall, the manuscript is promising, and these focused additions would substantially strengthen the presentation and conclusions.

Author Response

Reviewer 1

 

The study provides valuable data on the protective effects of capsanthin against glutamate-induced stress. Several additions, however, would help clarify the proposed mechanisms. Since mitochondrial protection is a major conclusion, an assessment of membrane potential (JC-1 or TMRE) would more directly support this point. Likewise, because cleaved caspase-3 and viability remain unchanged, an Annexin V/PI assay would help determine whether apoptosis truly progresses or whether the cells remain in a reversible stress state.

 

We fully agree that direct measurements of mitochondrial membrane potential (e.g., JC-1 or TMRE) and Annexin V/PI-based apoptosis assays would provide valuable complementary information and would significantly strengthen the mechanistic interpretation. Unfortunately, we are not in a position to perform these additional experiments within the current revision timeframe. At present, our laboratory does not have access to the required fluorescence imaging/flow cytometry equipment or to the necessary dyes and antibodies, and establishing and validating these protocols from scratch would extend well beyond the deadline of the major revision.

In response to your comment, we have therefore tempered our conclusions and expanded the Discussion and Limitations to state that explicitly:

Mitochondrial protection is inferred from ATP preservation, modulation of redox status, and caspase-9 signaling rather than from direct mitochondrial membrane potential measurements; and the lack of change in cleaved caspase-3 and overall viability suggests that apoptosis is not fully executed under these conditions, but definitive discrimination between reversible stress and progressing apoptosis will require future studies employing Annexin V/PI staining, mitochondrial membrane potential assays, and more detailed mitochondrial functional readouts.

We respectfully hope that, with these clarifications and explicitly acknowledged limitations, the current dataset can still be considered informative at the in vitro mechanistic level, while we plan to address these more advanced assays in a follow-up project.

 

The use of a single capsanthin concentration would be more convincing with a brief dose–response validation. Similarly, the glutamate concentrations employed are higher than those typically used in excitotoxicity models; a simple comparison with a physiologically relevant range (e.g., 300–500 µM) would improve interpretability. Finally, some supplementary figures are difficult to follow due to minimal labeling, and clearer annotation would enhance readability.

Overall, the manuscript is promising, and these focused additions would substantially strengthen the presentation and conclusions.

 

We agree that a full protective dose-response curve would be desirable. As described in the Methods and Supplementary data, we performed preliminary viability assays with several capsanthin concentrations and selected the highest non-cytotoxic concentration for the mechanistic experiments, anticipating that this would provide the best chance to detect protective effects on redox, cytokine, and mitochondrial parameters. In the revised manuscript, we now refer more explicitly to these preliminary data, and we emphasize in the Limitations section that we did not establish a complete protective dose-response relationship, nor did we define the minimal effective dose or potential hormetic effects. We highlight this as an important direction for future work.

 

Thank you for drawing attention to this point. In our RA-differentiated SH-SY5Y system, we carried out pilot colorimetric viability measurements with 100–500 µM glutamate. Under these conditions, 100 µM glutamate already reduced viability to under 50% after 24 h, and higher concentrations (200–500 µM) caused even more rapid loss of viability, leaving insufficient surviving cells for the planned 24 and 48 h measurements. As shown in the current Supplementary viability data, even 20 µM glutamate decreased the viability to ~88% at 6 h, ~79% at 24 h, ~73% at 48 h, and ~61% at 72 h.

Our glutamate paradigm (1-5 mM, 24 and 48 hours) aligns with previous studies using differentiated SH-SY5Y cells, where millimolar glutamate concentrations are applied to induce strong excitotoxic or oxidative glutamate toxicity (e.g., 20 mM in RA-differentiated SH-SY5Y neurons by Nampoothiri et al., 2014 (doi: 10.1155/2014/674164); reviewed by Kritis et al., 2015 (https://doi.org/10.3389/fncel.2015.00091)). In RA-differentiated SH-SY5Y models, lower, submillimolar doses (0.1–0.5 mM) have been used in specific paradigms such as hypoxia/reoxygenation (doi: 10.1038/s41419-018-0784-6), but these conditions did not produce sustained injury compatible with our 24 and 48-hour mechanistic measurements.

Based on these preliminary findings and on previous reports using millimolar glutamate in differentiated SH-SY5Y excitotoxicity models, we chose 1 and 5 mM glutamate as conditions that produced robust but still analysable injury over 24 and 48 h in our differentiation protocol. In the revised manuscript, we explicitly discuss this optimization and clearly acknowledge that these concentrations are higher than physiological extracellular glutamate levels. We now state that our model captures experimental excitotoxic stress rather than physiological synaptic glutamate dynamics, and that future work should explore lower, more disease-relevant exposure paradigms (including repeated or chronic low-dose stimulations) once conditions compatible with longer-term survival are established.

 

We appreciate this remark and have revised the Supplementary figures to improve clarity. Specifically, we revised the labels for the morphology images (Supplementary Figure 6, clarified figure legends for the Supplementary figures, and provided underlying raw numerical data (OD values for viability assays) in an Excel table, and added compound characterization/purity documentation as Supplementary Figure 8 (HPLC chromatogram at 450 nm with peak integration and UV-Vis spectrum of the main peak) and clarified the purity assessment in the Materials and Methods section and we added the 1H and 13C NMR spectra of the isolated capsanthin, as well.

These changes might make it easier for readers to understand how the figures relate to the original measurements and to follow the supplementary material without ambiguity.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised the manuscript; however, the following issues remain:

Comment 1:
No spectral data related to the compound have been provided, nor is its purity clearly documented. Since the authors consider this information to be routine, it should not be difficult to provide the requested data or similar characterization data.

Comment 2:
In Supplementary Figure S6. Representative phase-contrast images of undifferentiated and 5-day RA-differentiated SH-SY5Y, what is the difference between the two images in the upper panel and the two images in the lower panel? For a paper intended for formal publication, each image must be clearly labeled so that it is unambiguous what it represents.

In addition, there are still no images showing cell damage and apoptosis, nor any images illustrating the effects of the compound. As histograms are not raw data, it is indeed difficult to accept a manuscript that presents only histograms without any corresponding original images. Alternatively, the authors could provide some of the original images corresponding to the histograms in the Supplementary Materials to facilitate readers’ understanding.

Author Response

Reviewer 4

The authors have revised the manuscript; however, the following issues remain:

Comment 1:
No spectral data related to the compound have been provided, nor is its purity clearly documented. Since the authors consider this information to be routine, it should not be difficult to provide the requested data or similar characterization data.

 

Thank you for your comment. We have added compound characterization/purity documentation as Supplementary Figure S8 (HPLC chromatogram at 450 nm with peak integration and UV-Vis spectrum of the main peak) and clarified the purity assessment in the Materials and Methods section. We added the 1H and 13C NMR spectra of the isolated capsanthin, as well.

 

Comment 2:
In Supplementary Figure S6. Representative phase-contrast images of undifferentiated and 5-day RA-differentiated SH-SY5Y, what is the difference between the two images in the upper panel and the two images in the lower panel? For a paper intended for formal publication, each image must be clearly labeled so that it is unambiguous what it represents.

In addition, there are still no images showing cell damage and apoptosis, nor any images illustrating the effects of the compound. As histograms are not raw data, it is indeed difficult to accept a manuscript that presents only histograms without any corresponding original images. Alternatively, the authors could provide some of the original images corresponding to the histograms in the Supplementary Materials to facilitate readers’ understanding.

 

Thank you for your question. There is no experimental difference between the two images in the upper panel and the two images in the lower panel. They represent independent representative fields of view from the same condition (undifferentiated vs. 5-day RA-differentiated SH-SY5Y), photographed to illustrate typical morphology. We apologise, and we have revised Supplementary Figure S6 so that each panel is clearly labeled.

Regarding cell damage readouts, we would like to clarify that cell viability in this study was assessed using a plate-based colorimetric In Vitro Toxicology assay, which produces optical density (OD) values rather than microscopy images; therefore, there are no corresponding raw images for these endpoints. Although the kit can also be used in a fluorescence-based format, we performed the assay colorimetrically and recorded absorbance at 600 nm. We apologize if this was unclear in the previous version, and we have revised the Materials and Methods section accordingly by explicitly specifying the colorimetric readout. To improve transparency and facilitate interpretation, we will provide the raw OD values underlying all viability experiments as a Supplementary Table S1 (exported as an Excel file), together with a brief description of the plate layout and normalization steps used to generate the plotted values.

Author Response File: Author Response.pdf