Review Reports

Round 1

Reviewer 1 Report

introduction:

lines 73-77: the satements require stronger literature evidence

The introduction woul benefit from a explaination of wether C. corallina is genuinely competitive as a carotenoid source? Does its extract provide an unusual biological profile? Is its astaxanthin concentration distinct from related freshwater macroalgae?

Materials and methods:

• The biomass was collected from one pond during one period in March 2025. This is definitely a limitation to adress as  carotenoid composition can vary with season, light, temperature, nutrient status, developmental stage, and other environmental conditions. Please comment
• 129-138: inconsistency around the temperature of drying; 40C in the materials and methods but fig 1 says 50C; please correct.
• lines 143-146: please explain why 48% (v/v) ethanol was selected.
• lines 151-155: “periodically replaced with fresh water as required.” thsi could lead to variations
• 164-165: “All extraction experiments were performed in triplicate using independently prepared biomass samples.” - n = 3 represents true biological replicates??
• lines 173-179: The mobile phase is described as methanol/acetonitrile under gradient elution. please provide the gradient programme
• lines 180-188 Astaxanthin can occur as free astaxanthin, esterified forms, and E/Z isomers. - whyich form was reported?
• lines 200-208: In teh materials and methods, it is said that L-ascorbic acid was used as the positive control. However, Table 2 reports Trolox as the reference antioxidant. please correct
• DPPH is the only antioxidant method used, therefore you cannot stat a "strong or powerful" antioxidative...
• lines 229-233: Cells between passages 5 and 20 were used.For primary-like human dermal fibroblasts, this is a relatively broad passage window. please justify
• 246-261: MTT test concentration is inconsistent:  cells were exposed to 0-200 ug/ml, but fig 4 does not report results for 200 ug/lm. please comment/explain
• lines 342–346, 361–364, and 379–381: 3 technical replicates and 3independently repeated experiments were reported. please descrive the difference between technical and biological. Technical PCR triplicates should be averaged within each biological sample, with only the independent biological experiments treated as n for inferential statistical analyses. Individual technical wells should not be considered independent observations, as doing so would constitute pseudoreplication .
• lines 375–381: For the inflammatory gene-expression analysis, the LPS-stimulated vehicle control is used as the calibrator, whereas in the UV experiment the normal control is used as the reference condition. Although this choice is not incorrect,please justify using different calibrators . Importantly, the untreated normal control should also be included and presented in the results to allow readers to verify the magnitude of gene induction caused by LPS stimulation and to distinguish the effect of LPS itself from the effects of the tested treatments.
• lines 390–397:  The Methods state that epigallocatechin gallate (EGCG) was used as the positive control, whereas the Results and Figure 7 refer to astaxanthin at 20 μM. Please check and correct ensuring that the Methods, Results, figure, and figure legend are fully consistent.
• lines 390–393:  The Methods state that concentrations of 12.5, 25, 50, and 100 μg/mL were tested, whereas only 12.5, 25, and 50 μg/mL are reported in the Results and Figure 7. The authors should clarify whether the 100 μg/mL concentration was tested and, if so, explain why these data are not presented. 
• lines 408–414: The authors explicitly state that dose–response curves were generated and IC₅₀ values calculated; however, the corresponding enzyme IC₅₀ values are not subsequently reported. Moreover, at the highest concentration presented in Figure 7 (50 μg/mL), inhibition of collagenase and elastase remains below 50%, meaning that any IC₅₀ estimates derived solely from the displayed concentration range would require extrapolation beyond the experimental data and may therefore be unreliable. 
• Lines 491–513: Figure 3 appears to present only a single control group together with CCE at 12.5, 25, and 50 μg/mL, whereas the Methods describe a nonirradiated normal control, a UV-irradiated vehicle control, UV-irradiated cells treated with the extract, and a UV-irradiated positive control treated with ascorbic acid. Consequently, it is unclear which experimental condition is represented by “Control,” whether UV irradiation itself significantly altered SOD1, GPX1, or CAT expression relative to nonirradiated cells, and how the effects of CCE compare with those of the positive control. All experimental groups described in the Methods should therefore be clearly presented in Figure 3, with unambiguous labeling and appropriate statistical comparisons.
• lines 530–533: The Figure legend reports n = 6, whereas the Methods describe three independent biological replicates, each with three technical replicates. How n = 6 was derived and whether n refers to independent biological experiments, technical measurements, or another experimental unit? Please clarify this discrepancy 
• Lines 534–550; Figure 5;lines 552–557: The Methods describe untreated normal, LPS inflammatory, extract-treated, and ascorbic acid positive-control groups, whereas Figure 5 appears to show only a “Control” and extract concentrations. The untreated normal control should be included to demonstrate the magnitude of LPS-induced inflammation and allow proper interpretation of the extract effects.
• 584-600: Only 12.5, 25, and 50 μg/mL values are described even though Methods specify a 100 μg/mL condition
• Discussion:
• Lines 655–669: The claim that the selected UAE conditions preserved biological functionality is not adequately supported, as biological activity was not compared with ethanol, ethyl acetate, or conventional/non-UAE extracts. Without an experimental comparator, preservation of biological activity cannot be concluded and the statement should be revised accordingly

• The Discussion frequently explains the observations through known molecular activities of astaxanthin. However, the tested material was a complex extract, not purified astaxanthin.

• Lines 804–817: The claim of “comparable anti-photoaging activity” is not sufficiently supported, as no direct comparator study was performed. This statement should therefore be moderated or supported by appropriate comparative experimental data.
• The Discussion contains considerable repetition of similar statements regarding the selected astaxanthin-rich extract, its multifunctional biological activities, sustainability, and potential cosmeceutical applications. The section should be substantially condensed to improve clarity and focus without loss of scientific content.

• Lines 900–955: The authors appropriately acknowledge several study limitations; however, this section should be expanded to include the single sampling site and season, uncertainty regarding biomass replicate independence, absence of a non-UAE extraction control and direct comparison with established astaxanthin sources, lack of orthogonal astaxanthin confirmation and carotenoid stability analysis, absence of functional oxidative-stress and protein-level inflammatory measurements, and the lack of an experimental assessment of sustainability.
• conclusion:

• Lines 957–981: The Conclusion overstates the current evidence, particularly regarding “potent” activity, ECM preservation, sustainability, and cosmeceutical applicability. 

please see the above section

Author Response

Introduction:

1. lines 73-77: the satements require stronger literature evidence

Response to Reviewer:

We thank the Reviewer for this important comment. We agree that the previous statements regarding freshwater macroalgae and Chara corallina were too broadly stated and were supported by insufficient literature evidence. Accordingly, we have substantially revised this paragraph and incorporated additional references specifically addressing the phytochemical composition and previously reported biological activities of C. corallina, including its antioxidant and anti-tyrosinase activities. The revised text now more clearly distinguishes these earlier studies, which mainly focused on crude extracts, general phytochemical composition, and associated biological activities, from the specific knowledge gap addressed in the present study. In particular, we now emphasize the novelty of integrating solvent selection and ultrasound-assisted extraction with HPLC-based confirmation and quantification of astaxanthin, followed by cellular evaluation of antioxidant, anti-inflammatory, and photoaging-related activities in C. corallina-derived extracts. These revisions provide stronger literature support and more precisely define the novelty and scope of the present study. The revised text is provided in Lines 68–91 of the revised manuscript, as highlighted in yellow.

2. The introduction would benefit from a explaination of wether  corallinais genuinely competitive as a carotenoid source? Does its extract provide an unusual biological profile? Is its astaxanthin concentration distinct from related freshwater macroalgae?

Response to Reviewer:

We thank the Reviewer for this insightful comment. We agree that the original Introduction did not sufficiently place C. corallina within the broader context of established astaxanthin sources and related freshwater macroalgae. We have therefore expanded the Introduction to clarify these points and to avoid implying that C. corallina is already demonstrated to be commercially competitive with established astaxanthin-producing microalgae such as Haematococcus pluvialis.

Specifically, the revised manuscript now clarifies that the available evidence is insufficient to conclude that the astaxanthin concentration of C. corallina is intrinsically higher than, or distinct from, that of related freshwater macroalgae. Previous studies have demonstrated considerable variation in carotenoid composition among freshwater Charophyceae and Chara species, whereas quantitative information specifically concerning astaxanthin in C. corallina remains limited. We have therefore framed C. corallina more conservatively as an underexplored freshwater biomass rather than as a direct competitor to established commercial astaxanthin sources.

We have also clarified the biological relevance of the extract investigated in the present study. Previous studies of C. corallina have mainly reported phytochemical composition and antioxidant and anti-tyrosinase activities of crude extracts. In contrast, the present study evaluates an astaxanthin-containing extract across multiple complementary endpoints, including radical-scavenging activity, antioxidant- and inflammation-associated gene expression, COL1A2 expression, and inhibition of extracellular matrix-degrading enzymes. We therefore describe this as a broader biological profile rather than claiming that it is unique or unusual. Additional literature on carotenoid composition in freshwater Charophyceae and related macroalgae has also been incorporated to provide appropriate context. These clarifications have been added to the Introduction (Lines 93–112 of the revised manuscript).

Materials and methods:

1. The biomass was collected from one pond during one period in March 2025. This is definitely a limitation to adress as carotenoid composition can vary with season, light, temperature, nutrient status, developmental stage, and other environmental conditions. Please comment

Response to Reviewer:

We thank the Reviewer for highlighting this important limitation. We fully agree that the carotenoid composition of algal biomass may vary considerably with environmental and physiological factors, including season, irradiance, water temperature, nutrient availability, and developmental stage. In the present study, all C. corallina biomass was collected from a single freshwater pond during one sampling period in March 2025. We have therefore revised Section 2.1 of the Materials and Methods to explicitly clarify the spatial and temporal scope of biomass collection and that the material used for the extraction experiments originated from a single collected biomass batch.

More importantly, we have added this issue explicitly to the Study Limitations and Future Perspectives section. The revised manuscript now clarifies that the astaxanthin content and extract composition reported in this study should be interpreted as representative of the specific biomass batch examined rather than as species-wide values for C. corallina. We further note that future studies involving multiple sampling locations, seasons, and environmental conditions will be necessary to establish the natural variability and reproducibility of carotenoid accumulation in this species. These clarifications have been incorporated into Sections 2.1 and 4.6 of the revised manuscript (Lines 143–145 and 1017–1026, respectively).

2. 129-138: inconsistency around the temperature of drying; 40 C in the materials and methods but fig 1 says 50 C; please correct.

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. We apologize for the error in Figure 1. The correct drying temperature used for preparation of the C. corallina biomass was 40 °C, as originally described in Section 2.2 of the Materials and Methods. The value of 50 °C shown in the original Figure 1 was a graphical error and has now been corrected to 40 °C. We have also carefully checked the corresponding descriptions throughout the revised manuscript to ensure consistency between the Materials and Methods and Figure 1. No change to the experimental procedure or data was required, as this correction concerns only the erroneous temperature displayed in the original figure.

 

3. lines 143-146: please explain why 48% (v/v) ethanol was selected.

Response to Reviewer:

We thank the Reviewer for requesting clarification regarding the selection of the mixed-solvent composition. The 48% (v/v) ethanol in ethyl acetate composition was selected during preliminary method development as a candidate mixed-solvent system because it provided favorable astaxanthin recovery relative to the other mixed-solvent compositions examined during preliminary testing. It was subsequently evaluated in the main experiment together with ethanol and ethyl acetate as single-solvent comparators under otherwise identical UAE conditions. Among these three solvent systems, the 48% ethanol–ethyl acetate mixture provided the highest astaxanthin recovery and was therefore selected for subsequent biological evaluation.

We have revised Section 2.3 of the Materials and Methods to clarify this selection procedure. Importantly, we now explicitly state that the preliminary screening was used to identify a candidate mixed-solvent composition and was not intended to represent a formal optimization study. Corresponding terminology throughout the manuscript has also been revised where necessary to avoid implying that a systematic solvent-ratio optimization was performed. This clarification has been added to Section 2.3 of the revised manuscript (Lines 180–188).

 

4. lines 151-155: “periodically replaced with fresh water as required.” this could lead to variations

Response to Reviewer:

We thank the Reviewer for raising this important methodological concern. We agree that the phrase “periodically replaced with fresh water as required” was insufficiently defined and could imply uncontrolled variation among extraction treatments. The purpose of refreshing the bath water was solely to minimize heat accumulation during sonication and to maintain the extraction temperature within the predefined range of 30 ± 2 °C, rather than to introduce an additional variable into the extraction procedure. The same temperature-control criterion was applied consistently to all extraction treatments.

We have therefore revised Section 2.3 of the Materials and Methods to remove the ambiguous wording and to explicitly describe the temperature-control procedure. The revised text now states that the bath temperature was monitored throughout sonication and maintained within 30 ± 2 °C, with bath water refreshed only when necessary to maintain this specified temperature range. This clarification has been incorporated into Section 2.3 of the revised manuscript (Lines 193–197).

5. 164-165: “All extraction experiments were performed in triplicate using independently prepared biomass samples.” - n = 3 represents true biological replicates??

Response to Reviewer:

We thank the Reviewer for raising this important point. We agree that the original wording “independently prepared biomass samples” could incorrectly imply that the three replicates represented independent field-level biological samples. This was not the case. All biomass used in the extraction experiments originated from the same collected biomass batch obtained from the single sampling site and collection period described in Section 2.1. For each extraction condition, separate aliquots of this biomass batch were independently subjected to the complete extraction procedure. Therefore, n = 3 represents independent extraction replicates rather than true independent field-level biological replicates.

We have revised the Materials and Methods to explicitly clarify the nature of these replicates and have replaced the potentially misleading terminology throughout the manuscript. The corresponding limitation has also been acknowledged in the Discussion, where we now clarify that the reported variability primarily reflects extraction-level variability and does not capture spatial, temporal, or population-level biological variation in C. corallina. These revisions have been incorporated into Sections 2.3 and 4.6 of the revised manuscript (Lines 206–209 and 1038–1046, respectively).

 

6. lines 173-179: The mobile phase is described as methanol/acetonitrile under gradient elution. please provide the gradient programme

Response to Reviewer:

We thank the Reviewer for identifying this omission. We agree that the complete gradient program is necessary to ensure reproducibility of the HPLC–PDA analysis. Accordingly, we have revised Section 2.4 of the Materials and Methods to provide the full chromatographic gradient, including the initial mobile-phase composition, time-dependent changes in methanol (A) and acetonitrile (B), and the final return to the initial conditions for column re-equilibration. The flow rate, column temperature, injection volume, and detection wavelength are also explicitly stated. The complete gradient program has now been incorporated into Section 2.4 of the revised manuscript (Lines 221–228).

 

7. lines 180-188 Astaxanthin can occur as free astaxanthin, esterified forms, and E/Z isomers. - which form was reported?

Response to Reviewer:

We thank the Reviewer for raising this important analytical point. We agree that astaxanthin may occur in different molecular forms, including free and esterified derivatives as well as different E/Z geometric isomers. The HPLC–PDA method used in the present study was not specifically designed or validated to comprehensively resolve these different molecular forms. Therefore, no specific free, esterified, or E/Z isomeric form of astaxanthin can be assigned from the present data.

In the revised manuscript, we have clarified that the sample chromatographic peak was assigned based on its retention time and UV–visible spectral characteristics corresponding to those of the authentic astaxanthin reference standard, and quantification was performed using an external calibration curve prepared from this standard. We have therefore revised the terminology throughout the manuscript to avoid implying definitive structural identification of a specific astaxanthin molecular form. Where appropriate, the quantified amount is now described as astaxanthin-equivalent content/recovery. We have also explicitly acknowledged the inability to distinguish free, esterified, and individual E/Z isomeric forms as an analytical limitation of the present study. These clarifications have been incorporated into Section 2.4, the corresponding Results and Figure 2 legend, and the Study Limitations section of the revised manuscript (Lines 229–238, 508–514, and 1059–1064, respectively).

 

8. lines 200-208: In teh materials and methods, it is said that L-ascorbic acid was used as the positive control. However, Table 2 reports Trolox as the reference antioxidant. please correct

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. We apologize for the error in the original Materials and Methods. Trolox, rather than L-ascorbic acid, was the reference antioxidant used in the DPPH radical-scavenging assay, as correctly reported in Table 2. The reference to L-ascorbic acid in the Materials and Methods was erroneous and has now been corrected to Trolox. We have also carefully reviewed the corresponding Methods, Results, and Table 2 to ensure consistent terminology throughout the revised manuscript. The numerical data reported in Table 2 were not affected by this correction. This correction has been incorporated into Section 2.5 of the Materials and Methods (Lines 252–253).

 

9. DPPH is the only antioxidant method used, therefore you cannot stat a "strong or powerful" antioxidative...

Response to Reviewer:

We thank the Reviewer for this important comment and fully agree that a single DPPH assay is insufficient to support broad statements describing CCE as having “strong,” “powerful,” or “potent” antioxidant activity. We have therefore systematically revised the manuscript to remove such terminology and to restrict the interpretation of the DPPH results specifically to DPPH radical-scavenging activity.

In the revised manuscript, the corresponding Materials and Methods subsection has been clarified as a DPPH radical-scavenging assay, and the Results now report the experimentally determined IC₅₀ values without making broader claims regarding antioxidant potency. We have also revised the Discussion to distinguish the cell-free DPPH findings from the observed changes in antioxidant-related gene expression (SOD1, CAT, and GPX1). Importantly, these transcriptional responses are now described as modulation of antioxidant-related gene expression rather than as direct evidence of increased antioxidant enzyme activity or overall cellular antioxidant capacity. The use of a single cell-free antioxidant assay and the absence of direct measurements of intracellular ROS or antioxidant enzyme activities are also explicitly acknowledged as limitations of the present study. These revisions have been incorporated throughout the Materials and Methods, Results, Discussion, and Study Limitations sections of the revised manuscript (Lines 241–243, 550–553, 747–761, and 1069–1073, respectively).

 

10. lines 229-233: Cells between passages 5 and 20 were used. For primary-like human dermal fibroblasts, this is a relatively broad passage window. please justify

Response to Reviewer:

We thank the Reviewer for raising this important methodological point. We agree that passages 5–20 represent a relatively broad passage range for human dermal fibroblasts and that passage-dependent changes in cellular phenotype and transcriptional responses cannot be completely excluded. The passage range was predefined to provide sufficient cell availability across the independent experiments while avoiding extensively passaged cultures, and cells were routinely examined for the expected fibroblast-like morphology before experimental use.

To address the Reviewer’s concern, we have clarified the passage range and cell-selection criteria in the Materials and Methods and have explicitly acknowledged the relatively broad passage window as a methodological limitation in the revised Discussion. We now note that potential passage-related variability may have influenced subtle cellular responses and that future studies should employ a narrower and more standardized passage range. These revisions have been incorporated into Section 2.6 of the Materials and Methods and Section 4.6 of the revised manuscript (Lines 282–288 and 1076–1081, respectively).

 

11. 246-261: MTT test concentration is inconsistent:  cells were exposed to 0-200 ug/ml, but fig 4 does not report results for 200 ug/lm. please comment/explain

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. We apologize for the error in the original Materials and Methods. The reference to 200 μg/mL was erroneous and did not represent a concentration evaluated in the reported MTT experiments. The Materials and Methods have therefore been corrected to specify the actual concentrations tested for each cell type. HDF cells were evaluated at 0 (vehicle control), 12.5, 25, 50, and 100 μg/mL, whereas RAW264.7 cells were evaluated at 0 (vehicle control), 12.5, 25, and 50 μg/mL. These concentrations correspond to the data presented in Figure 4.

We have also reviewed the corresponding Results and Figure 4 legend to ensure consistency between the experimental concentrations described in the Materials and Methods and those presented in the figure. This correction concerns the description of the concentration range in the original manuscript and does not affect the experimental data, statistical analyses, or conclusions of the study. The revised information has been incorporated into Section 2.7 of the Materials and Methods and the Figure 4 legend (Lines 302–306).

 

12. lines 342–346, 361–364, and 379–381: 3 technical replicates and 3independently repeated experiments were reported. please describe the difference between technical and biological. Technical PCR triplicates should be averaged within each biological sample, with only the independent biological experiments treated as nfor inferential statistical analyses. Individual technical wells should not be considered independent observations, as doing so would constitute pseudoreplication.

Response to Reviewer:

We thank the Reviewer for this important clarification regarding the experimental unit and statistical treatment of technical replicates. We fully agree that individual technical replicate wells should not be treated as independent observations. We apologize that the description in the original manuscript did not clearly distinguish technical replication from independently performed experiments.

In the present study, the cellular experiments were independently performed three times (n = 3 independent experiments). Within each independent experiment, measurements, including RT-qPCR reactions, were performed in three technical replicate wells. These technical replicates were used to account for within-assay measurement variability and were not treated as independent experimental units. For RT-qPCR analyses, the technical replicate Ct values were averaged within each independent experiment before calculation of relative gene expression. Consequently, each independent experiment contributed a single value for each experimental condition, and only the three independent experiment-level values (n = 3) were used for inferential statistical analyses.

 

To avoid ambiguity, we have revised the relevant Materials and Methods sections, Statistical Analysis, and figure legends throughout the manuscript. We now consistently use the term “independent experiments” rather than implying that technical replicate wells represent biological replicates, and we explicitly state that technical replicates were averaged before statistical analysis. These revisions have been incorporated into Sections 2.10–2.12, and 2.14 and the corresponding figure legends of the revised manuscript (Lines 403–408, 428-433, 449-455, and 496-503; 600–602, 625-627, 651-653, and 682-684, respectively).

 

 

13. lines 375–381: For the inflammatory gene-expression analysis, the LPS-stimulated vehicle control is used as the calibrator, whereas in the UV experiment the normal control is used as the reference condition. Although this choice is not incorrect, please justify using different calibrators. Importantly, the untreated normal control should also be included and presented in the results to allow readers to verify the magnitude of gene induction caused by LPS stimulation and to distinguish the effect of LPS itself from the effects of the tested treatments.

Response to Reviewer:

We thank the Reviewer for this important and constructive comment. We agree that the rationale for using different calibrators in the UV-irradiation and LPS-induced inflammatory models was insufficiently explained in the original manuscript. The calibrator in each model was selected according to the primary biological comparison. In the UV model, the nonirradiated normal control was used as the calibrator to evaluate transcriptional changes associated with UV exposure relative to the nonirradiated baseline. In contrast, in the LPS-induced inflammatory model, the LPS-stimulated vehicle control was used as the calibrator to evaluate the effects of CCE relative to the induced inflammatory state.

 

We also fully agree that presentation of the untreated normal control is necessary to demonstrate the magnitude of gene induction caused by LPS stimulation and to distinguish the effect of LPS itself from the effects of CCE. Accordingly, the untreated normal control from the original experiments has now been included in the revised Results and Figure 5 for Nos2, Ptgs2, and Il1b. The revised presentation therefore allows direct visualization of the transcriptional response induced by LPS relative to untreated cells, followed by evaluation of the effects of CCE relative to the LPS-stimulated vehicle control.

 

We have revised the Materials and Methods, Results, and corresponding figure legend to clearly explain the rationale for the calibrator selection and the role of each control group. These revisions have been incorporated into Section 2.12 (Lines 444–457), Section 3.4 (Lines 643–647), and Figure 5 of the revised manuscript.

 

14. lines 390–397: The Methods state that epigallocatechin gallate (EGCG) was used as the positive control, whereas the Results and Figure 7 refer to astaxanthin at 20 μM. Please check and correct ensuring that the Methods, Results, figure, and figure legend are fully consistent.

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. We apologize for the error in the original manuscript. Epigallocatechin gallate (EGCG), rather than astaxanthin, was the positive inhibitory control used in the collagenase, elastase, and hyaluronidase inhibition assays. The references to astaxanthin in the original Results and Figure 7 were reporting and figure-labeling errors.

We have carefully reviewed the corresponding experimental records and revised the Materials and Methods, Results, Figure 7, and its legend to consistently identify EGCG (20 μM) as the positive inhibitory control. References to astaxanthin as the positive control have been removed from these sections. This correction concerns the reporting and labeling of the positive control and does not affect the experimental data, statistical analyses, or conclusions derived from the enzyme inhibition assays. The corresponding corrections have been incorporated into Section 2.13 (Lines 481–483), Section 3.5.2 (Lines 705-707), Figure 7, and its legend of the revised manuscript.

 

15. lines 390–393: The Methods state that concentrations of 12.5, 25, 50, and 100 μg/mL were tested, whereas only 12.5, 25, and 50 μg/mL are reported in the Results and Figure 7. The authors should clarify whether the 100 μg/mL concentration was tested and, if so, explain why these data are not presented. 

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. We apologize for the error in the original Materials and Methods. Upon re-examination of the experimental records, we confirmed that 100 μg/mL was not included in the enzyme-inhibition experiments. The concentrations actually evaluated were 12.5, 25, and 50 μg/mL, as correctly presented in the Results and Figure 7. The reference to 100 μg/mL in the original Materials and Methods was therefore a reporting error.

We have corrected the Materials and Methods to accurately reflect the experimentally evaluated concentration range and have carefully reviewed the corresponding Results, Figure 7, and figure legend to ensure consistency throughout the revised manuscript. This correction concerns only the description of the tested concentration range and does not affect the experimental data or statistical analyses. The revised information is provided in Lines 477 of the revised manuscript.

 

16. lines 408–414:The authors explicitly state that dose–response curves were generated and IC₅₀ values calculated; however, the corresponding enzyme IC₅₀ values are not subsequently reported. Moreover, at the highest concentration presented in Figure 7 (50 μg/mL), inhibition of collagenase and elastase remains below 50%, meaning that any IC₅₀ estimates derived solely from the displayed concentration range would require extrapolation beyond the experimental data and may therefore be unreliable. 

Response to Reviewer:

We thank the Reviewer for this important observation. We fully agree that reliable estimation of an IC₅₀ value requires an experimentally evaluated concentration range that adequately encompasses the 50% inhibition level. In the present study, the highest CCE concentration experimentally evaluated was 50 μg/mL, at which inhibition of collagenase and elastase remained below 50%. Therefore, estimation of IC₅₀ values for these enzymes would require extrapolation beyond the experimentally observed concentration range and could yield unreliable estimates.

Accordingly, we have removed the statement that dose–response curves were generated for the calculation of enzyme IC₅₀ values, and no extrapolated IC₅₀ values are reported in the revised manuscript. Instead, enzyme inhibitory activity is now reported as the experimentally observed percentage inhibition at each tested CCE concentration (12.5, 25, and 50 μg/mL). The Materials and Methods, Results, Discussion, and Figure 7 legend have been revised accordingly to ensure that the interpretation is restricted to the experimentally observed concentration range. This revision avoids unsupported extrapolation and provides a more conservative interpretation of the enzyme-inhibition data. These revisions have been incorporated into Section 2.13 (Lines 494–504), Section 3.5.2 (Lines 709–711), the corresponding Discussion, and Figure 7 legend of the revised manuscript (Lines 728–731).

 

17. Lines 491–513: Figure 3 appears to present only a single control group together with CCE at 12.5, 25, and 50 μg/mL, whereas the Methods describe a nonirradiated normal control, a UV-irradiated vehicle control, UV-irradiated cells treated with the extract, and a UV-irradiated positive control treated with ascorbic acid. Consequently, it is unclear which experimental condition is represented by “Control,” whether UV irradiation itself significantly altered SOD1, GPX1, or CAT expression relative to nonirradiated cells, and how the effects of CCE compare with those of the positive control. All experimental groups described in the Methods should therefore be clearly presented in Figure 3, with unambiguous labeling and appropriate statistical comparisons.

Response to Reviewer:

We thank the Reviewer for identifying this important inconsistency between the original Materials and Methods and Figure 3. Upon re-examination of the experimental records, we confirmed that the UV-irradiation experiment included a UV-irradiated vehicle control and UV-irradiated cells treated with CCE at 12.5, 25, and 50 μg/mL. A separate nonirradiated normal control and an ascorbic acid-treated positive-control group were not included in this experiment. We apologize that these groups were incorrectly described in the original Materials and Methods.

We have therefore revised the Materials and Methods to accurately reflect the experimental groups that were actually evaluated. In addition, the ambiguous label “Control” in Figure 3 has been replaced with “UV + Vehicle”, and the figure legend has been revised accordingly. The UV-irradiated vehicle control was used as the calibrator for the relative gene-expression analysis, and statistical comparisons of the CCE-treated groups were performed relative to this control.

We fully agree with the Reviewer that, in the absence of a nonirradiated normal control, the present experiment cannot determine whether UV irradiation itself significantly altered SOD1, CAT, or GPX1 expression relative to the nonirradiated state. Accordingly, we have revised the Results and Discussion to avoid interpreting the observed responses as UV-induced changes or as restoration toward normal expression levels. The findings are now interpreted more conservatively as CCE-associated changes in antioxidant-related gene expression relative to UV-irradiated vehicle-treated cells. The absence of a nonirradiated control has also been explicitly acknowledged as a limitation of the study.

Similarly, because an ascorbic acid-treated positive-control group was not included in the experiment, the erroneous reference to this group has been removed from the Materials and Methods and related text. These revisions have been incorporated into the Materials and Methods (Lines 414–431), Results (Lines 602–607), Discussion (Lines 1137–1142), Figure 3, and its legend (Lines 618–622).

18. lines 530–533: The Figure legend reports n= 6, whereas the Methods describe three independent biological replicates, each with three technical replicates. How n = 6 was derived and whether n refers to independent biological experiments, technical measurements, or another experimental unit? Please clarify this discrepancy

 Response to Reviewer:

We thank the Reviewer for identifying this discrepancy and apologize for the ambiguity in the original figure legend. Upon re-examination of the experimental design and data analysis, we confirmed that n = 6 reported in the original figure legend was a reporting error. The cellular gene-expression experiments were independently performed three times (n = 3 independent experiments), and within each independent experiment, qPCR measurements were performed in technical triplicate.

 

Importantly, the technical replicate measurements were not treated as independent experimental units. Technical replicate Ct values were averaged within each independent experiment before calculation of relative gene expression, such that each independently performed experiment contributed a single value for each experimental condition. Accordingly, the experimental unit used for statistical analysis was the independently performed experiment, and the correct sample size for inferential statistical analysis was n = 3, rather than n = 6.

 

We have therefore corrected the figure legend from n = 6 to n = 3 and revised the Materials and Methods and Statistical Analysis sections to explicitly distinguish independent experiments from technical replicates and to clarify the experimental unit used for statistical analysis. The corresponding figure legends have also been reviewed and corrected for consistency throughout the revised manuscript. These revisions have been incorporated into Sections 2.11 (Lines 432–437) and 2.13 and the corresponding figure legend(s) of the revised manuscript.

 

19. Lines 534–550; Figure 5; lines 552–557: The Methods describe untreated normal, LPS inflammatory, extract-treated, and ascorbic acid positive-control groups, whereas Figure 5 appears to show only a “Control” and extract concentrations. The untreated normal control should be included to demonstrate the magnitude of LPS-induced inflammation and allow proper interpretation of the extract effects.

Response to Reviewer:

We thank the Reviewer for identifying this important inconsistency and agree that inclusion of an unstimulated normal control would have allowed the magnitude of the LPS-induced transcriptional response to be directly assessed. Upon re-examination of the experimental records, however, we confirmed that the original experiment consisted of an LPS-stimulated vehicle control and LPS-stimulated cells treated with CCE at 12.5, 25, and 50 μg/mL. A separate unstimulated normal control and an ascorbic acid-treated positive-control group were not included in this experiment. We apologize that these groups were incorrectly described in the original Materials and Methods.

We have therefore revised the Materials and Methods to accurately reflect the experimental design actually employed. The ambiguous label “Control” in Figure 5 has also been replaced with “LPS + Vehicle”, and the LPS-stimulated vehicle control is now explicitly identified as the calibrator (relative expression = 1) for the relative gene-expression analysis. Statistical comparisons of the CCE-treated groups were performed relative to this control.

 

We agree with the Reviewer that, because an unstimulated normal control was not included, the present experiment cannot establish the magnitude of LPS-induced changes in Nos2, Ptgs2, and Il1b expression relative to an unstimulated baseline. We have therefore revised the Results and Discussion to restrict our interpretation to the experimentally supported comparison, namely, CCE-associated changes in inflammation-related gene expression relative to LPS-stimulated vehicle-treated cells. Statements implying restoration toward normal expression levels or direct quantification of the LPS-induced inflammatory response have been removed. The absence of an unstimulated normal control has also been explicitly acknowledged as a limitation of the study.

 

Accordingly, the Materials and Methods (Lines 448–464), Results (Lines 657–661), Discussion (Lines 1147–1152), Figure 5, and its legend have been revised to ensure that the presentation and interpretation are fully consistent with the experimental design and the available data.

 

20. 584-600: Only 12.5, 25, and 50 μg/mL values are described even though Methods specify a 100 μg/mL condition

Response to Reviewer:

We thank the Reviewer for identifying this inconsistency. Upon re-examination of the experimental records, we confirmed that the concentrations actually evaluated in the enzyme-inhibition experiments were 12.5, 25, and 50 μg/mL. The reference to 100 μg/mL in the original Materials and Methods was a reporting error, and no 100 μg/mL condition was included in these experiments. Therefore, the concentration range presented in the original Results and Figure 7 (12.5–50 μg/mL) correctly reflects the experimentally evaluated conditions.

We have corrected the Materials and Methods by removing the erroneous reference to 100 μg/mL and have carefully reviewed the corresponding Results, Figure 7, and figure legend to ensure consistent reporting of the tested concentration range throughout the revised manuscript. No additional 100 μg/mL data have been added because this concentration was not experimentally evaluated. This correction concerns the description of the experimental concentration range and does not alter the underlying experimental data. The corresponding revisions have been incorporated into Section 2.13 (Lines 484). and the related Results (Lines 718–720). and Figure 7 legend.

Discussion:

1. Lines 655–669: The claim that the selected UAE conditions preserved biological functionality is not adequately supported, as biological activity was not compared with ethanol, ethyl acetate, or conventional/non-UAE extracts. Without an experimental comparator, preservation of biological activity cannot be concluded, and the statement should be revised accordingly

Response to Reviewer:

We thank the Reviewer for this important comment and fully agree that the original statement regarding preservation of biological functionality was not adequately supported by the experimental design. Although the selected CCE exhibited measurable biological activities, the present study did not directly compare the biological activity of extracts obtained using different solvent systems or a conventional/non-UAE extraction method. Therefore, the data do not allow us to conclude that UAE itself preserved or enhanced biological functionality.

Accordingly, we have removed the statement that the selected UAE conditions “preserved biological functionality” and revised the Discussion to restrict the interpretation to the experimentally supported observation that the selected CCE exhibited measurable DPPH radical-scavenging, cellular gene-expression, and extracellular matrix-related enzyme inhibitory activities. We now explicitly clarify that these biological responses represent properties of the selected CCE and should not be interpreted as evidence of a UAE-mediated preservation effect. We have also noted that direct comparison with appropriately matched conventional/non-UAE extraction methods would be required to determine whether UAE influences the biological activity of the resulting extracts. The corresponding Discussion has been revised in Lines 823–838 of the revised manuscript.

 

 

2. The Discussion frequently explains the observations through known molecular activities of astaxanthin. However, the tested material was a complex extract, not purified astaxanthin.

Response to Reviewer:

We thank the Reviewer for this important comment and fully agree that the biological activities observed in the present study should not be attributed exclusively to astaxanthin because the tested material was a complex Chara corallina extract (CCE) rather than purified astaxanthin. We acknowledge that several statements in the original Discussion relied too strongly on the established molecular activities of purified astaxanthin when interpreting the responses observed with CCE.

Accordingly, we have systematically revised the Discussion to distinguish the experimentally observed effects of CCE from the previously reported biological and molecular activities of purified astaxanthin. Throughout the revised Discussion, the antioxidant-, inflammation-, and extracellular matrix-related responses are now explicitly described as effects associated with CCE. Previous studies of astaxanthin are used only to provide biological and mechanistic context and are no longer presented as evidence that astaxanthin itself was responsible for the observed responses.

 

We have also explicitly clarified that CCE is a chemically complex extract and that other carotenoids and unidentified constituents may have contributed independently or through additive or synergistic interactions. Statements implying direct astaxanthin-mediated activation of antioxidant signaling, suppression of inflammatory pathways, enhancement of collagen production, or inhibition of extracellular matrix-degrading enzymes have therefore been removed or substantially moderated. In addition, the absence of purified astaxanthin as a comparator has been explicitly acknowledged as a limitation, and the Conclusion has been revised accordingly.

 

These revisions ensure that the mechanistic discussion of astaxanthin is presented as contextual evidence rather than as direct attribution of the biological activities of CCE to astaxanthin. The corresponding revisions have been incorporated throughout the Discussion (Lines 785–813) and Study Limitations sections (Lines 1149–1156) and in the Conclusion (Lines 1186–1192).

 

3. Lines 804–817: The claim of “comparable anti-photoaging activity” is not sufficiently supported, as no direct comparator study was performed. This statement should therefore be moderated or supported by appropriate comparative experimental data.

Response to Reviewer:

We thank the Reviewer for this important comment and agree that the original statement describing the activity of CCE as “comparable anti-photoaging activity” was not adequately supported because the present study did not include a direct experimental comparator for the overall anti-photoaging response. We have therefore removed the term “comparable anti-photoaging activity” and revised the corresponding Discussion to avoid comparative efficacy claims.

In the revised manuscript, the findings are interpreted more conservatively on the basis of the endpoints directly evaluated in this study. Specifically, the increased COL1A2 mRNA expression and inhibition of collagenase, elastase, and hyaluronidase are now discussed as extracellular matrix-related biological activities that support the anti-photoaging potential of CCE, rather than as evidence of comparable or superior anti-photoaging efficacy. We have also clarified that increased COL1A2 mRNA expression does not necessarily demonstrate increased collagen protein synthesis or extracellular matrix deposition.

 

Accordingly, all comparative statements regarding anti-photoaging activity have been removed or moderated throughout the revised Discussion and Conclusion. The corresponding revisions are provided in Lines 983–991 of the revised manuscript.

 

4. The Discussion contains considerable repetition of similar statements regarding the selected astaxanthin-rich extract, its multifunctional biological activities, sustainability, and potential cosmeceutical applications. The section should be substantially condensed to improve clarity and focus without loss of scientific content.

Response to Reviewer:

We thank the Reviewer for this helpful comment and agree that the original Discussion contained unnecessary repetition, particularly regarding the description of CCE as an astaxanthin-rich extract, its multifunctional biological activities, sustainability, and potential cosmeceutical applications. We have therefore substantially condensed and reorganized the Discussion to improve clarity and scientific focus.

Specifically, repeated descriptions of the biological activities of CCE and redundant concluding statements at the end of individual subsections have been removed. Each subsection is now focused on the specific experimental endpoint being discussed: extraction and chemical recovery, antioxidant-related responses, inflammation-related responses, and extracellular matrix/photoaging-related responses. Mechanistic discussion has also been shortened and restricted to literature-supported context directly relevant to the observed findings.

 

In addition, repeated claims regarding sustainability and cosmeceutical applicability have been removed or substantially moderated. Because environmental sustainability and commercial feasibility were not directly evaluated in the present study, C. corallina is now described more conservatively as an underexplored freshwater biomass/source of carotenoid-containing extracts, and sustainability is explicitly identified as an aspect requiring future evaluation. Similarly, discussion of potential cosmeceutical applicability has been consolidated into the final implications/limitations section rather than repeated throughout the individual biological subsections.

 

Finally, limitations and future research needs that were previously repeated across several sections have been consolidated into a dedicated Study Limitations and Future Perspectives section. These revisions substantially reduce repetition while retaining the scientific interpretation and relevant mechanistic context of the findings. The revised Discussion is provided in Lines 1063–1137 of the revised manuscript.

5. Lines 900–955: The authors appropriately acknowledge several study limitations; however, this section should be expanded to include the single sampling site and season, uncertainty regarding biomass replicate independence, absence of a non-UAE extraction control and direct comparison with established astaxanthin sources, lack of orthogonal astaxanthin confirmation and carotenoid stability analysis, absence of functional oxidative-stress and protein-level inflammatory measurements, and the lack of an experimental assessment of sustainability.

Response to Reviewer:

We thank the Reviewer for this comprehensive and constructive comment. We agree that the limitations of the original study required more explicit and systematic consideration. Accordingly, we have substantially expanded and reorganized Section 4.6, “Study Limitations and Future Perspectives,” to address all of the issues raised by the Reviewer while consolidating previously dispersed limitations to reduce repetition elsewhere in the Discussion.

Specifically, the revised section now clarifies that (i) all C. corallina biomass originated from a single freshwater pond and a single sampling period in March 2025; (ii) the three extraction replicates were independently processed aliquots derived from the same collected biomass batch and therefore represent independent extraction replicates rather than independent field-level biological replicates; (iii) no conventional non-UAE extraction control or direct experimental comparison with an established commercial astaxanthin source was included; (iv) chemical characterization relied primarily on HPLC–PDA correspondence with an authentic astaxanthin reference standard without orthogonal structural confirmation or comprehensive assessment of astaxanthin molecular forms, other carotenoids, and potential degradation or oxidation products; (v) antioxidant-related cellular responses were not confirmed by direct measurements of intracellular oxidative stress, antioxidant protein abundance, or enzyme activity; (vi) inflammation-related responses were evaluated primarily at the transcriptional level without corresponding protein-level or upstream signaling measurements; and (vii) environmental sustainability and commercial feasibility were not experimentally assessed.

 

We have additionally clarified that CCE is a chemically complex extract rather than purified astaxanthin and that the observed biological responses therefore cannot be attributed exclusively to astaxanthin. The revised section also acknowledges the limitations associated with the HDF passage range, the absence of corresponding nonirradiated and unstimulated normal controls in the UV- and LPS-based experiments, and the fact that increased COL1A2 mRNA expression and cell-free enzyme inhibition do not directly demonstrate increased collagen production or preservation of extracellular matrix integrity in a biological skin model.

 

In parallel, claims elsewhere in the Discussion concerning UAE superiority, preservation of biological functionality, sustainability, and practical cosmeceutical applicability have been removed or moderated to maintain consistency with these limitations. Future research priorities are now explicitly directed toward independent multi-site and seasonal sampling, orthogonal and comprehensive chemical characterization, functional and protein-level mechanistic validation, appropriate experimental comparators, advanced skin models, and environmental and techno-economic assessment. These revisions are provided in Section 4.6, Lines 1063–1137 of the revised manuscript.

 

conclusion:

1. Lines 957–981: The Conclusion overstates the current evidence, particularly regarding “potent” activity, ECM preservation, sustainability, and cosmeceutical applicability. 

Response to Reviewer:

We thank the Reviewer for this important comment and agree that several statements in the original Conclusion extended beyond the evidence directly supported by the present study. We have therefore substantially revised the Conclusion to remove or moderate claims regarding potent biological activity, preservation of extracellular matrix integrity, sustainability, and practical cosmeceutical applicability.

 

Specifically, the revised Conclusion now describes the experimentally observed endpoints directly, including DPPH radical-scavenging activity, modulation of antioxidant- and inflammation-associated gene expression, increased COL1A2 mRNA expression, and inhibition of collagenase, elastase, and hyaluronidase within the experimentally evaluated concentration range, rather than collectively characterizing these effects as “potent” antioxidant, anti-inflammatory, or anti-photoaging activities. The previous implication of extracellular matrix preservation has also been removed because increased COL1A2 mRNA expression and inhibition of extracellular matrix-degrading enzymes do not directly demonstrate increased collagen protein synthesis, extracellular matrix deposition, or preservation of matrix integrity.

 

In addition, C. corallina is now described more conservatively as an underexplored freshwater source of carotenoid-containing bioactive extracts, rather than as an established sustainable source. We explicitly state that environmental sustainability and commercial feasibility were not experimentally evaluated and require dedicated assessment. Similarly, practical cosmeceutical applicability is now presented as a subject for future investigation requiring additional chemical characterization, mechanistic validation, advanced skin models, and formulation and translational studies.

We have also clarified that CCE is a chemically complex extract and that the observed biological responses cannot be attributed exclusively to astaxanthin. Overall, the revised Conclusion has been restricted to conclusions directly supported by the experimental evidence and is consistent with the corresponding revisions made throughout the Discussion. The revised Conclusion is provided in Lines 1166–1191 of the revised manuscript.

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors report the production of an astaxanthin-rich extract from Chara corallina and its
biological activities of interest in cosmetic applications.
The study of this carotenoid from the specific algae investigated here is new, but ultrasound-assisted
recovery of this metabolite is an efficient method already used for other natural sources.
1) In this regard, the authors should compare the quantities reported in Table 1 with those of other
studies, especially in the case of the commercial astaxanthin predominantly produced from the
microalga Haematococcus pluvialis, as they report in their Introduction.
2) The evaluation of the antioxidant, anti-inflammatory, and anti-photoaging activities was performed
according to well-defined and rigorous methodologies. Still, for all the assays, data for a sample of
pure astaxanthin are missing.
3) In Table 1, the volume of extraction solvent must be indicated, corresponding to the amount (mg)
of astaxanthin evaluated using the calibration curve.
3) On page 21, the authors write, "Utilization of ultrasound-assisted extraction preserves the
antioxidant functionality of astaxanthin-rich extracts." However, there is no evidence of this integrity.
The chromatogram in Figure 2B shows the presence of astaxanthin, but along with numerous other
peaks, some of which could be products of partial oxidation. This must be verified by HPLC-ESIMS
analysis, which can also indicate the presence of other carotenoids that may contribute to the
biological activities of the extract.
4) On page 22, the authors write, "Although astaxanthin was successfully identified and quantified in
the selected extract, other carotenoids, phenolic compounds, and minor bioactive constituents were
not comprehensively characterized." This cannot be a future perspective, but the chemical
characterization of the extract must be described in this work, which otherwise provides too little
information to indicate the algae studied here as a worthy source for naturally derived astaxanthin
already on the market.
The manuscript has the potential to become a suitable article, enriching it by detailed comparisons to
data already known in the literature, and focusing on the novelty of this study. However, its present
version is not acceptable.

Please, see the Major comments section.

Comments for author File: Comments.pdf

Author Response

• The authors report the production of an astaxanthin-rich extract from Chara corallinaand itsbiological activities of interest in cosmetic applications.
• The study of this carotenoid from the specific algae investigated here is new, but ultrasound-assistedrecovery of this metabolite is an efficient method already used for other natural sources.

 

1) In this regard, the authors should compare the quantities reported in Table 1 with those of other studies, especially in the case of the commercial astaxanthin predominantly produced from the microalga Haematococcus pluvialis, as they report in their Introduction.

Response to Reviewer:

We thank the Reviewer for this valuable suggestion. We agree that the astaxanthin-equivalent recovery reported in Table 1 should be interpreted in the context of established natural astaxanthin sources, particularly Haematococcus pluvialis. Accordingly, we have expanded Section 4.1 of the Discussion to include a quantitative comparison with previously reported astaxanthin contents in H. pluvialis. Ambati et al. (2014) reported astaxanthin contents ranging from approximately 2.7 to 3.8% of dry biomass among different H. pluvialis strains, while Ren et al. (2021) indicated that astaxanthin accumulation can approach approximately 5% of dry weight under favorable cultivation and stress conditions. In comparison, the highest astaxanthin-equivalent yield obtained from C. corallina in the present study was 0.2598 ± 0.0086% of dry biomass.

We have therefore clarified that the value obtained for C. corallina is approximately one order of magnitude lower than the levels commonly reported for astaxanthin-accumulating H. pluvialis. At the same time, we emphasize that direct numerical comparisons should be interpreted cautiously because astaxanthin content and recovery are influenced by strain, cultivation and stress conditions, physiological stage, biomass pretreatment, extraction procedure, solvent system, and analytical methodology. Importantly, the revised manuscript does not present C. corallina as quantitatively competitive with H. pluvialis. Instead, the present findings are positioned as initial quantitative evidence of an astaxanthin-corresponding component in an underexplored freshwater macroalgal biomass. The corresponding quantitative comparison and clarification have been incorporated into Section 4.1 of the revised manuscript (Lines 783–798).

2) The evaluation of the antioxidant, anti-inflammatory, and anti-photoaging activities was performed according to well-defined and rigorous methodologies. Still, for all the assays, data for a sample of pure astaxanthin are missing.

Response to Reviewer:

We thank the Reviewer for this important observation and appreciate the positive assessment of the methodologies used to evaluate the biological activities of the extract. We agree that inclusion of purified astaxanthin as a comparator would have strengthened the study by allowing direct comparison of biological potency and assessment of the relative contribution of astaxanthin to the observed responses.

Purified astaxanthin was not included as a biological comparator in the original experimental design. Therefore, rather than making unsupported compound-specific interpretations, we have substantially revised the manuscript to clearly define the biological evaluation as an assessment of the chemically complex Chara corallina extract (CCE) as a whole. HPLC–PDA analysis revealed a chromatographic component with retention-time and UV–visible spectral characteristics corresponding to those of the authentic astaxanthin reference standard, together with additional unresolved chromatographic components. Accordingly, the biological responses observed with CCE cannot be attributed exclusively to astaxanthin.

 

Throughout the revised Results and Discussion, the DPPH radical-scavenging activity, modulation of antioxidant- and inflammation-associated gene expression, increased COL1A2 mRNA expression, and inhibition of extracellular matrix-degrading enzymes are now explicitly described as CCE-associated responses. Statements implying that astaxanthin itself was responsible for these effects have been removed or moderated. Likewise, previous studies describing the biological and molecular activities of purified astaxanthin are now used only to provide relevant biological context and are not presented as direct mechanistic evidence for the activity of CCE.

 

We have also explicitly acknowledged the absence of purified astaxanthin as a comparator as a limitation of the present study. The revised manuscript now clarifies that the present data neither determine the relative contribution of astaxanthin to the observed biological responses nor permit direct comparison of the biological potency of CCE with purified astaxanthin. Other carotenoids and unidentified constituents of CCE may have contributed independently or through additive or synergistic interactions. Direct comparison with purified astaxanthin and activity-guided chemical characterization are therefore identified as important directions for subsequent investigation.

 

Accordingly, the Materials and Methods (Lines 239–243), Results (Lines 695–697), Discussion (Lines 953–961), Study Limitations (Lines 1114–1128), and Conclusion (Lines 1167–1175) have been revised to ensure that the biological interpretation is restricted to the experimentally evaluated CCE and does not imply astaxanthin-specific activity beyond the available evidence.

3) In Table 1, the volume of extraction solvent must be indicated, corresponding to the amount (mg) of astaxanthin evaluated using the calibration curve.

Response to Reviewer:

We thank the Reviewer for identifying this important omission. We agree that the extraction solvent volume should be explicitly reported to provide an appropriate basis for interpreting the astaxanthin-equivalent recovery expressed in milligrams in Table 1.

We have therefore revised Section 2.3 and Table 1 to clarify the complete extraction procedure and total solvent volume used. Each independent extraction replicate was performed using 5.0 g of dried Chara corallina biomass. The initial UAE cycle was conducted with 100 mL of extraction solvent, after which the residual biomass was re-extracted once with an additional 100 mL of fresh solvent of the same composition under identical conditions. The supernatants obtained from the two sequential extraction cycles were subsequently combined. Thus, a total extraction solvent volume of 200 mL was used per 5.0 g of dried biomass for each independent extraction replicate.

 

The caption and footnote of Table 1 have been revised accordingly to explicitly indicate the biomass amount, solvent volume used in each extraction cycle, total solvent volume, and the basis of the reported astaxanthin-equivalent recovery. These clarifications ensure that the quantitative values derived from the HPLC–PDA external calibration curve can be interpreted in relation to the complete extraction procedure.

These revisions have been incorporated into Section 2.3 (Lines 176–178, 198-206) and Table 1 and its footnote (Lines 579–592).

4) On page 21, the authors write, "Utilization of ultrasound-assisted extraction preserves the
antioxidant functionality of astaxanthin-rich extracts." However, there is no evidence of this integrity. The chromatogram in Figure 2B shows the presence of astaxanthin, but along with numerous other peaks, some of which could be products of partial oxidation. This must be verified by HPLC-ESIMS analysis, which can also indicate the presence of other carotenoids that may contribute to the biological activities of the extract.

Response to Reviewer:

We thank the Reviewer for this important and well-founded observation. We agree that the original statement, “Utilization of ultrasound-assisted extraction preserves the antioxidant functionality of astaxanthin-rich extracts,” was not sufficiently supported by the analytical evidence obtained in the present study. HPLC–PDA can demonstrate chromatographic and UV–visible spectral correspondence with an authentic reference standard, but it cannot by itself establish preservation of astaxanthin chemical integrity or definitively identify the additional components observed in the chromatogram.

 

Accordingly, we have removed the statement that UAE preserves the antioxidant functionality, chemical integrity, or stability of astaxanthin, together with other statements throughout the manuscript that could imply such preservation. The HPLC–PDA results are now described more conservatively as demonstrating a chromatographic component with retention-time and UV–visible spectral characteristics corresponding to those of the authentic astaxanthin reference standard.

 

We also agree with the Reviewer that the additional peaks observed in Figure 2B require structural characterization before their identities can be established. Because orthogonal mass-spectrometric analysis was not performed in the present study, we have explicitly stated that these additional chromatographic components remain unresolved and cannot be assigned to other carotenoids, astaxanthin derivatives, or potential degradation/oxidation products on the basis of HPLC–PDA alone. The possibility that some of these components represent degradation or oxidation products therefore cannot be excluded.

 

We further agree that HPLC–ESI-MS/LC–MS/MS would provide valuable complementary structural information for identifying these additional components and characterizing other carotenoids present in the extract. Rather than inferring structural identities that are not supported by the present analytical dataset, we have restricted the revised manuscript to the analytical evidence actually obtained and have explicitly acknowledged the absence of orthogonal mass-spectrometric characterization as a limitation.

 

Importantly, this revision has also been incorporated into the interpretation of the biological assays. CCE is now consistently described as a chemically complex extract, and the antioxidant-, inflammation-, and extracellular matrix-related responses are interpreted as properties of CCE as a whole. We explicitly acknowledge that other carotenoids and unidentified constituents represented among the unresolved chromatographic components may have contributed independently or through additive or synergistic interactions to the observed biological responses.

 

Accordingly, Section 2.4 (Lines 213–220), the HPLC–PDA Results (Lines 565–574)and Figure 2 legend (Lines 602–607), Section 4.1, the biological Discussion (Lines 780–798), Study Limitations (Lines 1127–1146), and Conclusion (Lines 1200–1208) have been revised to remove unsupported claims regarding chemical integrity and to clearly define the analytical limitations of the present study.

5) On page 22, the authors write, "Although astaxanthin was successfully identified and quantified in the selected extract, other carotenoids, phenolic compounds, and minor bioactive constituents were not comprehensively characterized." This cannot be a future perspective, but the chemical characterization of the extract must be described in this work, which otherwise provides too little information to indicate the algae studied here as a worthy source for naturally derived astaxanthin already on the market.

Response to Reviewer:

We thank the Reviewer for this important concern. We agree that comprehensive chemical characterization would be required to establish Chara corallina as a chemically defined, quantitatively competitive, or commercially relevant source of natural astaxanthin. We also agree that presenting the absence of such characterization merely as a future perspective, while simultaneously drawing strong conclusions regarding C. corallina as an alternative source of natural astaxanthin, was not sufficiently supported by the analytical dataset of the present study.

 

Accordingly, we have substantially revised the scope and positioning of the manuscript rather than retaining conclusions that would require analytical evidence not obtained in the present study. The revised manuscript no longer presents C. corallina as an established, commercially competitive, or market-ready alternative to currently available natural astaxanthin sources such as Haematococcus pluvialis. We have also removed or moderated the terms “astaxanthin-rich,” “commercial alternative,” and related statements implying that the present chemical characterization is sufficient to establish C. corallina as a defined natural astaxanthin source.

 

The chemical findings are now explicitly restricted to the analytical evidence obtained in this study. Specifically, HPLC–PDA revealed and quantified a chromatographic component showing retention-time and UV–visible spectral correspondence with an authentic astaxanthin reference standard. We have revised the manuscript to clarify that this evidence does not constitute comprehensive structural characterization of CCE. The additional chromatographic components remain unresolved and cannot presently be assigned to other carotenoids, astaxanthin derivatives, phenolic constituents, or potential degradation/oxidation products. Orthogonal mass-spectrometric confirmation and comprehensive carotenoid/phytochemical profiling were not performed, and this analytical limitation is now explicitly stated in the Results, Discussion, and Study Limitations.

 

Importantly, this revision also changes the interpretation of the biological data. CCE is now consistently described as a chemically complex extract, and the observed DPPH radical-scavenging, gene-expression, and extracellular matrix-related enzyme inhibitory responses are interpreted as properties of CCE as a whole rather than as activities attributable specifically to astaxanthin. Other unresolved constituents may have contributed independently or through additive or synergistic interactions.

 

The contribution of the revised study is therefore framed more conservatively as the integration of UAE-based recovery, HPLC–PDA-based assessment of an astaxanthin-corresponding component, and biological evaluation of the resulting chemically complex extract from an underexplored freshwater macroalgal biomass. The revised manuscript presents these findings as an initial basis for further investigation of C. corallina as a source of carotenoid-containing bioactive extracts, rather than as evidence establishing this species as a commercially competitive source of natural astaxanthin.

 

To ensure consistency with this revised scope, corresponding claims have been revised throughout the Title, Abstract (Lines 26–38), Introduction (Lines 134–137), Materials and Methods (Lines 213–220), Results (Lines 567–571), Discussion (Lines 764–849), Study Limitations (Lines 1115–1135), and Conclusions (Lines 1180–1199).

The manuscript has the potential to become a suitable article, enriching it by detailed comparisons to data already known in the literature, and focusing on the novelty of this study. However, its present version is not acceptable.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

the authors replied to all comments

the authors responded to all comments

Reviewer 2 Report

The authors have considered the comments, and  substantially revised the scope and positioning of the manuscript accordingly.

No detailed comments