Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript aims to explain combined action of some peptides and silybin on skin. The experiments include relevant cells and skin explants as well as clinical cosmetic investigation. The results are interesting and useful to motivate development of novel cosmetics. The presentation, however, needs improvement. 

1. Looking at Fig 1 and other data presented in the paper it is impossible to understand concentrations of components and exact compositions which were used in experiments. Authors should present tables of what exactly was in the treatment formulations and what were their concentrations in cellular or explant culture media. Listing in Table S1 is insufficient to derive the concentrations which were in the culture media. 
2.  Since the study includes clinical studies appropriate ethical permits should be disclosed. 
3. In the discussion § authors might including signalling diagram to summarise claimed synergy effects. 

Author Response

Comments 1: Looking at Fig 1 and other data presented in the paper it is impossible to understand concentrations of components and exact compositions which were used in experiments. Authors should present tables of what exactly was in the treatment formulations and what were their concentrations in cellular or explant culture media. Listing in Table S1 is insufficient to derive the concentrations which were in the culture media.

Response 1: We thanks for this comment of concentrations. The testing concentrations of multi-peptides in UV irradiation have been added in the figure panels (Figures 3–5, S1–S3), and the corresponding figure legends have been revised accordingly. As the ratio of the multi-peptides and silybin in the cream is identical to that used in the explant culture media, and to protect proprietary formulation details, we disclose the overall concentration of the active complex rather than the exact ratio of individual components. These modifications clarify the experimental conditions and improve the readability of the figures.

 

Comments 2: Since the study includes clinical studies appropriate ethical permits should be disclosed.

Response 2: We thank the reviewer for this important comment. The ethical approval details (approval number and date) have been disclosed in the Institutional Review Board Statement section of the manuscript. In addition, we have provided the official ethics approval letter to the editor for their records.

 

Comments 3: In the discussion § authors might including signalling diagram to summarise claimed synergy effects.

Response 3: We thank the reviewer for this constructive suggestion. In the revised Discussion, we have added a description of the functional synergy mechanism (page 19, Figure 10), highlighting that a combination of functionally diverse peptides (collagen stimulation, elastin enhancement, matrix protection) together with the antioxidant and photoprotective roles of silybin produces greater ECM benefits than peptides alone. The consistency of results across in vitro, ex vivo, and clinical evaluations further confirms the translational validity of these effects.

In addition, we have included a signaling diagram to summarize this proposed synergy, as suggested.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "Anti-aging Efficacy of a Multi-Peptides-Silybin Complex: Mechanistic Insights and a 56-Day Clinical Evaluation" is written and presented as per the journal style. The study includes in vitro mechanistic data with ex vivo UV-damage restoration and acceptable clinical efficacy. The result and discussion are supported by protein expression of ECM-related markers, ex vivo,, and a a 56-day clinical trial using advanced noninvasive instrumental methods. Data from a vehicle study (56 days) without using multi-peptide can be included for comparison if available. 

Based on supplied data and results, I recommend the acceptance of the manuscript in the present form. 

Author Response

Comments: The manuscript entitled "Anti-aging Efficacy of a Multi-Peptides-Silybin Complex: Mechanistic Insights and a 56-Day Clinical Evaluation" is written and presented as per the journal style. The study includes in vitro mechanistic data with ex vivo UV-damage restoration and acceptable clinical efficacy. The result and discussion are supported by protein expression of ECM-related markers, ex vivo, and a a 56-day clinical trial using advanced noninvasive instrumental methods. Data from a vehicle study (56 days) without using multi-peptide can be included for comparison if available.

Based on supplied data and results, I recommend the acceptance of the manuscript in the present form.

Response: We sincerely thank the reviewer for the thorough evaluation and the positive recommendation for acceptance. We also appreciate the thoughtful suggestion to include a 56-day clinical arm without the multi-peptide blend.

This study was conducted within a product-development program with a pre-specified, single-arm clinical design under ethics approval, focusing on within-participant baseline-to-post changes using standardized, noninvasive instrumentation. A no-multi-peptide clinical arm was therefore not included in the present protocol. To contextualize clinical findings mechanistically, our in vitro and ex vivo experiments incorporated appropriate comparators (including minimal vehicle and peptides-only conditions), which helped isolate the incremental contribution of silybin at the tissue level and demonstrated convergence across models.

We agree that a controlled clinical design (e.g., split-face or a no-active comparator arm) could further strengthen the evidence base. We will consider such designs in future work aimed at comparative clinical profiling, while the current dataset, in our view, provides sufficient and reliable support for the conclusions presented.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Title is ok

2. Please add future studies in the abstract

3. Why only use Silybum marianum only, is there any other plants that have synergistic effet with Silybum marianum? Add some explanation in the introduction.

4. Please add literature on all sections of methods

5. Since this study using human volunteer, please add ethical clearance of clinical study by committee from research institute or university

6. Result is ok

7. In the discussion part, please add some bioactive compound from S. marianum that probably involved in the mechanism of anti-ageing.

8. In conclusion, add some quantitative result of the study.

 

Author Response

Comments 1: Title is ok

Response 1: We thank the reviewer for this positive comment.

 

Comments 2: Please add future studies in the abstract

Response 2: Thank you for pointing this out. We have added a statement on future studies to the Abstract (page 1, lines 27–28) of the revised manuscript.

 

Comments 3: Why only use Silybum marianum only, is there any other plants that have synergistic effet with Silybum marianum? Add some explanation in the introduction.

Response 3: We thank the reviewer for this insightful comment. In the revised manuscript, we have added an explanation in the Introduction (page 2, lines 83-89 and lines 96-97) regarding our rationale for selecting Silybum marianum. Silybin is a retinoid-like botanical that can complement the lack of reactive oxygen species scavenging capacity of peptides. Moreover, published evidence indicates that its activity is superior to the well-known retinoid-like compound, bakuchiol. We have noted that future work may extend to other botanical candidates.

 

Comments 4: Please add literature on all sections of methods

Response 4: Thanks for your helpful advice. In the revised manuscript, we have added appropriate references to support each section of the Materials and Methods. Specifically:

 

Section 2.2 (Cell Culture, page 4, line 139): Added references [31] for the cell culture procedure.

Section 2.5 (UV Irritation and Drug Treatment, page 5, line 172): Added reference [32] for UVA and UVB irradiation.

Section 2.6 (MTT Assay, page 5, line 187): Added reference [33] for the cell viability evaluation procedure.

Section 2.7 (RNA Extraction and qRT-PCR, page 5, lines 196-197): Added reference [34] for RNA extraction and qRT-PCR procedure. 2^-△△CT method was shown in reference [35].

Section 2.8 (Immunofluorescence (IF) Staining, page 5, line 204): Added reference [34] for IF staining procedure.

Section 2.9 (Western Blot (WB) Assay, page 6, line 220): Added reference [36] for WB assay procedure.

Section 2.10 (Victoria Blue (VB) Staining, page 6, line 227): Added reference [37] for VB staining procedure.

Section 2.12 (Statistical Analysis, page 6, line 246): Added reference [38] for statistical approach.

 

These changes ensure that all methodologies are now appropriately referenced for clarity and reproducibility.

 

Comments 5: Since this study using human volunteer, please add ethical clearance of clinical study by committee from research institute or university

Response 5: We thank the reviewer for this important comment. The study involving human volunteers was reviewed and approved by the Ethic Committee for clinical research of SGS-CSTC Standards Technical Services Co., Ltd. The approval number and date have been disclosed in the Institutional Review Board Statement section of the manuscript, and the official ethics approval letter has also been provided to the editor for reference.

 

Comments 6: Result is ok

Response 6: We appreciate the reviewer’s confirmation.

 

Comments 7: In the discussion part, please add some bioactive compound from S. marianum that probably involved in the mechanism of anti-ageing.

Response 7: We thank the review for this suggestion. In the revised manuscript, we have added a description of the major bioactive constituents of S. marianum (silybin, isosilybin, silychristin, and silydianin) and their reported antioxidant and protective mechanisms in the Discussion (page 18, lines 426–432).

 

Comments 8: In conclusion, add some quantitative result of the study.

Response 8: We thank the reviewer for this valuable suggestion. In the revised manuscript, we have added quantitative results from both the in vitro and clinical studies to strengthen the conclusion. These additions can be found on page 20, lines 508-511 and line 516 of the revised version.