Guang Chenpi Extract as a Multifunctional Phytotherapeutic: Enhanced Effects with Ergothioneine and Polydeoxyribonucleotide on Redox Homeostasis and Tissue Resilience
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study presents interesting results, and the expression of the findings is generally clear and well demonstrated. However, there are several points that the authors could improve to further strengthen the manuscript:
- Organization and logical flow of results
Conventionally, experimental findings are presented in a progressive order from cell-based studies to tissue models, and subsequently to animal or human relevance (i.e., cell → tissue → animal/human). In the current study, however, the results are arranged starting from the in vivo zebrafish model, followed by melanocyte monoculture, and finally the MelaKutis® in vitro human 3D skin model. Reorganizing the sequence to present in vitro findings first, followed by in vivo validation, would provide a more logical scientific flow and improve readability. - Melanocyte toxicity assessment
Related to the previous point, melanocyte experiments in this study focus solely on cytotoxicity testing in human melanocytes. In its current form, the outcome does not strongly correlate with the depigmenting results observed in the 3D human skin model or the zebrafish model. If the authors could include functional melanogenesis assays—such as melanin content measurement or tyrosinase activity in melanocytes—and correlate these findings with depigmentation in the 3D skin model and melanogenesis inhibition in zebrafish, the overall mechanistic linkage would be significantly strengthened.
However, if such melanogenesis data are not available, presenting melanocyte cytotoxicity results only as supplementary material rather than in the main manuscript may be more appropriate. The primary results focusing on the 3D human skin model, followed by in vivo zebrafish validation, would already be sufficient and more coherent. - Clarification of model classification
Please revise the following statement:
“Owing to the barrier function of the stratum corneum, the tolerable concentration for topical application on ex vivo skin models is often more than tenfold greater than the cytotoxic concentration observed in conventional 2D cell-based assays.”
The authors are encouraged to verify the appropriateness of the term “ex vivo skin models” in this context. MelaKutis® is a reconstructed human pigmented skin equivalent and is generally classified as an advanced in vitro model rather than an ex vivo tissue model.
- Reference formatting
Please revise the reference format according to the journal Pharmaceuticals Based on the Instructions for Authors (https://www.mdpi.com/journal/pharmaceuticals/instructions#references) : “References must be numbered in order of appearance in the text.” And “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].” - Section 2.1 (Chemicals and Reagents)
Please correct the information provided in Section 2.1, regarding "Rehmannia glutinosa Libosch extract (batch number: N103002586) procured from Aladin Group".
Author Response
Comment 1. Organization and logical flow of results
Conventionally, experimental findings are presented in a progressive order from cell-based studies to tissue models, and subsequently to animal or human relevance (i.e., cell → tissue → animal/human). In the current study, however, the results are arranged starting from the in vivo zebrafish model, followed by melanocyte monoculture, and finally the MelaKutis® in vitro human 3D skin model. Reorganizing the sequence to present in vitro findings first, followed by in vivo validation, would provide a more logical scientific flow and improve readability.
Reply 1: Amended as suggested. Thank you.
Comment 2. Melanocyte toxicity assessment
Related to the previous point, melanocyte experiments in this study focus solely on cytotoxicity testing in human melanocytes. In its current form, the outcome does not strongly correlate with the depigmenting results observed in the 3D human skin model or the zebrafish model. If the authors could include functional melanogenesis assays—such as melanin content measurement or tyrosinase activity in melanocytes—and correlate these findings with depigmentation in the 3D skin model and melanogenesis inhibition in zebrafish, the overall mechanistic linkage would be significantly strengthened.
However, if such melanogenesis data are not available, presenting melanocyte cytotoxicity results only as supplementary material rather than in the main manuscript may be more appropriate. The primary results focusing on the 3D human skin model, followed by in vivo zebrafish validation, would already be sufficient and more coherent.
Reply 1: Suggestion accepted. Removed the melanocyte toxicity part to supplementary materials section.
Comment 3. Clarification of model classification
Please revise the following statement:
“Owing to the barrier function of the stratum corneum, the tolerable concentration for topical application on ex vivo skin models is often more than tenfold greater than the cytotoxic concentration observed in conventional 2D cell-based assays.”
Reply 3: Suggestion accepted. Due to the barrier function of the stratum corneum, the tolerable concentration for topical application on in vitro skin models can be over tenfold higher than the cytotoxic concentration observed in conventional 2D cell-based assays
Comment 4. The authors are encouraged to verify the appropriateness of the term “ex vivo skin models” in this context. MelaKutis® is a reconstructed human pigmented skin equivalent and is generally classified as an advanced in vitro model rather than an ex vivo tissue model.
Reply 4: Thanks for the suggestion. Ex vivo model” has been changed to “in vitro model”.
Comment 5. Reference formatting
Please revise the reference format according to the journal Pharmaceuticals Based on the Instructions for Authors (https://www.mdpi.com/journal/pharmaceuticals/instructions#references) : “References must be numbered in order of appearance in the text.” And “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].”
Reply 5: Thanks. We have revised the reference format according to the journal requirement.
Comment 6. Section 2.1 (Chemicals and Reagents)
Please correct the information provided in Section 2.1, regarding "Rehmannia glutinosa Libosch extract (batch number: N103002586) procured from Aladin Group".
Reply 6: Corrected. Thanks.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper by Zongxiao Cen reports data on the use of guang chenpi extract in zebrafish and skin models in response to oxidative and UV stress. Taken together the data support claims that the extract has cell benefits against stress. However, the authors overstate the effects and leap from zebrafish to antiaging in their interpretation.
The paper uses the term “synergistic” 18 times, but it is used each time incorrectly. Synergy means that the result is greater than the sum of the effects, that is, more than additive. However, the experiments reported here never test EGT or PDRN alone. Therefore, they cannot make the comparison to simply the additive effects of two antioxidants. They must remove the word synergy/synergistic from the title and the other text places and modify their claims to what was observed – an increase when adding additional antioxidants.
L441-2 The authors claim that they have no conflict of interest. However, the first three authors work for a company that sells cosmetic formulas containing PDRN and are likely to offer others with guang chenpi extract. This paper is designed to support the sale of these products. This is an obvious conflict of interest, and they should remove this sentence from the paper.
Zebrafish are an interesting model, but they are not human skin, and effects in these studies must be interpreted with caution. However, the authors leap to the conclusion that these studies with tailfin regeneration and skin lesions caused by acetic acid are the same as antiaging effects (e.g. L380-381). The authors should include in their paragraph L400 that the zebrafish model is not human skin and additional experiments in human skin models are needed.
L121: define hpf (hours post fertilization?)
Fig 3A: Line and dotted line are unexplained. The images do not obviously support the interpretation, and the authors should include a detailed explanation of what we are looking for in them.
Fig 5: The legends in Fig 5A (hatched boxes) are too small and hard match up with the data bars. It looks like there is a reverse dose-response relationship, but the authors do not comment on this.
Fig 6 & 7: The data show a statistically significant decrease at low doses, which is the opposite of what is observed in Fig 5. The authors should comment on this.
Author Response
Comment 1. The paper by Zongxiao Cen reports data on the use of guang chenpi extract in zebrafish and skin models in response to oxidative and UV stress. Taken together the data support claims that the extract has cell benefits against stress. However, the authors overstate the effects and leap from zebrafish to antiaging in their interpretation.
Reply 1: Thanks for raising this point. Each though lots of research have been performed to use zebrafish as human disease investigation and drug screening models, The authors bear in mind the differences between fish and human. Thus, we outlined the drawbacks of present in the last paragraph of Discussion.
Comment 2. The paper uses the term “synergistic” 18 times, but it is used each time incorrectly. Synergy means that the result is greater than the sum of the effects, that is, more than additive. However, the experiments reported here never test EGT or PDRN alone. Therefore, they cannot make the comparison to simply the additive effects of two antioxidants. They must remove the word synergy/synergistic from the title and the other text places and modify their claims to what was observed – an increase when adding additional antioxidants.
Reply 2: Thanks for pointing out this important issue. The words “synergistic/synergy” were replaced with “enhance”, “combined”, and “combination” throughout the manuscript.
Comment 3. L441-2 The authors claim that they have no conflict of interest. However, the first three authors work for a company that sells cosmetic formulas containing PDRN and are likely to offer others with guang chenpi extract. This paper is designed to support the sale of these products. This is an obvious conflict of interest, and they should remove this sentence from the paper.
Reply 3: thank you for the suggestion. The paragraph was replaced by below:
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Z.C. (Zongxiao Cen), Z.C. (Zhiyuan Chen), D.W., Y. Z. were employed by Guangzhou Luanying Cosmetics Co., LTD, J.C. and J.P. were employed by Vitargent (International) Biotechnology Limited, and X.C. was employed by Research Institute of Tsinghua, Pearl River Delta and Vitargent (International) Biotechnology Limited.
Comment 4. Zebrafish are an interesting model, but they are not human skin, and effects in these studies must be interpreted with caution. However, the authors leap to the conclusion that these studies with tailfin regeneration and skin lesions caused by acetic acid are the same as antiaging effects (e.g. L380-381). The authors should include in their paragraph L400 that the zebrafish model is not human skin and additional experiments in human skin models are needed.
Reply 4: Please see our first reply.
Comment 5. L121: define hpf (hours post fertilization?)
Reply 5: Thanks for point it out. Yes, added the definition of hpf as hours post fertilization.
Comment 6. Fig 3A: Line and dotted line are unexplained. The images do not obviously support the interpretation, and the authors should include a detailed explanation of what we are looking for in them.
Reply 6: Thanks for point it out. Added the explanation as “Dotted and solid lines indicate the amputation plane and the regenerated fin edge, respectively.”
Comment 7. Fig 5: The legends in Fig 5A (hatched boxes) are too small and hard match up with the data bars. It looks like there is a reverse dose-response relationship, but the authors do not comment on this.
Reply 7: Thanks for the comments. The hatched boxes were enlarged in Fig 5A. The title was amended as “GCE upregulated the expression of type I collagen (col1a1a, col1a1b, and col1a2) and elastin (elna) genes in zebrafish embryos, with a more potent effect observed at lower concentrations.”
Comment 8. Fig 6 & 7: The data show a statistically significant decrease at low doses, which is the opposite of what is observed in Fig 5. The authors should comment on this.
Reply 8: Thanks for raising the point. The authors noticed the trend differences too, while current knowledge can’t provide a solid explanation and thus not able to comment on it.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis work investigates the bioactivity of a standardized Guang Chenpi extract (the aged pericarp of Citrus reticulata) and synergistic potential with ergothioneine and polydeoxyribonucleotide in vitro and in vivo.
The manuscript is clear, well-structured, and relevant to the pharmaceutical and cosmeceutical sector, as there is a current need to source natural raw materials to formulate organic and sustainable formulations with dermatological applications.
The experimental design is adequate to test the hypothesis, as are the figures, tables, and graphs. The cited references are relevant and up to date, and the conclusions are consistent with the evidence and arguments presented.
However, a minor revision is required for approval.
Specific comments:
- Line 106 and following: Please correct the Celsius degree format (100 °C).
Symbols typesetting: The general rule of the International Bureau of Weights and Measures (BIPM) is that the numerical value always precedes the unit, and a space is always used to separate the unit from the number, e.g. "30.2 °C" (not "30.2°C" or "30.2° C")
Author Response
Comment 1. Specific comments:
- Line 106 and following: Please correct the Celsius degree format (100 °C).
Symbols typesetting: The general rule of the International Bureau of Weights and Measures (BIPM) is that the numerical value always precedes the unit, and a space is always used to separate the unit from the number, e.g. "30.2 °C" (not "30.2°C" or "30.2° C")
Reply 1: The typo was corrected. Thank you.
