Holobiont Rebalancing by a Natural Gentian Extract on a Skin Dehydration Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is a novel subject with high scientific significance.

Author Response

No comments

==>  Authors warmly thank reviewer 1 for his/her appreciation of their work.

Reviewer 2 Report

Comments and Suggestions for Authors

This research article effectively introduces the concept of skin homeostasis and the skin holobiont, but it suffers from several issues that could be beneficial from clarification and improvement. 

Abstract

The description of the development and benefits of the gentiana-fermented extract (GFE) is promising. 

1.     However, the abstract could benefit from a more concise conclusion that succinctly ties together the key findings and their implications for skin health.

 

Introduction

1.     The introduction does not clearly outline the specific objectives or hypotheses of the study. While it mentions a metaproteomic analysis and the development of an active cosmetic ingredient (GFE), the linkage between these elements and the overarching research goal is not explicitly stated.

2.     Line 85-86, the presenting information is not clear. It should be rewritten.

3.     Line 89-90, The phrase “regains the holobiotic characteristics of younger and hydrated skin” seems to be somewhat overclaiming. Suggest presenting the information in a more measured and evidence-based way, avoiding strong claims and emphasizing the need for additional research to validate the potential effects of the product.

 

Methodology

1.     Line 104-106, Kindly state rationale for combining the fermented extract with the purified ethanolic extract to form the final product.

2.     Line 107, The rationale for the final stabilization in water/propanediol is not fully explained.

3.     Line 116-119, The incubation time of 72 hours with GFE 0.5% lacks a justification or reference to support its appropriateness for measuring HA release (about the biological relevance of this timeframe). 

4.     Line 131, The description of the replication strategy is inconsistent, with six replications for GFE and four for D-panthenol, which may affect the robustness of comparative analysis. 

5.     Line 133-135, While the primary and secondary antibodies for CD44 detection are specified, the protocol lacks details on the duration and conditions of antibody incubation, which are crucial for reproducibility.  

6.     Line 139-142, The quantification process, although described, could benefit from more details on the image analysis techniques and software used.   

7.     Line 151-155, The study claims exemption from Ethics Committee approval and Competent Authority authorization based on certain regulatory requirements. However, it's essential to ensure that the exemption criteria are clearly justified and appropriate for the nature of the study, especially when human subjects are involved. Transparency in regulatory compliance should be explicitly stated.

8.     Line 158-159, While a sub-panel of sebum-deficient skin was included, the rationale for this subgroup and its size relative to the main group should be clearly justified.

9.     Line 183-185, The study design includes two groups: older women with dehydrated skin and younger women with various skin types as controls. However, it's not explicitly clear why the younger group with all skin types serves as an appropriate control for metaproteomic analysis of dehydrated skin.  The rationale behind this should be clarified.

10.  Line 192, While the use of nano LC coupled with High Resolution Accurate Mass Spectrometry (HRAM MS) is appropriate for proteomic analysis, specific details such as the chromatographic conditions and mass spectrometry settings (e.g., resolution, scan range) are missing.

 

Results

1.     Figure 5. The presentation of Figure 5 is confusing to the reader, especially on the right panel.  Do the authors mean up-regulation of several categories can be seen in dehydrated older D0? 

2.     Figure 6. What would be the title of Y axis in 6a and 6b?

 

Discussion

1.     While the discussion explains the role of GFE in enhancing hyaluronic acid (HA) and CD44 synthesis, the mechanistic pathway of how GFE achieves these effects should be discussed. 

2.     The claim that GFE preserves microbiota diversity while increasing certain beneficial species is significant. The broader implications of altering the microbiome composition should be discussed.

3.     It is suggested to include a comparison with other similar studies or established treatments.

4. Line 346, spelling error: “Morevover”.

 

Conclusion

 

1.     It is suggested to add on future research directions, particularly in understanding the long-term effects of GFE on skin health and its potential therapeutic applications.

Comments on the Quality of English Language

Some parts should be improved for spelling checks.

Author Response

This research article effectively introduces the concept of skin homeostasis and the skin holobiont, but it suffers from several issues that could be beneficial from clarification and improvement. 

• Abstract

The description of the development and benefits of the gentiana-fermented extract (GFE) is promising. 

1. However, the abstract could benefit from a more concise conclusion that succinctly ties together the key findings and their implications for skin health.

==> As requested by reviewer 2, the last sentence of the abstract was modified accordingly (lines 22-23 in revised version of the manuscript)

 

• Introduction

1. The introduction does not clearly outline the specific objectives or hypotheses of the study. While it mentions a metaproteomic analysis and the development of an active cosmetic ingredient (GFE), the linkage between these elements and the overarching research goal is not explicitly stated.

==> Authors thank the reviewer for his/her comment. In their opinion, this point is already addressed in lines 81-84: “In the present study, age and dehydration were taken here as putative modulators or stressors of the holobiont functions to determine the impact of stresses on skin holobiont protein interactions and find a natural topical solution to rebalance the observed modulations.”

2. Line 85-86, the presenting information is not clear. It should be rewritten.

==> Authors apologize for their lack of clarity. As requested, the sentence was rewritten (lines 85-87 in revised version of the manuscript): “In vitro, GFE acts on hyaluronic acid production and protection, as well as epidermal lipid synthesis. Clinically GFE increases, on dehydrated volunteers, skin hydration and skin surface lipids versus placebo.”

3. Line 89-90, The phrase “regains the holobiotic characteristics of younger and hydrated skin” seems to be somewhat overclaiming. Suggest presenting the information in a more measured and evidence-based way, avoiding strong claims and emphasizing the need for additional research to validate the potential effects of the product.

==> As requested, sentence was rewritten as follow (lines 87-90 in revised version of the manuscript): “Thanks to a metaproteomic study aiming at deciphering the main biological functions modulated between aged dehydrated and young skins, the restorative properties of GFE on skin holobiont were demonstrated.”

• Methodology

1. Line 104-106,Kindly state rationale for combining the fermented extract with the purified ethanolic extract to form the final product.

==> As requested, authors completed the material and method section in order to detail the process that led them to obtain GFE (lines 101-103 in revised version of the manuscript): “In order to enrich amino acids and lipids, a fermentation has been conducted in parallel, as antioxidant molecules present in gentian roots are consumed by the bacteria (data not shown).”

2. Line 107, The rationale for the final stabilization in water/propanediol is not fully explained.

==> In order to detail the choice of stabilizer, authors completed the following sentence (lines 108-110 in revised version): “The latter is finally stabilized in water/propanediol (50/50) to solubilize both antioxidant molecules from the ethanolic extract and the fermented aqueous one.”

3. Line 116-119, The incubation time of 72 hours with GFE 0.5% lacks a justification or reference to support its appropriateness for measuring HA release (about the biological relevance of this timeframe).

==> Previous internal studies have shown the relevancy of the timing for HA measurement (ELISA). Furthermore, some published datas showed the detection of HA after 72h of treatment with a cosmetic extract (Zeichner et al, J. Clin. Aesthet. Dermatol., 2024) 

4. Line 131, The description of the replication strategy is inconsistent, with six replications for GFE and four for D-panthenol, which may affect the robustness of comparative analysis. 

==> As requested, the sentence was rewritten for more consistency in the methodology (lines 130-132 in revised version): Immunodetection and quantification of CD44 were performed on NHEK (n4) from 55-year-old donors treated 48h with GFE 0.5% or D-panthenol 0.3% or nothing (control). Topical D-pantenol is a well-known molecule that acts like a moisturizer (Ebner et al., 2002).

5. Line 133-135, While the primary and secondary antibodies for CD44 detection are specified, the protocol lacks details on the duration and conditions of antibody incubation, which are crucial for reproducibility.

==> As requested, details of antibody incubation were added to the text (lines 135-136 in revised version)

6. Line 139-142, The quantification process, although described, could benefit from more details on the image analysis techniques and software used.

==> Software used for image analysis was added in the text (line 142 in revised version)

7. Line 151-155, The study claims exemption from Ethics Committee approval and Competent Authority authorization based on certain regulatory requirements. However, it's essential to ensure that the exemption criteria are clearly justified and appropriate for the nature of the study, especially when human subjects are involved. Transparency in regulatory compliance should be explicitly stated.

==> Authors agree with reviewer and apologize for the unwanted lack of transparency. The paragraph was modified accordingly (lines 152-160 in revised version);

“This study was performed by EUROFINS Dermscan/Pharmascan, which is authorized as a clinical investigation center by the French Health Authorities, on cosmetic products defined by the article L. 5131-1 of the French Public Health Code, in accordance with Decree n° 2017-884 of May 9, 2017 modifying some regulatory requirements concerning researches involving human subjects. Thus, this trial adhered to the principles of good clinical practices and the declaration of Helsinki. According to local and European regulatory guidelines (Official journal of EU of March 10th 2010, paragraph 1.2.9), this type of trial testing on active cosmetics does not require ethics committee approval or the com-petent authority authorization.”

8. Line 158-159, While a sub-panel of sebum-deficient skin was included, the rationale for this subgroup and its size relative to the main group should be clearly justified.

==> Authors apologize for the lack of clarity. The paragraph was modified for a better understanding of the methodology (lines 163-165 in revised version):

“Among them, a sub-panel of sebum-deficient skin was identified (n=10), with sebum <100mg/cm2 on forehead, measured by Sebumeter® [18].”

9. Line 183-185, The study design includes two groups: older women with dehydrated skin and younger women with various skin types as controls. However, it's not explicitly clear why the younger group with all skin types serves as an appropriate control for metaproteomic analysis of dehydrated skin.  The rationale behind this should be clarified.

==> Sorry for the confusion. “all skin types” means multiple phototypes. In order to avoid any confusion, “all skin types” was removed from the sentence (lines 185-187 in revised version)

10. Line 192, While the use of nano LC coupled with High Resolution Accurate Mass Spectrometry (HRAM MS) is appropriate for proteomic analysis, specific details such as the chromatographic conditions and mass spectrometry settings (e.g., resolution, scan range) are missing.

==> As requested, supplementary details were added in the paragraph (lines 196-200 in revised version):

“Resulting peptidic mixtures (500 ng) were injected and analyzed using nano-LC HRAM MS technology (nano chromatography PepMap100 C18 column; 2.5% to 35% acetonitrile gradient; coupled with High Resolution Accurate Mass Spectrometer Q-Exactive Plus, Thermo at a resolution of 70,000 for MS scan and 17,500 for MS/MS scan).”

• Results

1. Figure 5.The presentation of Figure 5 is confusing to the reader, especially on the right panel.  Do the authors mean up-regulation of several categories can be seen in dehydrated older D0? 

==> Authors deeply apologize for this mistake. The new figure 5 replaces the old one in revised version of the manuscript

2. Figure 6. What would be the title of Y axis in 6a and 6b?

==> Sorry for this omission. Figures 6a and 6b were modified

• Discussion

1. While the discussion explains the role of GFE in enhancing hyaluronic acid (HA) and CD44 synthesis, the mechanistic pathway of how GFE achieves these effects should be discussed. 

==> According to reviewer’s comment, a supplementary paragraph was added in the discussion (lines 373-383 in revised version of the manuscript):

“GFE contains active molecules which have demonstrated their efficiency in biological assays. Indeed, swertiamarin (STM) was shown to reduce ROS and superoxide anion levels and reduce interferon and inflammatory factors via the cGAS-STING pathway [33]. STM inhibits lipid peroxidation and reduces the level of inflammatory mediators by activating the defense system of nuclear factor erythroid 2 related factor 2 (Nrf2) and inhibiting nuclear factor-κ B (NF-κB) [34]. Interestingly, GFE also contains loganic acid, which was also shown to exhibit a protective effect through its anti-inflammatory and anti-oxidant effects via inactivating the TLR4/NF-κB signaling pathway and activating the SIRT1/Nrf2 pathways [35]. Thus, it is highly likely that GFE acts on HA/CD44 pathway through NF-κB downregulation and Nrf2 upregulation.”

Bibliographic references were modified accordingly:

33. Zhao, Z.; He, D.; Wang, J.; Xiao, Y.; Gong, L.; Tang, C.; Peng, H.; Qiu, X.; Liu, R.; Zhang, T.; et al. Swertiamarin Relieves Radiation-Induced Intestinal Injury by Limiting DNA Damage. Mol. Cell. Biochem. 2024, doi:10.1007/s11010-024-05030-z.
34. Zhou, Q.; Zhou, Q.; Xia, R.; Zhang, P.; Xie, Y.; Yang, Z.; Khan, A.; Zhou, Z.; Tan, W.; Liu, L. Swertiamarin or Heat-Transformed Products Alleviated APAP-Induced Hepatotoxicity via Modulation of Apoptotic and Nrf-2/NF-κB Pathways. Heliyon 2023, 9, e18746, doi:10.1016/j.heliyon.2023.e18746.
35. Prakash, A.N.; Prasad, N.; Puppala, E.R.; Panda, S.R.; Jain, S.; Ravichandiran, V.; Singh, M.; Naidu, V.G.M. Loganic Acid Protects against Ulcerative Colitis by Inhibiting TLR4/NF-κB Mediated Inflammation and Activating the SIRT1/Nrf2 Anti-Oxidant Responses in-Vitro and in-Vivo. Int. Immunopharmacol. 2023, 122, 110585, doi:10.1016/j.intimp.2023.110585.

 

2. The claim that GFE preserves microbiota diversity while increasing certain beneficial species is significant. The broader implications of altering the microbiome composition should be discussed.

==> According to reviewer’s comment, a supplementary paragraph was added in the discussion (lines 421-427 in the revised version): “Preserving microbiota diversity at genus level is key to maintain skin homeostasis. Indeed, a disruption to that delicate balance (due to barrier alteration or imbalance between symbionts and pathogens for example) may lead to an impaired skin function or even to diseases including atopic dermatitis, psoriasis or cutaneous lupus [50]. Further investigations would be worth to address GFE efficacy towards such skin pathologies in which microbiota, hydration and lipid imbalance have been highlighted [51–53].”

Bibliographic reference was modified accordingly:

50. Zhu, Y.; Yu, X.; Cheng, G. Human Skin Bacterial Microbiota Homeostasis: A Delicate Balance between Health and Disease. mLife 2023, 2, 107–120, doi:10.1002/mlf2.12064.
51. Wu, J.; Li, L.; Zhang, T.; Lu, J.; Tai, Z.; Zhu, Q.; Chen, Z. The Epidermal Lipid-Microbiome Loop and Immunity: Important Players in Atopic Dermatitis. J. Adv. Res. 2024, S2090-1232(24)00088-2, doi:10.1016/j.jare.2024.03.001.
52. Filatov, V.; Sokolova, A.; Savitskaya, N.; Olkhovskaya, M.; Varava, A.; Ilin, E.; Patronova, E. Synergetic Effects of Aloe Vera Extract with Trimethylglycine for Targeted Aquaporin 3 Regulation and Long-Term Skin Hydration. Mol. Basel Switz. 2024, 29, 1540, doi:10.3390/molecules29071540.
53. Kosik-Kozioł, A.; Nakielski, P.; Rybak, D.; Frączek, W.; Rinoldi, C.; Lanzi, M.; Grodzik, M.; Pierini, F. Adhesive Antibacterial Moisturizing Nanostructured Skin Patch for Sustainable Development of Atopic Dermatitis Treatment in Humans. ACS Appl. Mater. Interfaces 2024, 16, 32128–32146, doi:10.1021/acsami.4c06662

 

3. It is suggested to include a comparison with other similar studies or established treatments.

==> According to reviewer’s comment, a supplementary paragraph was added in the discussion (lines 345-354 in the revised version of the manuscript):

“Numerous studies demonstrated the interest of using fermented plant extracts for cosmetic purposes. For example, the bio-fermented Aframomum angustifolium extract allowed better hydration of the stratum corneum and improved dermal-epidermal connectivity in treated skins [24]. Fermented grape stem extracts can be used in moisturizing cosmetic formulations and also to complement the treatment of dry and sensitive skin [25]. Noteworthy, our study enhanced also the interest of the use of fermentation in cosmetic application as it participates to extend potential applications. Indeed, our group previously demonstrated the efficacy of gentian extracts as anti-aging eye contour [26]. The present study demonstrates for the first time the impact of fermentation on extract properties, especially on skin holobiont.”

Bibliographic references were modified accordingly:

24. Albouy, M.; Aubailly, S.; Jeanneton, O.; Marteau, C.; Sobilo, L.; Boulgana, R.; Bru, G.; Bellanger, M.; Leblanc, E.; Dos Santos, M.; et al. Skin-Protective Biological Activities of Bio-Fermented Aframomum Angustifolium Extract by a Consortium of Microorganisms. Front. Pharmacol. 2023, 14, 1303198, doi:10.3389/fphar.2023.1303198.
25. Stanek-Wandzel, N.; Zarębska, M.; Wasilewski, T.; Hordyjewicz-Baran, Z.; Zajszły-Turko, E.; Tomaka, M.; Bujak, T.; Ziemlewska, A.; Nizioł-Łukaszewska, Z. Kombucha Fermentation as a Modern Way of Processing Vineyard By-Products into Cosmetic Raw Materials. Int. J. Cosmet. Sci. 2023, 45, 834–850, doi:10.1111/ics.12891.
26. Berthon, J.-Y.; Cabannes, M.; Bouton, C.; Carre, M.; Bridon, E.; Filaire, E. In Vitro, Ex Vivo and Clinical Approaches to Evaluate the Potential Effect of Gentiana Lutea Extract on Skin. Int. J. Cosmet. Sci. 2023, 45, 688–698, doi:10.1111/ics.12878

4. Line 346, spelling error: “Morevover”.

==> Typo was corrected

 

• Conclusion

1. It is suggested to add on future research directions, particularly in understanding the long-term effects of GFE on skin health and its potential therapeutic applications.

==> According to reviewer’s comment, a supplementary paragraph was added in the discussion (lines 421-427 in revised version of the manuscript):

“Preserving microbiota diversity at genus level is key to maintain skin homeostasis. Indeed, a disruption to that delicate balance (due to barrier alteration or imbalance between symbionts and pathogens for example) may lead to an impaired skin function or even to diseases including atopic dermatitis, psoriasis or cutaneous lupus [50]. Further investigations would be worth to address GFE efficacy towards such skin pathologies in which microbiota, hydration and lipid imbalance have been highlighted [51–53].”

Bibliographic references were modified accordingly:

50. Zhu, Y.; Yu, X.; Cheng, G. Human Skin Bacterial Microbiota Homeostasis: A Delicate Balance between Health and Disease. mLife 2023, 2, 107–120, doi:10.1002/mlf2.12064.
51. Wu, J.; Li, L.; Zhang, T.; Lu, J.; Tai, Z.; Zhu, Q.; Chen, Z. The Epidermal Lipid-Microbiome Loop and Immunity: Important Players in Atopic Dermatitis. J. Adv. Res. 2024, S2090-1232(24)00088-2, doi:10.1016/j.jare.2024.03.001.
52. Filatov, V.; Sokolova, A.; Savitskaya, N.; Olkhovskaya, M.; Varava, A.; Ilin, E.; Patronova, E. Synergetic Effects of Aloe Vera Extract with Trimethylglycine for Targeted Aquaporin 3 Regulation and Long-Term Skin Hydration. Mol. Basel Switz. 2024, 29, 1540, doi:10.3390/molecules29071540.
53. Kosik-Kozioł, A.; Nakielski, P.; Rybak, D.; Frączek, W.; Rinoldi, C.; Lanzi, M.; Grodzik, M.; Pierini, F. Adhesive Antibacterial Moisturizing Nanostructured Skin Patch for Sustainable Development of Atopic Dermatitis Treatment in Humans. ACS Appl. Mater. Interfaces 2024, 16, 32128–32146, doi:10.1021/acsami.4c06662

 

Comments on the Quality of English Language: Some parts should be improved for spelling checks.

==>  The revised manuscript has been entirely revised and corrected by a native English speaker

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, I have read and reviewed the article entitled Holobiont rebalancing by a natural gentian extract on a skin dehydration model. 

The study focuses on gentiana-fermented extract (GFE) and how it can influence the skin holobiont for a reinforced and rehydrated skin with an optimal lipid content.

-please write Latin names in italics (line 63, 71, 84, 97)

-please rephrase sentence in line 102-103, there is smtg missing

-please use full stop instead of commas in numbers 

 

Comments on the Quality of English Language

Please use passive voice and refrain from using "I" or "we" in scientific text. 

Author Response

-please write Latin names in italics (line 63, 71, 84, 97)

==> Done

-please rephrase sentence in line 102-103, there is smtg missing

==> Done

-please use full stop instead of commas in numbers 

==> Done

Comments on the Quality of English Language

Please use passive voice and refrain from using "I" or "we" in scientific text. 

==> Done