Evaluating the Effect of Fresh and Aged Antioxidant Formulations in Skin Protection Against UV Damage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for the opportunity to read your work. I write you in regard to your manuscript titled "Evaluating the effect of fresh and aged antioxidant formulations in skin protection against UV damage". 

• Please add to the abstract the storage conditions under which the samples were stored during the study.
• Please add the temperature at which samples were irradiated (item 2.1).
• Describe the qualitative and quantitative composition of the samples and the type of packaging material. How was the staorage conditions?
• How was the sample collected in item 2.4?
• To provide an in vivo assay regarding lipid peroxidation, the authors could consult the specialized literature about the HPLC-TBARS-EVSC protocol.
• Please add the remaining concentrations of the antioxidant actives from the storage period. They should have been quantified by an analytical tool. 
• Add a paragraph about strengths and limitations.

Author Response

Reviewer 1

Dear Authors,

Thank you for the opportunity to read your work. I write you in regard to your manuscript titled "Evaluating the effect of fresh and aged antioxidant formulations in skin protection against UV damage". 

• Please add to the abstract the storage conditions under which the samples were stored during the study.

R- We have added a sentence regarding the storage of formulations in abstract— “AOX formulations were stored in cool, dry and dark place at approximately 20-22°C during the whole study.”

• Please add the temperature at which samples were irradiated (item 2.1).

R- Samples were irradiated at room temperature, circa 20-22°C. We have now added this information to the revised manuscript in the methods section.

• Describe the qualitative and quantitative composition of the samples and the type of packaging material. How was the storage conditions?

R- We have included the requested information in the methods section of the revised text

• How was the sample collected in item 2.4?

R- We have implemented the method section with the requested information

• To provide an in vivo assay regarding lipid peroxidation, the authors could consult the specialized literature about the HPLC-TBARS-EVSC protocol.

R- we agree with the reviewer in regards to the use of HPLC-TBARS-EVSC protocol to determin peroxidation levels. We have chosen the 4HNE immunofluorescence staining because this technique allows us to understand the tissue distribution of the oxidative damage, as we want to understand eventual differences between the dermis and epidermis.

• Please add the remaining concentrations of the antioxidant actives from the storage period. They should have been quantified by an analytical tool. 

R- The loss of the antioxidant concentration was evaluated by HPCL and results in mess than 10%. We have added this information in the revised text/

• Add a paragraph about strengths and limitations.

R- We thank the reviewer for the  good suggestion and indeed we have now added the requested paragraph at the end of discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

The present study aimed to investigate the effectiveness of a differently aged (up to 36 months) topical antioxidant mix containing 15% l-ascorbic acid, 1% tocopherol, and 0.5% ferulic acid on UV induced skin damage. The study was well-designed, and the efficacy of the formulation was confirmed through multiple parameters. The findings are both novel and significant, offering valuable contributions to the field of cosmetic science. There are only a few minor concerns that need to be addressed.

First, the authors refer to an 'antioxidant formulation' throughout the manuscript and in the title, yet its exact composition is not specified. Based on Figure 1, it appears to be a simple solution; however, the whole formulation composition, including all excipients, should be clearly detailed, as these components may also influence overall effectiveness.

While the effects of the differently aged antioxidant mix were assessed through various skin parameters, it remains unclear whether ferulic acid effectively stabilized vitamins C and E over time. Was the content of these compounds measured in formulations stored for up to 36 months (especially considering the conclusion that ferulic acid may act as a stabilization agent for topical antioxidant mixtures)? This point should be clarified and elaborated upon.

The term ‘photoaging’ should be defined in the Introduction.

Line 103: the term ‘oxinflammatory’ is not commonly used; I recommend describing the concept as having dual antioxidant and anti-inflammatory effects for clarity.

The abbreviations EtOH and AOX are used in the manuscript but are not introduced. Please ensure that all abbreviations are defined upon first use to improve clarity for the reader.

IL1α should be used instead of IL1a or IL1alpha.

The reference numbers are presented in superscript format; please verify whether this aligns with the journal’s formatting guidelines.

Author Response

Reviewer 2

The present study aimed to investigate the effectiveness of a differently aged (up to 36 months) topical antioxidant mix containing 15% l-ascorbic acid, 1% tocopherol, and 0.5% ferulic acid on UV induced skin damage. The study was well-designed, and the efficacy of the formulation was confirmed through multiple parameters. The findings are both novel and significant, offering valuable contributions to the field of cosmetic science. There are only a few minor concerns that need to be addressed.

First, the authors refer to an 'antioxidant formulation' throughout the manuscript and in the title, yet its exact composition is not specified. Based on Figure 1, it appears to be a simple solution; however, the whole formulation composition, including all excipients, should be clearly detailed, as these components may also influence overall effectiveness.

R- It should be taking into account that the AOX formulation used has been in the market for more than 20 years and its efficiency again pollution has been clearly demonstrated previously. Considering that the aim of this study was not to evaluate the efficiency of a new formulation but to understand the loss of efficiencty over time we did not take into consideration the eventual role of excipients. We have anyway added some new information in the revised text.

While the effects of the differently aged antioxidant mix were assessed through various skin parameters, it remains unclear whether ferulic acid effectively stabilized vitamins C and E over time. Was the content of these compounds measured in formulations stored for up to 36 months (especially considering the conclusion that ferulic acid may act as a stabilization agent for topical antioxidant mixtures)? This point should be clarified and elaborated upon.

R- We understand the question of the reviewer and indeed there was a slight loss in antioxidant content overtime as reported in the text:

“In fact, in manufactured samples, ascorbic acid concentration decreased from 15% to 13.6% over six months of real-time use. A further reduction to approximately 12% was observed in extremely aged samples equivalent to 3 years (internal L’Oreal analytical data on file). “

The term ‘photoaging’ should be defined in the Introduction.

R- We have now included the definition of photoaging in the introduction section.

Line 103: the term ‘oxinflammatory’ is not commonly used; I recommend describing the concept as having dual antioxidant and anti-inflammatory effects for clarity.

R- We have clarified the term “oxinflammation” in the revised manuscript.

The abbreviations EtOH and AOX are used in the manuscript but are not introduced. Please ensure that all abbreviations are defined upon first use to improve clarity for the reader.

R- Abbreviations are now correctly introduced in materials to provide clarity.

IL1α should be used instead of IL1a or IL1alpha.

R- We agree with the reviewer and we have now corrected the text.

The reference numbers are presented in superscript format; please verify whether this aligns with the journal’s formatting guidelines.

R- Reference numbers have been modified as indicated in the journal guideline

 

 

 

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Auhtors,

Thank you for the improved version of your manuscript. From the first peer review round, some questions remained.

• explain the concentrations of the actives more clearly after 6 months of storage. Was there a maintenance? Diminution? This information is relevant because the investigation focused on stability. 
• it was not found the composition of the samples. please, describe them.
• strenghts and limitations must be improved.
• in the conclusions, revise the term biostability. It was not clear.

Author Response

Dear Auhtors,

Thank you for the improved version of your manuscript. From the first peer review round, some questions remained.

• explain the concentrations of the actives more clearly after 6 months of storage. Was there a maintenance? Diminution? This information is relevant because the investigation focused on stability. 

We have determined that the decrease of the antioxidants compounds after 6 month was circa 10%. i.e. a 6 months, ascorbic acid levels was 13.6% +/- 0.3%

• it was not found the composition of the samples. please, describe them.

Please find below the composition of the serum used, now added to the revised manuscript:

WATER, ASCORBIC ACID, GLYCERIN, AQUEOUS GLYCOL SYSTEM, LAURETH-23, PHENOXYETHANOL, TOCOPHEROL, FERULIC ACID, SODIUM HYALURONATE

• strenghts and limitations must be improved.

We have now modified the limitations paragraph

• in the conclusions, revise the term biostability. It was not clear. This information is relevant because the investigation focused on stability. 

We have modified the text accordingly to the suggestion