Effect of an Oral Formulation on Skin Lightening: Results from In Vitro Tyrosinase Inhibition to a Double-Blind Randomized Placebo-Controlled Clinical Study in Healthy Asian Participants

Round 1

Reviewer 1 Report

Oral supplementation for skin problem is increasing topic in skin care. Results are promising but several concerns are raised in this paper.

First of all, author present that combined effects of ingredients for tyrosinase inhibition are bigger than that of sum of each ingredients although vitamin C is not included. However, it can’t be a full evidence that Belight are effective for brightening effects. It can ben presented but this results is not much important in this paper. 

Describe more information how randomization was done.

Describe information about ethical review. 

ITA may be another parameter based on Lab value. Provide ITA is a good parameter for skin brightness in addition to L. Is there additional value to analyze ITA?

Add more information how 58 selected among 103 screened.  

There are very small difference and big SD. Is there statistical difference even with these changes? Provide calculation as supplementary data. 

Fig 3). In lower part, author used changes of data (measured data - baseline data) for analysis. Provide reference that these calculation are acceptable.

In left panel of upper part, author cut the lower part of figure. Show all column.  

Fig 4). In left panel, author cut the lower part of figure to exaggerate difference. Show all column.

Fig 4) In right panel, auhtor used changes of data (measured data - baseline data) for analysis. Provide reference that these calculation are acceptable. 

Author Response

We would like to inform that during the peer-review process, Spincontrol Asia Co. Ltd. has changed its official name to DERMAPROOF ASIA Co. Ltd. So, we have changed the affiliation and e-mails of Kunyanatt Chalotorn² and Fabrice Perin² :

²Dermaproof Asia Co., Ltd. Bangkok, Thaïland; [email protected] (K.C.); [email protected]

Oral supplementation for skin problem is increasing topic in skin care. Results are promising but several concerns are raised in this paper.

We thank the reviewer for this positive feedback.

First of all, author present that combined effects of ingredients for tyrosinase inhibition are bigger than that of sum of each ingredients although vitamin C is not included. However, it can’t be a full evidence that Belight are effective for brightening effects. It can ben presented but this results is not much important in this paper. 

Thank you for this feedback. We agree that it can’t be full evidence that Belight^3TM are effective for brightening effects since the changes in skin color are statistically significant but clinically weak. We have replaced “evidence” by “results” in the title of the publication:  

Effect of an oral formulation on skin lightening: results from in vitro tyrosinase inhibition and a double-blind randomized placebo controlled clinical study in healthy Asian participants.

We also have modified some sentences of the discussion and conclusions to tone down our interpretation.

Describe more information how randomization was done.

As requested, we have now added more information about the randomization in the section 2.3. Clinical study – Products under investigation:

The products under investigation were hard-shell capsules packaged in bottles and labelled in accordance with a computer-generated randomization list generated by a statistician using a study sponsor’s proprietary software and stratified with a 1:1 allocation. The randomization list was stored in a location not accessible neither to the staffs in charge of the evaluations nor to the subjects of the study. Active and placebo capsules were undistinguishable, matched for color, shape, size, smell, and taste. Participants, the investigator and all the study staff remained blinded until the completion of the data analysis. Each participant received two bottles according to their randomization number, these bottles being dispensed by one independent technician.  

Describe information about ethical review. 

As requested in the “Instruction for authors” provided by the Cosmetics editorial board (https://0-www-mdpi-com.brum.beds.ac.uk/journal/cosmetics/instructions#ethics), information about the ethical review was described in the section “Institutional Review Board Statement”:

The study was conducted in accordance with the Decla-ration of Helsinki, and approved by the Human Research Ethics Committee of Thammasat University (Faculty of Medicine) on August 16, 2022 (certificate of approval 160/2022; project No. MTU-EC-IM-5-115/65). The trial was performed according to the most recent recommendations given by the World Medical Association (Helsinki Statement 1964, amended in Fortaleza, Brazil, 2013), and in accordance with Standard Operating Procedures of the contract-testing laboratory. Good Clinical Practices and Personal Data Protection Act B.E. 2562 (2019) of Thailand were respected.  

ITA may be another parameter based on Lab value. Provide ITA is a good parameter for skin brightness in addition to L. Is there additional value to analyze ITA?

The reviewer is right. As explained in the method section, the luminance L* represents the relative brightness from total darkness (L*=0) to absolute white (L*=100).  As stated by Petit et al.[1], the best description of a lightening effect is given by ITA° = Arctg [(L*-50)/b*] x (180/π). The lighter the skin is, the higher the L* and ITA° values are. We have added the reference of Petit and al. in the section 2.5. Clinical study - Measurement of the skin color by image analysis:

As stated by Petit and al. [18], the best description of a lightening effect is given by the so-called Individual Typology Angle ITA°, where ITA° = Arctg [(L*-50)/b*] x (180/π). The lighter the skin is, the higher the L* and ITA° values are.

In addition, there are also other color parameters such as a* and b* chromaticity values that are red-green and yellow-blue color coordinates respectively. The a* and b* are often used for the evaluation of erythema and melanin changes respectively. However, b* is not a good indicator of melanin variations, especially in subjects with relatively dark skin as is the case with the Asian subjects included in this study[2]. As the aim of the study was to investigate the lightening efficacy of the oral supplement on skin color and dark spots, no statistical comparison was done on a* and b* parameters since they were not directly relevant to the objectives of the study. 

Add more information how 58 selected among 103 screened.  

We have modified the figure 2 to explain how the population was selected. We also added this information in the section 3.2. Clinical study – Baseline characteristics

Among the 103 screened participants, 24 participants did not meet the inclusion criteria (they had a dark spot with a diameter < 3 mm), 16 participants met exclusion criteria (10 participants presented melasma on the face, 2 participants presented facial erythema, and 2 others presented freckles) and 5 were not eligible for other reasons. Thus, fifty-eight healthy Asian subjects were randomized into the study, including 44 females and 14 males (Figure 2).

 There are very small difference and big SD. Is there statistical difference even with these changes? Provide calculation as supplementary data. 

We have checked the calculations of the statistical comparisons. No error was detected in the statistical comparisons although they are small differences and big SD. As stated in the section statistical analyses, to assess the significance of the observed values or variations for a given product, two-tailed paired Student’s t-test or non-parametric Wilcoxon test (if data were not normally distributed using the Shapiro-Wilk test at 1% threshold) were used. Comparisons between product groups were performed using two-tailed unpaired Student t-test (if the data were normally distributed and the variances were homogeneous) or using Mann-Whitney Rank Sum Test. As suggested by the reviewer, we have now provided calculations of the statistical analyses of skin color and color of dark spots in supplementary materials 1 and 2, respectively.

Fig 3). In lower part, author used changes of data (measured data - baseline data) for analysis. Provide reference that these calculations are acceptable.

Despite the use of appropriate methods of randomization, imbalances in baseline measurements between treatment groups may be observed by chance. Using changes parameters (measured data – baseline data) in the statistical analyses allows to get rid of potential differences at baseline.  Such statistical comparisons were largely used in clinical trial analyses and were in line with previous publications comparing efficacy of oral supplements on skin lightening in clinical trials [3–5]. These references were added in the section 2.8 Statistical analyses.

In left panel of upper part, author cut the lower part of figure. Show all column.  

The figure 3a has been modified to show all columns.

Fig 4). In left panel, author cut the lower part of figure to exaggerate difference. Show all column.

The figure 4a has been modified to show all columns.

Fig 4) In right panel, author used changes of data (measured data - baseline data) for analysis. Provide reference that these calculations are acceptable.

Despite the use of appropriate methods of randomization, imbalances in baseline measurements between treatment groups may be observed by chance. Using changes parameters (measured data – baseline data) in the statistical analyses allows to get rid of potential differences at baseline.  Such statistical comparisons were largely used in clinical trial and were in line with previous publications comparing efficacy of oral supplements on skin lightening between treatment groups in clinical trials [3–5]. These references were added in the section 2.8 Statistical analyses.

References

1. Petit, L.; Piérard, G.E. Skin-Lightening Products Revisited. Int J Cosmet Sci 2003, 25, 169–181, doi:10.1046/J.1467-2494.2003.00182.X.
2. Chardon, A.; Cretois, I.; Hourseau, C. Skin Colour Typology and Suntanning Pathways. Int J Cosmet Sci 1991, 13, 191–208, doi:10.1111/J.1467-2494.1991.TB00561.X.
3. Duperray, J.; Sergheraert, R.; Chalothorn, K.; Tachalerdmanee, P.; Perin, F. The Effects of the Oral Supplementation of L-Cystine Associated with Reduced L-Glutathione-GSH on Human Skin Pigmentation: A Randomized, Double-Blinded, Benchmark- and Placebo-Controlled Clinical Trial. J Cosmet Dermatol 2022, 21, 802–813, doi:10.1111/JOCD.14137.
4. Juturu, V.; Bowman, J.P.; Deshpande, J. Overall Skin Tone and Skin-Lightening-Improving Effects with Oral Supplementation of Lutein and Zeaxanthin Isomers: A Double-Blind, Placebo-Controlled Clinical Trial. Clin Cosmet Investig Dermatol 2016, 9, 325–332, doi:10.2147/CCID.S115519.
5. Tsuchiya, T.; Fukui, Y.; Izumi, R.; Numano, K.; Zeida, M. Effects of Oligomeric Proanthocyanidins (OPCs) of Red Wine to Improve Skin Whitening and Moisturizing in Healthy Women - a Placebo-Controlled Randomized Double-Blind Parallel Group Comparative Study. Eur Rev Med Pharmacol Sci 2020, 24, 1571–1584, doi:10.26355/EURREV_202002_20215.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript by Pouchieu studied the skin lightening effect of an oral formulation of Belight^3TM, containing polyphenol-rich extracts and vitamin c. Overall the manuscript is well-written, and the results are clear and convincing. However, there are a few issues the authors need to addressed, and please see specific comments below:

1.      The author showed that Belight^3TM potently inhibited the tyrosinase activity in vitro, but will the use of an oral formulation may lead to the development of systemic inhibition of tyrosinase activity? Can this formulation lead to systemic side effect, such as graying/whitening of the hair, as hair melanin production may also be inhibited? Additional data showing effect on hair graying/whitening should be added and discussed.

2.      Can topical application of this formulation be developed to avoid systemic side effect? Please discuss.

3.      For clinical study, please add the IRB reference # and approval committee information in the method section.

4.      What is the species of the tyrosinase used in assay for in vitro tyrosinase activity? Please specify in the method section.

Author Response

We would like to inform that during the peer-review process, Spincontrol Asia Co. Ltd. has changed its official name to DERMAPROOF ASIA Co. Ltd. So, we have changed the affiliation and e-mails of Kunyanatt Chalotorn² and Fabrice Perin²:

²Dermaproof Asia Co., Ltd. Bangkok, Thaïland; [email protected] (K.C.); [email protected]

This manuscript by Pouchieu studied the skin lightening effect of an oral formulation of Belight^3TM, containing polyphenol-rich extracts and vitamin c. Overall the manuscript is well-written, and the results are clear and convincing. However, there are a few issues the authors need to addressed, and please see specific comments below:

We thank the reviewer for this positive feedback.

1. The author showed that Belight^3TM potently inhibited the tyrosinase activity in vitro, but will the use of an oral formulation may lead to the development of systemic inhibition of tyrosinase activity? Can this formulation lead to systemic side effect, such as graying/whitening of the hair, as hair melanin production may also be inhibited? Additional data showing effect on hair graying/whitening should be added and discussed.

Thank you for highlighting this limitation. As we have shown that Belight^3TM potently inhibited the tyrosinase activity in vitro, we hypothesized that this oral formulation may lighten the skin color by a potential systemic inhibition of tyrosinase activity in vivo. In the study design, we did not plan to specifically assess hair graying/whitening before and after product intake. To our mind, it seems unlikely that this oral formulation including nutritional dose of botanical extracts and vitamin C lead to hair graying or whitening because the nutritional doses of the ingredients may be commonly ingested through a healthy diet including the consumption of fruits and vegetables.

As mentioned in the results and discussion, no side effect imputable to the product has been reported during the study. At the end of the study, participants were interviewed by a dermatologist about tolerance signs with a questionnaire including the following question:

All the participants taking Belight^3TM answered NO to this question. No participant has reported graying or whitening of the hair after 12 weeks of supplementation.

As requested, we have now discussed this limitation at the end of the discussion section:

Although hair graying has never been associated with the administration of skin lightening food supplements including polyphenols or vitamin C [1–3], one limitation of the study design is the absence of hair graying or whitening assessment before and after product intake. As this oral formulation may lead to systemic inhibition of tyrosinase activity, we can hypothesize that melanogenesis may also be inhibited in the hair leading to hair whitening. However, neither participant nor the investigator has observed hair whitening nor graying associated to the product administration during 12 weeks.

2. Can topical application of this formulation be developed to avoid systemic side effect? Please discuss.

We would like to thank the reviewer for this suggestion. As previously discussed, it’s unlikely that hair graying may be induced by this oral formulation including nutritional doses of grape seed extract, licorice extract, grape pomace extract and coated vitamin C. To develop topical application, it would be necessary to assess the cutaneous permeability of our formulation that has never been tested since our expertise is based on nutraceutical ingredients and formulations.

3. For clinical study, please add the IRB reference # and approval committee information in the method section.

As requested in the “Instruction for authors” provided by the Cosmetics editorial board (https://0-www-mdpi-com.brum.beds.ac.uk/journal/cosmetics/instructions#ethics), the IRB reference and approval committee information were mentioned in Section “Institutional Review Board Statement”:

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Human Research Ethics Committee of Thammasat University (Faculty of Medicine) on August 16, 2022 (certificate of approval 160/2022; project No. MTU-EC-IM-5-115/65). The trial was performed according to the most recent recommendations given by the World Medical Association (Helsinki Statement 1964, amended in Fortaleza, Brazil, 2013), and in accordance with Standard Operating Procedures of the contract-testing laboratory. Good Clinical Practices and Personal Data Protection Act B.E. 2562 (2019) of Thailand were respected.  

4. What is the species of the tyrosinase used in assay for in vitro tyrosinase activity? Please specify in the method section.

As detailed below, we have included this information in the section 2.1. Assay for in vitro tyrosinase activity:

Briefly, microplate wells were filled with 40 µL of sample, 40 µL of mushroom tyrosinase (Sigma-Aldrich, ref T3824-25KU, 125 U/mL) and 80 µL of phosphate buffer (50 mM, pH 6.5).

References

1. Handog, E.B.; Galang, D.A.V.F.; De Leon-Godinez, M.A.; Chan, G.P. A Randomized, Double-Blind, Placebo-Controlled Trial of Oral Procyanidin with Vitamins A, C, E for Melasma among Filipino Women. Int J Dermatol 2009, 48, 896–901, doi:10.1111/J.1365-4632.2009.04130.X.
2. Nobile, V.; Schiano, I.; Peral, A.; Giardina, S.; Spartà, E.; Caturla, N. Antioxidant and Reduced Skin-Ageing Effects of a Polyphenol-Enriched Dietary Supplement in Response to Air Pollution: A Randomized, Double-Blind, Placebo-Controlled Study. Food Nutr Res 2021, 65, doi:10.29219/FNR.V65.5619.
3. Yamakoshi, J.; Sano, A.; Tokutake, S.; Saito, M.; Kikuchi, M.; Kubota, Y.; Kawachi, Y.; Otsuka, F. Oral Intake of Proanthocyanidin-Rich Extract from Grape Seeds Improves Chloasma. Phytother Res 2004, 18, 895–899, doi:10.1002/PTR.1537.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have adequately answered my questions

Author Response

.