ANTI-POLLUTION 83
TABLE 3: EFFECT OF H-EPS ON ROS PRODUCTION. EFFECT WAS STUDIED IN KERATINOCYTES AND IN THE POLLUTION MODEL
Mitochondrial membrane potential
UFA per 104
cells
Control (vehicle)
H-EPS
Control (UPM)
UPM + H-EPS
2017 ± 109
1093 ± 180
2560 ± 342
1923 ± 299
% Change Reference -45,8% p<0,01 Reference -24,9% p<0,01
Aldrich) staining. Immunostaining was also performed to determine quantity of involucrin and corneodesmosin.
S. epidermidis growth S. epidermidis were seeded at 4x105 CFU/mL and cultivated with increasing concentrations of H-EPS. Bacteria were then incubated at 37°C for 24 hours. Growth was monitored using a plate reader (Cytation5, BioTEK) using optical density at 600 nm.
Sphingomyelin gene expression S. epidermidis was seeded in 12-well plates (Falcon) and cultivated with H-EPS or its control. Bacteria were then incubated at 37°C for 24 hours. RNA was extracted using a commercial RNA extraction kit (Maxwell) and quantified Nanodrop. cDNA was prepared using iScript cDNA Synthesis kit (Biorad, USA) and 100 ng of RNA. Gene expression was assessed using
SYBRGreen (Biorad, USA) and CFX technology (Biorad, USA). Fold-change of gene expression was calculated using ΔΔCt method
Ceramide quantification Keratinocytes (HaCaTs) were treated with H-EPS or co-cultured with S. epidermidis. Cell lysates and supernatants were used to quantify total amount of ceramides using human ceramide (CER) Elisa kit (Mybiosource).
Clinical evaluation Two independent studies were conducted: one in France (Paris area) and one in China (Guangzhou). The first study was conducted in Paris area on 54 volunteers that applied either a serum containing 3% H-EPS (27 volunteers) or a placebo (27 volunteers) on their face twice a day for two months. Hydration, skin roughness, radiance and firmness were measured after one month and two months. The second study was performed in
Guangzhou on 127 volunteers that applied a sheet mask containing 3% H-EPS twice daily for two weeks. Skin tone and rejuvenation were evaluated at 15 minutes and two weeks, using a self-assessment form.
Statistical analysis For in vitro assays, one-way analysis of variance (ANOVA) was used to determine whether there
www.personalcaremagazine.com UPM UPM + H-EPS Control H-EPS
Figure 2: Effect of UPM and/or H-EPS in skin equivalent models
was any significant difference between the means of two or more independent groups. Difference between two means with similar variances was performed with Student’s t test. A p-value p<0.05 or p<0.01 were considered statistically significant. For clinical studies, statistical analysis were
performed using Student or Wilcoxon test; two tailed, paired series. For the self-assessment study, a Chi-squared test was performed. A p-value p<0.05 or p<0.01 were considered statistically significant.
Results Mitochondrial activity Since studies have shown that pollution disrupts mitochondrial metabolism [2-6], we wanted to identify an active ingredient that could fight against pollution. We therefore established an in vitro pollution model for keratinocytes and studied the effect of UPM in the following parameters: mitochondrial membrane potential; ATP production and mitochondrial oxygen (O2) consumption rates (respiration). UPM inhibited all these parameters validating
our protocol (Table 1). We then tested the effect of H-EPS in mitochondrial metabolism both in basal conditions and the in vitro pollution model. H-EPS was able to induce mitochondrial parameters in basal conditions and to restore all three mitochondria parameters studied, making H-EPS a good candidate to fight pollution in keratinocytes (Table 2).
ROS & NRF2 ROS are a by-product of the mitochondrial respiratory chain. Because H-EPS induced mitochondrial activity in keratinocytes, we wanted to study its effect on ROS. Keratinocytes were treated with H-EPS and ROS were quantified. Despite the activation of mitochondrial metabolism, H-EPS inhibited ROS production basally (Table 3). Furthermore, H-EPS was also able to reduce ROS generated by UPM (Table 3). The NRF2 pathway is involved in cellular
detoxification. NRF2 translocation to the nuclei activates HMOX and NQO-1 enzymes that scavenge intracellular ROS. NRF2 is activated by both H-EPS and UPM (Figure 1A). H-EPS induced moderate NRF2 activation (36%- 41%) that remained stable over 24 hours. NRF2 activation was significantly higher in
UPM treated keratinocytes and increased over 24 hours to maximum of 401% relative to untreated controls (Figure 1B). We conclude that NRF2 activation by H-EPS allows keratinocytes to reduce ROS production. In pollution treated cells, NRF2 activation may
be explained by a protective defence mechanism. Because ROS levels continue growing, NRF2 remained activated. Altogether these data suggest that H-EPS reduces ROS production by efficiently activating NRF2.
Skin structure We then set up an equivalent skin model treated
May 2026 PERSONAL CARE MAGAZINE
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