SACRAN PROTECTS SKIN AGAINST POLLUTANTS 27 DISCUSSION This study aimed to investigate the antipollution effects of sacran using tobacco smoke as a representative air pollutant. In a previous study, we identifi ed the unique characteristics of sacran that result in the formation of a gel-like sheet. That gel-like sheet suppresses water evaporation and penetration by chemicals (23). The application of a sacran solution improves itching and facial rashes in patients with atopic dermatitis based on a question- naire survey (24). In addition, sacran showed improvement of corneocyte maturation in healthy volunteers who had a history of atopic dermatitis (24). These effects have been considered to be based on the shielding effects of the skin against external stimuli due to the physicochemically unique properties of sacran. Thus, sacran is also expected to have protective effects against air pollutants. To identify the potential effects of sacran against air pollutants, we conducted various examinations focusing on oxidative stress using tobacco smoke as a representative air pollutant. Furthermore, to demonstrate whether sacran ex- hibits specifi c effects, we also examined the effects of HA, which is commonly formulated as an anionic polysaccharide in cosmetic products. In this study, to evaluate the effects of sacran, the effects of HA were compared as a representative polysaccharide. Figure 6. Amelioration of ROS generation induced by tobacco smoke in HaCaT keratinocytes. HaCaT kerati- nocytes were cultured in DMEM containing PBS diffused with tobacco smoke through fi lters treated with or without sacran or HA for 24 h at 37°C. Intracellular ROS levels were measured using H2DCFDA. Each value represents the mean ± SD of six experiments. Wilcoxon rank-sum test, ***p 0.001. Control(-) denotes sham-treated cells and Control(+) denotes cells treated with tobacco smoke diffused through a nontreated fi lter.

JOURNAL OF COSMETIC SCIENCE 28 First, to examine whether sacran functions as a shield, we monitored the localization of sacran topically applied on the surface of RHEEs from a histochemical viewpoint. More than 99% of sacran stayed in or on the stratum corneum of RHEEs, and HA also exhibited a similar behavior (Figure 2). Based on those results, it seemed likely that sacran and HA would function as an artifi cial barrier because they remained in or on the stratum corneum. In the next examination, to investigate whether sacran remaining on the skin surface func- tions as a barrier against penetration from the chemical aspect, we determined the amount of ACs or BaP in PBS diffused with tobacco smoke through membrane fi lters treated with sacran or HA. Sacran or HA reduced levels of ACs or BaP in PBS, and the amounts of ACs and BaP in PBS were signifi cantly lower after passing through sacran-treated membrane fi lters (Figure 3). Furthermore, although tape-stripped corneocytes treated with tobacco smoke had increased levels of CPs, topical treatment with sacran or HA reduced the level of CPs in corneocytes treated with tobacco smoke (Figure 8). Regarding that ability to reduce levels of CPs, sacran was signifi cantly superior to HA. The sum of these results Figure 7. Amelioration of protein carbonylation induced by tobacco smoke on HaCaT keratinocytes. HaCaT keratinocytes were cultured in DMEM containing PBS diffused with tobacco smoke through fi lters treated with or without sacran or HA. After 24 h, intracellular CP levels were estimated by FI of FTSC labeling. (A) CP levels were quantifi ed by image analysis, and each value represents the mean ± SD of six experiments. Wilcoxon rank-sum test, *p 0.05, ***p 0.001. (B) Representative images of CPs in HaCaT keratinocytes after each treatment (scale bar, 100 μm). Control(-) denotes sham-treated cells and Control(+) denotes cells treated with tobacco smoke diffused through a nontreated fi lter.