The skin hydration level is a key factor that influences the physical and mechanical properties of the skin. The stratum corneum (SC), the outermost layer of the epidermis, is responsible for the skin’s barrier function. In this study, we investigated the role of a unique composition of Viola yedoensis extract for its ability to activate CD44, a cell-surface receptor of hyaluronic acid, and aquaporin-3, a water-transporting protein, in human keratinocytes (HaCaT). An ELISA assay evaluating the protein expression levels of CD44, aquaporin-3 (AQP3), filaggrin, and keratin-10 revealed that V. yedoensis extract upregulated the levels of CD44 and AQP3 by 15% and 78%, respectively. Additionally, V. yedoensis extract demonstrated a comparative effect on water vapor flux in TEWL and lipid perturbation in DSC versus the reference, glycerin. In light of this new biological efficacy, a detailed phytochemical characterization was undertaken using an integrated LC-HRMS/MS-based metabolomics approach, which provided further insights on the chemistry of V. yedoensis. This led to the identification of 29 secondary metabolites, 14 of which are reported here for the first time, including esculetin, aesculin, apigenin and kaempferol C-glycosides, megastigmane glycosides, roseoside, platanionoside B, and an eriojaposide B isomer, along with the rare, calenduloside F and esculetin diglucoside, which are reported for the first time from the genus, Viola. Notably, two active components identified in the V. yedoensis extract, namely, aesculin and schaftoside, showed an upregulation of the protein expression of CD44 in HaCaT cells by 123% and 193% within 24 h of treatment, respectively, while aesculin increased AQP3 levels by 46%. Aesculin and schaftoside also significantly upregulated the expression of K-10 levels by 299% and 116%, which was considerably higher than sodium hyaluronate, the positive control. The rationale used to characterize the new structures is outlined along with the related biosynthetic pathways envisioned to generate roseoside and Eriojaposide B. These findings provide new molecular insights to deepen the understanding of how V. yedoensis extract, along with the biomarkers aesculin and schaftoside, restores the skin barrier and skin hydration benefits.

Skin sensitization is a critical endpoint in cosmetic safety assessment, necessitating reliable animal-free testing alternatives. Current established in chemico assays, such as the Direct Peptide Reactivity Assay and Amino acid Derivative Reactivity Assay, are limited by prolonged 24 h incubation periods and their inability to distinguish between direct electrophilic sensitizers and pro-electrophiles requiring metabolic activation or spontaneous oxidation. This study presents the design, synthesis, and validation of NNDNAC (N,N-dimethyl N-(2-(1-naphthyl)acetyl)-l-cysteine), a novel nucleophilic reactivity probe synthesized via a seven-step pathway. A modified naphthalene structure featuring N,N-dimethylamino substituent enhances nucleophilicity of the cysteine sulfur atom, enabling rapid reactivity assessment within an hour incubation using LC-DAD quantification. Comparative validation studies demonstrated that NNDNAC rapidly identified strong electrophilic sensitizers, achieving 100% and 98% depletion rates for p-benzoquinone and 2-methyl-4-isothiazolin-3-one, respectively, within 1 h. Critically, the NNDNAC assay successfully differentiated pro-electrophiles like p-phenylenediamine and 4-aminophenol, which showed negligible depletion at 1 h but significant depletion after 24 h due to auto-oxidation. Furthermore, NNDNAC classified farnesal as a weak sensitizer, aligning with established KeratinoSens™ and LLNA data. The NNDNAC probe represents a significant advancement in skin sensitization assessment, offering a time-efficient, high-throughput platform that not only accelerates screening processes but also provides crucial mechanistic insights through electrophile/pro-electrophile differentiation, significantly improving animal-free toxicological evaluations.