Passion Fruit Seed Oil as a Natural Tyrosinase Inhibitor: Extraction Optimization, Multi-Mechanism Elucidation, and...

Tyrosinase promotes excessive deposition of melanin, which may lead to severe skin diseases. Passiflora edulis f. edulis seeds have been reported to be rich in diverse bioactive constituents exhibiting potential tyrosinase inhibitory activity. However, the principal bioactive constituents responsible for tyrosinase inhibitory activity and its underlying mechanisms remain largely unclear. Therefore, this study aimed to: (1) optimize SC-CO₂ extraction of Passiflora edulis f. edulis seed oil (PFSO) for maximum yield and bioactive preservation; (2) comprehensively characterize its physicochemical and phytochemical profile; (3) elucidate the tyrosinase inhibition mechanism through kinetic, spectroscopic, and computational approaches; and (4) validate its safety, antioxidant, and anti-pigmentation efficacy in a zebrafish model. PFSO exhibited a yield of 24.96%, with a high content of unsaturated fatty acids (88.03%, mainly linoleic acid at 74.40%). The oil inhibited tyrosinase via a reversible mixed-type mechanism (IC₅₀ = 1.12 mg/mL). Fluorescence spectroscopy and molecular docking revealed that linoleic acid binds to LYS180 and β-sitosterol binds to TYR78, mainly driven by hydrogen bonding and hydrophobic interaction, which changed the microenvironment of tryptophan residues and indicated static quenching. Further validation experiments revealed that the major constituent, linoleic acid, exhibited only weak inhibitory activity against tyrosinase (IC₅₀ = 29.44 mg/mL), whereas the key component β-sitosterol markedly suppressed tyrosinase activity (IC₅₀ = 46.43 μg/mL). In vitro assays demonstrated PFSO's significant efficacy in reducing the melanin content and tyrosinase activity in α-MSH-stimulated B16F10 murine melanoma cells. In vivo experiments in zebrafish that received dietary supplementation with PFSO confirmed that PFSO (≤5 mg/mL) reduced ROS production, suppressed melanin deposition, inhibited tyrosinase activity, and downregulated the expression of melanogenesis-related genes (TYR, TYRP1, TYRP2, MITF). This study provides, for the first time, a comprehensive elucidation of PFSO's potential as a natural tyrosinase inhibitor, integrating extraction optimization, multicomponent characterization, multimodal inhibition analysis, and in vivo validation.