Enzyme-Assisted Extraction of Bioactive Compounds from Rosa canina L. Pseudofruit in Natural Deep Eutectic Solvents: Protease Stability and Biological Activities

Enzyme-Assisted Extraction (EAE) in Natural Deep Eutectic Solvents (NaDES) was investigated as a green approach to extract bioactive compounds from the pseudofruit of <i>Rosa canina</i> L. Initially, the thermal stability of protease (Neutrase^®) was evaluated at different temperatures (30-80 °C) in the NaDES Choline Chloride: Glycerol (1:2 molar ratio) (ChCl: Gly) with 20% (<i>w</i>/<i>w</i>) water as a cosolvent and in a buffer solution of the same pH. Kinetic and thermodynamic analyses revealed that ChCl:Gly markedly enhanced enzyme stability, extending half-life by up to 13-fold at 30-50 °C by increasing the enthalpic barrier to deactivation. EAE in NADES parameters, including enzyme loadings and extraction time, were optimized based on total phenolic (TPC) and flavonoid content (TFC), yielding maximum values of 135.75 ± 0.33 mg GAE/g DW and 65.05 ± 0.58 mg CAE/g DW, respectively. Extracts obtained under optimal conditions exhibited enhanced antioxidant, antidiabetic (α-amylase and α-glucosidase inhibition), anti-aging (tyrosinase inhibition), and antibacterial (inhibition of <i>Escherichia coli</i> growth) activities, outperforming enzyme-free extracts in all cases. The optimum extract also significantly reduced A431 cell viability (27-40%, <i>p</i> < 0.05). Overall, EAE in NaDES improved both enzyme stability and extraction efficiency, offering a sustainable and effective alternative for producing bioactive plant extracts.