Isorhamnetin Derivatives from Opuntia ficus-indica Oil-Extraction By-Products: NADES-Based Ultrasound-Assisted Extraction Optimization, Phytochemical Profiling, and Bioactivity Assessment

Prickly pear (Opuntia ficus-indica (L.) Mill.) generates substantial agro-industrial by-products, such as press cake, seed, and oil, that remain underexploited despite their recognized phytochemical richness. This study reports the systematic optimization, characterization, and bioactivity profiling of flavonoid-rich extracts recovered from these three matrices. A Box–Behnken design (BBD) coupled with response surface methodology (RSM) was applied to optimize the ultrasound-assisted extraction (UAE) of total flavonoid content (TFC) from press cake using a natural deep eutectic solvent (NADES: fructose–glycerol–sorbitol–water and FGSH), selected through an initial screening of fifteen solvent systems. The quadratic polynomial model showed excellent fit (R2 = 0.9852; R2adj = 0.9687; MAPE = 1.31%; Durbin–Watson = 1.857), and optimal extraction conditions were established at 37.6 min extraction time, 35.6% ultrasonic power, and 29.4 °C, yielding a maximum predicted TFC of 54.78 ± 0.49 mg quercetin equivalents (QE)/mL. HPLC-DAD analysis of the press cake extract revealed five isorhamnetin derivatives as the dominant flavonoids, with isorhamnetin-3-O-glucoside (23.18 ± 0.12 mg/g DW) and isorhamnetin-3-O-rutinoside (13.80 ± 0.28 mg/g DW) as the most abundant. Comprehensive bioactivity assessment demonstrated significant antioxidant capacities (CUPRAC: 191.35 ± 3.22 µM AAE; ORAC: 184.44 ± 3.44 µM TE; DPPH: 103.47 ± 9.98 µM TE for press cake extract), potent in cellulo ROS/RNS suppression in a yeast UV-stress model (85.9 ± 1.0% inhibition for press cake), and differential tyrosinase inhibition across fractions (press cake: 32.8%; seed: 57.5%; oil: 83.8%), highlighting the oil as a potent anti-melanogenic ingredient. In silico safety prediction (ProTox-II/pkCSM) confirmed the favorable toxicity profiles of all identified isorhamnetin derivatives (LD50 > 5000 mg/kg; Toxicity Class V). These results collectively position Opuntia ficus-indica by-products as high-value natural sources of bioactive flavonoids with applications in cosmetic, nutraceutical, and dermatological formulations.