Effect-directed extraction of grape pomace: optimizing antioxidant and antibrowning efficacy

Abstract

The increasing interest in valorizing agricultural by-products has positioned grape pomace as a rich source of bioactive compounds. This study developed an effect-directed extraction (EDE) approach guided by bioactivity quantification on thin layer chromatography (TLC). Twelve grape pomaces were screened based on antioxidant and tyrosinase inhibitory properties. Using hydroalcoholic solvent (ethanol:water, 1:1), the two most promising sources (Malbec from San Rafael) were subjected to response surface methodology (RSM) to optimize extraction of anti-browning and antioxidant compounds visualized as TLC spots. Temperature and time were optimized (76 °C, 45 min), and samples were analyzed using TLC coupled with DPPH and laccase inhibition bioautography. Antioxidant compounds showed retention factor values on TLC plates of 0.37 and 0.75 (DPPH/ABTS-active), while laccase inhibition occurred at Rf 0.35, coinciding with the primary tyrosinase inhibition zone. However, subsequent bioassay-guided HPLC fractionation and HRMS/MS analysis revealed that tyrosinase and laccase inhibitions are mediated by distinct compounds within this bioactive zone, highlighting a synergistic multi-target effect in the optimized extract that is retained throughout the process. The primary tyrosinase inhibitor at Rf ~0.35 was tentatively elucidated as an acylated anthocyanin, consistent with malvidin-3-O-(p-coumaroyl)glucoside. Optimized extracts were evaluated on Pink Lady apple slices at different timepoints. The browning index was reduced by 25% versus the control at 15 h, confirmed by significantly lower ΔE values (p < 0.05). The process requires only food-grade solvents and conventional equipment, facilitating scale-up for grape pomace generated worldwide. Validating the EDE strategy, this TLC-guided approach successfully tracked and preserved the primary anti-tyrosinase activity from the crude waste matrix down to the tentatively identified molecule, contributing to circular economy objectives in the wine industry.