Green extraction and stabilization of bioactive compounds from annatto (Bixa orellana L.) seeds using hydrophobic natural deep eutectic solvents

Abstract

Hydrophobic natural deep eutectic solvents (HNADES) have emerged as promising green alternatives to conventional organic solvents for the extraction of lipophilic bioactive compounds. In this study, saturated fatty acid–based HNADES were employed as multifunctional media for the simultaneous extraction and stabilization of bioactive compounds from annatto (Bixa orellana L.) seeds, with particular emphasis on bixin and geranylgeraniol. HNADES composed of dodecanoic acid combined with decanoic, octanoic, or heptanoic acids were formulated and physicochemically characterized. Extraction parameters were optimized using a one-factor-at-a-time approach by evaluating solvent composition, seed concentration, temperature, and extraction time. Among the evaluated systems, C12:C8 (1:3 M ratio) showed the highest extraction efficiency, yielding greater total carotenoid content and antioxidant capacity than conventional solvents (ethanol and methanol). High-performance liquid chromatography confirmed significantly higher concentrations of bixin and geranylgeraniol in the optimized extract. Furthermore, the HNADES-based system improved the stability of carotenoids, antioxidant activity, and color over 90 days of storage, exhibiting substantially lower degradation rates than the control extracts, likely due to the physicochemical properties of the eutectic matrix. The optimized extract also demonstrated relevant antibacterial activity against selected foodborne bacteria. Together, these findings demonstrate that fatty acid-based HNADES act as multifunctional and sustainable media that simultaneously enhance extraction efficiency, improve the stability of oxidation-sensitive lipophilic bioactive compounds, and support the valorization of annatto seeds through a green extraction strategy.