Experimental Design-Driven Optimization of Choline-Based Natural Deep Eutectic Solvents for the Extraction of...

The development of sustainable and efficient sample-preparation strategies remains a key challenge in analytical workflows for plant-derived bioactive compounds. In this study, choline chloride (ChCl)-based natural deep eutectic solvents (NADES) were systematically investigated as green extraction media and combined with chemical and enzyme assays to identify the bioactive effect of phenolic compounds from Bellevalia dubia, an understudied Mediterranean plant. A design of experiments approach was applied to evaluate the influence of hydrogen bond donors (lactic, malic, and citric acid) and their molar ratios on extraction performance. Among the tested systems, the ChCl-malic acid (MA) mixture at a molar ratio of 1:2.5 (precisely 1:2.47) was identified as the best overall solution according to the desirability approach, which integrated both phytochemical and enzyme-inhibitory responses. This system also showed strong enzyme-inhibitory effects compared to conventional extractants (water and 80% methanol). To identify the extract composition, a suspect screening workflow was applied based on ultra-high-performance liquid chromatography hybrid quadrupole Orbitrap mass spectrometry (UHPLC-q-Orbitrap-MS). Twenty-one compounds were detected and annotated, mainly belonging to the flavonoid class. Principal component analysis (PCA) showed that ChCl-MA extracts were discriminated, particularly at molar ratio 1:2.5, from the rest, indicating a distinct chemical and bioactivity profile. PCA further suggested a possible association of the ChCl-MA 1:2.5 system with vitexin and AChE inhibition, which requires further investigation. Overall, the proposed NADES-based approach offers a tunable, promising, and potentially more sustainable alternative for the extraction of bioactive compounds from plant-based sources.