3.5. Preparation of NADES

The synthesis of all NADES used in the present work was based on previous research [20,51]. In brief, lactic acid, choline chloride and L-proline (hydrogen bond donor —HBD) were mixed with sodium acetate, ammonium acetate, glycerol, 1, 2-propanediol (hydrogen bond acceptors —HBA) at proper molar ratios and the mixtures were heated under stirring, until a homogeneous liquid was reached. NADES were kept in the dark inside capped flasks at ambient temperature. The codes of the NADES used in this study, along with details regarding their synthesis are shown in Table 4.

All NADES were used as 80% (v/v) aqueous solutions, incorporating water 20% to reduce the viscosity. Extractions were carried out according to a previously described methodology [44,51]. Plant material (0.1 g) was placed in a 50 mL conical tube and 10 mL of NADES was added. As control, we use methanol under the same extraction conditions used for the NADES. The mixtures were shaken vigorously manually for a few seconds to form a slurry and then extracted through heating and stirring (Büchi Syncore Polyvap R24, Switzerland). Extraction conditions were: 340 rpm, 60 °C for 50 min. Then, samples were centrifuged at 8000 rpm for 10 min (Eppendorf 5804 R, USA). The supernatants were diluted four times with mobile and filtered through a 0.22 μm cellulose acetate membrane filter prior to analysis. Extraction yields, were determined after recover the sample extract from the NADES. So, the solvent was removed using solid-phase extraction (SPE) on HLB cartridges and following the method of Lui and coworkers [55] with slight modifications. In brief, HBL cartridge were equilibrated with 5 mL of methanol, followed by 5 mL of water. After loading the extract solution (2 mL), the cartridge was subsequently rinsed with 8 mL of water twice and then eluted with 8 mL of methanol in pre-weighted vials. Methanol was evaporated in heating block under nitrogen stream to obtain the extracts. Yields per gram of dry extract were calculated as follows:

where Y is the yield and m is the mass in milligrams of dry extract.

The yield of Total Polyphenols (TP) was determinate using an adapted Folin-Ciocalteau procedure [56]. In brief, 20 μL of properly diluted samples were mixed with 780 μL of distilled water and 50 μL of Folin-Ciocalteu reagent in 1.5 mL vials. After 1 min, 150 μL of 7.5% sodium carbonate solution were added and mixed. After incubation in the dark (25 °C) for 1 h, aliquots of 200 μL were loaded in 96 well microplates. Absorbances were measured at λ 750 nm using a microplate reader (EPOC, Biotek). The analysis were performed in triplicate and normalized against negative controls (distilled water or diluted NADES) according to the Table 4 and expressed as mg of gallic acid equivalents (GAE) per gram of dried extract. The equation of gallic acid calibration curve was y = 0.089x + 0.0221 (R² = 0.9981).