The total polyphenol contents (TPC) of the extracts were determined following a Folin–Ciocalteu procedure (Singleton, Joseph, & Rossi, 1965). The appropriate dilutions of extracts were oxidised with Folin–Ciocalteu reagent and its reaction was neutralised with sodium carbonate. The absorbance

of the resulting blue colour was measured at 765 nm, after 60 min, with a UV–Vis spectrophotometer (Model U-1800; Hitachi, Tokyo, Japan). The TPC was expressed as gallic acid equivalents (GAE) in mg per mL of extract (mg GAE/mL). The determination of chlorogenic acid was performed using an HPLC this website system (Shimadzu LC-10, Kyoto, Japan) equipped with a reverse-phase column (Shim-pack C18, 4.6 mm Ø × 250 mm), thermostated at 40 °C, and a UV–Vis detector (Shimadzu SPD 10A, λ = 280 nm). An isocratic mobile phase of water:acetic acid:n-butanol (350:1:10 v/v/v) was used at a flow rate of 0.8 mL/min. The

injection volume was 10 μL. For the quantitative analysis, a standard calibration curve was obtained by plotting the peak area against different concentrations of chlorogenic acid. The curve showed see more a good linearity and followed Beer’s Law (r2 = 0.99). Similarly, the final concentration of chlorogenic acid present in the samples was determined as average content after three consecutive injections. A volume of 15 mL of each extract was treated with 60 mL of dichloromethane for 1 h and the organic phase was concentrated to 2 mL under reduced pressure. The HPLC analysis was performed on a Shimadzu LC-10A system equipped with a UV–Vis detector SPD 10A set at 272 nm. The Thalidomide experiments were carried out on a reversed-phase Shim-pack C18 (4.6 mm Ø × 250 mm) column. The system was operated isocratically at 30 °C using a mobile phase composed of acetonitrile:0.1% formic

acid (15:85 v/v), with a flow rate of 1.0 mL/min. Prior to injection, all the samples were centrifuged at 2800g for10 min (Hermle, Z 200A, Wehingen, Germany) and filtered through 0.22-μm micropore membranes. The injection volume was 10 μL for the mate extract and 5 μL for the concentrated mate extract. For quantitative analysis, standard calibration curves were obtained for caffeine (1.0–100.0 μg/mL; r2 = 0.99) and for theobromine (2.5–50 μg/mL; r2 = 0.99). The final concentrations of each sample were determined by three consecutive injections ( Strassmann et al., 2008). The extraction procedure was performed according to Gnoatto, Shenkel, and Bassani (2005). The saponins present in 10 mL of each of the two extracts were hydrolysed with 5 mL of HCl (12 M) for 2 h under reflux. The saponins were extracted with 6 mL of chloroform. The organic phase was evaporated in a rotary evaporator and the residue was resuspended in 10 mL of ethanol.