Determination of total flavonoids
Total flavonoids content was determined by a pharmacopeia method (Miliauskas, Venskutonis & van Beek 2004). Rutin hydrate (purity ≥ 94%, Sigma Aldrich; Italy) was used as reference compound. Briefly, 160 μL of sample or a standard solution of rutin was mixed with 160 μL AlCl3 (20 g L-1 in ethanol; Sigma Aldrich; Italy) and 3.68 mL ethanol (Sigma Aldrich; Italy). Samples and standards were incubated at 20 °C for 40 min, and absorbance read at 415 nm using a spectrophotometer (Agilent Technologies, Cary Series 100 UV-VIS Spectrophotometer, Italy). Results were expressed µmol rutin equivalent.