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.