When total flavonoids were determined, the filtrate (1 mL) wasadded to a 10 mL flask, 0.3 mL 5% NaNO2 and 0.3 mL 10% Al (NO3)3added, shaken for 5 min at room temperature, then 2 mL 1 mol L−1NaOH solution added. The final volume of 10 mL was made up withdistilled water, kept at 4◦C 15 min for coloring. Total flavonoidswere measured at 500 nm and calculated with standard catechinsample