Total flavonoids
Total flavonoids were determined according to the
method of Šarić et al. (2012) with some modifications.
All samples were centrifuged at 3,000 rpm and 4°C
for 10 min. Four milliliters of supernatant were mixed
for 5 min with a mixture solution of 4 ml deionized
water and 2 ml sodium nitrite (Sigma-Aldrich).
Afterward, 2 ml of 10% (w/v) aluminium chloride
(Sigma-Aldrich) were transferred into the mixture
and stirred for 6 min before adding 12 ml of 1M
sodium hydroxide (Merck, Germany). The solution
was shaken and the absorbance was measured at 510
nm. Total flavonoids were expressed as mg quercetin
equivalent per 100 ml sample (mg QE/100 ml).