2.4. Determination of TFs
The TFs were measured by the colorimetric-based method assay
(Christ & Mueller, 1960) with a slight modification. Briefly, 1.0 ml
TFs solution was mixed with 4.0 ml 70% aqueous ethanol, and
0.5 ml NaNO2 (5%, w/v) was added. After 6 min, 0.5 ml AlCl3
(10%, w/v) and 3.0 ml NaOH (1 M) were added, followed by the
addition of distilled water to reach 10.0 ml. The solution was
mixed and incubated for 15 min at room temperature. The solutions
were scanned by a UV-1700 spectrophotometer using quartz
cuvettes (1.0 cm) (Shimadzu, Japan) at 510 nm against the control.
The control contained all the reaction reagents except for the test
sample. Standard curve regression equations were: A = 0.0152C +
0.0024 R2 = 0.9997 (where A is the absorption and C is the rutin
concentration in lg/ml). The flavonoid content was calculated
from the calibration curve and expressed as rutin equivalents (RE).