Quantifying Total Flavonoid Content

Total flavonoid content (TFC) was determined by colorimetric method using aluminum chloride (AlCl₃) as reported by Chandra et al.^{16 (link)} with minor modifications. 25 mg of ethanolic extracts of O. stamineus leaf or stem was dissolved in 25 mL ethanol. 1 mL of the solution was mixed with 0.5 mL of 10% AlCl₃ and incubated at room temperature for 74 min. The absorbance was measured at 410 nm with a Uv–Vis spectrophotometer (Shimadzu Uv–Vis 1800, Japan). Quercetin standard was measured ranging from 5 to 20 µg/mL as the standard for total flavonoid concentration. TFC was calculated as μg/g quercetin equivalent (QE) of dried extract. All measurements were carried out in triplicates.