2.12. EGCG and Curcumin Determination

The EGCG was determined using HPLC performed with LC 20AD (SHIMADZU, Tokyo, Japan) using a Diamonsil C18 column (5 μm, 150 × 4:6 mm) at 30 °C. A linear gradient elution program was referred to a previous study [28] with modification performed as follows: the injection volume was 10 μL and the mobile phase was 0.4% (v/v) water–formic acid (A) and acetonitrile (B) by firstly using 10–25% (v/v) B from 0 to 12 min, linear gradient to 10% (v/v) B from 12 to 14 min and then kept at 10% (v/v) B for 6 min. The flow rate was 1.0 mL/min and total run time was 20 min. The compounds were then detected at 280 nm and quantified based on the calibration curve of EGCG (Y = 3836.9X − 28,244, R² = 0.9967). The curcumin concentration (mg/mL) was determined with a UV-Vis spectrophotometer at 425 nm using a calibration curve (Y = 0.0859X − 0.0144, R² = 0.998).