*3.2. Extraction and Determination of Flavonoids in PUL*

PUL were dried and crushed up to 50 mesh. The crushed leaves (30 g) were extracted by stirring for 2 h at 30 ◦C with 600 mL of 70% ethanol, followed by filtration. The extract was obtained in powder form by concentrating and freeze-drying. The extract was subjected to HPLC analysis on a Waters 2695 separation module equipped with a Mightysil RP-18GP column (KANTO CHEMICAL, Japan). The sample was examined at a wavelength of 270 nm (UV) using a detector (Tunable Absorbance Detector, Waters, USA). Two solution systems were used in the HPLC mobile phase: the A solution (acetonitrile containing 0.1 wt.% acetic acid) and B solution (distilled water containing 0.1 wt.% acetic acid). The analysis was performed at a flow rate of 1.0 mL/min. The mobile phase B was eluted from 0 min to 100% and to be 70% to 40 min, 40% from 40 to 45 min, 10% to 50 min, and 100% to 60 min.
