Flavonoid Extraction and Quantification

The extraction and quantification of flavonoid were carried out according to previous studies with minor modification [48 (link)]. Briefly, 0.5 g of dried powder was added to 10 mL of 65% ethanol and then extracted with ultrasonic wave at 50 °C for 30 min. After centrifuged at 10,000× g for 10 min, the supernatant was collected and the absorbance was measured with an ultraviolet spectrophotometer (MAPADA, Shanghai, China) at 340 nm. The quantification of luteoloside was conducted using Dionex U3000 HPLC system (Dionex, Sunnyvale, CA, USA) with a Welch LP-C18 column (5 μm, 150 mm × 4.6 mm) at 25 °C. The flow rate was 1 mL/min and the mobile phase consisted of acetonitrile (eluent A) and water: acetic acid (999:1, v/v, eluent B). The elution program was 0–5 min, 8–10% A; 5–25 min, 10–20% A; 25–45 min, 20–30% A; 45–55 min, 30–100% A. 10 μL of the extracted sample was injected and fractions were monitored at 350 nm. Commercial luteoloside was used as the standard to identify and quantify the luteoloside contents in all samples. All results were representative of three independent experiments.