2.3. HUVECs extraction and sample preparation
HUVECs line was purchased from Shanghai Institute of Cell
Biology, Chinese Academy of Sciences (Shanghai, China). HUVECs
were grown at 37 C and 5% CO2 cultured in 10% FBS DMEM medium.
YQFM prepared as afore mentioned was added when the cell
density reached 90%. The drug solution was removed after incubation
for 1 h and the 10-cm dish was washed with 8 mL PBS for 5
times to remove unbound components, collected the last 1.5 mL
PBS eluate as a control for HPLC-MS analysis (sample A1 and B1).
Finally, HUVECs were denatured and extracted with 4 mL of 80%
methanol by ultrasonic extraction to liberate components bound to
the cells. The desorption eluate was centrifugated and the supernatant
was condensed to 100 mL for HPLC-MS analysis (sample A4
and B4). The control sample, in which YQFM was replaced by
DMEM, was prepared using the same procedures as described
above (sample A2 and B2).