Approximately 50 mL of methanol was added to 2 g of leaf powder and oleuropein was removed by extraction in an ultrasonic bath at approximately 55°C for 45 minutes. The supernatant was discarded and procedure repeated. The leaves were then dried for two hours at 105°C and maintained in a lowhumidity environment before use. The quantity of oleuropein remaining in the blank after double extraction acted as the first point on the calibration curve.