One gram of powdered samples was mixed with 10 ml 95% ethanol in glass vial and shake
well with vortex mixer and the extract left at room temperature overnight. Each sample was
centrifuged at 1,000 rpm for 3 min at 20o
C to settling. The 100 µl suspension was filtered
through syringe filter with 0.2 µm membrane, and the sample was kept at -20o
C until analyzed