Apoptosis Evaluation of Plant Extracts

The apoptosis effect of the plant crude extracts was primarily determined by fluorescent dye staining and DAPI to identify the condensation and fragmentation of nuclear DNA [14 (link), 15 (link)]. Briefly, the cancer cells (HepG2 or U937) were treated with various concentrations of the test compounds (6 to 500 µg/mL of P. kesiya and 0.75 to 15 µg/mL of melphalan) for 24 h, after which the culture medium was removed and the cells were washed with fresh medium. Cells were then fixed by cold methanol. DAPI dye (Sigma–Aldrich Chemie GmbH, Germany) was then added to stain the nuclei DNA for 1 h. The excess dye was removed and 1X PBS (pH 7.4; 10 mM) was added to glycerin (at a 1:1 ratio). The DAPI staining assay was performed in triplicate in independent experiments. An inverted fluorescence microscope ​(Nikon eclipse 80i, Kanagawa, Japan) was used to record images of the DAPI staining. The average percentage of apoptotic cells was calculated from three independent wells with 10 eye views per well under the inverted fluorescence microscopy at a magnitude of 40×.