In Annexin V-PI assays, a time-dependent increase in Annexin V positive cells was observed in cells treated with S. artemisiae MCCB 248 extract, implying that there was an increased PS translocation to the outer leaflet of the plasma membrane. At the beginning of treatment (0 h), a majority of the cells were negative for both Annexin V and propidium iodide (PI) staining. After 6 h of treatment, 40% of the cells were Annexin V positive and only 9% of cells were both Annexin V and PI positive. A similar observation was observed after 12 h (42% Annexin V and 13% both Annexin V and PI positive reaction), which was indicative of the early stages of apoptosis. After 24 h, 51% of cells were Annexin V positive and more cells had become positive for PI (34%), indicative of the later stages of apoptosis and signified more dead cells (Figs. 6, 7).Fig. 6Fluorescent microscope image (20×) of Annexin-V/PI double-staining assay. After treating with S. artemisiae MCCB 248 extract, cells were stained with Annexin V-FITC and propidium iodide and analyzed after 0, 6, 12 and 24 h of treatment Fig. 7Percentage of H-460 cells showing early and late apoptotic cell death at 0, 6, 12, 24 h after exposure to the extract of S. artemisiae MCCB 248 (data shown are mean of four independent observations and its standard deviation)