GC–MS Metabolite Profiling and Induction of Cell Death by Sphaeranthus amaranthoides Leaf Extracts

ABSTRACT Sphaeranthus amaranthoides , a lesser‐known species of the Asteraceae family, has shown promising medicinal properties, prompting investigation into its cytotoxic and pro‐apoptotic effects on human cancer cells. This study evaluated aqueous, methanol, ethyl acetate, and hexane leaf extracts for selective anticancer activity. Normal Vero cells and cancer cell lines AGS (gastric) and HT‐29 (colorectal) were exposed to Sa‐EAE extract, and GC–MS analysis identified key bioactive compounds, including methyl 10,13‐dimethyltetradecanoate, 3,7,11,15‐tetramethyl‐2‐hexadecen‐1‐ol, and 1,3,5‐triazine derivatives. The extract showed selective cytotoxicity against AGS cells while sparing normal Vero cells. Apoptosis was evidenced by mitochondrial‐membrane depolarization (ΔΨm), cytochrome c release, activation of Caspase‐9 and Caspase‐3, LDH leakage, and modulation of the Bcl‐2/Bax ratio. Apoptotic morphology was confirmed via AO/EB and PI staining. Flow cytometry revealed G 0 /G 1 cell cycle arrest, with downregulation of Cyclin D1 and CDK4 and upregulation of p21 and p27. These findings indicate that Sa‐EAE exerts dual effects of mitochondrial‐mediated apoptosis and G 0 /G 1 arrest in gastric cancer cells, with minimal toxicity to normal cells, supporting its potential as a therapeutic candidate for further in vivo validation.