This study focuses on key genes (Caspase-3, JAK2, BCL2L1 and MAPK8) and their modulation in response to hypoxia-induced stress using Methyl Glycoside (MG), a small molecule spectroscopically screened from Aganosma dichotoma. Hypoxia/reoxygenation (H/R) induced H9C2 cells, pre- treated with MG, were subjected to cell viability assay, free radical scavenging activities (catalase, GST, GSH-Px, SOD), caspase activity, mitochondrial membrane potential, and gene expression profiling through standard assays and molecular techniques. Results indicated that MG treatment, has potential protective effects against H/R induced stress in H9C2 cell lines. Cell viability assays showed that MG maintained cellular viability with significant protection (P <
0.05) observed from 10 µM. Free radical scavenging assays revealed that MG, enhanced detoxification mechanisms and exhibited potential antioxidant effect in a significantly (P <
0.05) in a dose dependant manner. MG pre-treatment in H9C2 cells protected cellular damage from caspase activity, cells exhibited high mitochondrial membrane potential, and gene expression profiles, including upregulation of anti-apoptotic BCL2L1 and modulation of stress-responsive genes like CASP3, JAK2 and MAPK8. Hence, MG exhibited concentration-dependent protective effects on viability, oxidative stress, and apoptosis-related pathways, laying the foundation for further exploration and translational applications in cardiovascular interventions.