Abstract
Calotropis procera, a drought-tolerant shrub widely used in folk medicine, was evaluated for its antimicrobial potential and safety using an integrative in vitro/in silico workflow. Ethanolic leaf extract (EE-CP) displayed a dose-dependent inhibition of Staphylococcus aureus ATCC 2913 and Escherichia coli ATCC 35218, reaching 93% and 52% of the amoxicillin control, respectively (MIC 207 µg mL(-1) and 149 µg mL(-1)). GC-MS and LC-HRMS profiling revealed cardenolides (strophanthidin, gitoxigenin) and indole derivatives as major constituents. Pharmacophore mapping highlighted the essential glycosyltransferase MurG as a likely bacterial target; molecular docking showed that strophanthidin and NCGC00384918 bind MurG more strongly than the native substrate UDP-GlcNAc (ΔG ≤ -9.4 kcal mol(-1)), a result corroborated by 100 ns molecular dynamics simulations and MM-PBSA binding energies (-96.4 and -49.3 kcal mol(-1)). EE-CP caused <10% hemolysis up to 1.5 mg mL(-1) and exhibited LC(50) values of 302 µg mL(-1) (human lymphocytes) and 247 µg mL(-1) (BHK-21 cells), indicating a narrow but exploitable therapeutic window. Collectively, these findings constitute the first report on Colombian C. procera demonstrating potent anti-Staphylococcus activity, MurG-targeted cardenolides, and acceptable erythrocyte compatibility. This study supports EE-CP as a promising source of lead molecules and antibiotic adjuvants, warranting guided fractionation and in vivo validation to optimize efficacy and mitigate cytotoxicity.