Crocus sativus (Saffron) Aqueous Extract Mediated Green Synthesis of Iron Oxide Nanoparticles and Their In Vitro and In Vivo Biological Investigations

ABSTRACT The present study reports the green synthesis of iron oxide nanoparticles (IONPs) using the aqueous extract of Crocus sativus (saffron) as a natural reducing and stabilizing agent, followed by comprehensive in vitro and in vivo biological investigations. IONPs were confirmed by UV–visible spectroscopy (peak at 430 nm), FTIR analysis (peaks at 3400, 2900, 1650, 1380, and 1050 cm −1 indicating polyphenols, flavonoids, and proteins), and SEM imaging, which revealed a highly aggregated, irregular morphology. ImageJ analysis features gave a mean equivalent diameter of 111.98 ± 83.70 nm. EDS analysis confirmed iron (44.74 wt%) and oxygen (24.82 wt%) as the major elements with organic capping (28 wt% carbon). The phytochemical constituents of C. sativus are rich in phenolics and flavonoids, which effectively facilitated the reduction of Fe 3+ ions to stable nanoscale iron oxide particles. The in vitro enzyme inhibition assays revealed significant inhibitory effects on α ‐glucosidase and xanthine oxidase, with IC 50 values of 33.08 ± 1.29 and 2.96 ± 0.49 μg/mL, respectively, and moderate inhibition of carbonic anhydrase‐II (49%). Antibacterial evaluation demonstrated an important zone of inhibition ZOI (21.45 mm) against Klebsiella pneumoniae , indicating the antimicrobial potential of the IONPs compared to the crude extract. In vivo pharmacological screening further supported their biological efficacy. IONPs showed potent analgesic activity (78% inhibition at 10 mg/kg), pronounced anti‐inflammatory effects in carrageenan‐ and histamine‐induced paw edema models (up to 95% inhibition), and marked sedative properties at low doses. These results suggest that Crocus sativus ‐mediated IONPs may possess strong therapeutic potential due to their enhanced biochemical, antimicrobial, anti‐inflammatory, and analgesic activities. The study highlights saffron‐assisted green synthesis as an efficient, eco‐friendly, and biocompatible approach for developing multifunctional nanomaterials with possible applications in pharmaceutical, biomedical, and health‐related fields.