Network Pharmacology-Guided Identification of Orientin from Passiflora incarnata Reveals Neuroprotective Effects Against Glutamate-Induced Excitotoxicity Through Regulation of Calcium Homeostasis

Epilepsy is a chronic neurological disorder in which current antiseizure medications are limited by incomplete seizure control and variable therapeutic responses, highlighting the need to identify new bioactive candidates. Passiflora incarnata (P. incarnata) has demonstrated neuropharmacological and anticonvulsant properties. However, the individual phytochemicals and molecular mechanisms underlying these effects remain incompletely characterized. In this study, we integrated network pharmacology with cellular validation to identify epilepsy-relevant phytochemicals of P. incarnata and evaluate their neuroprotective effects. Eleven phytochemicals underwent target prediction and were compared with epilepsy-associated genes, yielding 147 common targets. Protein–protein interaction analysis identified 134 interacting proteins and 590 interactions, with GABAA receptor subunits prominently represented among the top hub genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses further identified GABA-related functions and neuroactive ligand–receptor signaling. Integration of compound–target functional profiling with structural analysis prioritized the luteolin-derived flavonoid lineage, leading to the selection of orientin, a C-glycosylflavone constituent of P. incarnata, and luteolin, its structurally related core flavone scaffold, for experimental validation. In differentiated SH-SY5Y cells, pretreatment with orientin or luteolin attenuated L-glutamic acid-induced cellular injury in the presence of glycine, as evidenced by improved cell viability and reduced lactate dehydrogenase release. Both compounds also markedly reduced glutamate-induced intracellular Ca2+ accumulation. Notably, thapsigargin substantially reversed these Ca2+-lowering effects, suggesting a potential role for ER-associated Ca2+ regulatory mechanisms. The predicted GABAergic targets were not directly validated, and the cellular findings demonstrate neuroprotection against glutamate-induced excitotoxicity rather than direct GABAA receptor modulation or antiseizure efficacy. Collectively, these findings demonstrate the utility of network pharmacology-guided prioritization for identifying bioactive constituents of P. incarnata and support orientin as a neuroprotective candidate for further mechanistic and in vivo investigation.