Integrated metabolomic, nanoformulation, and network pharmacology approach reveals multifunctional bioactivities of an Ocimum sanctum nanoemulsion

Background Although Ocimum sanctum (Holy Basil) exhibits broad pharmacological potential, its therapeutic application is constrained by poor solubility, instability, and limited bioavailability. Previous nanoemulsion studies have not integrated comprehensive metabolomic profiling with mechanistic and multi-bioactivity validation. This study developed and characterized an O. sanctum nanoemulsion to overcome these barriers and enhance biological activity. Methods The ethanolic extract was profiled by GC–MS and LC–QTOF–MS/MS, identifying diverse phytochemicals. The optimized oil-in-water nanoemulsion (NE) was prepared using high-energy emulsification followed by high-pressure homogenization and ultrasonication. Physicochemical characterization included droplet size, polydispersity index (PDI), zeta potential, entrapment efficiency, and release profile. Biological activities were evaluated through cytotoxicity assays against Caco-2, HepG2, MDA-MB-231, and A549 cancer cell lines, antibacterial screening, antioxidant testing, and anti-inflammatory validation in LPS-stimulated RAW 264.7 macrophages. Network pharmacology analysis was performed to predict potential anti-inflammatory targets associated with the identified metabolites. Results The optimized NE displayed nanoscale spherical droplets (51–73 nm), narrow polydispersity (PDI = 0.264), high negative zeta potential (−42.1 mV), and strong entrapment efficiency (96.2 ± 3.1%). A biphasic release profile reached ∼60% over eight days. The NE showed potent cytotoxicity against Caco-2, HepG2, MDA-MB-231, and A549 cells (IC 50 = 13–25 μg/mL), demonstrating higher cytotoxic potency than the crude extract (∼200 μg/mL) while maintaining lower toxicity in normal fibroblasts (IC 50 = 102 μg/mL). Antibacterial screening revealed inhibition zones up to 14.8 ± 0.3 mm, and antioxidant testing demonstrated enhanced radical-scavenging activity (SC 50 = 16.4 μg/mL) compared with the crude extract (20.8 μg/mL). Network pharmacology analysis of identified metabolites predicted anti-inflammatory targets, highlighting AKT1, STAT3, PTGS2, and TLR4 as key regulators. Guided by these predictions, anti-inflammatory efficacy was experimentally validated in LPS-stimulated RAW 264.7 macrophages, where both the extract and NE significantly reduced TNF-α and IL-6 secretion (p < 0.0001), with stronger suppression by the NE. Conclusion Collectively, this metabolomics-guided O. sanctum nanoemulsion represents a promising plant-derived nanosystem. This preliminary study demonstrates its potential anticancer, antibacterial, antioxidant, and anti-inflammatory activities in vitro. However, further detailed investigations, including more extensive in vitro studies as well as in vivo evaluations, are required to better elucidate its mechanisms, safety, and potential biological applications.