Abstract

Azadirachta indica A. Juss. (neem) leaf ethanolic extract was characterized using multi-platform analytical and bioinformatics approaches to establish its phytochemical and pharmacological profile. Soxhlet extraction with 98% ethanol yielded 10.0 ± 0.5% (w/w) dark green viscous extract. Comprehensive analysis employed Thin-Layer Chromatography (TLC), High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD), Gas Chromatography-Mass Spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR) spectroscopy. Bioinformatics included PASS prediction, SwissTargetPrediction, MetaboAnalyst 5.0 pathway enrichment, and Cytoscape network construction. TLC revealed optimal separation using hexane:ethyl acetate:acetic acid (50:49:1) with 4-5 distinct bands. HPLC-DAD confirmed azadirachtin (Rt = 7.51 min) and identified additional limonoids including salannin and gedunin. GC-MS identified 30 compounds, with chlorinated terpenoid ester (36.41%), polycyclic terpenoid acetate (21.60%), and 2,4-di-tert-butylphenol (21.17%) as major constituents. NMR provided structural confirmation for limonoids, flavonoids, and fatty acid derivatives. The extract demonstrated significant antimicrobial activity against Staphylococcus aureus (15.3 ± 0.6 mm) and Escherichia coli (12.7 ± 0.5 mm), correlating with 2, 4-di-tert-butylphenol and was complemented by gedunin despite its minimal concentration. Bioinformatics predicted insecticidal (Pa = 0.845), antimicrobial (Pa = 0.912), anti-inflammatory (Pa = 0.789), and antioxidant (Pa = 0.802) activities for key compounds. Pathway enrichment identified terpenoid backbone biosynthesis (p = 3.2×10⁻⁶), steroid biosynthesis (p = 1.8×10⁻⁵), and NF-κB signaling (p = 4.5×10⁻⁴) as significantly enriched pathways. Network pharmacology demonstrated multi-target interactions involving ecdysone receptor, NF-κB, COX-2, and antioxidant enzymes. This integrated approach provides validated protocol for neem standardization and mechanistic insights into its polypharmacological effects.

Key words: Azadirachta indica, Phytochemical profiling, GC-MS, NMR, Bioinformatics, PASS prediction, Network pharmacology, Standardization