Integrative metabolomic and pharmacological profiling of Nyctanthes arbor-tristis L. tissues using HPTLC ¹H-NMR molecular docking and anti-inflammatory assays

Nyctanthes arbor-tristis L. (NAT) is a cornerstone of traditional Ayurvedic medicine for treating chronic inflammation. However, the precise distribution of bioactives across different plant tissues and their specific molecular targets remains insufficiently characterized. This study investigates the tissue-specific chemical architecture of NAT and integrates its in silico multi-target inhibitory potential with in vitro biological validation. Methanolic extracts of NAT leaf and bark were analyzed using HPTLC and 1 H-NMR spectroscopy to identify major secondary metabolites (alkaloids, steroids, and tannins). In silico molecular docking evaluated the thermodynamic stability of these bioactives against key inflammatory mediators (COX-2, TNF-α, and IL-6). Finally, the in vitro anti-inflammatory efficacy of both extracts was validated using egg albumin and bovine serum albumin (BSA) denaturation assays (100–800 µg/mL). 1 H-NMR analysis confirmed the presence of indole mixed alkaloids, phytosterols, and polyphenolic tannins, with signals in the aromatic (δ 6.1–8.6 ppm), aliphatic (δ 2.0–3.5 ppm), and methyl/side-chain regions (δ 0.8–1.3 ppm) correlating with reserpine-type alkaloids and β-sitosterol. In vitro validation demonstrated dose-dependent protein stabilization: leaf extracts consistently outperformed bark extracts. In the egg albumin assay, the leaf extract achieved an IC 50 of 168.34 µg/mL compared to 182.76 µg/mL for the bark. Similarly, in the BSA assay, the leaf extract showed superior potency with an IC 50 of 194.82 µg/mL versus 208.34 µg/mL for the bark. While diclofenac sodium remained the most potent agent (IC 50 69.42–72.18 µg/mL), the high inhibitory capacity of the crude NAT leaf extract (reaching 89.16% at 800 µg/mL) confirms its significant anti-inflammatory potential. This research establishes a definitive molecular and biological basis for the anti-inflammatory efficacy of NAT. The congruence between identified structural motifs and superior in vitro performance of foliar tissues suggests that NAT leaves are a potent source of diverse scaffolds for the development of green-chemistry-based, dual-action anti-inflammatory agents. While synthetic NSAIDs remain more potent, the efficacy of these crude extracts warrants further isolation of target fractions.