Validation, quantification, and molecular docking of isolated eupalitin 3-O-β-D-galactopyranoside in Boerhavia diffusa Linn for hepatoprotective and immunomodulatory activity

Boerhavia diffusa is traditionally used for liver disorders and immunomodulation, but the mechanisms of its active flavonoid glycoside, eupalitin-3-O-β-D-galactopyranoside (EGP), remain incompletely defined. EGP was isolated by bioactivity-guided fractionation, and an ICH-aligned HPLC (High-performance liquid chromatography) method was developed and validated for its quantification. Mechanistic plausibility was probed by docking EGP to KEAP1 (NRF2 pathway; PDB: 6QMK) and the NF-κB p52–DNA complex (PDB: 1A3Q), benchmarking against silymarin and levamisole. Hepatoprotection was assessed in rats with D-galactosamine (GalN, 400 mg/kg, i.p.)–induced injury following prophylactic EGP (100 mg/kg, p.o.) via serum transaminases (ALT, AST), ALP, bilirubin, hepatic antioxidants (SOD, catalase, GSH), and histology. In vitro cytoprotection was evaluated in hepatocytes challenged with CCl4 (MTT assay), and immunostimulation was screened by LPS-induced NO release in RAW 264.7 macrophages. HPLC resolved a single EGP peak (Rt 2.79 min) with excellent linearity (R2 = 0.999), precision (RSD < 2%), and sensitivity (LOD 3 ng; LOQ 5 ng). Docking supported target engagement: for KEAP1 (6QMK), EGP achieved Glide scores of −7.29/−7.00 kcal·mol⁻1 versus silymarin −6.31/−6.16; for NF-κB p52–DNA (1A3Q), EGP scored −5.20/−4.53 versus levamisole −0.11. In vivo, EGP markedly ameliorated GalN hepatotoxicity, reducing ALT by 74%, AST by 63%, ALP by 38%, and bilirubin by 68%, while restoring antioxidant defenses (SOD +422%, catalase +190%, GSH +255%); histology corroborated near-normal lobular architecture with minimal periportal inflammation. In vitro, EGP improved hepatocyte viability in a dose-dependent manner (58% at 100 μg/mL; 67% at 200 μg/mL), comparable to silymarin (100 μg/mL). EGP also increased NO output in LPS-stimulated RAW 264.7 cells, consistent with immunostimulatory activity. EGP is a quantifiable B. diffusa constituent that exhibits convergent hepatoprotective and immunomodulatory effects across in silico, in vivo, and in vitro assays. These findings motivate pharmacokinetic studies and pathway-level validation (NRF2/ARE, NF-κB, MAPKs, iNOS/COX-2) to enable translation.