Exploring the anti-Helicobacter pylori activity of Guluchyadi Kashayam through in-vitro and in-silico analysis.

UNLABELLED: Helicobacter pylori infection, a driver of chronic gastritis and gastric cancer, requires new therapeutic strategies due to rising antibiotic resistance. Guluchyadi Kashayam, a traditional Ayurvedic formulation, serves as a potential source of anti-H. pylori compounds. Anti-H. pylori activity was evaluated using solvent-fractionated extracts and in vitro assays. GC-MS-identified constituents were screened for ADME properties. Promising compounds underwent molecular docking against essential H. pylori proteins, with lead complexes analyzed via 200 ns molecular dynamics simulations and MM/GBSA calculations. Multiple solvent fractions showed inhibitory activity. GC-MS identified 129 phytochemicals, with four showing favorable ADME profiles. Molecular docking identified Benzenemethanimine, N-(1,1-dimethylethyl)-α-[(formylamino)methyl]-4-methyl- (CID: 546407) as the top candidate, demonstrating high affinity for the β-clamp protein (docking: - 7.479 kcal/mol; MM/GBSA ΔGbind: - 32.94 kcal/mol). Molecular dynamics and MM-GBSA analyses identified it as the sole viable lead (ΔG = - 10.94 kcal/mol). Driven by robust Van der Waals forces, it avoids the massive entropic penalties hindering other compounds. Structurally, it induces a stable, highly compact, yet uniquely solvent-exposed conformation without triggering localized flexibility disruptions. This study supports the anti-H. pylori potential of Guluchyadi Kashayam. We identify the β-clamp protein as a therapeutic target and CID 546,407 as a promising lead for drug development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-026-00709-1.