Inhibition of β-glucuronidase by amide alkaloids isolated from the fruits of Piper longum L. Enzyme kinetics, molecular docking, and molecular dynamics simulations

Piper longum L., also known as long pepper, is commonly cultivated in tropical and subtropical areas. In addition to its use as spice and seasoning, the fruit of P. longum is an important medicinal plant in traditional medicine. Amide alkaloids are a major class of phytochemicals found in P. longum. In this study, the inhibitory effects of 43 amide alkaloids isolated from the fruits of P. longum on β-glucuronidase were evaluated using in vitro assays. Compounds piperlongumamide F (32), 1-(eicosa-2E,14Z-dienoyl)piperidine (33), N-isobutyl-(2E,4E)-undeca-2,4-dienamide (38), and (2E,4E,12Z)-N-isobutyloctadeca-2,4,12-trienamide (41), (2E,4E,14Z)-N-isobutyleicosa-2,4,14-trienamide (42), and (2E,4E,16Z)-N-isobutyldocosa-2,4,16-trienamide (43) strongly inhibited β-glucuronidase with IC₅₀ values of 4.9–8.1 μM, and piperlongumamide E (19) exhibited moderate β-glucuronidase inhibitory activity. Kinetic studies revealed that compounds 19, 32, and 33 acted as noncompetitive β-glucuronidase inhibitors, and compounds 38 and 41−43 were uncompetitive inhibitors. Molecular docking and dynamics simulations were performed to investigate the interactions, binding mechanisms, and dynamics behavior of these active compounds with the binding sites of β-glucuronidase. Moreover, modern computational techniques, including principal component analysis, dynamic cross-correlation maps, Gibbs free energy landscape, and free energy surface, were conducted to further analyze the active compounds.