Identification of narcissoside from Anoectochilus roxburghii as an alcohol dehydrogenase inhibitor with protective effects against ethanol-induced HepG2 cell injury.

Alcohol dehydrogenase (ADH)-mediated ethanol oxidation and oxidative stress are closely associated with alcohol-induced hepatocellular injury. In this study, an integrated bioactivity-guided strategy combining microfractionation, affinity ultrafiltration, and HPLC was developed to screen hepatoprotective constituents from Anoectochilus roxburghii. Narcissoside was identified as the leading dual-active candidate and was successfully isolated by one-step high-speed countercurrent chromatography (HSCCC) with a purity of 98.32%. Narcissoside showed strong ABTS radical-scavenging activity and apparent ADH-binding activity. Enzyme assays demonstrated that narcissoside inhibited ADH activity in a concentration-dependent manner, with an IC50 of 39.73 μM. Kinetic analysis indicated that narcissoside acted as a reversible mixed-type inhibitor, with Ki lower than Kis, indicating a higher apparent affinity for free ADH than for the ADH-substrate complex. Fluorescence spectroscopy indicated changes in the local microenvironment of ADH, whereas far-UV circular dichroism indicated changes in its secondary structure. Molecular docking and 200-ns molecular dynamics simulations further provided a plausible structural model involving hydrogen-bonding and hydrophobic interactions between narcissoside and ADH. In an ethanol-injured HepG2 cell model, narcissoside improved cell viability and reduced ALT and AST activities in the culture supernatant, indicating protective effects against alcohol-induced cellular injury. These findings identify narcissoside as a bioactive flavonoid glycoside from A. roxburghii with antioxidant, ADH-inhibitory, and hepatocellular protective activities in vitro, although whether the cellular protection is mechanistically related to ADH inhibition remains to be determined.