NADH-Activated Near-Infrared Fluorescent Probe for Efficient Screening of ADH/ALDH2 Dual Agonists for Alcohol Detoxification

Alcohol-induced liver injury remains a major global health concern, while effective strategies that facilitate alcohol metabolism are still limited. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase 2 (ALDH2) play central roles in hepatic ethanol metabolism, their catalytic reactions are stoichiometrically coupled to nicotinamide adenine dinucleotide (NADH) production. However, many existing NADH probes suffer from short excitation/emission wavelengths, high background interference, and limited dynamic ranges, restricting their application in enzyme-related screening. Herein, we report a near-infrared fluorescent probe, BOD-NH (Ex/Em = 715/760 nm), featuring low background fluorescence, high photostability, and a broad linear response toward NADH (0–200 μM). Leveraging these properties, an enzyme-coupled screening strategy was established to evaluate NADH generation associated with ADH- and ALDH2-catalyzed reactions. Screening of a natural product library identified Citri Grandis Exocarpium as an extract exhibiting pronounced activity in both enzyme systems. Subsequent spectrum–effect correlation analysis combined with HPLC-Q-TOF-MS/MS characterization identified naringin as a key bioactive constituent. Enzyme-based assays showed that naringin was associated with enhanced ADH- and ALDH2-related NADH generation. In vivo studies further demonstrated that naringin administration was associated with accelerated ethanol and acetaldehyde clearance, improved behavioral recovery following acute alcohol exposure, and attenuation of alcohol-induced liver injury accompanied by reduced oxidative stress and inflammation. Collectively, this work establishes BOD-NH as a practical NADH-responsive analytical tool for enzyme-coupled screening applications and identifies naringin as a bioactive natural product with potential value for further investigation in alcohol metabolism–related modulation.