Optimization of deep eutectic solvent extraction of Piper nigrum L. and evaluation of hepatoprotective effects against acute ethanol-induced liver injury in mice.

INTRODUCTION: Alcohol-associated liver disease (ALD) represents a growing global health burden, for which safe and accessible therapeutic options remain limited. As a widely used medicinal and food plant, Piper nigrum L. contains piperine as its core hepatoprotective alkaloid. However, conventional organic solvent extraction poses environmental risks, leaves solvent residues, and causes loss of synergistic endogenous bioactives. As sustainable green extraction media, deep eutectic solvents (DESs) show great potential for phytochemical enrichment, yet their impacts on the hepatoprotective potency and gut microbiota-regulatory activity of black pepper extracts remain unclear. METHODS: A choline chloride-1,4-butanediol-based ultrasonic-assisted DES extraction system was optimized via single-factor experiments and Box-Behnken design. We systematically compared the phytochemical profiles, in vitro antioxidant activities, and in vivo hepatoprotective efficacies of DES extracts versus conventional 60% ethanol extracts using a mouse model of acute ethanol-induced liver injury, and employed 16S rRNA gene sequencing to analyze gut microbiota composition. RESULTS: Under the optimized conditions (extraction time of 60 min, water content of 30%, solid-to-liquid ratio of 1:28 g/mL, molar ratio of choline chloride to 1,4-butanediol of 1:3, ultrasonic power of 400 W, and temperature of 40 °C), the DES process achieved a piperine yield of 44.40 ± 0.84 mg/g. In the DPPH radical scavenging assay, DES extract exhibited better in vitro antioxidant activity than the ethanol extract. In mice with acute ethanol-induced liver injury, DES extract reduced hepatic lipid accumulation, oxidative stress, and hepatocellular injury. 16S rRNA sequencing revealed that DES extract treatment was correlated with altered gut microbial composition under ethanol stimulation, including decreased relative abundance of Muribaculaceae and Clostridia UCG-014, and increased relative abundance of Lactobacillus murinus. DISCUSSION: Collectively, these findings indicate that DES-derived black pepper extract is associated with favorable hepatoprotective phenotypes in the acute ethanol-induced liver injury model, alongside distinct correlational changes in gut microbiota composition. The study provides technical support for green high-value processing of plant-based spice resources, and scientific insights for developing microbiota-targeted plant-derived functional foods.