Enhanced bioactivity of honeysuckle-Cassia seeds extracts through Lactobacillus acidophilus and Bacillus subtilis co-fermentation: Impact on alcoholic liver disease and gut microbiota

Abstract

This study investigated the hepatoprotective potential of Honeysuckle-Cassia seeds extracts co-fermented with Lactobacillus acidophilus and Bacillus subtilis against alcoholic liver disease (ALD) and gut microbiota dysbiosis. Through network pharmacology analysis, 209 overlapping targets between Honeysuckle-Cassia seeds bioactive components and ALD-related targets were identified, with 39 core targets subsequently determined. Comparative analysis of aqueous extract (AE), Lactobacillus acidophilus fermentation broth (LAF), and mixed bacteria fermentation broth (MBF) revealed that MBF significantly enhanced the content of bioactive compounds: total polysaccharides (72.6 ± 3.8 mg/g), flavonoids (34.7 ± 2.5 mg/g), and saponins (15.2 ± 1.1 mg/g), representing 275 %, 72 %, and 62 % increases over AE, respectively (p < 0.05). In a murine ALD model, MBF intervention (12.5 mL/kg, 30 days) significantly reduced serum markers of liver injury (ALT: 35 %, AST: 28 %, TC: 42 %, TG: 39 %; (p < 0.05) and hepatic oxidative stress (MDA ↓52 %, GSH ↑156.55 %, SOD ↑76.71 %). Mechanistically, MBF suppressed pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) by 40-50 % while elevating anti-inflammatory mediators (IL-4, IL-10, PGE2) 1.6-2.0-fold via AMPK/ACC/SREBP1c signaling modulation. Gut microbiota analysis revealed that MBF restored α-diversity indices (Shannon ↑10.06 %, ACE ↑32.34 %) and reversed alcohol-induced dysbiosis by enriching Lachnospiraceae and Blautia while suppressing Alloprevotella. Structural degradation of plant residues (SEM) confirmed microbial synergy in releasing insoluble-bound phytochemicals (100-400 m/z range). These findings validate co-fermentation as a potent strategy to amplify the hepatoprotective and microbiota-modulating activities of traditional herbs, offering a scientific foundation for developing functional foods against ALD.

Keywords: Alcoholic liver disease; Enhanced bioactivity; Gut-liver axis; Honeysuckle-Cassia seeds; Mixed bacterial fermentation.