Novel prolyl endopeptidase inhibitor from <i>Myricaria germanica</i> alleviates steatohepatitis

Prolyl endopeptidase (PREP), a serine protease, plays a critical role in the progression of hepatic steatosis and thereby contributes to metabolic dysfunction-associated fatty liver disease (MAFLD). Its inhibition has been shown to reverse disease progression. This study aimed to identify effective PREP inhibitors derived from <i>Myricaria germanica</i>, a deciduous shrub widely used in folk and traditional Chinese medicine, and to assess their potential therapeutic role in steatohepatitis. A bioassay-guided approach was employed to isolate PREP inhibitors from <i>M. germanica</i> crude extracts. The most active inhibitor was assessed through kinetic and computational studies. Moreover, its protective effects were evaluated using palmitic acid (PA) induced lipotoxicity in HepG2 cells and a high-fat diet (HFD)-induced steatohepatitis mice model. We identified and isolated a novel PREP inhibitor, (±)-2-pentacosylcyclohexanol (PREPi), with an IC₅₀ value of 20.05 ± 1.6 μM. Kinetic and computational studies confirmed that PREPi acts as a competitive inhibitor. Furthermore, PREPi protected against PA-induced lipotoxicity and oxidative stress in HepG2 cells. In HFD-induced steatohepatitis mice, PREPi administration revealed improved liver function conditions (ALT, AST and ALP), quantitative scoring of steatosis and inflammation, and serum lipid profile, as well as the efficacy in weight gain and glucose tolerance. Mechanistically, PREP inhibition disrupts cascades linked to lipid accumulation and oxidative damage, suppresses lipogenic genes (SREBP-1c/FASN), and enhanced antioxidant defences positioning a novel natural PREPi as a potential candidate for steatosis and steatohepatitis treatment. These results also validate <i>M. germanica</i> as a bioactive source for intervening metabolic disorder and MAFLD.