Pueraria lobata antioxidant extract ameliorates non-alcoholic fatty liver by altering hepatic fat accumulation and oxidative stress

Pueraria lobata is essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata is widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata has remarkable biological activities in protecting liver, relieving alcoholism, antioxidation, anti-tumor and anti-inflammation in clinic. However, the potential mechanism of ethyl acetate extract of Pueraria lobata after 70% alcohol extraction(APL) ameliorating nonalcoholic fatty liver disease (NAFLD) has not been clarified. This study aimed to investigate the ameliorative effect of P. lobata extract on human hepatoma cells and injury in rats, and to evaluate its therapeutic potential for ameliorating NAFLD. Firstly, the effective part of P. lobata extract was determined as APL by measuring its total substances and antioxidant activity. And then the in vitro and in vivo models of NAFLD were adopted., HepG2 cells were incubated with palmitic acid (PA) and hydrogen peroxide (H2O2). In order to evaluate the effect of APL, Simvastatin and Vitamin C (VC) were used as positive control. Various parameters related to lipogenesis and fatty acid β-oxidation were studied, such as intracellular lipid accumulation, reactive oxygen species (ROS), Western Blot, mitochondrial membrane potential, apoptosis, and the mechanism of APL improving NAFLD. The chemical components of APL were further determined by HPLC and UPLC-MS, and molecular docking was carried out with Keap1/Nrf2/HO-1 pathway related proteins. APL significantly reduced lipid accumulation and levels of oxidative stress-related factors in vitro and in vivo. Immunohistochemical、Western Blot and PCR analysis showed that the expressions of Nrf2 and HO-1 were up-regulated in APL treatment. The Nrf2 inhibitor ML385 can block the rescue by APL of cellular oxidative stress and lipid accumulation induced by H2O2 and PA, demonstrating its dependence on Nrf2. UPLC/MS analysis showed that there were 3'-hydroxyl puerarin, puerarin, 3'-methoxy puerarin, daidzein, genistin, ononin, daidzin and genistein. This study further clarified the mechanism of P. lobata extract in improving NAFLD, which provided a scientific basis for developing new drugs to protect liver injury and laid a solid foundation for developing P. lobata Chinese herbal medicine resources.