Extraction, Purification of Polyphenols from Phellinus baumii Pilát and Lipotoxicity Alleviating Effect on AML12 Hepatocytes

This study aimed to optimize the extraction process of polyphenols from Phellinus baumii Pilát (PBP), evaluate their antioxidant activity, and examine their alleviative effect on palmitic acid (PA)-induced lipotoxicity in AML12 hepatocytes. Ultrasonic-assisted ethanol extraction was optimized using response surface methodology. The crude extract was purified through liquid-liquid extraction and silica gel column chromatography. The composition of purified PBP fractions was identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and their antioxidant capacity was evaluated. A PA-induced lipotoxicity model was established in AML12 cells. The effects of PBP intervention were assessed by oil red O staining and measuring triglyceride, malondialdehyde, and superoxide dismutase levels. Protein expression of SREBF1, ACC1, CD36, ACSL4, GPX4 and caspase 3 were analyzed by Western blotting. Results: The highest polyphenol content (7.98±0.25 mg/g) was achieved under conditions of 1:32 g/mL, 56 ℃, and 0.85 h extraction time. Five specific polyphenols were identified in the purified PBP fraction, including Hispolon, Hispidin, Phelligridin B, 2,3-dihydroxycinnamic acid, and rosmarinic acid. The purified PBP exhibited significantly higher antioxidant activity in vitro compared to the crude extract (P<0.05). Cell experiments demonstrated that PBP intervention significantly reduced intracellular triglyceride and malondialdehyde levels (P<0.001), enhanced superoxide dismutase activity (P<0.01), and decreased lipid accumulation. Additionally, PBP significantly downregulated ACSL4 and caspase 3 expression, upregulated GPX4 (P<0.05), thus alleviating PA-induced ferroptosis and apoptosis. In conclusion, this study optimized the extraction process of PBP and identified five polyphenolic compounds. PBP exhibited significant antioxidant activity and alleviated PA-induced lipotoxicity in AML12 hepatocytes through the inhibition of ferroptosis and apoptosis, suggesting its potential for development as a functional food ingredient.