暴马丁香桑黄多酚的提取纯化及缓解AML12肝细胞脂毒性的效果

孙嫚; 范勇; 杜加利; 李永萍; 周岚; 李宏彬; 李良玉; 孟奥格; 高雅; 曹荣安

doi:10.13386/j.issn1002-0306.2025100293

暴马丁香桑黄多酚的提取纯化及缓解AML12肝细胞脂毒性的效果

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

摘要

摘要: 为探究暴马丁香桑黄多酚（Phellinus baumii Pilát polyphenols，PBP）的提取工艺，评价其抗氧化活性及对AML12肝细胞脂毒性的缓解作用。本研究采用响应面法优化超声辅助乙醇提取工艺，通过萃取和硅胶柱层析进行分离纯化，采用超高效液相色谱-串联质谱（Ultra performance liquid chromatography-Tandem mass spectrometry，UPLC-MS/MS）技术对纯化的PBP进行组成鉴定，并分析其抗氧化能力。建立棕榈酸（Palmitic acid，PA）诱导的AML12肝细胞脂毒性模型，通过油红O染色、甘油三酯、丙二醛、超氧化物歧化酶含量检测以及Western Blotting分析SREBF1、ACC1、CD36、ACSL4、GPX4和caspase 3蛋白表达，研究PBP对脂毒性的影响。结果表明，最佳提取工艺为：料液比1:32 g/mL、温度56 ℃、时间0.85 h，此条件下多酚含量为7.98±0.25 mg/g，并从纯化后的PBP中鉴定出Hispolon、Hispidin、Phelligridin B、2,3-二羟基肉桂酸以及迷迭香酸5种多酚。与未纯化样品相比，纯化后的PBP体外抗氧化活性显著增加（P<0.05）。细胞实验表明：PBP降低细胞内甘油三酯和丙二醛含量（P<0.001），提高超氧化物歧化酶活性（P<0.01），减少脂质积聚，同时通过下调ACSL4、caspase 3蛋白表达，上调GPX4蛋白表达（P<0.05），缓解PA诱导的铁死亡及凋亡。综上所述：本研究优化了PBP提取工艺，从中鉴定出5种多酚成分，证实其具有显著的抗氧化活性，并能通过抑制铁死亡和凋亡缓解PA诱导的AML12肝细胞脂毒性，表明PBP具有作为功能性食品成分的开发潜力。

Abstract: 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.