目的 对苦参Sophora flavescens醋酸乙酯部位的化学成分进行研究，并进行抗肝纤维化活性评价。方法 采用硅胶、聚酰胺、葡聚糖凝胶LH-20及C₁₈反相柱色谱等多种柱色谱法进行分离纯化，并运用现代波谱技术及理化鉴定法对化合物进行结构鉴定。以转化生长因子-β1（transforming growth factor beta-1，TGF-β1）诱导的LX-2细胞为肝纤维化细胞模型，运用CCK-8法筛选化合物的抗肝纤维化活性。通过细胞划痕实验、实时荧光定量多聚核苷酸链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）实验、Western blotting实验、流式细胞术等实验，对化合物14（苦参醇T）的抗肝纤维化活性及作用机制进行深入研究。结果 从苦参醋酸乙酯部位分离得到28个化合物，分别鉴定为2,4-二羟基苯甲酸乙酯（1）、三叶紫檀苷（2）、2,4-二羟基苯甲酸（3）、苦参啶（4）、苦参醇N（5）、苦参黄酮A（6）、苦参醇C（7）、8-异戊烯基-5-甲氧基-7,2¢,4¢-三羟基二氢黄酮醇（8）、2¢-羟基-异黄腐酚（9）、苦参酮（10）、2,4-二羟基苯甲酸甲酯（11）、sophoflavescenol（12）、伞形花内酯（13）、苦参醇T（14）、sophophenoside B（15）、6ʺ-木糖-染料木素葡萄糖苷（16）、3′,4′-methylenedioxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)-O-β-D-glucopyranoside（17）、染料木素-7-O-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷（18）、苦参醇O（19）、4′-羟基-3′-甲氧基异黄酮-7-O-β-D-木吡喃糖基-(l→6)-O-β-D-葡萄吡喃苷（20）、3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside（21）、染料木素（22）、异黄腐酚（23）、8-异戊烯基-5-甲氧基-7,4¢-二羟基二氢黄酮醇（24）、2-(2¢,4¢-二羟基苯基)-5,6-亚甲二氧基苯并呋喃（25）、calycosin（26）、daidzein（27）、柚皮芸香苷（28）。活性筛选结果表明，化合物9、11～14、17、20、23～25及28对LX-2细胞具有显著的抑制作用。其中化合物14活性最为显著，其在基因水平和蛋白水平均显著抑制肝纤维化标志物α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、纤连蛋白（fibronectin，FN）和I型胶原蛋白（Collagen I）的表达，同时促进细胞凋亡，并能抑制Toll样受体4（Toll-like receptors 4，TLR4）/核因子-κB（nuclear factor kappa-B，NF-κB）信号通路中TLR4、髓样分化蛋白（myeloid differentiation primary response 88，MyD88）、TGF-β活化激酶1（TGF-β-activated kinase 1，TAK1）、肿瘤坏死因子-α（tumor necrosis factor- α，TNF-α）和白细胞介素-1β（interleukin-1β，IL-1β）的蛋白表达水平。结论 化合物18、25、28为首次从苦参中分离得到。化合物9、11～14、17、20、23～25及28能显著抑制LX-2细胞增殖，其中，化合物14活性最为显著，并通过TLR4/NF-κB信号通路发挥抗肝纤维化作用。

Objective To investigate the chemical compositions of the ethyl acetate (EtOAc) fraction of Sophora flavescens and evaluate their anti-liver fibrosis activity. Methods The EtOAc extraction of S. flavescens was separated and purified by various column chromatography methods, including silica gel, polyamide, Sephadex LH-20 and C₁₈reversed-phase column chromatography. The structures of the isolated compounds were elucidated based on modern spectroscopic techniques and physical and chemical identification method. Transforming growth factor beta-1 (TGF-β1)-induced LX-2 cells were used as an in vitro model of hepatic fibrosis, and the anti-fibrotic activities of the compounds were evaluated using the CCK-8 assay. Additionally, cell wound scratch assay, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and flow cytometry assays were employed to investigate the anti-liver fibrosis activity and mechanism of compound 14 (kushenol T). Results A total of 28 compounds were isolated from the EtOAc of S. flavescens, and they were identified as follows: ethyl 2,4-dihydroxybenzoate (1), trifohrhizin (2), 2,4- dihydroxybenzoic acid (3), kuraridin (4), kushenol N (5), kurarinol A (6), kushenol C (7), 8-isopentenyl-5-methoxy-7,2′,4′-trihydroxy flavanonol (8), 2′-hydroxy-isoxanthohumol (9), kurarinone (10), methyl 2,4-dihydroxybenzoate (11), sophoflavescenol (12), umbeliferone (13), kushenol T (14), sophophenoside B (15), 6′′-β-D-xylosegenistin (16), 3′,4′-methylenedioxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)-β-D-glucopyranoside (17), ambocin (18), kushenol O (19), 4′-hydroxy-3′-methoxyisoflavone-7-O-β-D-xylopyranosyl-(l→6)-β-D-glucopyranoside (20), 3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (21), genistein (22), isoxanthohumol (23), 8-isopentenyl-5-methoxy-7,4′-dihydroxyflavanonol (24), 2-(2¢,4¢-dihydroxyphenyl)-5,6-methylenedioxy- benzofuran (25), calycosin (26), daidzein (27), narirutin (28). Bioactivity screening revealed that compounds 9, 11—14, 17, 20, 23—25 and 28 exhibited significant inhibitory effects on LX-2 cells. Among these, 14 demonstrated the most potent activity, markedly downregulating the expression of hepatic fibrosis markers, such as α-smooth muscle actin (α-SMA), fibronectin (FN) and Collagen I, at both the gene and protein levels. Furthermore, 14 promoted cell apoptosis and suppressed TLR4 in the Toll-like receptors 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway, myeloid differentiation primary response 88 (MyD88), and TGF-β-activated kinase 1 (TGF-β-activated kinase 1), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β). Conclusion Compounds 18, 25 and 28 were isolated from this species for the first time. Compounds 9, 11—14, 17, 20, 23—25 and 28 significantly inhibited LX-2 cell proliferation. Among them, compound 14 had the most significant activity and exerts an anti-liver fibrosis effect through the TLR4/NF-κB signaling pathway.

R284.1

贵州省科技厅科技计划项目（黔科合基础-ZK[2024]重点047）；贵州道地药材慢性病防治工程研究中心（2023-035）；中国大学生创新创业训练计划（S2024106601432X）；贵州省卫生健康委员会科技计划项目（gzwkj-2025-538）；中药功效成分发掘与利用全国重点实验室自主研发项目（GMUSKL-202537）