Herbacetin is a bioactive flavanol compound that has various pharmacological effects. However, the pharmacokinetic characteristics have not been thoroughly investigated. Previously, we screened a natural compound library and identified herbacetin as a potent CYP blocker. Herein, we aimed to mechanistically determine the inhibitory effects of herbacetin on CYP450 and its potential application. A human liver microsome incubation system was developed based on a UPLC-MS/MS method. Moreover, an in silico docking assay and a human CYP recombinase reaction system were developed and used to investigate binding affinity and inhibitory efficacy. Subsequently, the effects of the combination of herbacetin and sorafenib on HepG2 cells were assessed by MTT and immunoblotting assays. The concentration of sorafenib and its main metabolite were measured by UPLC-MS/MS after incubation with or without herbacetin. As a result, we found herbacetin almost completely inhibited the functions of major CYPs at 100 µM. Moreover, through analysis of the structure-activity relationship, we found 4-, 6-, and 8-hydroxyl were essential groups for the inhibitory effects. Herbacetin inhibited CYP3A4, CYP2B6, CYP2C9, and CYP2E1 in a mixed manner, but non-competitively blocked CYP2D6. These results are in good agreement with the recombinase reaction in vitro results, with an IC50 < 10 µM for each tested isoenzyme. Interestingly, the stimulatory effects of sorafenib on HepG2 cell apoptosis were significantly enhanced by combining with herbacetin, which was associated with increased sorafenib exposure. In summary, herbacetin is a potent inhibitor of a wide spectrum of CYP450s, which may enhance the exposure of drugs in vivo.

中文翻译：

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Herbacetin 通过不同的抑制机制广泛阻断 CYP450 的活性

Herbacetin是一种具有多种药理作用的生物活性黄烷醇化合物。然而，尚未彻底研究药代动力学特征。此前，我们筛选了一个天然化合物库，并将草本素鉴定为一种有效的 CYP 阻滞剂。在此，我们旨在从机制上确定草本素对 CYP450 的抑制作用及其潜在应用。基于UPLC-MS/MS方法开发了人肝微粒体培养系统。此外，还开发了计算机对接试验和人 CYP 重组酶反应系统，并用于研究结合亲和力和抑制功效。随后，通过MTT和免疫印迹测定评估了草药和索拉非尼组合对HepG2细胞的影响。在与或不与除草剂一起孵育后，通过 UPLC-MS/MS 测量索拉非尼及其主要代谢物的浓度。结果，我们发现 Herbacetin 在 100 µM 时几乎完全抑制了主要 CYP 的功能。此外，通过分析构效关系，我们发现4-、6-和8-羟基是抑制作用的必要基团。Herbacetin 以混合方式抑制 CYP3A4、CYP2B6、CYP2C9 和 CYP2E1，但非竞争性阻断 CYP2D6。这些结果与体外重组酶反应结果非常吻合，每种测试的同工酶的 IC50 < 10 µM。有趣的是，索拉非尼对 HepG2 细胞凋亡的刺激作用通过与草本素组合显着增强，这与索拉非尼暴露量增加有关。总之，