补阳还五汤黄酮类有效成分在Caco-2细胞单层模型的转运特征及对CYP450酶活性的影响

目的 研究补阳还五汤3个黄酮类有效成分（羟基红花黄色素A、毛蕊异黄酮、芒柄花素）在Caco-2细胞单层模型中的转运特征及其在大鼠肝微粒体中对CYP450酶亚型活性的影响。方法 MTT法研究药物在Caco-2细胞单层模型的安全浓度范围，以Caco-2细胞单层模型研究黄酮类有效成分的双向转运机制，以表观渗透系数（Papp）为检测指标，考察时间、浓度以及P-gp抑制剂维拉帕米对转运的影响，利用Cocktail探针法研究药物对CYP450亚型的体外抑制作用。结果 从顶侧（apical, AP）到底外侧（basolateral, BL）（AP→BL），芒柄花素、毛蕊异黄酮的Papp>10-6 cm/s，表明其吸收性良好，羟基红花黄色素A的Papp为10-6 cm/s，表明其吸收性一般；且三者的转运量随浓度升高和时间延长而显著升高，BL→AP的转运量与AP→BL的转运量的比值小于1.5，其中芒柄花素、毛蕊异黄酮的转运都受到P-gp外排蛋白的外排作用；补阳还五汤黄酮类有效成分明显降低了CYP2E1、CYP1A2酶探针底物的代谢量(P<0.05)。结论 3种黄酮类有效成分在Caco-2细胞模型的转运方式均为被动转运，毛蕊异黄酮与芒柄花素明显受到P-gp的外排作用，补阳还五汤黄酮类有效成分可以增加主要通过CYP2E1、CYP1A2酶代谢的药物浓度。

Transport Characteristics of Flavonoids in Buyang Huanwu Decoction across Caco-2 Cell Monolayer Model and Their Effect on Cytochrome P450 Activity

Objective To investigate the transport characteristics of flavonoids (hydroxysafflor yellow A, formononetin, and calycosin) in Buyang Huanwu Decoction (BYHWD) across Caco-2 cell monolayer model and their effect on cytochrome P450 (CYP450) activity in rat liver microsomes. Methods The safe concentration range of flavonoids in Caco-2 cell monolayer model was determined by MTT assay. The mechanism of bidirectional transport of flavonoids was investigated using the Caco-2 cell monolayer model. The influence of time, concentration, and P-gp inhibitor verapamil on the transport of flavonoids was studied using apparent permeability coefficient (Papp) as the index. The in vitro inhibitory effect of flavonoids on CYP450 activity was studied by the Cocktail method. Results The Papp of formononetin and calycosin in the transport from apical (AP) side to basolateral (BL) side were more than 10-6 cm/s, which showed a good absorption, but the Papp of hydroxysafflor yellow A was 10-6 cm/s, which showed an ordinary absorption. In the Caco-2 cell monolayer model, the transport of the three compounds was positively correlated with time and concentration, and the ratio of the transport from BL side to AP side to that from AP side to BL side was lower than 1.5. The transport of formononetin and calycosin was rejected by P-gp, and the flavonoids in BYHWD significantly reduced the metabolism of CYP1A2 and CYP2E1 probe substrates (P<0.05). Conclusion The transport of the three flavonoids across Caco-2 cell monolayer model is passive, and formononetin and calycosin are rejected by P-gp. The three flavonoids in BYHWD can increase the concentration of the drug metabolized through CYP1A2 and CYP2E1.