HPLC考察白头翁汤4种标志成分在Caco-2细胞模型的转运特征

Study on Transport Characteristics of Four Marker Constituents of Baitouweng Decoction in Caco-2 Cell Monolayer Model by HPLC

目的研究白头翁汤中标志成分秦皮甲素、秦皮乙素、小檗碱、白头翁皂苷B4在Caco-2细胞模型的转运特征及其机制。方法通过HPLC建立白头翁汤指纹图谱以及4种标志成分的同时定量分析方法,随后以Caco-2细胞模型和P-gp抑制剂维拉帕米考察复方标志成分的双向转运机制,通过HPLC检测药物浓度计算其表观渗透系数(Papp)。结果①秦皮甲素在顶端(AP)和底端(BL)双向转运大致相同,为被动转运机制;②秦皮乙素在AP侧转运大于BL侧,维拉帕米可抑制AP侧向BL侧转运,为主动转运机制;③小檗碱在AP侧和BL侧转运大致相同,对于BL→AP侧外排Papp值显著低于单体的文献报道值,在复方中可能存在化学成分抑制小檗碱BL侧向AP侧外流载体,促使外流减少,增加小檗碱的摄取;④白头翁皂苷B4紫外响应弱,未能检测出。结论白头翁汤中秦皮甲素、秦皮乙素、小檗碱可通过HPLC考察其在Caco-2细胞模型的转运特征。

OBJECTIVE To study on transport characteristics of four marker constituents of Baitouweng decotion, namely aesculin, esculetin, anemoside B4 and berberine in Caco-2 cells by HPLC. METHODS Primarily, fingerprint and quantitative determination of four compounds was established. Subsequently, the double direction transport characteristics for those compounds were researched by Caco-2 cell monolayer model and verapamil, a selective P-gp inhibitor. Drug concentration determined through HPLC was applied to calculate the apparent partition coefficient （Papp）. RESULTS The testing results for four compounds were shown below： ①The transport of aesulin from AP to BL was similar to BL to AP, which indicated its main mechanism in Caco-2 cells was passive transportation; ②The transport contents of esculetin from AP to BL was much more than that of BL to AP, and further experiments showed that the contents from AP to BL could be reduced by P-gp inhibitor verapamil. So, for those evidences, the mechanism of esculetin was active transference involved with P-gp; ③The transport of berberine from AP to BL was similar to BL to AP. However, Papp（BL→AP） was significantly lower than that of reference reported. One reason may be that the interaction between compounds or some one could restrained P-gp（BL→AP） activity to augment the absorption. ④It was a pity that anemoside B4 was unable to be detected due to its poor response to UV. CONCLUSION The transport characteristics of acsulin, esculetin and berberine can be detected by HPLC in Caco-2 cell model.