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目的通过大鼠原代肝细胞模型,明确小檗碱、巴马汀和药根碱的肝细胞摄取特征。方法 Seglen两步法分离大鼠原代肝细胞并悬浮培养,应用已知底物瑞舒伐他汀评价转运体的功能,将药物在4和37℃分别与肝细胞孵育不同时间,UPLC-MS/MS测定肝细胞摄取量,考察温度和时间对药物摄取的影响,并计算转运参数。通过浓度依赖的摄取实验,计算得到K_m、V_(max)和主动摄取率等参数。在肝细胞模型上,将转运体的特异抑制剂与小檗碱、巴马汀和药根碱共孵育,观察阳性抑制剂对受试药物转运的影响。结果小檗碱、巴马汀和药根碱在37℃条件下的摄取量随着底物浓度的增高而增加,4℃条件下摄取量较低且变化很小,3种生物碱进入肝细胞的CL_(active)/CL_(uptake)分别为85.26%、74.90%和57.74%。阳性抑制剂布洛芬、地高辛、甘草酸和盐酸哌唑嗪能显著降低小檗碱和巴马汀的摄取,甘草酸和盐酸哌唑嗪能减少药根碱的摄取。结论小檗碱、巴马汀和药根碱进入肝细胞均以主动转运为主,Oatp1a1、Oatp1a4、Oatp1b2和Oct1可能介导了小檗碱和巴马汀的肝脏摄取,Oatp1b2和Oct1参与了药根碱的主动转运。
Objective To determine the hepatocyte uptake characteristics of berberine, palmatine and jatrorrhizine by rat primary hepatocyte model. Methods Seglen two-step primary hepatocytes were isolated and cultured in vitro. Rosuvastatin, a known substrate, was used to assess the function of the transporter. The hepatocytes were incubated at 4 and 37 ℃ for different times. UPLC-MS / MS determination of liver cell uptake, study temperature and time on the impact of drug intake, and calculate the transport parameters. Through concentration-dependent uptake experiments, parameters such as K_m, Vmax and active uptake rate were calculated. In the hepatocyte model, specific inhibitors of transporters were co-incubated with berberine, palmatine and jatrorrhizine to observe the effect of the positive inhibitor on the transport of the test drug. Results The uptake of berberine, palmatine and jatrorrhizine at 37 ℃ increased with the increase of substrate concentration. The intakes of berberine, palmatine and jatrorrhizine at 37 ℃ were lower and changed little at 4 ℃. The three alkaloids entered into hepatocytes The CL / uptake was 85.26%, 74.90% and 57.74% respectively. Positive inhibitors ibuprofen, digoxin, glycyrrhizin and prazosin hydrochloride can significantly reduce the uptake of berberine and palmatine, glycyrrhizic acid and prazosin hydrochloride can reduce the jatrorrhizine uptake. Conclusions Berberine, palmatine and jatrorrhizine enter the liver cells actively. Oatp1a1, Oatp1a4, Oatp1b2 and Oct1 may mediate hepatic uptake of berberine and palmatine. Oatp1b2 and Oct1 are involved in the drug delivery Caustic soda initiative transport.