Miao Qing, Wang Zhiyong, Zhang Yuanyuan, Miao Peipei, Zhao Yuanyuan, Zhang Yujie, Ma Shuangcheng
a School of Chinese Materia Medica , Beijing University of Chinese Medicine , Beijing , P.R. China ;
b National Institutes for Food and Drug Control , Beijing , P.R. China.
Pharm Biol. 2016 Sep;54(9):1548-56. doi: 10.3109/13880209.2015.1107744. Epub 2016 Jan 25.
Context Previous studies have shown that Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi (Labiatae), has a certain inhibitory effect on P-glycoprotein (P-gp), but the effects of its main active constituents on P-gp are still ambiguous. Objectives In vitro studies were performed to investigate the effects of its main active constituents (baicalin and its aglycone, baicalein) on the activity and expression of P-gp in intestine using Caco-2 cells and rat gut sacs. Materials and methods In Caco-2 cell experiments, the effects of baicalin and baicalein on P-gp activity were investigated using a P-gp substrate, rhodamine 123 and non-substrate fluorescein Na, by determining their intracellular fluorescence accumulation, and their effects on P-gp expression were determined using flow cytometry. In addition, rat gut sac model was selected to investigate the effects of baicalin and baicalein on the transport of verapamil, a classical P-gp substrate. The gut sacs of male Sprague-Dawley rats were filled with 0.4 mL the test solution contained verapamil (0.2575 mg/mL) and the drugs [baicalin and baicalein, at concentrations of 1/8 IC50 (59.875, 41.5 μg/mL), 1/4 IC50 (119.75, 83 μg/mL) and 1/2 IC50 (239.5, 166 μg/mL)], and then incubated in Tyrode's solution for a period of time. After termination of the incubation, the incubated solution was processed for the subsequent detection. Results According to the results of MTT assay, the IC50 values of verapamil, baicalin and baicalein were 104, 479, 332 μg/mL, respectively. The obtained results from the two models were confirmed mutually. As a result, baicalin exhibited no obvious effect on intracellular accumulation of Rh-123, and almost had no effect on P-gp expression and verapamil transportation, while baicalein significantly increased intracellular accumulation of Rh-123 (p < 0.01), down-regulated P-gp expression (p < 0.01) and increased the transport of verapamil (p < 0.05). Discussion and conclusion The results indicated that baicalein may be a P-gp inhibitor, which presented obvious inhibitory effects on P-gp activity and expression level. A comparison of the structures of baicalin and baicalein indicates that the existence of glucosyl plays a decisive role in influencing the activity and expression of P-gp.
背景 以往研究表明，黄芩（唇形科植物黄芩的干燥根）对P-糖蛋白（P-gp）有一定抑制作用，但其主要活性成分对P-gp的影响仍不明确。目的 进行体外研究，以考察其主要活性成分（黄芩苷及其苷元黄芩素）对肠中P-gp活性及表达的影响，采用Caco-2细胞和大鼠肠囊模型。材料与方法 在Caco-2细胞实验中，通过测定罗丹明123和非底物荧光素钠在细胞内的荧光积累，考察黄芩苷和黄芩素对P-gp活性的影响，采用P-gp底物罗丹明123和非底物荧光素钠；采用流式细胞术测定它们对P-gp表达的影响。此外，选用大鼠肠囊模型考察黄芩苷和黄芩素对经典P-gp底物维拉帕米转运的影响。将雄性Sprague-Dawley大鼠的肠囊填充0.4 mL含维拉帕米（0.2575 mg/mL）和药物[黄芩苷和黄芩素，浓度分别为1/8 IC50（59.875、41.5 μg/mL）、1/4 IC50（119.75、83 μg/mL）和1/2 IC50（239.5、166 μg/mL）]的测试溶液，然后在台氏液中孵育一段时间。孵育结束后，对孵育液进行后续检测。结果 根据MTT法结果，维拉帕米、黄芩苷和黄芩素的IC50值分别为104、479、332 μg/mL。两个模型得到的结果相互印证。结果显示，黄芩苷对Rh-123的细胞内积累无明显影响，对P-gp表达和维拉帕米转运几乎无影响，而黄芩素显著增加Rh-123的细胞内积累（p < 0.01），下调P-gp表达（p < 0.01）并增加维拉帕米的转运（p < 0.05）。讨论与结论 结果表明黄芩素可能是一种P-gp抑制剂，对P-gp活性和表达水平呈现明显抑制作用。黄芩苷和黄芩素结构比较表明，葡萄糖基的存在对影响P-gp的活性和表达起决定性作用。
