Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma
Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma
复制标题
补肾益智方CYP3A4抑制剂增强大鼠血浆蛇床子素生物利用度的筛选与验证
DOI：
10.1016/j.jep.2021.114643
复制
发表时间：
2021-09-17
影响因子：
5.4
通讯作者：
Liang, Yong
中科院分区：
文献类型：
--
作者：
Liu, Jin-man;Chen, Jun-mei;Liang, Yong
民族药理学相关性：中医药具有多成分、多靶点、多功能的特点，长期以来被广泛应用于各种疾病的预防和治疗。在中医药的应用过程中，提高方剂中活性成分生物利用度的研究日益活跃。补肾益智方是一种以蛇床子素为主要活性成分的中药方剂，被广泛用于治疗肾虚、智力低下和阿尔茨海默病。然而，OST的生物学机制和化合物-酶相互作用介导的生物利用度提高仍不清楚。本研究的目的：探讨补肾益肾丸提高骨钙素生物利用度的物质基础和分子机制。通过理论预测筛选潜在的CYP 3A 4抑制剂并进行体外验证，以及筛选出的CYP 3A 4抑制剂与BSYZ联合给药时OST在大鼠血浆中的药代动力学研究也鲜见报道。材料与方法：利用系统药理学数据库中的分子对接方法，从BSYZ中筛选CYP 3A 4抑制剂。通过使用P450-Glo筛选系统验证筛选的化合物。建立了多反应监测（MRM）质谱法定量OST。将雄性Sprague-Dawley大鼠分为4组，每组6只大鼠用于OST的药代动力学研究。给药条件为I组，OST（20 mg/kg）; II组，BSYZ（含OST 1 mg/mL，剂量为BSYZ中OST 20 mg/kg）; III组，酮康唑（Ket，75 mg/kg）和OST（20 mg/kg）联合给药; IV组，CYP 3A 4抑制剂（10 mg/kg）和OST（20 mg/kg）联合给药。通过使用HPLC-MS/MS（MRM）测定它们，并使用学生t检验进行统计分析，p < 0.05作为显著性水平。结果：从BSYZ化合物库中筛选出21个潜在的CYP 3A 4抑制剂。从体外验证的结果中，我们发现了4个化合物具有较好的CYP 3A 4抑制效率，包括油酸，1，2，3，4，6 O-五没食子酰葡萄糖，大黄素，和芍药苷B。经进一步验证，在5 μ M浓度下，芍药苷B对CYP 3A 4的抑制作用最好（IC 50 = 0.339 μ M），甚至优于临床使用的药物（Ket）。在药代动力学研究中，曲线下面积口服BSYZ、Ket和芍药苷B后OST的AUC（ng/L*h）（2196.23 +/- 581.33，462.90 +/- 92.30和1053.03 +/- 263.62，分别为227.89 ± 107.90，p < 0.01。结论：从BSYZ中筛选出一种高效的CYP 3A 4抑制剂--黄芩苷B，通过抑制活性的方式拮抗OST对CYP 3A 4的代谢，从而显著提高OST在大鼠体内的生物利用度。本研究结果将有助于解释中药方剂配伍的合理性，也有助于开发配伍更合理的中药新方剂。
Ethnopharmacological relevance: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. Aim of the study: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. Materials and methods: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. Results: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 mu M), and even better than the clinically used drug (Ket) at the concentration of 5 mu M. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 +/- 581.33, 462.90 +/- 92.30 and 1053.03 +/- 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 +/- 107.90, p < 0.01). Conclusions: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.