College of Pharmacy and International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education, Jinan University, Guangzhou 510632, PR China.
State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), The Hong Kong Polytechnic University Shenzhen Research Institute, Shenzhen 518057, PR China.
J Pharm Biomed Anal. 2020 Jan 5;177:112836. doi: 10.1016/j.jpba.2019.112836. Epub 2019 Aug 26.
Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.
仙灵骨葆胶囊（XLGB）是一种在中国用于预防和治疗骨质疏松症的有效中药方剂（TCMP）。本研究建立了一种基于 UPLC-MS/MS 技术的快速、简单、高效、稳定的方法，用于同时测定大鼠血浆中 XLGB 的多种成分。质谱检测采用电喷雾电离（ESI）的多反应监测（MRM）模式进行。对于 21 种选定的定量原型，所有校准曲线在线性范围内均表现出良好的线性（r>0.9932）。下定量限（LLOQ）分别为：补骨脂素（PL）2ng/mL、虎杖苷 VI（AS）2.5ng/mL、异补骨脂素（IPS）和蛇床子素（SW）1ng/mL、厚朴碱（MA）0.5ng/mL、补骨脂甲素（BVN）、丹参酮 IIA（TA）、知母皂苷 BII（TBII）和淫羊藿苷（ICT）0.1ng/mL、淫羊藿苷 B（EB）和淫羊藿苷 C（EC）0.05ng/mL、淫羊藿苷（IC）0.01ng/mL、异甘草素（IBC）、补骨脂定（PD）、虎杖苷（BV）、异补骨脂定（BC）、淫羊藿苷 A（EA）和异甘草素（IBV）、新异甘草素（NEO）0.02ng/mL、淫羊藿苷 I（ICI）和淫羊藿苷 II（ICII）0.01ng/mL。所有分析物的日内和日间（低、中、高）精密度（相对标准偏差）均小于 8.63%，准确度（相对误差）在-12.45%至 8.91%范围内。分析物和内标物的提取回收率和基质效应可接受。在血浆样品中，所有分析物在检测和储存过程中均稳定。该验证方法成功应用于药效剂量（0.3 和 1g/kg/天）下 21 种原型的药代动力学（PK）研究。此外，还通过其面积/内标面积-时间曲线监测了 28 种代谢物（Ⅱ相共轭物：23 种葡萄糖醛酸轭合物、2 种硫酸轭合物和 3 种葡萄糖醛酸或硫酸轭合物）的动态特征。结果表明，由于其高暴露量和适当的药代动力学特征，补骨脂素、来自补骨脂属的倍半萜类黄酮、来自淫羊藿属的生物碱和倍半萜类黄酮苷以及环烯醚萜苷、三萜皂苷类来自川续断的甾体，被认为是 XLGB 的关键有效物质。这是首次通过反向药效学（PD）-药代动力学（PK）研究揭示药效成分的报告。
