Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China.
J Ethnopharmacol. 2015 Jul 1;169:401-6. doi: 10.1016/j.jep.2015.04.033. Epub 2015 Apr 25.
Radix Polygala has a long history of use as a sedative in traditional Chinese medicine and its major ingredients are saponins, which are recognized effective in memory improvement but highly toxic to gastricintestinal mucosa. Polygala saponin hydrolysates (PSH), an alkaline hydrolysis product and also the intestinal metabolites of the saponins, exhibited stronger effects in improving memory of mice and had less toxicity than its original saponins. The present study aims to develop a sensitive LC-MS/MS method for simultaneously determining PSH three major active components, 3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), in rat plasma and apply the method to a pharmacokinetic study.
The acidic plasma (100μl) was treated by liquid-liquid extraction with ethyl acetate and reconstituted sample was analyzed on a C18 column eluted with acetonitrile-water (50:50) containing 0.2% formic acid at 0.4ml/min. The mass detection in negative electrospray ionization was used. The ion pairs for multiple reaction monitoring were set at m/z 237.0/103.0, 177.0/116.6 and 679.5/425.3 for TMCA, PMCA and TF, respectively. Their pharmacokinetic profiles were studied in rats after intravenous and oral dose of PSH at 20 and 100mg/kg, respectively.
The calibration curves had good linearity (r(2)>0.99) for TMCA, PMCA and TF within the tested concentration ranges. The limits of detection and quantification were 1, 10, 0.5ng/ml and 10.0, 20.0, 1.0ng/ml, respectively. The intra-day and inter-day precisions were less than 18.9% and accuracies between 93.2% and 113.3%, and the extraction recovery ranged from 91.2% to 112.1% for all analytes. The pharmacokinetic study showed that TMCA, PMCA and TF could be rapidly absorbed into the circulation and reached their peak concentrations at about 9.1, 9.0 and 24.0min, respectively. TF had a lower oral bioavailability (2.0%) than TMCA (90.1%) and PMCA (96.5%), but it remained in the body much longer (t1/2, λz, 4.8h, oral dose) than TMCA (0.6h) and PMCA (0.9h).
A sensitive LC-MS/MS method was developed and applied to a pharmacokinetic study of TMCA, PMCA and TF of PSH in rats. The three components are proved to be bio-available active components of PSH and might display their in vivo pharmacological activities at different levels and different time periods after oral administration.
远志作为传统中药的镇静剂已有悠久的历史，其主要成分是皂甙，已被证实对改善记忆力有效，但对胃-肠黏膜有高度毒性。远志皂甙水解产物（PSH）是一种碱性水解产物，也是皂甙的肠道代谢物，在改善小鼠记忆力方面效果更强，毒性比其原皂甙低。本研究旨在开发一种灵敏的 LC-MS/MS 方法，同时测定大鼠血浆中 PSH 的三种主要活性成分 3,4,5-三甲氧基肉桂酸（TMCA）、对甲氧基肉桂酸（PMCA）和远志皂苷元（TF），并将该方法应用于药代动力学研究。
用乙酸乙酯进行液-液萃取处理酸性血浆（100μl），用含 0.2%甲酸的乙腈-水（50:50）在 0.4ml/min 的流速下洗脱，在 C18 柱上进行分析。采用负电喷雾电离进行质量检测。用于多重反应监测的离子对分别为 TMCA、PMCA 和 TF 的 m/z 237.0/103.0、177.0/116.6 和 679.5/425.3。分别于 20mg/kg 和 100mg/kg 静脉和口服给予 PSH 后，研究其在大鼠体内的药代动力学特征。
TMCA、PMCA 和 TF 的校准曲线在测试浓度范围内均具有良好的线性（r²>0.99）。检测限和定量限分别为 1、10、0.5ng/ml 和 10.0、20.0、1.0ng/ml。日内和日间精密度均小于 18.9%，准确度在 93.2%至 113.3%之间，所有分析物的提取回收率在 91.2%至 112.1%之间。药代动力学研究表明，TMCA、PMCA 和 TF 可迅速被吸收到循环系统中，分别在约 9.1、9.0 和 24.0min 时达到峰值浓度。TF 的口服生物利用度（2.0%）低于 TMCA（90.1%）和 PMCA（96.5%），但它在体内的半衰期（t1/2，λz）较长（口服剂量为 4.8h），而 TMCA（0.6h）和 PMCA（0.9h）较短。
本研究建立了一种灵敏的 LC-MS/MS 方法，用于测定大鼠远志皂甙水解产物中 TMCA、PMCA 和 TF 的药代动力学。这三种成分被证实是远志皂甙水解产物的生物有效活性成分，在口服给药后可能在不同水平和不同时间发挥其体内药理活性。
