UHPLC-MS/MS联用微透析法研究牛膝引栀子苷关节靶向在佐剂性关节炎大鼠体内药动学的影响

Pharmacokinetics of Achyranthes bidentata on adjuvant arthritis rats by microdialysis and UHPLC-MS/MS

建立了UHPLC-MS/MS测定栀子苷(GE)和3种牛膝皂苷成分(姜状三七皂苷R1、人参皂苷Ro、竹节参皂苷Ⅳa)在大鼠关节、血液透析液中药物浓度的分析方法,并应用该法研究牛膝总皂苷-栀子苷(ABS-GE)配伍前后在佐剂性关节炎(AA)大鼠体内的药动学过程,以探讨ABS"引药下行"作用的活性部位。AA大鼠模型单用或联用ABS,GE后,微透析探针植入大鼠关节腔及颈静脉血管中收集样品,采用Waters Acquity HSS C_(18)色谱柱分离,以乙腈-0. 1%甲酸水为流动相梯度洗脱,采用电喷雾离子源(ESI),负离子模式扫描,多反应监测模式(MRM)检测上述4种成分。结果显示,大鼠关节及血液透析液中GE、姜状三七皂苷R1、人参皂苷Ro及竹节参皂苷Ⅳa,分别在2～4 000,16～4 096,14～3 584,23～5 888μg·L-1线性良好;上述4种成分精密度、准确度、稳定性及基质效应结果均符合生物样品定量分析要求。ABS与GE联用(60 mg·kg^(-1)+60 mg·kg^(-1))后,使GE的AUCjoint/AUCplasm达到GE单用(60 mg·kg^(-1))的2倍,表明ABS可以提高GE向关节腔靶向分布的程度。同时3种牛膝皂苷成分在关节腔的药动学变化趋势与配伍后GE的变化趋势无显著差别。此外,ABS与GE联用后对AA大鼠足爪肿胀、关节炎指数及关节滑膜病理组织的改善作用均强于GE单用。表明ABS对GE具有靶向引导作用,增加GE在关节腔浓度,并在药效上协同增强。

To investigate the ' drug-guide' effect of Achyranthes bidentata saponins( ABS) and geniposide( GE) in the treatment on adjuvant arthritis( AA) rats. A UHPLC-MS/MS method for the quantitative determination of GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa in rat blood and joint dialysate was established. After single or combined administration with ABS and GE was given to AA rat model,a microdialysis sampling method for rat joint cavity and jugular vein blood vessels was established to collect microdialysis samples. Waters Acquity HSS C18 column was used to separate the above four components,with mobile phase as acetonitrile-0. 1% formic acid water as mobile phase for gradient elution. ESI source was adopted for mass spectra in a negative ion scanning mode. Multiple reaction monitoring( MRM) mode was applied to detect the above four components. The methodological results showed that GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa demonstrated a good linear relationship within the concentration ranges of 2-4 000,16-4 096,14-3 584,23-5 888 μg·L-1 respectively. The precision,accuracy,stability and matrix effect of these four ingredients reached the requirements of quantitative analysis of biological samples. The pharmacokinetic results demonstrated that the combined administration of ABS and GE( 60 mg·kg-1+60 mg·kg-1) can increase the degree of GE in joint cavity distribution,and the AUCjoint/AUCplasmwere twice of that of single administration of GE( 60 mg·kg-1),which indicated that ABS might played a vital role in GE’s distribution to joint cavity. Moreover,there was no significant difference between the distribution trend of total three ABS and GE in rats. The pharmacodynamics results showed that the combined administration of ABS and GE has stronger effects on paw swelling,arthritis index and synovial pathomorphology of AA rats than single administration of GE,which suggested that ABS might improve GE’s anti-inflammatory effect in AA rats. Based on the above results,ABS has a targeting effect in increasing GE’s concentration in joint cavity,with a synergy in efficacy.