正交试验法优化生脉散中有效成分组方亲和介质的制备工艺

Optimization of Preparation Technology for Affinity Medium with Four Active Ingredients From Shengmai Powder as Ligands by Orthogonal Test

目的:制备以生脉散中有效成分人参皂苷Rg1、人参皂苷Rb1、麦冬皂苷DT-13及五味子醇甲为配基的亲和介质。方法:采用4个有效成分与环氧活化琼脂糖凝胶6B(EAS 6B)偶联,制备亲和介质。建立HPLC-DAD对4个有效成分同时在线分离与检测,以偶联率为指标,通过L9(34)正交试验考察反应温度,反应时间和投料比对偶联反应的影响,优化制备工艺条件。结果:4个成分均可与EAS 6B发生偶联,最佳制备工艺为反应温度25℃,反应时间24 h,添加10倍量生脉散有效成分组方。人参皂苷Rg1、人参皂苷Rb1、麦冬皂苷DT-13及五味子醇甲亲和介质的偶联率分别为27.4%,22.3%,50.0%,24.5%。结论:成功制备了以生脉有效成分组为配基的亲和介质,为进一步识别该方的作用靶点提供技术手段,同时也为制备类似结构中药复方的亲和介质提供实验依据。

Objective： To prepare affinity medium with four effective ingredients of ginsenoside Rg 1,ginsenoside Rb 1,ophiopogonin DT-13 and schisandrin from Shengmai powder as ligands.Method： Affinity medium was prepared by coupling with four effective ingredients and epoxy-activated-sepharose TM 6B（EAS 6B）.A HPLC-DAD method for simultaneous separation and determination of four ingredients was established,with coupling rate as index,effect of feeding rate,reaction time and temperature on coupling reaction was investigated by orthogonal test,then optimized preparation technology.Result： Optimum preparation technology was as following： reaction time 24 h,reaction temperature 25 ℃,added 10 times the amount of active ingredients ligands.Four ingredients could coupling with EAS 6B,coupling rate with four ingredients and affinity medium was ginsenoside Rg 1 of 27.40%,ginsenoside Rb 1 of 22.30%,ophiopogonin DT-13 of 50%,schizandrin of 24.50%.Conclusion： We successfully prepared affinity medium with four effective compounds deprived from Shengmai powder as ligands.It provided technical means to further identify target of this compound and some references for preparing affinity medium of other multiple natural compounds with similar active groups.