参芪抗炎方中调控TNF-α表达治疗类风湿关节炎成分的筛选和HPLC-ELSD同时测定

Selection of Active Components Treating Rheumatoid Arthritis via Regulating TNF-αExpression in Shenqi Kangyan Formula and their HPLC-ELSD Simultaneous Determination

目的找到参芪抗炎方中调控TNF-α表达治疗类风湿关节炎的成分并建立基于含量同时测定的质量评价方法。方法活性筛选使用ELISA法,根据文献报道,对方中7个成分进行测定,根据结果,选择TNF-α表达调控活性最为显著的成分,作为评价参芪抗炎方活性的主要指标;以党参炔苷和黄芪甲苷为指标,使用HPLC-ELSD法进行含量同时测定研究,使用Agilent Eclipse C18色谱柱(250mm×4.6mm,5μm),流动相为甲醇-水,梯度洗脱,流速1.0mL·min-1,柱温30℃,漂移管温度40℃。结果 7个候选成分中,党参炔苷和黄芪甲苷有明显的TNF-α表达调控活性,其余成分相较模型组无明显区别;对参芪抗炎方(汤剂)的含量测定结果显示,两个指标性成分党参炔苷和黄芪甲苷的含量分别为0.0283-0.0295mg·mL-1和1.045-1.181mg·mL-1。结论参芪抗炎方可能通过调控TNF-α表达治疗类风湿关节炎,党参炔苷和黄芪甲苷可作为评价参芪抗炎方疗类风湿关节炎的功效指标。

OBJECTIVE To selection active components treating rheumatoid arthritis via regulating TNF-αexpression in Shenqi Kangyan Formula,and establish a simultaneous determination method using HPLC-ELSD.METHODS ELISA was used to evaluate regulatory activities for each component candidates reported by literature in the formula,compounds with obvious effect were chosen among 7 candidates;as a result,lobetyolin and astraloside were set as index for HPLC-ELSD based simultaneous determination,the column used was Agilent Eclipse C 18(250mm×4.6mm,5μm),mobile phase was methanol-water in gradient elution,the flow rate was 1.0mL·min-1,column temperature was 30℃,drifting tube temperature was 40℃.RESULTS Among 7 candidates,lobetyolin and astraloside had obvious TNF-αregulation activities,while the others were not significant;the result of Shenqi Kangyan Formula(in form of decoction)showed that the two index compounds namely lobetyolin and astraloside had concentrations of 0.0283-0.0295mg·mL-1 and 1.045-1.181mg·mL-1.CONCLUSION Shenqi Kangyan Formula probably presents therapeutic effect for rheumatoid arthritis via regulating TNF-αexpression,and lobetyolin and astraloside can be used as indexes to evaluate such effect.