半夏及掌叶半夏毒针晶中共性毒蛋白的研究

目的:寻找天南星科半夏属有毒中药半夏及掌叶半夏毒性物质基础毒针晶中的共有蛋白,研究刺激性相关蛋白的致炎效应。方法:采用溶液内双酶解方法将半夏与掌叶半夏毒针晶中的蛋白降解为混合肽段,采用液相色谱与串联质谱偶联分析降解后的肽段,将质谱分析数据与BIOWORKS软件搜索的NCBI绿色植物Viridiplantae蛋白库比对进行蛋白质检索分析。采用聚丙烯酰胺凝胶电泳对半夏、掌叶半夏毒针晶蛋白进行分析,同时采用胶内酶解后液相色谱与串联质谱偶联的方法,分析并鉴定约13kDa蛋白。提取分离半夏中的凝集素蛋白,并采用中性粒细胞迁移试验和测定腹腔渗出液中PGE2的含量,考察毒针晶及凝集素的炎症刺激性效应。结果:半夏及掌叶半夏毒针晶中均含约13kDa的蛋白条带,此条带经鉴定为凝集素类蛋白,且此13kDa蛋白条带为毒针晶中的主要蛋白之一,凝集素类蛋白可诱导中性粒细胞向大鼠腹腔迁移,可显著增加小鼠腹腔渗出液中PGE2的含量。结论:天南星科有毒中药半夏及掌叶半夏毒针晶蛋白中均含有凝集素类蛋白,且此类凝集素蛋白具有显著的致炎作用,是半夏与掌叶半夏毒针晶中的共性毒蛋白。

Study of toxic proteins on raphides from Pinellia ternata and Pinellia pedatisecta Schott

Objective:To investigate the irritation of the proteins on the raphides from Pinellia ternata(Thunb.) Breit.and Pinellia pedatisecta Schott.Methods:The proteomic approach was used to analyze the proteins on the surface of the raphides and the data was against the Viridiplantae database.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) was employed to analyze the raphides’ 13kDa protein bands.Pinellia ternata agglutinin(PTA) was purified,and the model of neutrophil migration into rat peritoneal cavities and the determination of the content of PGE2 in mice peritoneal cavities was used to study the inflammatory activity of agglutinin.Results:Database searching results showed that the agglutinin presented on both raphideds from P.ternata and P.pedatisect,and 13kDa protein bands was the agglutinin protein.PTA induced significant neutrophil migration into rat peritoneal cavities and enhanced the content of PGE2 in mice peritoneal cavities.Conclusion:The agglutinin,which had obvious effect of pro-inflammation,was the common toxic protein on the surface of the raphides from P.ternata and P.pedatisect.