甘草汁炮制半夏对毒性成分凝集素蛋白的影响  

Effects of toxic component lectin protein in Pinelliae Rhizoma by processing with licorice juice

作　　者：陶兴宝 曹纬国 吴皓[3,4,5] TAO Xing-bao;CAO Wei-guo;WU Hao(College of Pharmacy,Chongqing College of Traditional Chinese Medicine,Chongqing 402760,China;Post-Doctoral Research Center,Chongqing College of Traditional Chinese Medicine,Chongqing 400011,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Key Laboratory of Chinese Medicine Processing,Nanjing 210023,China;China Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing,Nanjing 210023,China)

机构地区：[1]重庆中医药学院中药系,重庆402760 [2]重庆中医药学院博士后工作站,重庆400011 [3]南京中医药大学药学院,南京210023 [4]江苏省中药炮制重点实验室,南京210023 [5]国家教育部中药炮制规范化及标准化工程研究中心,南京210023

出　　处：《中华中医药杂志》2023年第5期2245-2250,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：重庆市技术创新与应用发展专项(No.cstc2020jscx-dxwtBX0054)。

摘　　要：目的:研究法半夏炮制过程中炮制辅料甘草汁浸泡对其毒性成分凝集素蛋白的影响。方法:以WesternBlot法考察甘草汁(醇沉上清液、醇沉沉淀、甘草酸溶液)浸泡对凝集素蛋白含量的影响。采用SDS-PAGE法结合银染技术测定甘草汁及甘草酸溶液浸泡凝集素蛋白后上清液与沉淀中的蛋白组成;以MALDITOF MS/MS技术检测甘草汁及甘草酸溶液浸泡凝集素蛋白后上清液及沉淀中肽段分子量分布;以等电点沉淀法测定凝集素蛋白等电点,并测定其Zeta电位变化考察甘草汁及甘草酸溶液浸泡凝集素蛋白后对其等电点的影响;同时,采用透射电镜观察甘草汁及甘草酸溶液浸泡对凝集素蛋白聚合作用的影响。结果:甘草汁、甘草汁醇沉上清液及甘草酸溶液浸泡均可使凝集素蛋白含量显著降低(P<0.05)。甘草汁及甘草酸溶液浸泡凝集素蛋白后的上清液和沉淀均在12 kDa位置出现凝集素蛋白对应的条带,且上清液对应的凝集素蛋白条带与沉淀对应的凝集素蛋白条带相比明显变浅。凝集素蛋白的等电点pH约为5~6,甘草汁及甘草酸溶液的pH分别为5.25、5.62。甘草酸溶液浸泡凝集素蛋白后其Zeta电位接近于0,达到等电点而沉淀。甘草汁、甘草酸溶液及甘草汁石灰水共同浸泡凝集素蛋白后出现大量聚集体,形成超分子自组装复合物结构而加速其沉淀。结论:甘草汁在法半夏炮制过程中解毒机制为甘草汁中甘草酸浸泡凝集素蛋白可使其达到等电点,促进其超分子自组装,改变溶解度而沉淀。甘草汁与石灰水在炮制过程中以协同作用形成超分子自组装复合物而解毒。Objective:To investigate the effects of immersion in licorice juice on toxic component lectin protein during the processing of Pinelliae Rhizoma.Methods:Western Blot was used to investigate the effects of immersion in licorice juice(alcohol-precipitated supernatant,alcohol-precipitated precipitation,glycyrrhizic acid solution)on the content of lectin protein.SDS-PAGE combined with silver staining was used to determine the protein composition in the supernatant and precipitation of the lectin protein soaked in licorice juice and glycyrrhizic acid solution.MALDI-TOF MS/MS technique was used to detect the molecular weight distribution of peptide fragments in supernatant and precipitation of lectin protein,which soaked in glycyrrhizin juice and glycyrrhizin solution.The isoelectric point of lectin protein was determined by isoelectric point precipitation method,and then the effects of soaking in licorice juice and glycyrrhizin solution on the isoelectric point of lectin protein was investigated by measuring the change of Zeta potential.Meanwhile,the effects of soaking in licorice juice and glycyrrhizin solution on the polymerization of lectin protein was observed by transmission electron microscopy.Results:The content of lectin protein was significantly decreased by soaking in licorice juice,supernatant of licorice juice and glycyrrhizin.The lectin protein bands in the supernatant and precipitation after soaking the lectin protein in licorice juice and glycyrrhizin solution appeared at the position of 12 kDa,and the lectin protein bands corresponding to the supernatant became significantly lighter than the lectin protein bands corresponding to the precipitation.The isoelectric pH of lectin protein was about 5~6,and the pH of licorice juice and glycyrrhizin solution were 5.25 and 5.62,respectively.The lectin protein was precipitated in glycyrrhizic acid solution due to the fact that the Zeta potential of the protein was close to 0,reaching the isoelectric point.A large number of aggregations of lectin protein were for

关 键 词：半夏 甘草汁 凝集素蛋白 炮制 解毒机理 

分 类 号：R283[医药卫生—中药学]