石灰水炮制半夏对毒性成分凝集素蛋白的影响研究  

Effect of lime water processing of Pinelliae Rhizoma Praeparatum on toxic component lectin protein

作　　者：陶兴宝 吴皓[1,2,3] TAO Xing-bao;WU Hao(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Key Laboratory of Traditional Chinese Medicine Processing in Jiangsu Province,Nanjing 210023,China;Engineering Research Center for Standardization of Traditional Chinese Medicine Processing,Ministry of Education,Nanjing 210023,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]江苏省中药炮制重点实验室,江苏南京210023 [3]国家教育部中药炮制规范化及标准化工程研究中心,江苏南京210023

出　　处：《中国中药杂志》2023年第4期951-957,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81173549,81573605);2021年江苏省研究生科研创新计划面上项目(KYCX21_1756)。

摘　　要：为探究法半夏炮制过程中炮制辅料石灰水浸泡对其毒性成分凝集素蛋白的影响,阐释法半夏炮制过程中石灰水解毒的科学内涵。以Western blot法考察不同pH石灰水(pH 10、11、12.4)、饱和氢氧化钠及碳酸氢钠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不同pH石灰水浸泡凝集素蛋白后上清与沉淀中蛋白组成;以MALDI-TOF-MS/MS技术检测不同pH石灰水浸泡凝集素蛋白后上清及沉淀中肽段相对分子质量分布,圆二色谱检测浸泡过程中凝集素蛋白二级结构比例变化。结果显示,pH>12的石灰水及饱和氢氧化钠溶液浸泡均可使凝集素蛋白含量显著下降,而pH<12的石灰水及饱和碳酸氢钠溶液浸泡对凝集素蛋白含量无显著影响。pH>12的石灰水浸泡凝集素蛋白后其上清和沉淀均在12 kDa位置未检测到对应的凝集素蛋白条带和分子离子峰。这是由于pH>12的石灰水浸泡可使凝集素蛋白的二级结构比例发生明显改变,出现了不可逆变性过程,而pH<12的石灰水浸泡未使凝集素蛋白的二级结构比例发生明显改变,未出现变性过程。pH>12是法半夏炮制过程中石灰水解毒的关键条件。石灰水(pH>12)浸泡可使凝集素蛋白发生不可逆变性,致使半夏致炎毒性显著下降,起到炮制解毒的关键作用。The present study investigated the effect of immersion in the excipient lime water on the toxic component lectin protein and explained the scientific connotation of lime water detoxication during the processing of Pinelliae Rhizoma Praeparatum. Western blot was used to investigate the effects of immersion in lime water with different pH(pH 10, 11, and 12.4), saturated sodium hydroxide, and sodium bicarbonate solution on the content of lectin protein. The protein compositions of the supernatant and the precipitate after immersing lectin protein in lime water of different pH were determined by the SDS-PAGE method combined with the silver staining technique. The MALDI-TOF-MS/MS technique was used to detect the molecular weight distribution of peptide fragments in the supernatant and precipitate after immersing lectin protein in lime water of different pH, and circular dichroism spectroscopy was used to detect the ratio changes in the secondary structure of lectin protein during the immersion. The results showed that immersion in lime water at pH>12 and saturated sodium hydroxide solution could significantly reduce the content of lectin protein, while immersion in lime water at pH<12 and sodium bicarbonate solution had no significant effect on lectin protein content. The corresponding lectin protein bands and molecular ion peaks were not detected at the 12 kDa position in the supernatant and precipitate after immersing the lectin protein in lime water at pH>12, which was attributed to the fact that lime water immersion at pH>12 could significantly change the ratio of the secondary structure of lectin protein, resulting in irreversible denaturation, while lime water immersion at pH<12 did not change the ratio of the secondary structure of lectin protein. Therefore, pH>12 was the key condition for the detoxication of lime water during the processing of Pinelliae Rhizoma Praeparatum. Lime water immersion at pH>12 could cause irreversible denaturation of lectin protein, resulting in a significant decrease in the inflamma

关 键 词：半夏 石灰水 凝集素蛋白 炮制 解毒机制 

分 类 号：R283.1[医药卫生—中药学]