利用iTRAQ技术分析续断“发汗”前后三萜皂苷类成分含量变化的机制  

Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis

作　　者：何华 徐娇 周涛 周太敏 郭娟 江维克 肖承鸿 龚安慧 HE Hua;XU Jiao;ZHOU Tao;ZHOU Tai-min;GUO Juan;JIANG Wei-ke;XIAO Cheng-hong;GONG An-hui(Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;National Resource Center for Chinese Materia Medica,Chinese Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]贵州中医药大学,贵州贵阳550025 [2]中国中医科学院中药资源中心,北京100700

出　　处：《中国中药杂志》2021年第18期4730-4735,共6页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81860675);中央本级重大增减支项目(2060302);贵州省科技创新人才团队项目(黔科合平台人才[2019]5611);贵州省高层次创新型人才项目(黔科合平台人才[2018]5638-2);贵州省教育厅创新群体重大研究项目(黔教合KY字[2018]022);贵州省科技计划成果应用及产业化项目(黔科合成果[2021]一般136)。

摘　　要：为了阐释续断"发汗"加工导致三萜皂苷类成分含量变化的机制,该研究对续断"发汗"加工前后总三萜皂苷以及指标性成分川续断皂苷Ⅵ的含量进行了检测,同时运用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)技术结合LC-MS/MS对续断"发汗"加工前后的差异表达蛋白进行了分析。结果显示,"发汗"加工后,续断总三萜皂苷的含量显著下降,川续断皂苷Ⅵ的含量显著升高;经iTRAQ-LC-MS/MS方法分析得到140个显著差异表达蛋白,"发汗"加工后显著上调蛋白为50个,显著下调蛋白为90个。GO分析发现差异蛋白中存在多种水解酶、氧化还原酶及转移酶,进一步检测2类差异表达的氧化还原酶的活性,结果与iTRAQ-LC-MS/MS分析一致。通过分析差异蛋白中与三萜皂苷生物合成途径相关的酶,发现参与三萜皂苷结构修饰的CYP450及UGT家族各有1个成员在"发汗"加工后表达显著下调,表明"发汗"加工可能通过影响续断三萜皂苷生物合成途径下游修饰酶CYP450及UGT的表达进而导致三萜皂苷类化学成分含量的变化。The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D.asper,and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ)combined with LC-MS/MS.After sweating,the content of total triterpene saponins decreased manifestly,while that of asperosaponin Ⅵ increased significantly.As revealed by the iTRAQ-LC-MS/MS analysis,140 proteins with significant differential expression were figured out,with 50 up-regulated and 90 down-regulated.GO analysis indicated a variety of hydrolases,oxido-reductases,and transferases in the differential proteins.The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis.As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway,two enzymes(from CYP450 and UGT families,respectively,which are involved in the structural modification of triterpene saponins)were significantly down-regulated after sweating.The findings suggested that sweating of D.asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450s and UGTs in the biosynthesis pathway of triterpene saponins of D.asper.

关 键 词：续断 “发汗”加工 三萜皂苷类 差异蛋白 同位素标记相对和绝对定量技术(iTRAQ) 

分 类 号：R284.1[医药卫生—中药学]