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.
Zhongguo Zhong Yao Za Zhi. 2021 Sep;46(18):4730-4735. doi: 10.19540/j.cnki.cjcmm.20210311.303.
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 CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.
本研究旨在通过检测生地黄和发汗地黄中总三萜皂苷及指标成分梓醇皂苷Ⅵ的含量变化，结合等压标签相对和绝对定量技术（iTRAQ）与液相色谱-串联质谱（LC-MS/MS）分析差异表达蛋白，探讨地黄发汗对三萜皂苷含量变化的影响机制。发汗后，总三萜皂苷含量明显降低，而梓醇皂苷Ⅵ含量显著增加。iTRAQ-LC-MS/MS分析结果显示，共鉴定出140个差异表达显著的蛋白，其中上调50个，下调90个。基因本体（GO）分析表明，差异蛋白中存在多种水解酶、氧化还原酶和转移酶。对两个差异表达的氧化还原酶的活性测试结果与iTRAQ-LC-MS/MS分析结果一致。通过对三萜皂苷生物合成途径相关酶的分析表明，发汗后两种酶（分别来自CYP450和UGT家族，参与三萜皂苷的结构修饰）显著下调。研究结果提示，地黄发汗可能通过影响地黄三萜皂苷生物合成途径中下游CYP450和UGT的表达来调控三萜皂苷的含量。
