快速分离与鉴定天山堇菜中的天然环肽.doc

快速分离与鉴定天山堇菜中的天然环肽 项 斌1, 2, 杜国华1, 王煦辰1, 张书香1, 秦咸蕴1, 孔建强1, 程克棣1, 李永吉2, 王 伟1* (1. 中国医学科学院北京协和医学院 (卫生部天然药物生物合成重点实验室、中草药物质基础与资源利用教育部重点实验室), 北京 100050) 摘要: 为快速准确地分离鉴定天山堇菜的天然环肽分子, 采用50% 乙醇浸提、不同极性溶剂萃取, 利用Sephadex LH-20对含有环肽的正丁醇部分进行色谱分离, 然后直接利用DTT还原分离的组分, 结合反相高效液相色谱 (RP-HPLC) 和质谱跟踪确定含有环肽的组分差异洗脱峰, 进而分离纯化获得单体分子; 利用DTT还原二硫键碘乙酰胺烷化保护被还原产生的巯基蛋白酶Endo-Glu-C、Endo-Lys-C和胰蛋白酶酶解结合RP-HPLC分离获得肽段; 用基质辅助激光解析电离飞行时间质谱 (MALDI TOF/TOF MS) 进行质谱分析, 鉴定了2个环肽分子, 其中一个新的环肽分子命名为cycloviolacin T1, 另一个为已知结构的varv E。通过此研究建立了一套直接利用DTT还原色谱分离组分、结合MALDI TOF/TOF MS检测进而鉴定天然环肽分子的解析方法。 关键词: 天山堇菜; 植物环肽; MALDI TOF/TOF质谱鉴定 中图分类号: R284.1 文献标识码: A 文章编号: 0513-4870 (2010) 11--08 Elucidating the structure of two cyclotides of Viola tianshanica Maxim by MALDI TOF/TOF MS analysis XIANG Bin1, 2, DU Guo-hua1, WANG Xu-chen1, ZHANG Shu-xiang1, QIN Xian-yun1, KONG Jian-qiang1, CHENG Ke-di1, LI Yong-ji2, WANG Wei1* (1. Institute of Materia Medica, Chinese Academy of Medical Sciences Peking Union Medical College, Key Laboratory ofBiosynthesis of Natural Products, Ministry of Health of PRC Key Laboratory of Bioactive Substances andResources Utilization of Chinese Herbal Medicine, Ministry of Education of PRC, Beijing 100050, China; 2. Institute of Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, China) ( Abstract: The cyclotides are a family of cyclic “mini” proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V. tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase H