快速分离与鉴定天山堇菜中的天然环肽  

Elucidating the structure of two cyclotides of Viola tianshanica Maxim by MALDI TOF/TOF MS analysis

作　　者：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] 

机构地区：[1]中国医学科学院北京协和医学院药物研究所(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 [2]黑龙江中医药大学中医药研究院,黑龙江哈尔滨150040

出　　处：《药学学报》2010年第11期1402-1409,共8页Acta Pharmaceutica Sinica

基　　金：国家科技重大专项综合平台子课题"天然产物化学"平台(2009ZX09301-003-4-1);中央级公益性科研院所基本科研业务费专项资助(2007ZD06)

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、碘乙酰胺烷基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。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 HPLC.The peaks with different retention times were shown on the profile of RP-HPLC and collected.The cyclotides were speculated based on masses range from 3 000 to 3 500 Da.The purified cyclotides were reduced with DTT,alkylated with iodoacetamide,and then were cleaved with endoproteinase Glu-C,endoproteinase Lys-C and Trypsin,separately.The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer.A new cyclotide,cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system.So the method for the isolation and characterization of cyclotides was quickly built up in succession.

关 键 词：天山堇菜 植物环肽 MALDITOF/TOF质谱鉴定 

分 类 号：R284.1[医药卫生—中药学]