反相高效液相色谱法制备HMC毒素纯品

PREPARATIVE SEPARATION OF HMC-TOXIN FROM HELMINTHOSPRIUM MAYDISRACE C BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMOTOGRAPHY

本文采用反相高效液相色谱纯化制备了从玉米小斑病病菌Ｃ小种（ＨｅｌｍｉｎｔｈｏｓｐｒｉｕｍｍａｙｄｉｓＲａｃｅＣ）分离出来的毒素ＴｏｘｉｎＩ，经ＮＭＲ的结构分析证实此制备物纯度较高，达到结构分析要求。用氯仿提取含有ＨＭＣ的毒素培养滤液，经过多次ＴＬＣ展层层析（ＣＨＣｌ_３：ＭｅＯＨ＝９：１），刮取具有侵染活性的部分（Ｒ_ｆ＝０．４）溶解在小体积甲醇中备用。色谱柱为Ｗａｔｅｒｓ（制备型２５０ｍｍ×１０ｍｍ）填料为ＹＷＧ－Ｃ１８反相柱，粒度１０ｕｍ，流动相选用甲醇：水５５：４５，流速为０．８ｍｌ／ｍｉｎ，检测波长２７２ｎｍ，进样量每次１００ｕｌ，当主要峰出现时，收集合并冻干保存（保留时间９.５ｍｉｎ）。

A reversed-phase HPLC method has been successfully used to separate and prepare the HMC-Toxin fromHelminthosprium maydis Race C It is evidenced that the separated sample by HYLC is highly purified and suitable for theNMR structural analysis. The filtered toxin culture solution containing HMC-toxin was extracted by chloroform andmult-separated through TLC by using the development reagent of CHCL3:MeOH(9:1). Then bioactive fraction of toxin toC-cytoplasm corn was collected and dissolved in 500ul methanol for HPLC analysis and preparation. The optimum conditions were observed by using a 10um YMG-C18, analytical column (250mm×4.6mm) and methanol;water eluent in PrimaryexperimentS. A preparative columns 250nun× 10mm, with the same pekings and methanol;water(55:45) as mobile phase at aflow-rate of 0.8ml / min and UV detector at 272nm were used for preparation. 100ul of sample were injected into HPLCsystem with 100ul loop of Aheodyne injection valve every time, The eluate containing purified sample was collected whenthe main peak is appeared. The retention time of main componemt (Toxin I). from HMC-toxin is 9.5min.This method couldbe used for preparation for structure analysis of natural products.