球等鞭金藻生长抑制物的高效液相色谱分析

Analysis of Growth-inhibitor in Isochrysis Galbana by High Performance Liquid Chromatography

采用硅烷化键合相C18反相色谱柱，以V（乙腈）：V（水）=63：37为流动相，0．3mL／min恒流洗脱，检测波长UV-235nm等条件，建立了一种利用反相高效液相色谱分析球等鞭金藻生长抑制物的粗提物、SephadexG-15凝胶柱层析分离获得的3个组分、硅胶G薄层层析分离获得的4个组分等8个分离纯化产物中具有抑制活性组分的方法，并确定了具有抑制活性组分的保留时间。通过乙酸乙酯萃取球等鞭金藻老化培养液，得到了具有抑制活性的粗提物；粗提物经SephadexG-15凝胶柱层析分离后，获得3个具有抑制活性的组分；通过硅胶G薄层层析对此3个组分进行分离，在获得的4个组分中，仅有一个组分具有抑制活性。将粗提物与SephadexG-15凝胶柱层析分离获得组分的高效液相色谱进行对比，确定具有抑制活性组分的保留时间在3．827～6．002min范围内；通过与硅胶G薄层层析分离获得组分的高效液相色谱的再次对比，发现仅具有抑制活性的组分Ⅱ（Rt为0．637）中出现保留时间为4．875min的峰，并且相同保留时间的峰同样出现在具有抑制活性的粗提物以及SepbadexG-15凝胶柱层析分离获得的3个组分的高效液相色谱中。结果表明：在优化的实验条件下，高效液相色谱中保留时间为（4．872±0．005）min的峰对应的组分正是具有抑制活性的组分。

A method for the determination of acetate ethyl extract of growth-inhibitor, three fractions by gelfiltered on Sephadex G-15 column and four bands by preparative thin-layer chromatography on silica gel, in lsochrysis galbana by reversed phase high performance liquid chromatography with ZORBAX Eclipse XDB C18 column and UV detector was developed. The selected chromatographic conditions were ZORBAX Eclipse XDB C18 with aqueous acetonitrile at a flow rate of 0.3 mL/min, UV detection at 235 nm. The growth-inhibitor, a kind of ethyl acetate extract, was isolated from the old culture liquid of lsochrysis galbana. The extract dissolved in diluted aqueous NaOH was gel-filtered on Sephadex G-15 column with water, and three fractions that inhibited the growth of lsochrysis galbana were obtained. Those fractions were extracted with ethyl acetate, and then the extracts were separated by preparative thin layer chromatography on silica gel with chloroform. The extracts were separated into four bands, but only the band at Rf 0.63 was active. And then all fractions were finally analyzed by high performance liquid chromatography on ODS with aqueous acetonitrile. The retention times of inhibitory activity fraction in acetate ethyl extract, three fractions by gel-filtered on Sephadex G-15 column and the band at Rf 0. 637 by preparative thin layer chromatography on silica gel were 4.867, 4. 866, 4. 878, 4. 874 and 4. 875 min, respectively. Therefore, the compounds whose retention time ranged among 4. 872 ± 0.05 min were the activity-inhibitor under the chromatographic conditions.