高速逆流色谱分离小孢拟盘多毛孢PM-1 菌株中的除草活性物质

Preparative separation of the herbicidal substance from Pestalotiopsis microspora PM-1 by high-speed counter-current chromatography

摘要:

【目的】应用高速逆流色谱法(High-speed counter-current chromatography, HSCCC)实现对小孢拟盘多毛孢Pestalotiopsis microspora PM-1菌株代谢产物中除草活性物质的首次分离。【方法】以正己烷:乙酸乙酯:甲醇:水(4:5:4:5, V/V/V/V)为最佳的两相溶剂体系, 上相(水相)为固定相, 下相(有机相)为流动相, 正相洗脱。【结果】在流速2 mL/min、转速900 r/min、检测波长254 nm的条件下进行分离, 得到4个馏分, 其中馏分Ⅱ对马唐的活性较强。馏分Ⅱ经高效液相色谱法(High performance liquid chromatography, HPLC)以乙腈:水=75:25 (V/V)为流动相经过C18柱进一步分离检测, 所得馏分A对马唐种子萌发有较强的抑制作用。其保留时间为7.954 min, 该峰经二极管阵列光谱检测为单一组分。【结论】利用最佳的高速逆流色谱条件和高效液相色谱条件对小孢拟盘多毛孢Pestalotiopsis microspora PM-1菌株代谢产物进行分离, 可获得除草活性化合物——馏分A。

Abstract:

[Objective] The separation of a herbicidal active compound from Pestalotiopsis microspora PM-1 was to be accomplished by high-speed counter-current chromatography (HSCCC) for the first time. [Methods] The compound was separated by HSCCC from the ethyl acetate crude extract of Pestalotiopsis microspora PM-1 with the two-phase solvent system consisting of n-hexane:ethyl acetate:methanol:water:solution (4:5:4:5, V/V/V/V). The upper phase (aqueous phase) was chosen as the stationary phase and the lower phase (organic phase) as the mobile phase. [Results] Under the HSCCC conditions of a flow rate of 2.0 mL/min, the apparatus rotated at 900 r/min and the effluent detected at 254 nm, four fractions were obtained. FactionⅡ had the most intensive herbicidal activity on Digitaria sanguinalis, which was further analyzed by high performance liquid chromatography (HPLC) on C18 column with acetonitrile:water=75:25 (V/V) as the mobile phase under isocratic elution. Fraction A obtained at the retention time of 7.954 min showed an effective herbicidal activity on seed germination of Digitaria sanguinalis. The spectral analysis by diode array detection (DAD) for fraction A showed that fraction A is a pure component. [Conclusion] The results showed that the separation methods of high-speed countercurrent chromatography combined with high performance liquid chromatography was effective to obtain the herbicidal compound-fraction A from the metabolites of Pestalotiopsis microspora PM-1.