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

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.