Preparative Separation of Main Ustilaginoidins from Rice False Smut Balls by High-Speed Counter-Current Chromatography

Ustilaginoidins are bis-naphtho-γ-pyrone mycotoxins isolated from the rice false smut balls (FSBs) infected by the pathogen Villosiclava virens in rice spikelets on panicles. In order to obtain large amounts of pure ustilaginoidins to further evaluate their biological activities and functions, phytotoxicity on rice, security to human and animals as well as to accelerate their applications as pharmaceuticals, preparative high-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of seven bis-naphtho-γ-pyrone mycotoxins, namely ustilaginoidins A (1), G (2), B (3), H (4), I (5), C (6), and J (7) from the ethyl acetate crude extract of rice FSBs. Both 1 and 2 were prepared by HSCCC from the low-polarity fraction of the crude extract using the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at the volume ratio of 6.5:3.5:5.0:5.0. Similarly, 3, 4 and 5 were prepared from the medium-polarity fraction using the system at the volume ratio of 4.0:5.0:5.0:6.0, and 6 and 7 were prepared from the higher-polarity fraction using the system at volume ratio of 3.0:5.0:4.0:6.7. A total of 6.2 mg of 1, 5.1 mg of 2, 3.9 mg of 3, 1.2 mg of 4, 5.7 mg of 5, 3.5 mg of 6, and 6.1 mg of 7 with purities of 88%, 82%, 91%, 80%, 92%, 81% and 83%, respectively, were yielded from total 62 mg fraction samples in three independent HSCCC runs. The structures of the purified ustilaginoidins were characterized by means of physicochemical and spectrometric analysis.