Bioactivity-guided chromatographic methods are of great significance for the isolation of the active compounds in complex samples. In this study, four antifungal compounds were located by activity screening and successfully isolated from a microbial fermentation sample by preparative high-performance liquid chromatography. Separation performance of columns including C18, positively charged C18, negatively charged C18 and C8 were firstly investigated. And it showed a better capacity of mixed-mode stationary phases for retention and separation. Therefore, the positively charged C18 column was used to separate the sample into several fractions, among which the active one was identified by the antifungal test. And then the active fraction was enriched and separated again by successively using the negatively charged C18 and C8 columns to obtain four compounds, which were identified as polyoxins A, K, F and H. With activity verification, four polyoxins were found to have good inhibitory effects against the three fungal plant diseases including rice sheath blight, tomato grey mould disease, and apple spot leaf disease.
Developing methods for the isolation of highly polar compounds from complex samples is of great significance. In this study, three polyoxins were successfully isolated from a complex sample (PN1-1 # ) by preparative high-performance liquid chromatography. Separation was carried out on five polar reversed-phase stationary phases, using pure aqueous as mobile phase, where the C18HC column can provide the best performance for PN1-1 # . Next, the effects of the mobile phase composition were studied. It was found that adding NaClO 4 can enhance the retention and resolution, and adding NaH 2 PO 4 was beneficial to maintain good peak shapes when the sample loading increased. Therefore, the optimized mobile phase consisting of 20 mmol NaH 2 PO 4 and 20 mmol NaClO 4 (adding H 3 PO 4 to adjust pH 2) was used to separate PN1-1 # . This method of using 100% aqueous phase can effectively improve both the retention and the solubility of polar samples. Eventually, through further purification, three compounds, namely, polyoxins B, D, and G, were obtained. This paper provided an effective and eco-friendly strategy for the preparative-scale separation of polar samples.
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