Bis-naphtho-γ-pyrones From Rice False Smut Pathogen Ustilaginoidea Virens UV-2 And Their Biological Activities

The rice false smut is one of the most destructive diseases in rice cultivation area, which is caused by the pathogenic fungus Ustilaginoidea virens Takahashi (teleomorph:Villosiclava virens Tanaka and Tanaka). This fungus infects the spikes of rice plant and results in the formation of the false smut balls. Ustiloxins and ustilaginoidins are two kinds of mycotoxins produced by the false smut pathogen. Ustiloxins belong to cyclopeptides, and ustilaginoidins belong to bis-naphtho-y-pyrones. Bis-naphtho-y-pyrones showed various biological activities, such as:cytotoxicity to the human epidermoid carcinoma (KB) cells, impairing effects on mitochondria respiration, inhibitory activities against HIV-1 integrase, antibacterial activities, and immunological inhibitory activity. The study of the ustilaginoidins and their biological activities have important significance for the development and utilization of this kind of substances, for the follow-up study of biosynthetic pathway of ustilaginoidins and for the prevention and control of False smut of rice. As far as we know, there has not been any research on ustilaginoidins from the false smut pathogen Ustilaginoidea virens. This paper focuses on the research of the bis-naphtho-y-pyrones, their biological activities and the preparation process of the rice pathogenic fungus U. virens UV-2. The main results are as follows:1. Ustilaginoidins were isolated from Ustilaginoidea virens UV-2, including seven new compounds, namely ustilaginoidins K-P, and El, together with six known ustilaginoidins A, D, E, F, and G, and isochaetochromin B2.2. The isolated compounds were evaluated for their antibacterial, cytotoxic, and radicle elongation inhibitory activities. Ustilaginoidins K and L showed cytotoxic activities on the A2780 human ovarian cancer cell line with IC50 values of 4.18 and 7.26 μM, respectively. Ustilaginoidins N, D, E, and G were active against the tested pathogenic bacteria with MIC values in the range of 16-64 μg/mL. Ustilaginoidins O, E, and F, and isochaetochromin B2 displayed moderate inhibitory activity on the radicle elongation of rice seeds.3. Six ustilaginoidins were successfully separated by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.01% oxalic acid aqueous solution (5:4:7:4,v/v/v/v), with purities between 74.0% to 94.2%, as determined by high-performance liquid chromatography (HPLC), respectively. Among six main ustilaginoidins (i.e., ustilaginoidins D, E, G, M, N, and O), both ustilaginoidina N and O were separated as a mixture just by one-step HSCCC, which were further purified by semi-preparative HPLC.4. The enrichment conditions of macroporous resin for ustilaginoidins D-O were studied. Resin HP-20 was selected from 7 different macroporous resins. Through the static and dynamic adsorption and desorption experimentes, the optimum conditions were established as follows:initial pH of the sample solution was 4; the sample volume is about 24 BV, with flow rate at 3 BV/h; optimum adsorption solution was 60% acetone, the analytical volume is about 4.5 BV with flow rate at 3 BV/h.5. The antioxidant activity of mycelium polysaccharide (WPS), mycelium alkaline polysaccharides (SPS) and extracellular polysaccharide (EPS) from Ustilaginoidea virens UV-2 were evaluated by two kinds of antioxidant models. Among them, EPS showed the best antioxidant activity. In order to improve the yield of EPS from the fermentation, central composite experimental design and response surface analysis methods were used. When the concentration of glucose in the medium was 84.08 g/L, peptone 14.11 g/L, the yield of EPS reached the maximum,881.40 mg/L.The results in this study provide the basis for the the follow-up study of biosynthetic pathway、 regulation of metabolism、detection and analysis、 metabonomics of ustilaginoidins. Also, these results lay the foundation for the development and utilization of active substances, prevention and control of False smut of rice in the application.