Microgram-scale, in vivo natural product discovery using zebrafish bioassays, UHPLC-TOF-MS and microflow NMR: Identification of anticonvulsants in the Philippine medicinal plant Solanum torvum
The rapid acquisition of structural and bioactivity information on natural products at the sub-milligram scale is key for performing efficient bioactivity-guided isolations. We have recently established zebrafish as an ideal in vivo system for natural product discovery1,2. Zebrafish offer the possibility of rapid in vivo bioactivity analysis at the microgram scale, an attractive feature when combined with high-resolution fractionation technologies and microgram-scale analytical methods such as UHPLC-TOF-MS and microflow NMR3. Using this platform, we have performed high-resolution in vivo bioactivity profiling of Solanum torvum Schltdl., one of several Solanaceae species used as medicinal plants for the treatment of epilepsy4–6. The crude methanolic extract of S. torvum stem bark exhibited strong anti-convulsant activity in a microtiter plate-based zebrafish seizure assay. UHPLC-TOF-MS profiling revealed the presence of numerous steroid glycosides. An initial enrichment step localized the bioactivity to the steroid fraction. High resolution microfractionation by semi-preparative LC-MS enabled the resolution of most constituents directly in a single 96-well microtiter plate. The analysis of these wells by microflow NMR in combination with LC-MS profiling allowed dereplication of the active compounds, identifying them as spirostanol glycoside derivatives and estimating their microquantities for quantitative assessment of anticonvulsant activity in zebrafish. These results indicate the potential of zebrafish bioassay-guided microfractionation, in combination with UHPLC-TOF-MS and microflow NMR, to rapidly identify bioactive natural products.
References: 1. Crawford AD et al. (2008) Planta Med 6: 624. 2. Crawford AD et al. (2011) PLoS ONE 6:e14694. 3. Glauser G et al. (2009)J Agric Food Chem 57: 1127. 4. Quisumbing E (1951) Medicinal Plants of the Philippines, Bureau of Print, Manila. 5. Barber CF (1895) JAMA 25: 1023. 6. Trusch D (1904) Journal de Mèdecine de Paris 8: 74.