Abstract
Herein is detailed the development and validation of an ultra-micro-scale-fractionation (UMSF) technique for the discovery of plant-based, bioactive molecules, coupling the advantages of ultra-performance liquid chromatography mass spectrometry (UPLC-MS) separations with microtiter plate-based bioassay screens. This novel one-step approach simultaneously uses UPLC to collect chemical profile information, while performing high-resolution fractionation, greatly improving workflow compared to methods relying on high-performance liquid chromatography (HPLC), solid phase extraction or flash systems for chromatographic separations. Using the UMSF technique, researchers are able to utilize smaller quantities of starting materials, reduce solvent consumption during fractionation, reduce laborious solvent dry down times, replace costly single-use solid-phase-extraction cartridges with reusable analytical-sale UPLC columns, reduce fractionation times to less than 10 min, while simultaneously generating chemical profile data of active fractions and enjoying superior chromatographic resolution. Using this technique, individual bioactive components can be readily purified, identified, and bioassayed in one step from crude extracts, thereby eliminating ambiguous synergistic effects often reported in plant-based natural products research. A successful case-study is presented illustrating the versatility of this technique in identifying lupulone as the principal cytotoxic component from hops (Humulus lupulus L.), using a brine shrimp (Artemia franciscana) model. These results confirm and expand upon previous cell-based bioassay studies using a more complex, multicellular organism, and add to our understanding of structure-function activity relationships for secondary metabolites in hops and the Cannabaceae plant family.
Highlights
Assay guided fractionation, a directed and iterative process of chemical extraction, purification, and bioassay, has arguably been the paradigm technique for pharmacognosists across decades of study.the plant-derived chemotherapy agents paclitaxel [1], vinca alkaloids [2], and camptothecin [3], were all discovered using this approach, saving thousands of lives through applied clinical practice
Combining solvent pumps to pressure-resistant columns led to the advent of flash-chromatography (FC), the first step towards efficient and automated separations, resulting in the development of computer-controlled high-performance liquid chromatography (HPLC) in the 1980s and ultra-performance liquid chromatography (UPLC/UHPLC) in the 2000s
Both HPLC and UPLC have been widely adopted in natural products discovery laboratories, where minute quantities of extract could be efficiently and rapidly separated, and simultaneously chemically characterized (UV-Vis absorption, mass spectra)
Summary
A directed and iterative process of chemical extraction, purification, and bioassay, has arguably been the paradigm technique for pharmacognosists across decades of study. Combining solvent pumps to pressure-resistant columns led to the advent of flash-chromatography (FC), the first step towards efficient and automated separations, resulting in the development of computer-controlled high-performance liquid chromatography (HPLC) in the 1980s and ultra-performance liquid chromatography (UPLC/UHPLC) in the 2000s. As analytical platforms, both HPLC and UPLC have been widely adopted in natural products discovery laboratories, where minute quantities of extract (μg on column) could be efficiently and rapidly separated, and simultaneously chemically characterized (UV-Vis absorption, mass spectra). We illustrate how UMSF workflow supplants the conventional assay approach, detail theoretical considerations underlying the technique, make cost comparisons with traditional assay-guided fractionation strategies, and present case-study data illustrating the utility of the method
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