Ultra-Micro-Scale-Fractionation (UMSF) as a powerful tool for bioactive molecules discovery
Citation
McCallum, J.L., Vacon, J.N.D., Kirby, C.W. (2020). Ultra-Micro-Scale-Fractionation (UMSF) as a powerful tool for bioactive molecules discovery. Molecules, [online] 25(16), http://dx.doi.org/10.3390/molecules25163677
Plain language summary
A new approach to bioassay guided fractionation (isolating a pure chemical agent from a natural origin) was developed and validated with 6 different botanical extracts. This novel method combines the advantages of ultra-performance liquid chromatography (UPLC) with microtiter-based bioassays , (a technique where mixtures of chemicals are separated and tested), with microscale fractions being generated using a UPLC column for the first time. The novel method improves research workflow, simultaneously reducing: sample size, the separation and analysis time per sample, required solvent volumes, the volumes of chemical wastes generated, and decreases dry down times; while improving chromatographic performance and resolution. Using this approach, microfractions containing pure compounds can be obtained from crude botanical extracts in a single step, with a single leaf/flower/fruit often providing enough extract to conduct bioassay analysis.
Using this approach, we tested the effects of extracts prepared from Rocky Mountain Juniper berry (Juniperus scopulorum Hook), St. John’s Wort flowers (Hypericum perforatum), Nannyberry fruits (Viburnum lentago), Bunchberry Dogwood fruits (Cornus canadensis), Western Snowberry fruits (Symphoricarpos occidentalis), and Hops cones (inflorescences) (Humulus lupulus), on brine shrimp (Artemia franciscana), an established model organism for the discovery of anti-cancer (cytotoxic) natural products. Hops extracts possessed potent bioactivity, with the beta acids (lupulones) identified as the principal cytotoxic components. Among the major beta acids in hops, lupulone was the most active, followed by adlupulone, and colupulone, with both lupulone and adlupulone showing LC50 values (a measure of potency), in the single uM range. These results confirm the anti-cancer activity of hops components, in an intact, multicellular organism for the first time.
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 usesUPLCto 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.