Recent advances in countercurrent chromatography (CCC) have created novel spiral-design separation columns that hold a very high stationary phase of all the solvent systems, such that now all molecules of any size and water solubility can be successfully separated in high-speed countercurrent chromatography. With spiral CCC there is inherent versatility, more than any type of chromatography. Preliminary studies with the new spiral tubing support rotor succeeded in purifying Sutherlandioside B, a major water soluble cycloartane glycoside from Lessertia frutescent (Sutherlandia) is using an n- butanol solvent system. With the small volume of the coil (~100 ml) and a stationary phase at 50% (~50 ml), it is impressive that a mass of 1.2 g of crude plant extract could be efficiently fractionated in one run with high recovery of the target compound. In addition, the active iridoid glycoside, harpagoside was isolated from an extract of Harpagophytum procumbens (Devil's Claw) in one step using a similar method. The first specific aim for Phase I am the design and manufacture of a preparative-scale spiral CCC instrument that will serve to rapidly separate complex water-soluble natural products with high- throughput capability. The studies will involve testing of a higher volume separation rotor and then its incorporation into a scaled-up planetary centrifuge. The pre-commercial prototype testing will be performed in conjunction with Dr. William Folk, the consultant from the University of Missouri. The second specific aim is the preliminary process development of purifying significant amounts of water-soluble extracts to obtain each of the sutherlandiosides and harpagoside metabolites from the medicinal African plants, L. frutescens and H. procumbens, respectively. A strategy of modifying a suitable solvent system to recover a target compound will be identified. With the compounds successfully purified, the studies will be continued in phase II to characterize their properties in cell culture and animal models, in collaboration with Dr. Folk and the University of Missouri. The successful outcome of this research will be a commercial instrument, the preparative spiral high-speed countercurrent chromatograph, an important technology for natural products chemistry that meets a demand in the global research market. The result will be increased productivity in drug discovery, enabling alternative medicines to be characterized and new therapies for many diseases and disorders. Finally, the application of butanol solvents so readily in spiral high-speed countercurrent chromatography is a promising example of green chemistry as these solvents are derived from biofuels.
This SBIR project submitted in response to the RFA-AT-14-001 Methods Development in Natural Products Chemistry proposes to apply recent innovations in countercurrent chromatography to create a large-scale high-throughput fractionation machine for rapid de- replication and removal of nuisance compounds in the crude extracts of natural products. The separation rotor in a flow-through purification centrifuge is specially designed to apply any dual phase solvent system such that all types of molecules can be purified rapidly out of complex extracts in high recovery without the need of special, expensive solid support phases. This project will apply the new commercial instrument to isolate new active metabolites from the plants, Lessertia frutescens and Harpagophytum procumbens and identify their medicinal effects in a partnership (in phase II) between this small business and the University of Missouri.
| Chen, Cuiping; Folk, William R; Lazo-Portugal, Rodrigo et al. (2017) Isolation of sutherlandins A, B, C and D from Sutherlandia frutescens (L.) R. Br. by counter-current chromatography using spiral tubing support rotors. J Chromatogr A 1508:7-15 |
