Large-Scale Separation of Bioactive Compounds from African Medicinal Plants by pH-Zone-Refining Counter-Current Chromatography
As part of our ongoing anti-infective discovery program based on plants employed in African Traditional Medicine, bioactive plants extract were selected for preparative separations of their secondary metabolites using High-Speed Countercurrent Chromatography (HSCCC) . These phytochemicals were separated by a type-J multilayer coil planet centrifuge including the Spiral Tube Support (STS) and a newly developed flat-twisted tubing . This paper describes the fundamentals of CCC separations, various CCC methods, particularly pH-zone refining countercurrent chromatography (pH-zone-refining CCC), trends in CCC instrument development in Dr. Ito's Bioseparation Laboratory at NIH as well as successful preparative separation and purification of three endemic plants known in African ethnomedicine for their various therapeutic properties. A broad selection of applications of pH-zone-refining CCC involving a wide variety of compounds is described. Separation of alkaloids from Picralima nitida (Stapf) T. Durand & H. Durand seeds using a new flat-twisted tubing in the pH-zone-refining CCC mode yielded six major indole alkaloids including akuammine, akuammicine, alstonine, picraline, Melinonine A and akuammidine with purities ranging from 90% (Melinonine A) to 98% (akuammine). A leaf extract (10.0 g) from Larrea tridentata Coville yielded Anti-HIV lignans, NDGA (nordihydroguaiaretic acid, 1.9 g) and its monomethyl esters (3 – 0-methyl-NDGA and 4 – 0-methyl-NDGA 1.4 g). Likewise, pH-zone-refining CCC was applied to scale up the separation of five major biflavanones including 3”,4',4”',5,5”,7,7”-heptahydroxy-3,8”-biflavanone (GB1), GB-I-glucoside, GB-1a, GB-2 and kolaflavonone from the extracts of Garcinia kola Heckel seeds. In comparison to earlier CCC methods, the new STS system yielded very high partition efficiency with the best stationary phase retention in a shorter elution time. References:  Yang Y, Aisa HA, Ito Y (2009)J Chromatogr A, 1216(27): 5265 – 5271.  Ito Y, (1986) Crit Rev Anal Chem, 17(1): 65 – 143.