Rebiogram enhances the search of bioactive natural products

Finding active component(s) from natural sources using traditional chromatographic methods is a time consuming and tedious task. Especially with solid support material, potential loss due to irreversible absorption cannot be avoided during the separation procedure. In contrast, countercurrent separation (CCS), a liquid-liquid chromatography, can achieve loss free separation as it does not apply solid support material. This special characteristic deserves more attention when searching for bioactives from complex natural sources via bioassay guided fractionation (BGF) procedures. Considering recent progresses in CCS methodology, a considerable amount of sample can be loaded into a centrifugal partition chromatography (CPC) instrument with a single injection. ReBioGram is a new approach that enhances BGF for the search of bioactive natural products. It takes full advantage of CCS ability to swap the mobile phase in two separated runs to generate a pair of fractions that are exact normal- and reserved-phase mirror images of each other in K-plot. Almost a symmetric ReBioGram could be delineated based on bioactivities of these fractions, which could be used further to validate and prioritize active fraction.

Pueraria montana var. lobata (Willd.) (PL) has an isoflavone profile similar to that of Trifolium pratense (L.) (TP). However, the content of active components in PL is much lower than in TP. Guided by the alkaline phosphatase in Ishikawa cell assay, and using CPC fractionation and HPLC purification, we demonstrate the efficiency of ReBioGram method in the isolation of active component (ca 0.2 mg) from a DCM extract of PL aerial parts in just two separation steps.