Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts

P. Coulerie, Y. Ratinaud, S. Moco, L. Merminod, M. Naranjo Pinta, J. Boccard, L. Bultot, M. Deak, K. Sakamoto, E.F. Queiroz, J.-L. Wolfender, D. Barron

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

© 2016 The American Chemical Society and American Society of Pharmacognosy.To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.
Original languageEnglish
Pages (from-to)2856-2864
JournalJournal of Natural Products
Volume79
Issue number11
DOIs
Publication statusPublished - 23 Nov 2016
Externally publishedYes

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