title: Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts.
abstract: 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.
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.|School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU , 1, Rue Michel Servet, 1211, Geneva 4, Switzerland.
authors: Paul
authors: P
authors: Coulerie
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Yann
authors: Y
authors: Ratinaud
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Sofia
authors: S
authors: Moco
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Loraine
authors: L
authors: Merminod
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Martine
authors: M
authors: Naranjo Pinta
authors: School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU , 1, Rue Michel Servet, 1211, Geneva 4, Switzerland.
authors: Julien
authors: J
authors: Boccard
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Laurent
authors: L
authors: Bultot
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Maria
authors: M
authors: Deak
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Kei
authors: K
authors: Sakamoto
authors: School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU , 1, Rue Michel Servet, 1211, Geneva 4, Switzerland.
authors: Emerson Ferreira
authors: EF
authors: Queiroz
authors: School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU , 1, Rue Michel Servet, 1211, Geneva 4, Switzerland.
authors: Jean-Luc
authors: JL
authors: Wolfender
authors: Nestle Institute of Health Sciences , EPFL Innovation Park, H, CH-1015, Lausanne, Switzerland.
authors: Denis
authors: D
authors: Barron
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000465:Algorithms; D001688:Biological Products / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D019833:Genistein / Q000737:chemistry; D015394:Molecular Structure; D012015:Reference Standards; D013557:Switzerland; D029921:Trifolium / Q000737:chemistry*
chemical_list: D001688:Biological Products; D019833:Genistein; D055372:AMP-Activated Protein Kinases; C004541:biochanin A