TY - JOUR

T1 - Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts

AU - Coulerie, Paul

AU - Ratinaud, Yann

AU - Moco, Sofia

AU - Merminod, Loraine

AU - Naranjo Pinta, Martine

AU - Boccard, Julien

AU - Bultot, Laurent

AU - Deak, Maria

AU - Sakamoto, Kei

AU - Queiroz, Emerson Ferreira

AU - Wolfender, Jean Luc

AU - Barron, Denis

PY - 2016/11/23

Y1 - 2016/11/23

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84999122228&partnerID=8YFLogxK

U2 - 10.1021/acs.jnatprod.6b00628

DO - 10.1021/acs.jnatprod.6b00628

M3 - Journal article

C2 - 27792327

AN - SCOPUS:84999122228

VL - 79

SP - 2856

EP - 2864

JO - Journal of Natural Products

JF - Journal of Natural Products

SN - 0163-3864

IS - 11

ER -