High-Resolution α‑Amylase Assay Combined with High-Performance Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance Spectroscopy for Expedited Identification of α‑Amylase Inhibitors: Proof of Concept and α‑Amylase Inhibitor in Cinnamon

Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-d-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC–​HRMS–​SPE–​NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compoundsof which three are known α-amylase inhibitorsshowed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase inhibition assay/HPLC–​HRMS–​SPE–​NMR platform allowed identification of cinnamaldehyde as the α-amylase inhibitor in cinnamon (<i>Cinnamomum verum</i> Presl.)

Dual High-Resolution α‑Glucosidase and Radical Scavenging Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of Minor and Major Constituents Directly from the Crude Extract of <i>Pueraria lobata</i>

The crude methanol extract of <i>Pueraria lobata</i> was investigated by dual high-resolution α-glucosidase inhibition and radical scavenging profiling combined with hyphenated HPLC-HRMS-SPE-NMR. Direct analysis of the crude extract without preceding purification was facilitated by combining chromatograms from two analytical-scale HPLC separations of 120 and 600 μg on-column, respectively. High-resolution α-glucosidase and radical scavenging profiles were obtained after microfractionation of the eluate in 96-well microplates. This allowed full bioactivity profiling of individual peaks in the HPLC chromatogram of the crude methanol extract. Subsequent HPLC-HRMS-SPE-NMR analysis allowed identification of 21 known compounds in addition to two new compounds, i.e., 3′-methoxydaidzein 8-<i>C</i>-[α-d-apiofuranosyl-(1→6)]-β-d-glucopyranoside and 6″-<i>O</i>-malonyl-3′-methoxydaidzin, as well as an unstable compound tentatively identified as 3′-de-<i>O</i>-methylpuerariafuran

Advancing HPLC-PDA-HRMS-SPE-NMR Analysis of Coumarins in <i>Coleonema album</i> by Use of Orthogonal Reversed-Phase C₁₈ and Pentafluorophenyl Separations

A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-HRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLC-PDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C₁₈ columns for separation. Herein is reported the use of an analytical-scale pentafluorophenyl column as an orthogonal separation method following fractionation of a crude ethyl acetate extract of leaves of <i>Coleonema album</i> on a preparative-scale C₁₈ column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-<i>O-</i>β-d-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (<b>4</b>), (<i>Z</i>)-6-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (<b>6</b>), 6-(4-β-d-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (<b>8</b>), (<i>Z</i>)-7-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yloxy)­coumarin (<b>13</b>), (<i>S</i>)-8-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)-7-methoxycoumarin (<b>19</b>), and 7-(3-chloro-2-hydroxy-3-methylbut-1-yloxy)­coumarin (<b>20</b>). The use of the pentafluorophenyl column even allowed separation of several regioisomers that are usually difficult to separate using reversed-phase C₁₈ columns. The phytochemical investigation described for <i>C. album</i> in this report demonstrates the potential and wide applicability of HPLC-PDA-HRMS-SPE-NMR for accelerated structural identification of natural products in complex mixtures