3 research outputs found
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 compoundsof
which three are known α-amylase inhibitorsshowed 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 C<sub>18</sub> 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 C<sub>18</sub> 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 C<sub>18</sub> 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 C<sub>18</sub> 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