Magnetic Ligand Fishing as a Targeting Tool for HPLC-HRMS-SPE-NMR: α‑Glucosidase Inhibitory Ligands and Alkylresorcinol Glycosides from <i>Eugenia catharinae</i>

A bioanalytical platform combining magnetic ligand fishing for α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for structural identification of α-glucosidase inhibitory ligands, both directly from crude plant extracts, is presented. Magnetic beads with N-terminus-coupled α-glucosidase were synthesized and characterized for their inherent catalytic activity. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of caffeine, ferulic acid, and luteolin before proof-of-concept with the crude extract of <i>Eugenia catharinae</i>. The combination of ligand fishing and HPLC-HRMS-SPE-NMR identified myricetin 3-<i>O</i>-α-l-rhamnopyranoside, myricetin, quercetin, and kaempferol as α-glucosidase inhibitory ligands in <i>E. catharinae</i>. Furthermore, HPLC-HRMS-SPE-NMR analysis led to identification of six new alkylresorcinol glycosides, i.e., 5-(2-oxopentyl)­resorcinol 4-<i>O</i>-β-d-glucopyranoside, 5-propylresorcinol 4-<i>O</i>-β-d-glucopyranoside, 5-pentylresorcinol 4-<i>O</i>-[α-d-apiofuranosyl-(1→6)]-β-d-glucopyranoside, 5-pentylresorcinol 4-<i>O</i>-β-d-glucopyranoside, 4-hydroxy-3-<i>O</i>-methyl-5-pentylresorcinol 1-<i>O</i>-β-d-glucopyranoside, and 3-<i>O</i>-methyl-5-pentylresorcinol 1-<i>O</i>-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside

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.)

Combined Use of High-Resolution α‑Glucosidase Inhibition Profiling and High-Performance Liquid Chromatography–High-Resolution Mass Spectrometry–Solid-Phase Extraction–Nuclear Magnetic Resonance Spectroscopy for Investigation of Antidiabetic Principles in Crude Plant Extracts

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC₅₀ values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (<b>1</b>) and its dimer (<b>2</b>), trimer (<b>3</b>), and tetramer (<b>4</b>) were identified as main α-glucosidase inhibitors in A. ascalonicum peel, whereas (<i>E</i>)-piceatannol 3′-<i>O</i>-β-d-glucopyranoside (<b>5</b>), (<i>E</i>)-rhapontigenin 3′-<i>O</i>-β-d-glucopyranoside (<b>6</b>), (<i>E</i>)-piceatannol (<b>8</b>), and emodin (<b>12</b>) were identified as main α-glucosidase inhibitors in R. palmatum root

Potential of Polygonum cuspidatum Root as an Antidiabetic Food: Dual High-Resolution α‑Glucosidase and PTP1B Inhibition Profiling Combined with HPLC-HRMS and NMR for Identification of Antidiabetic Constituents

The worldwide increasing incidence of type 2 diabetes has fueled an intensified search for food and herbal remedies with preventive and/or therapeutic properties. Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) is used as a functional food in Japan and South Korea, and it is also a well-known traditional antidiabetic herb used in China. In this study, dual high-resolution α-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) inhibition profiling was used for the identification of individual antidiabetic constituents directly from the crude ethyl acetate extract and fractions of P. cuspidatum. Subsequent preparative-scale HPLC was used to isolate a series of α-glucosidase inhibitors, which after HPLC-HRMS and NMR analysis were identified as procyanidin B2 3,3″-<i>O</i>-digallate (<b>3</b>) and (−)-epicatechin gallate (<b>5</b>) with IC₅₀ values of 0.42 ± 0.02 and 0.48 ± 0.0004 μM, respectively, as well as a series of stilbene analogues with IC₅₀ value in the range from 6.05 ± 0.05 to 116.10 ± 2.04 μM. In addition, (<i>trans</i>)-emodin-physcion bianthrone (<b>15b</b>) and (<i>cis</i>)-emodin-physcion bianthrone (<b>15c</b>) were identified as potent PTP1B inhibitors with IC₅₀ values of 2.77 ± 1.23 and 7.29 ± 2.32 μM, respectively. These findings show that P. cuspidatum is a potential functional food for management of type 2 diabetes

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

High-Resolution Screening Combined with HPLC-HRMS-SPE-NMR for Identification of Fungal Plasma Membrane H⁺‑ATPase Inhibitors from Plants

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H⁺-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H⁺-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H⁺-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (<b>1</b>) and tellimagrandin II (<b>2</b>) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC₅₀ values for PM H⁺-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H⁺-ATPase with inhibitory effect on the growth of S. cerevisiae

Commercially obtained compounds that were tested in this study.

<p>Commercially obtained compounds that were tested in this study.</p

The effect of anti-VEGF and various concentrations of N-methyllevamisole (7a) and suramin (5) recorded in the in vitro angiogenesis assay performed with HUVECs growing on a fibroblast monolayer.

<p>The images show HUVECs visualized by immunostaining for CD31 after treatment with: <i>A)</i> 1 mM <i>N</i>-methyllevamisole triflate (7a); <i>B)</i> 0.5 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>C)</i> 0.25 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>D)</i> 0.13 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>E)</i> 0.06 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>F)</i> 0.03 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>G)</i> 0.02 mM <i>N</i>-methyllevamisole triflate (<b>7a</b>); <i>H)</i> 0.1 % DMSO (the control was diluted 1∶1000 corresponding the concentration of DMSO present in A); <i>I)</i> 5 μg/mL goat anti-recombinant human VEGF; <i>J)</i> 12 μM suramin (<b>5</b>); <i>K)</i> 1.5 μM suramin (<b>5</b>); <i>L)</i> medium (control); <i>M)</i> 10 mM BIS-TRIS; <i>N)</i> 10 mM BICINE; <i>O)</i> 10 mM <i>N</i>-methylimidazole; <i>P)</i> 1 mM <i>N</i>-methylimidazole.</p

Results of in vitro angiogenesis inhibition of levamisole and its derivatives in comparison with suramin (5), vehicle (DMSO), and medium alone, as observed by HUVEC number and morphology.

a<p>Percent of the area covered by HUVEC as observed by CD31 staining.</p>b<p><i>Clusters</i> refer to morphologies where several cells that form round or elongated clusters; <i>Cords</i> refer to morphologies where single to a few cells form cord-like structures without forming a network.</p>c<p>(++++)  =  clusters with area <4%; (+++)  =  cords with area <4%; (++)  =  clusters with area >4%; (+)  =  intermediate morphologies and varying area percentages.</p