HPLC–NMR Revisited: Using Time-Slice High-Performance Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance with Database-Assisted Dereplication

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC–NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC–SPE–NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting ¹H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized

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

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