Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop): a comparison of MS and NMR methods in metabolomics
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Hop (Humulus lupulus L. Cannabaceae) is an economically important crop. In addition to its role in beer brewing, its pharmaceutical applications have been of increasing importance in recent years. Bitter acids (prenylated polyketides), prenylflavonoids and essential oils, are the primary phytochemical components that account for hop medicinal value. An integrated approach utilizing nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques was used for the first large-scale metabolite profiling in Humulus lupulus. Resins and extracts prepared from 13 hop cultivars were analysed using NMR, liquid chromatography (LC)-MS and fourier transform ion cyclotron resonance (FTICR)-MS in parallel and subjected to principal component analysis (PCA). A one pot extraction method, compatible with both MS and NMR measurement was developed to help rule out effects due to differences in extraction protocols. Under optimised conditions, we were able to simultaneously quantify and identify 46 metabolites including 18 bitter acids, 12 flavonoids, 3 terpenes, 3 fatty acids and 2 sugars. Cultivars segregation in PCA plots generated from both LC-MS and NMR data were found comparable and mostly influenced by differences in bitter acids composition among cultivars. FTICR-MS showed inconsistent PCA loading plot results which are likely due to preferential ionisation and also point to the presence of novel isoprenylated metabolites in hop. This comparative metabolomic approach provided new insights for the complementariness and coincidence for these different technology platform applications in hop and similar plant metabolomics projects.
KeywordsHumulus lupulus L. Hop 1H NMR-based metabolomics ESI-FTICR MS LC-MS Humulones Lupulones Principal component analysis Flavonoids Isoprenoids Plant secondary metabolites
Fourier transform ion cyclotron resonance.
Tandem mass spectrometry
Nuclear magnetic resonance
Photodiode array detection
Principal component analysis
Dr. M. A. Farag thanks the Alexander von Humboldt-foundation, Germany for financial support. We are grateful to Hopsteiner Inc. (and here especially Dr. Martin Biendl, Hallertau, and Harald Schwarz) for hop resins, cones, and hop acid reference samples. We also thank Dr. Christoph Böttcher for assistance with the UPLC-MS, Prof. Barbara Seliger for support with HT29 measurements, and Orgentis Chemicals for reference samples of pure hop constituents. We are grateful to Dr. Steffen Neumann and Dr. Tilo Lübken for providing R scripts for NMR and MS data analysis.
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