Identification of constituents of the Alpine plant Rhinanthus glacialis by 13C nuclear magnetic resonance pattern recognition

Rhinanthus glacialis (Orobanchaceae) is an Alpine plant whose phytochemical and pharmacological profile has not yet been explored. There are also no reports about an ethnobotanical usage of this species. However, since plants from higher altitudes often accumulate substances that protect them against UV radiation, R. glacialis was selected for a screening approach aiming to identify extracts with anti-ageing properties. Preliminary results showed that the ethylacetate extract of the aerial parts of R. glacialis inhibits NADPH oxidase 4 (NOX4) in HEK 293 cells. In order to rapidly identify constituents of this extract, a recently developed dereplication method [1] was applied. The extract was fractionated using centrifugal partition chromatography (CPC), producing simplified mixtures of metabolites. The obtained fractions were subjected to a ¹³C nuclear magnetic resonance (¹³C NMR) analysis and the major constituents were directly identified by a pattern recognition method, which combines alignment of ¹³C NMR signals and hierarchical clustering analysis (HCA) [1]. Using an in-house ¹³C NMR chemical shift database, obtained clusters were assigned to molecular structures, and seven compounds were unambiguously identified, i.e. the iridoids shanzhiside methyl ester, mussaenoside, and melampyroside, the phenylpropanoid verbascoside, the triterpenoid β-sitosterol, and the fatty acids palmitic acid and oleic acid. The presence of further fatty acids in minor amounts was suggested. They were identified as linolenic acid, linoleic acid, heptadecanoic acid, stearic acid, and arachidic acid using HPLC-ELSD in comparison with reference compounds.

In conclusion, the combination of CPC fractionation, ¹³C NMR analysis and HCA for pattern recognition of spectral data resulted in the rapid, simultaneous, and successful identification of the main constituents of R. glacialis. Future studies will address the identification of the compounds responsible for the activity of the extract.

Acknowledgements: Supported by the Austrian Forschungsförderungsgesellschaft (FFG) (BRIDGE 1 project “Alpine plants in cosmetics”) and the EU-Marie-Curie Action (IAPP/FP7-PEOPLE project “Natprotec”).

Keywords: Rhinanthus glacialis, ¹³C nuclear magnetic resonance, pattern recognition, iridoids, fatty acids, NADPH oxidase 4.

References:

[1] Hubert J, Nuzillard JM, Purson S, Hamzaoui M, Borie N, Reynaud R, Renault JH. Identification of Natural Metabolites in Mixture: A Pattern Recognition Strategy Based on ¹³C NMR. Anal Chem 2014; 86: 2955 – 2962