Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography (MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to verify known and previously not published polyphenol structures. In addition the chemistry of dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized to the corresponding flavanone- C-glycosides (( R)/( S)-eriodictyol-6- C-beta- D-glucopyranoside and ( R)/( S)-eriodictyol-8- C-beta- D-glucopyranoside). Flavanone-6- C-beta- D-glucopyranosides were further degraded to flavones isoorientin and orientin.

Aspalathin and nothofagin are typical ingredients of unfermented rooibos (Krafczyk, N.; Glomb, M. A. J. Agric. Food Chem. 2008, 56, 3368). During oxidation these dihydrochalcones were degraded to higher molecular weight browning products under aerated and nonenzymatic conditions. In the early stages of browning reactions aspalathin formed two dimers. These two compounds were unequivocally established as atropisomers stemming from oxidative A to B ring coupling. Multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC) were applied to obtain pure substances. The purity and identity of isolated dimers were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS. In parallel to the formation of chromophores during the fermentation of black tea, the formation of aspalathin dimers implies an important mechanistic channel for the generation of color during the processing of rooibos.

Rooibos tea (Aspalathus linearis) contains different bioactive phenolic compounds such as dihydrochalcones, flavonols, flavanones, flavones, and flavanols. Flavonoids isolated from rooibos were subjected to different in vitro assays: Trolox equivalent antioxidant activity, LDL oxidation and Fremy's salt assays to determine the total antioxidant activity (TAA). Assay results were compared, and the structure-antioxidant relationship was investigated. A decoupled LDL oxidation test was established with the objective of having an assay adapted more to an in vivo situation. The different in vitro methods were coupled offline to HPLC-DAD. Results from these coupled offline methods were compared to the TAA to assess the usefulness of the coupling setup.

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