de Beer D., Malherbe C.J., Beelders T., Willenburg E.L., Brand D.J., Joubert E.
Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa; Food Science Department, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa; Nuclear Magnetic Resonance Laboratory, Central Analytical Facility, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa
de Beer, D., Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa; Malherbe, C.J., Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa; Beelders, T., Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa, Food Science Department, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa; Willenburg, E.L., Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa; Brand, D.J., Nuclear Magnetic Resonance Laboratory, Central Analytical Facility, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa; Joubert, E., Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, South Africa, Food Science Department, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa
Aspalathin and nothofagin, the major dihydrochalcones in rooibos (Aspalathus linearis), are valuable bioactive compounds, but their bioactivity has not been fully elucidated. Isolation of these compounds using high-performance countercurrent chromatography (HPCCC), a gentle, support-free, up-scalable technique, offers an alternative to synthesis for obtaining sufficient amounts. An HPLC-DAD method was adapted to allow rapid (16min from injection to injection) quantification of the four major compounds (aspalathin, nothofagin, isoorientin, orientin) during development of the isolation protocol. The traditional shake-flask method, used to determine distribution constants (KD) for target compounds, was also adapted to obtain higher repeatability. Green rooibos leaves with a high aspalathin and nothofagin content were selected as source material. Sample loading of the polyphenol-enriched extract was limited due to constituents with emulsifying properties, but could be increased by removing ethanol-insoluble matter. Furthermore, problems with degradation of aspalathin during HPCCC separation and further processing could be limited by acidifying the HPCCC solvent system. Aspalathin was shown to be fairly stable at pH 3 (91% remaining after 29h) compared to pH 7 (45% remaining after 29h). Aspalathin and nothofagin with high purities (99% and 100%, respectively) were obtained from HPCCC fractions after semi-preparative HPLC. © 2015 Elsevier B.V.
Chromatography; Emulsification; Liquid chromatography; Aspalathin; Countercurrent chromatography; Nothofagin; Rooibos; Semi-preparative HPLC; Loading; alcohol; aspalathin; chemical compound; isoorientin; nothofagin; orientin; phenol derivative; polyphenol; unclassified drug; aspalathin; chalcone derivative; flavonoid; glucoside; luteolin; nothofagin; plant extract; Article; chemical procedures; controlled study; counter current chromatography; degradation kinetics; diode array detection; emulsion; high performance countercurrent chromatography; high performance liquid chromatography; measurement repeatability; pH measurement; phase separation; structure analysis; Aspalathus; chemistry; counter current chromatography; electrospray mass spectrometry; isolation and purification; plant leaf; Aspalathus linearis; Aspalathus; Chalcones; Chromatography, High Pressure Liquid; Countercurrent Distribution; Flavonoids; Glucosides; Luteolin; Plant Extracts; Plant Leaves; Polyphenols; Spectrometry, Mass, Electrospray Ionization