title: Isolation of aspalathin and nothofagin from rooibos (Aspalathus linearis) using high-performance countercurrent chromatography: sample loading and compound stability considerations.
abstract: 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 (16 min 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 (K(D)) 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 29 h) compared to pH 7 (45% remaining after 29 h). Aspalathin and nothofagin with high purities (99% and 100%, respectively) were obtained from HPCCC fractions after semi-preparative HPLC.
authors.affiliation: Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa. Electronic address: dbeerd@arc.agric.za.
authors.forename: Dalene
authors.initials: D
authors.lastname: de Beer
authors.affiliation: Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa.
authors.forename: Christiaan J
authors.initials: CJ
authors.lastname: Malherbe
authors.affiliation: Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa; Food Science Department, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa.
authors.forename: Theresa
authors.initials: T
authors.lastname: Beelders
authors.affiliation: Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa.
authors.forename: Elize L
authors.initials: EL
authors.lastname: Willenburg
authors.affiliation: Nuclear Magnetic Resonance Laboratory, Central Analytical Facility, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa.
authors.forename: D Jacobus
authors.initials: DJ
authors.lastname: Brand
authors.affiliation: Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa; Food Science Department, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Joubert
mesh_terms: D036903:Aspalathus / Q000737:chemistry*; D047188:Chalcones / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003377:Countercurrent Distribution; D005419:Flavonoids / Q000302:isolation & purification; D005960:Glucosides / Q000302:isolation & purification; D047311:Luteolin / Q000302:isolation & purification; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D059808:Polyphenols / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D047188:Chalcones; D005419:Flavonoids; D005960:Glucosides; D010936:Plant Extracts; D059808:Polyphenols; C517016:aspalathin; C528728:nothofagin; C057912:homoorientin; C065886:orientin; D047311:Luteolin
keywords: Aspalathin; Countercurrent chromatography; Nothofagin; Rooibos; Sample stability; Semi-preparative HPLC