Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Rooibos tea, produced from the endemic South African fynbos plant Aspalathus linearis, has various healthpromoting
benefits which can largely be attributed to its phenolic composition. In this study, the reversedphase
liquid chromatographic (RP-LC) separation of the principal phenolic constituents of aqueous rooibos
infusions was optimised on conventional high-performance liquid chromatography (HPLC) instrumentation.
The phenolic constituents comprised dihydrochalcones (aspalathin and nothofagin), flavones (orientin,
isorientin, vitexin, isovitexin, luteolin, luteolin-7-O-glucoside and chrysoeriol), flavonols (quercetin,
isoquercitrin, hyperoside and rutin), a hydroxycinnamic acid (ferulic acid) and a phenylpropanoid
(phenylpyruvic acid glucoside, PPAG). A systematic approach towards method development was adopted:
resolution was optimised by simultaneous optimisation of gradient conditions and temperature on a sub-2 6m
phase to exploit the benefits of this phase for fast routine analyses. The developed HPLC method, utilising
photodiode-array (PDA) detection, yielded complete separation of the 15 target analytes on the 1.8 µm C18
column, thermostatted to 37°C, within 37 min (total analysis time: 50 min). The method was successfully
validated and demonstrated its suitability for the fast, quantitative analysis of aqueous infusions of
unfermented and fermented rooibos. Mass spectrometric (MS) and tandem MS detection confirmed peak
purity and permitted the tentative identification of 13 additional phenolic compounds, including a flavonol Odiglycoside
(quercetin-3-O-robinobioside), a luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl
derivative of aspalathin reported here for the first time.
The HPLC-PDA method was subsequently applied to a large number of fermented rooibos samples
representative of different production seasons (2009, 2010 and 2011) and quality grades (grades A, B, C
and D) to capture as much potential variation in the phenolic composition as possible. Production season
had no clear effect on the levels of the individual phenolic compounds in ‘cup-of-tea’ rooibos infusions, whilst
high quality tea (grades A and B) was associated with higher levels of phenolic compounds and soluble
solids than low quality tea (grades C and D). Steam-pasteurisation of the plant material, required to obtain a
product of high microbiological quality, induced significant reductions in the mean values of most of the
phenolic compounds in rooibos infusions. The major phenolic constituents of steam-pasteurised, fermented
rooibos were isoorientin and orientin, whilst quercetin-3-O-robinobioside, PPAG and aspalathin were also
present in high concentrations. Representative content values of the major phenolic compounds present in a
typical ‘cup-of-tea’ rooibos infusion were thus obtained and the generated data are suitable for inclusion in
food composition databases.
The application of comprehensive two-dimensional liquid chromatography (LCxLC) was investigated
as an alternative approach for the detailed investigation of rooibos phenolics. The combination of hydrophilic
interaction chromatography (HILIC) in the first dimension and RP-LC in the second dimension offered
different separation selectivities and hence a high degree of orthogonality. HILICxRP-LC provided a
significant improvement in resolution, as is evident from practical peak capacities in excess of 2000 and 800
for the off-line and on-line methods, respectively. Further optimisation, particularly of the first dimension
separation, is however required to improve the LCxLC separation of complex rooibos phenolic fractions. / AFRIKAANSE OPSOMMING: Gebruik van rooibostee, berei vanaf die eg Suid-Afrikaanse fynbosplant Aspalathus linearis, hou verskeie
gesondheidsvoordele in wat grootliks toegeskryf kan word aan sy fenoliese samestelling. Die skeiding van
die hoof fenoliese verbindings van ’n koppie rooibos is in hierdie studie deur middel van omgekeerde-fase
vloeistof chromatografie (RP-LC) op konvensionele hoë-druk vloeistof chromatografiese (HPLC) toerusting
geoptimiseer. Die fenoliese verbindings was verteenwoordigend van dihidrogalkone (aspalatien en
notofagien), flavone (orientien, isoorientien, viteksien, isoviteksien, luteolien, luteolien-7-O-glukosied en
krisoeriol), flavonole (kwersetien, isokwersetrien, hiperosied en rutien), ‘n hidroksiekaneulsuur (ferulasuur) en
‘n fenielpropanoied (fenielpirodruiwesuurglukosied, PPAG). Die ontwikkeling van die metode was sistematies
benader: resolusie is op ‘n geselekteerde 1.8 6m stationêre fase met welombekende kinetiese voordele
geoptimiseer deur die gradiëntkondisies en kolomtemperatuur gelyktydig te optimiseer. Die ontwikkelde
HPLC metode, gekoppel aan ultraviolet-fotodiode deteksie (PDA), het binne 37 min (totale analiese tyd: 50
min) volledige skeiding van die 15 standaard verbindings op die 1.8 µm C18 kolom teen 37°C bewerkstellig.
Die metode is suksesvol gevalideer en het sy toepaslikheid vir vinnige, kwantitatiewe analiese van
ongefermenteerde en gefermenteerde rooibos gedemonstreer. Piek suiwerheid is deur middel van massa
spektrometrie (MS) en tandem MS bevestig, wat ook die identifikasie van 13 addisionele verbindings
toegelaat het, insluitende ‘n flavonol O-diglukosied (kwersetien-3-O-robinobiosied), ‘n luteolien-6-Cpentosied-
8-C-heksosied en ‘n unieke C-8-heksosiel afgeleide van aspalatien wat vir die eerste keer hier
gemeld is.
Die geoptimiseerde HPLC-PDA metode is gevolglik toegepas vir die analiese van ‘n groot aantal
gefermenteerde rooibos monsters, verteenwoordigend van verskillende produksie seisoene (2009, 2010 en
2011) en kwaliteitsgrade (A, B, C en D). Hierdie lukraak-geselekteerde monsters het soveel as moontlik
potensiële variasie in die fenoliese samestelling verseker. Produksie seisoen het geen definitiewe effek op
die vlakke van die individuele fenoliese verbindings in ’n koppie rooibos gehad nie, terwyl hoë kwaliteit
rooibos (grade A en B) geassosieër was met hoër vlakke van die individuele fenoliese verbindings en
oplosbare vastestowwe in vergelyking met lae kwaliteit rooibos (grade C en D). Stoompasteurisasie van
rooibos plantmateriaal, noodsaaklik om ‘n produk van hoë mikrobiologiese gehalte te verseker, het gelei tot
‘n betekenisvolle afname in meeste fenoliese verbindings in ’n koppie rooibos. Die hoof fenoliese verbindings
van ‘n koppie stoom-gepasteuriseerde, gefermenteerde rooibos was orientien en isoorientien, terwyl
kwersetien-3-O-robinobiosied, PPAG en aspalatien ook in noemenswaardige hoeveelhede aanwesig was.
Verteenwoordigende waardes van die hoof fenoliese verbindings aanwesig in ‘n tipiese koppie rooibos is
derhalwe verkry en die data is geskik vir insluiting in voedsel-samestelling databasisse.
Die analiese van rooibos fenole met omvattende twee-dimensionele vloeistof chromatografie
(LCxLC) is bestudeer as ‘n alternatiewe metode om verdere insig tot hierdie komplekse fenoliese fraksie te
verkry. Die kombinasie van hidrofiliese interaksie chromatografie (HILIC) in die eerste dimensie en RP-LC in
die tweede dimensie het ‘n uiters gesogte lae graad van korrelasie verskaf. HILICxRP-LC het ‘n besonderse
toename in resolusie teweeg gebring, gekenmerk deur praktiese piek kapasiteite hoër as 2000 en 800 vir die
af-lyn en aan-lyn metodes, onderskeidelik. Verdere optimisering, veral van die eerste dimensie skeiding, is
egter nodig om die LCxLC skeiding van rooibos fenole te verbeter.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17818 |
| Date | 12 1900 |
| Creators | Beelders, Theresa |
| Contributors | De Villiers, A. J., Joubert, E., De Beer, D., Sigge, G. O., Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Format | 167 p. : ill. |
| Rights | Stellenbosch University |
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