Development of advanced chromatographic techniques for the in-depth phenolic profiling of rooibos

Walters, Nico Albertus (2016-12)

Thesis (MSc Food Sc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Rooibos (Aspalathus linearis) is a South African fynbos plant with known health-promoting properties, consumed mainly as a herbal tea. The health-promoting properties of rooibos are associated with its phenolic composition. During herbal tea production, the plant material is “fermented”, which reduces the phenolic content. This led to development of unfermented (green) rooibos tea with increased phenolic content. Conventionally the phenolic compounds are quantitatively and qualitatively analysed using reversed phase (RP) high performance liquid chromatography (HPLC) with diode array detection (DAD) and mass spectrometry (MS). This thesis reports the design of a validated quantitative RP-HPLC-DAD method to enable quantification of four eriodictyol-glucopyranoside isomers that have not been previously quantified in rooibos. External authentic reference standards were used to identify and quantify phenolic compounds with the help of MS, which also confirmed peak purity for the quantified compounds. The plant material of 10 rooibos plants were sub-divided before processing prepare green, semi-fermented and fermented products from each. Mixtures of acetonitrile and ethanol with water (0, 20, 40, 60, 80 and 100%) were evaluated for maximal extraction of phenolics from the plant material. Extracts prepared with 40% acetonitrile representing maximal extraction from the plant material, as well as water extracts (food ingredient extracts), were analysed. For the first time aspalathin was quantified in rooibos with its known degradation products, the eriodictyol-glucopyranoside isomers, iso-orientin and orientin. In addition, a phenylpropanoid and eight other phenolic compounds were also quantified. Complex natural samples such as rooibos contain a range of phenolic compounds, some of which remain unidentified due to challenges in their separation. This led to the development of a comprehensive two dimensional (2D) separation technique to gain indepth qualitative information on the phenolic composition of rooibos. Normal phase (NP) high performance countercurrent chromatography (HPCCC) was used to develop the first dimension (1D) separation. A gradient elution using an ethyl acetate, n-butanol, water solvent system was used to separate the phenolic compounds followed by an extrusion step (60 min analysis time, 48 fractions collected). The second dimension (2D) separation used ultra (U)HPLC to ensure rapid analysis and maximum efficiency. The 2D separation method was developed from the quantitative method with further development aimed at obtaining a high practical peak capacity in a reasonable analysis time. The practical peak capacity was determined as a function of the 2D flow-rate and gradient time, as well as the 1D fraction collection time. The off-line NP-HPCCC×RP-UHPLC method was applied to green and fermented rooibos samples. DAD was used to construct contour plots to elucidate quantitative and qualitative differences, while MS detection was used for tentative identification of previously unidentified compounds. A total of 39 compounds, 18 of which were not previously identified in rooibos, were identified using MS detection in positive ionisation mode. Most of the newly identified compounds were very polar. The combination of NP-HPCCC with RP-UHPLC separations was characterised by a high degree of orthogonality (~80%), contributing to a high practical peak capacity (3293) and improved separation of especially the novel polar phenolic compounds.

AFRIKAANS OPSOMMING: Rooibos (Aspalathus linearis) is „n Suid-Afrikaanse fynbosplant met gesondheidsvoordele wat merendeels gebruik word as „n kruietee. Die gesondheidvoordele van rooibos word geassosieer met sy fenoliese samestelling. Tydens produksie van die tee word die plant materiaal ge-“fermenteer”, wat die fenoliese inhoud verminder. Dit het gelei tot ontwikkeling van ongefermenteerde (groen) rooibostee met hoër fenoliese inhoud. Konvensionele kwalitatiewe en kwantitatiewe analiese van fenoliese verbindings behels omgekeerde fase (RP) hoë-druk vloeistof chromatografie (HPLC) met ultraviolet-fotodiode deteksie (DAD) en massa spektrometrie (MS). Hierdie tesis beskryf die ontwerp van „n gevalideerde RP-HPLC-DAD tegniek om vier eriodictyol-glukopiranosiel isomere vir die eerste keer in rooibos te kan kwantifiseer. Die verbindings is geïdentifiseer en gekwantifiseer deur gebruik te maak van eksterne outentieke standaarde m.b.v. MS, wat ook die suiwerheid van die pieke bevestig het. Plant materiaal van tien rooibos plante is voor prosessering onderverdeel om „n groen, semi-gefermenteerde en gefermenteerde produk van elk te berei. Mengsels van asetonitriel en etanol met water (0, 20, 40, 60, 80 en 100%) is ondersoek om maksimale ekstraksie van fenole uit die plant materiaal te verkry. Ekstrakte gemaak met 40% asetonitriel wat maksimale fenoliese ekstraksie uit die plant materiaal verteenwoordig, asook water ekstrakte (voedselbestandeel ekstrakte), is geanaliseer. Aspalatien is vir die eerste keer saam met bekende oksidasieprodukte, naamlik vier eriodictyol-glukopiranosiel isomere, iso-orientin en orientin, in rooibos gekwantifiseer. „n Fenielpropanoïed en agt ander fenoliese verbindings is ook gekwantifiseer. Komplekse natuurlike monsters soos rooibos bevat verskeie fenoliese verbindings, waarvan sommige nog nie geïdentifiseer is nie a.g.v. uitdagings om hulle te skei. Gevolglik is „n kwalitatiewe, omvattende, af-lyn, twee-dimensionele (2D) skeidingsmetode ontwikkel om in-diepte kwalitatiewe inligting te verskaf. „n Normale fase (NP) hoë werkverrigting vloeistofvloeistof chromatografie (HPCCC) metode is as eerste dimensie (1D) skeiding ontwikkel. Gradiënt eluering met „n oplosmiddelstelsel bewtaande uit etielasetaat, n-butanol en water is gebruik om die fenoliese verbindings te skei gevolg deur verplasing van die stasionêre fase (60 min analisetyd, 48 fraksies opgevang). RP ultra (U)HPLC is gebruik vir die tweede dimensie (2D) skeiding om vinnige analisetyd en effektiewe skeiding te verseker. Die 2D metode is ontwikkel vanaf die kwantitatiewe metode met verdere ontwikkeling met die doel om „n hoë praktiese piekkapasiteit in „n redelike analisetyd te behaal. Die praktiese piekkapasiteit is bepaal as „n funksie van die 2D vloeispoed en gradiënttyd sowel as die 1D fraksie opvangtyd. Die af-lyn NP-HPCCC×RP-UHPLC metode is toegepas op groen en gefermenteerde rooibos monsters. DAD is gebruik om kontoerplotte te genereer waarmee kwantitatiewe en kwalitatiewe verskille bepaal kon word, terwyl MS deteksie gebruik is vir tentitatiewe identifikasie van voorheen onbekende verbindings. Identifikasie van „n totaal van 39 verbindings, waarvan 18 nog nie vantevore in rooibos geïdentifiseer is nie, is moontlik deur gebruik van MS analiese met positiewe ionisasie. Meeste van die nuut geïdentifiseerde verbindings was baie polêr. Die kombinasie van NP-HPCCC en RP-UHPLC skeidings is gekenmerk deur „n hoë graad van ortogonaliteit (~80%), wat bygedra het tot „n hoë praktiese piekkapasiteit (3293) en verbeterde skeiding van veral nuwe polêre fenoliese verbindings.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100168
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