Xanthones and benzophenones from Cyclopia genistoides (honeybush) : chemical characterisation and assessment of thermal stability

Beelders, Theresa (2016-03)

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

Thesis

ENGLISH ABSTRACT: Numerous health-promoting benefits may be derived from the consumption of honeybush tea, a herbal infusion prepared from the leaves and fine stems of the endemic Cape fynbos genus, Cyclopia. These health-promoting benefits are attributed to its phenolic constituents and therefore insight into the nature, quantities and biological activities of individual compounds are required. Information regarding the thermal stability of these compounds is also crucial, as the plant material is subjected to a high-temperature chemical oxidation process (“fermentation”) to develop the sought-after characteristic sensory attributes of the herbal tea product. In this study, the phenolic composition of Cyclopia genistoides, a commercially important species, was comprehensively characterised by high-performace liquid chromatography (HPLC) coupled with diode-array and mass spectrometric detection. A species-specific HPLC method was developed for the qualitative analysis of aqueous extracts prepared from “unfermented” and “fermented” C. genistoides plant material and was subsequently validated for the quantification of 18 phenolic compounds in these types of extracts. The major phenolic constituents included the C-glucosyl xanthone mangiferin (1) and its regio-isomer isomangiferin (2), and the benzophenone 3-β-D-glucopyranosyliriflophenone (3). The presence of novel benzophenone and xanthone derivatives in C. genistoides was demonstrated for the first time, including an iriflophenone-di-O,C-hexoside derivative, present in large quantities. This compound was isolated and unambiguously identified by nuclear magnetic resonance spectroscopy as 3-β-D-glucopyranosyl-4-β-D-glucopyranosyloxyiriflophenone (4) – a novel benzophenone unique to Cyclopia. 3-β-D-Glucopyranosylmaclurin (5), present in small quantities, was also isolated. The isolated benzophenones (4 and 5) exhibited mammalian α-glucosidase inhibitory activity, while 4 and 3 were also marginally effective in increasing glucose uptake in vitro. Compound 4 was ineffective as antioxidant in the DPPH assay, but the most effective in the ORAC assay, compared to the other compounds tested (1, 2, 3, 5). Degradation of compounds 1-4 in C. genistoides plant material under simulated fermentation conditions (80 °C/24 h and 90 °C/16 h) followed first-order degradation kinetics and their thermal stability decreased in the order 4 > 2 > 3 > 1. An increase in the degree of glucosylation significantly increased the thermal stability of the benzophenones, whereas glucosylation at C-4 of the dibenzo-γ-pyrone structure, as opposed to C-2, increased the stability of the tetrahydroxyxanthones in the plant material matrix. This was also confirmed for individual compounds (1-5) in aqueous model solutions (pH 5). Inclusion of 5 in the model systems provided additional insight into structure-stability relationships. Increased B-ring hydroxylation significantly increased the first-order degradation rate constants of the benzophenones. Oxidative coupling of the polyhydroxybenzophenone 5 with the formation of its corresponding xanthones (1 and 2) led to substantial increases in the thermal stability of 1 and 2 compared to that of 5. Increased temperatures increased the degradation rates of all compounds in both the plant material matrix and model solutions. The thermal stability of 1, tested at pH 3-7, was found to be pH-dependent, with increased degradation rates observed at higher pH. Thermally-induced reactions principally included isomerisation, dimerisation and cleavage of O-linked sugar moieties; conversion of all benzophenones to the xanthones occurred to varying degrees. Of special interest was the rapid and predominant conversion of 5 to 1 and 2.

AFRIKAANSE OPSOMMING: Die gebruik van heuningbostee, berei van die eg Suid-Afrikaanse fynbosgenus, Cyclopia, mag verskeie gesondheidsvoordele inhou wat grootliks toegeskryf kan word aan sy fenoliese samestelling. Inligting aangaande die aard, hoeveelhede en biologiese aktiwiteite van individuele verbindings word dus benodig. Die hitte-stabiliteit van die fenoliese verbindings is ook van kardinale belang, aangesien hulle blootgestel word aan hoë temperature tydens chemiese oksidasie (“fermentasie”) van die plantmaterial – ’n proses wat noodsaaklik is om die hoogs-gesogte, karakteristieke sensoriese eienskappe van die kruie tee produk te ontwikkel. In hierdie studie is die fenoliese samestelling van C. genistoides, ’n kommersieël-belangrike spesie, in diepte gekarakteriseer deur middel van hoë-druk vloeistof chromatografie (HPLC) gekoppel aan ultraviolet-diode deteksie en massa spektrometrie. ’n Spesie-spesifieke HPLC metode is ontwikkel vir die kwalitatiewe analise van water ekstrakte wat van “ongefermenteerde” sowel as “gefermenteerde” C. genistoides plantmateriaal voorberei is. Die metode is vervolgens vir die kwantifisering van 18 fenoliese verbindings in hierdie tipe ekstrakte gevalideer. Die hoof fenoliese verbindings is geïdentifiseer as die C-glukosidiese xantoon mangiferien (1) en sy posisionele isomeer isomangiferien (2), sowel as die bensofenoon iriflofenoon-3-C-glukosied (3). Die aanwesigheid van unieke bensofenoon en xantoon-derivate is vir die eerste keer in C. genistoides aangedui, insluitende ‘n iriflofenoon-di-O,C-glukosied afgeleide wat in hoë konsentrasies aanwesig was. Hierdie verbinding is geïsoleer en met behulp van kern magnetiese resonans spektroskopie as iriflofenoon-3-C-glukosied-4-O-glukosied (4) geïdentifiseer – ’n nuwe bensofenoon uniek aan Cyclopia. Maklurien-3-C-glukosied (5), aanwesig in klein hoeveelhede, is ook geïsoleer. Die geïsoleerde verbindings (4 en 5) het inhiberende aktiweite teenoor α-glukosidase, geïsoleer uit rotte, getoon, terwyl 4 en 3 ook marginaal effektief was om glukose-opname in selsisteme te bevorder. Verbinding 4 was onreaktief as antioksidant in die DPPH toets, maar die mees reaktiefste in the ORAC toets, in vergelyking met al die ander verbindings wat getoets is (1, 2, 3, 5). Degradasie van die verbindings 1-4 in C. genistoides plantmateriaal onder gesimuleerde “fermentasie” kondisies (80 °C/24 h en 90 °C/16 h) het eerste-orde degradasie kinetika gevolg en hul hitte-stabiliteit het afgeneem in die volgorde 4 > 2 > 3 > 1. ‘n Toename in die graad van glukosilering het die hitte-stabiliteit van die bensofenone in die plantmateriaal matriks aansienlik verbeter, terwyl glukosilering op C-4 van die dibenso-γ-piroon struktuur, in plaas van C-2, die stabiliteit van die tetrahidroksiexantone verbeter het. Dit is ook vir individuele verbindings (1-5) in waterige model oplossings (pH 5) bevestig. Die insluiting van 5 in die modelsisteme het addisionele insig ten opsigte van struktuur-stabiliteitverwantskappe gebied. ‘n Toename in die aantal hidroksie-groepe op die B-ring van die bensofenone het hul eerste-orde degradasie tempo’s aansienlik verhoog. Oksidatiewe koppeling van die polihidroksiebensofenoon 5 met die vorming van sy ooreenkomstige xantone (1 en 2) het die hitte-stabiliteit van 1 en 2, in vergelyking met 5, merkbaar verbeter. ‘n Toename in reaksietemperatuur het die eerste-orde degradasie tempo’s van al die fenoliese verbindings in die plantmateriaal en in die modelsisteme laat toeneem. Die hitte stabiliteit van 1, getoets by pH 3-7, was ook afhanklik van die pH van die waterige model sisteem, met hoër reaksie tempo’s waargeneem by hoër pH-vlakke. Hitte-geïnduseerde reaksies het hoofsaaklik dimerisasie, isomerisasie en afsplitsting van O-gekoppelde suiker eenhede behels. Omskakeling van die bensofenone na die xantone is ook tot verskeie mates waargeneem, waar die snelle en oorheersende omskakeling van 5 na 1 en 2 veral opvallend was.

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