Thesis (MScFoodSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The phenolic composition of Cyclopia species is believed to be partially responsible for the numerous health
promoting properties associated with their extracts. Current quality control measures do not accommodate
variation in phenolic profiles of Cyclopia species. In this study, comprehensive high performance liquid chromatography
(HPLC) methods were developed for the improved characterisation of the phenolic composition of
aqueous extracts of two Cyclopia species (C. subternata and C. maculata). The methods were developed to be
suitable for both routine quantitative analysis on conventional HPLC instrumentation, and the construction of
chromatographic fingerprints for further data analysis. The latter entailed similarity analysis and prediction of
total antioxidant capacity (TAC).
Using a methodical approach, two separate HPLC methods, using diode array detection (DAD), were developed
and validated for the analysis of aqueous extracts prepared from unfermented (green) and fermented
plant material of C. subternata and C. maculata. Separation was achieved using the same method parameters
(column, temperature, mobile phases), except for differing mobile phase gradients. Hyphenation of
the developed HPLC methods with mass spectrometry (MS) and tandem MS allowed the confirmation of
phenolic compounds previously identified in Cyclopia, and the tentative identification of several additional
compounds in Cyclopia species, which are reported here for the first time. These included apigenin-6,8-di-
C-glucoside, 3-hydroxyphloretin-30,50-di-C-hexoside, eriodictyol-di-C-glucoside, iriflophenone-di-O,C-hexoside,
hydroxymangiferin and hydroxyisomangiferin. Subsequently, a large number of aqueous extracts of randomly
selected green C. subternata (n = 64) and C. maculata (n = 50) plant material samples were analysed. Large
quantitative variations were observed on intra- and inter-species levels. Cyclopia maculata extracts contained
almost six times more mangiferin than extracts from C. subternata.
HPLC-DAD analysis produced duplicate fingerprints for each extract which were consequently used for
further analysis. The chromatographic fingerprint of a bioactive extract of each species was included in the
respective data sets. Similarity analysis was conducted between the fingerprints from the randomly selected
extracts and the corresponding active extract. For each species several extracts were determined to have similar
“activity” as that of the active extract (n = 15 for C. subternata and n = 45 for C. maculata). Compounds
potentially responsible for the activity were tentatively identified with the aid of principal component analysis
(PCA) in combination with similarity analysis. PCA was more effective in identifying small differences between
fingerprints than similarity analysis based on the correlation coefficients (r) alone.
Furthermore, multivariate data analysis was used to construct partial least squares (PLS) regression models
for the prediction of TAC from fingerprint data of each species, and available data from two microplate TAC
assays. The construction of the models was successful with reasonable errors (< 10%), and permitted the
determination of compounds of interest for future research. These included compounds of known identity that
had large positive contributions toward the predictions of TAC, or unknown compounds that had small UV signals, but relatively large positive contributions to the models. / AFRIKAANSE OPSOMMING: Die talle gesondheidbevorderingseienskappe van ekstrakte van Cyclopia spesies word gedeeltelik geassosieer met
hul fenoliese samestelling. Huidige kwaliteitskontrolemaatreëls is nie in staat om die variasie wat in die fenoliese
profiele van die spesies voorkom, te akkommodeer nie. Omvattende hoë druk vloeistof chromatografiese (HPLC)
metodes is vir twee Cyclopia spesies, naamlik C. subternata en C. maculata, in hierdie studie ontwikkel vir beter
karakterisering van die fenoliese samestelling van waterekstrakte van dié spesies. Die metodes moes ook geskik
wees vir roetine analise van C. subternata en C. maculata ekstrakte op konvensionele HPLC instrumentasie,
en vir die opstel van chromatografiese vingerafdrukke (fenoliese samestellingsprofiele) vir verdere data analise,
soos gelykvormigheidsanalise en die voorspelling van die totale antioksidantkapasiteit (TAC).
Twee HPLC metodes, wat van ’n ultraviolet-diode detektor (DAD) gebruik maak, is ontwikkel deur ’n
sistematiese benadering te volg. Die onderskeie metodes is vir die ontleding van waterekstrakte van groen (ongefermenteerde)
en gefermenteerde plantmateriaal van C. subternata en C. maculata gevalideer. Ongeag die
spesie is optimale skeiding met dieselfde kolom, mobiele fase en kolom-temperatuur bereik, maar met verskillende
mobiele fase gradiënte. Analise met massaspektrometrie (MS) en tandem MS het die teenwoordigheid
van fenoliese verbindings, wat voorheen in Cyclopia spesies geidentifiseer is, bevestig. Verder is ook ’n aantal
verbindings vir die eerste keer in Cyclopia tentatief geidentifiseer. Dit sluit apigenien-6,8-di-C-glukosied, 3-
hidroksiefloretien-30,50-di-C-heksosied, eriodiktiol-di-C-glukosied, iriflofenoon-di-O,C-heksosied, hidroksiemangiferien
en hidroksie-isomangiferien in. Vervolgens is ’n groot aantal ewekansig gekose waterekstrakte van beide
groen C. subternata (n = 64) en C. maculata (n = 50) plantmateriaal geanaliseer, en groot kwantitatiewe variasie
op intra- en inter-spesievlak waargeneem. Cyclopia maculata ekstrakte het byvoorbeeld byna ses maal die
mangiferieninhoud van C. subternata ekstrakte gehad.
HPLC-DAD analise van die ekstrakte het duplikaat vingerafdrukke van elke ekstrak geproduseer, wat vir
verdere data analise gebruik is. Die chromatografiese vingerafdruk van ’n bioaktiewe ekstrak van elke spesie
was by die onderskeie datastelle ingesluit. Gelykvormigheidsanalise is tussen vingerafdrukke van die ewekansig
gekose ekstrakte en die ooreenstemmende aktiewe ekstrak uitgevoer. Vir elke spesie is ’n aantal “aktiewe”
ekstrakte aangewys (n = 15 vir C. subternata en n = 45 vir C. maculata). Die verbindings wat potensieel
verantwoordelik kan wees vir die aktiwiteite is met behulp van hoofkomponentontleding (PCA) in kombinasie
met gelykvormigheidsanalise, tentatief aangewys. PCA was egter meer effektief om klein verskille tussen vingerafdrukke
aan te dui, in vergelyking met gelykvormigheidsanalise wat slegs op die korrelasie koëffisiënt (r)
gebaseer is.
Meerveranderlike data analiese is gebruik om “gedeeltelike kleinste kwadrate” (PLS) regressiemodelle, vir
die voorspelling van die TAC van beide spesies te bou. Die voorspelling is gebaseer op hul vingerafdruk data en
TAC data van twee TAC mikroplaat metodes. Die model-konstruksie was suksesvol met aanvaarbare voorspellingsfoute
(< 10%). Verbindings van belang kon ook bepaal word. Dit sluit bekende verbindings in wat groot positiewe bydraes ten opsigte van die voorspelling van TAC getoon het, asook ongeidentifiseerde verbindings
wat klein UV-seine getoon het, maar relatiewe groot bydraes tot die modelle gehad het.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85773 |
| Date | 12 1900 |
| Creators | Schulze, Alexandra Elizabeth |
| Contributors | De Beer, D., De Villiers, A., Manley, M., Joubert, E., 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 |
| Type | Thesis |
| Format | xvii, 132 p. |
| Rights | Stellenbosch University |
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