Neurological disorders, such as epilepsy, often result from inherited or newly acquired genetic mutations. However, individuals possessing the exact same disease-causing mutations can exhibit dramatic differences in the severity of their symptoms. These differences can be explained in part by environmental factors, such as the microbes in our gut, that play an important role in the manifestation of disease symptoms. Within the last decade, microbes living in the gut have established themselves as an environmental factor with profound effects on our health and well-being. Of special interest is the relationship between the gut microbiota and neurological disease. The goal of my thesis was to: 1) characterize the gut microbiota composition and 2) understand how the gut microbiota modulates seizure-like behavior using Shudderer, a fruit fly (Drosophila melanogaster) model of epilepsy. Shudderer flies possess a mutation in the voltage-gated sodium channel gene and display seizure-like behavioral abnormalities including spontaneous tremors and heat-induced seizures. We identified differences in the microbial composition of the gut microbiota between Shudderer and control (healthy) flies. We also found that by removing the gut microbiota we could improve seizure-like behavior of Shudderer flies as well as another Drosophila mutant harboring a similar genetic mutation. Together, these findings provide evidence that a bidirectional interaction exists between the gut microbiota and neurological function. Since the molecular and cellular mechanisms controlling basic biological processes are highly conserved between fruit flies and humans, these findings are expected to be applicable to mammalian systems, including humans, and may lead to the future development of novel therapeutics to treat epilepsy and other neurological disorders.

Lifestyle diseases represent a large burden on developed societies and account for much morbidity worldwide. Research has shown that eating a diet rich in fruit and vegetables helps to ameliorate and prevent some of these diseases. Antioxidants found in fruits and vegetables may provide a substantial benefit in reducing disease incidence. This thesis examines the antioxidant properties of resveratrol, piceatannol, and pterostilbene, and the ability of Burkitt's Lymphoma (Raji) cells to uptake these three antioxidants. It also studies the effect of the antioxidants in protecting against DNA damage and their role in DNA repair following oxygen radical exposure in Raji cells. The Oxygen Radical Absorbance Capacity (ORAC) assay was used to measure overall antioxidant contribution as well as the ability of Raji cells to uptake antioxidant following exposure to 2,2’-Azobis(2-methyl-propionamide) dihydrochloride (AAPH). The single cell gel electrophoresis (Comet) assay was used to assess DNA damage and DNA repair rates of cells. Results showed that Raji cells, following oxygen radical exposure, significantly uptake pterostilbene (p < 0.0001), but not piceatannol or resveratrol. Piceatannol provided protection against hydrogen peroxide induced DNA damage, but pterostilbene and resveratrol increased DNA damage following hydrogen peroxide treatment. None of the compounds showed any effect on DNA repair. Overall, this study indicates there is merit for further research into the bioactive roles, including antioxidant capacity, of all three compounds. Such research may provide evidence for the more widespread use of these and other food based compounds for preventing lifestyle diseases.

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a long-lived, redox-active product of protein oxidation, is capable of functioning as both a pro- and anti-oxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Furthermore, as free DOPA is currently the major treatment available for Parkinson?s disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. The hypothesis presented in this thesis is that under physiological conditions, when little redox active transition metal is available, PB-DOPA can function as a redox signalling molecule, triggering an enhancement of cellular antioxidant defences, with a potentially specific role in the regulation of defences targeted against protein oxidation. Physiological levels of PB-DOPA are very low, however the level on individual proteins can change to a proportionally large degree during oxidative stress, an appropriate property for a signalling molecule. In addition, remarkably elevated levels occur in some pathologies, including atherosclerosis. As an initial and commonly formed product of protein oxidation, PB-DOPA is well placed for a signalling role, promoting a significant up-regulation of antioxidant defences in the early stages of oxidative stress, before extensive damage has occurred. As an initiator of antioxidant defences, PB-DOPA would be potentially useful as a therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage. The main objective of this thesis was, therefore, to examine the effect of PB-DOPA on the cellular antioxidant defence system using monocytic and macrophage-like cells, key cells involved in the formation of atherosclerotic plaques. The incorporation of free DOPA into protein during protein synthesis, a process previously shown to occur both in vitro and in vivo, was used to generate PB-DOPA. Neither free nor PB-DOPA were found to be toxic to monocytic or macrophage-like cells in culture, but rather were both capable of protecting these cells from oxidative stress. Free DOPA was shown to be capable of directly scavenging radicals, a process that was thought to be in part responsible for the protection induced during oxidative stress. The presence of free and PB-DOPA up-regulated the activity of catalase and NAD(P)H:quinone oxidoreductase, two enzymatic antioxidants, however the activity of superoxide dismutase and the concentration of oxidised and reduced glutathione were not affected. Whilst it was thought that PB-DOPA would have a specific effect on the activity of antioxidant defences targeted against protein oxidation, proteolysis and bulk chaperone activity were not affected by a combination of free and PB-DOPA. Oxidatively-induced protein aggregation, however, was inhibited by the presence of free and PB-DOPA, suggesting that a more specific chaperone regulation may be taking place. The regulation of gene and protein expression was thought to be one possible mechanism by which PB-DOPA could function as a signalling molecule. To test this hypothesis, the effect of free and PB-DOPA on transcription factor activation and protein expression were investigated. Free and PB-DOPA did not induce the expression or activation of Nrf2, AP-1 or NFJB, three transcription factors thought to be involved in the expressional regulation of genes involved in the antioxidant defence system. However, the expression of a number of proteins, including antioxidants, chaperones and proteins involved in cell cycle progression, were regulated in monocytic and macrophage-like cells following the administration of free DOPA under conditions that resulted in either a high or low level of PB-DOPA generation. The regulated proteins differed between the two conditions, suggesting that the level of PB-DOPA may be a key factor in determining the specific defences targeted. The results presented in this thesis support the hypothesis that PB-DOPA can function as a signalling molecule, triggering an enhancement of cellular antioxidant defences, with a specific role in the regulation of the chaperone system, a key defence targeted against protein oxidation. This thesis may provide the basis for the potential use of free or PB-DOPA as a therapeutic for diseases known to involve oxidative stress or oxidative damage, however more research will be required to determine if the effects demonstrated in this thesis are also capable of occurring in vivo.

Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

The overall aims of this project were to assess the pharmaceutical quality and consistency of commercially available solid dose Sutherlandia frutescens containing products (viz. tablets & capsules) by exploring the use of monitoring the pharmaceutical presentation, flavonoid profile and antioxidant activity levels and to develop/or adapt methods and specifications that may be used for the quality control of such products.Stability tests were conducted on all of the selected SCP. The products were stored under elevated temperatures and environmental humidity conditions and total phenol, antioxidant and chromatographic analysis was conducted on these samples. Samples of each of the SCP were hydrolyzed using HCL and then analyzed using HPLC to test the stability of the flavonoids present in each product. The SCP investigated in this study physically appeared to be of quite good “pharmaceutical” quality, but generally lacked information on the date of manufacture and lacked package inserts, or when these were present they contained insufficient information.Based on the results obtained, it is recommended that, the manufacturers of SCP pay more attention to the information provided on the package inserts and the storage conditions for their products. Further the levels of antioxidant activity, total phenols and flavonoid (sutherlandins A to D) be used as specifications to control the quality of commercially available solid dose Sutherlandia frutescens containing preparations on an individual basis.

Cyclolinopeptides (CLs) are hydrophobic cyclic peptides found in flaxseed. They show immunosuppressive activity, but the biological function of these compounds is largely unknown. This thesis presents the results of studies that were conducted to determine whether CLs could act as antioxidants. In the first study, flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. Individual polar fractions were then added back to the silica-treated flaxseed oil and the oxidative stability index of these samples was determined at 100 °C. A polar fraction containing mainly CLA, β/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.3 ± 0.28 h to 3.2 ± 0.41 h. A positive effect of the polar fraction containing a mixture of CLA and CLD-CLG on the oxidative stability of oil was also observed. The antioxidant mechanism of CLs was investigated in several model systems using electron spin resonance spectroscopy. The concentration of radicals in a DMPO (5,5-dimethyl-1-pyrroline-N-oxide) radical-CLs reaction mixture was monitored. All CLs exhibited dose dependent scavenging activities. CLA–CLC reactions with DMPO-OH at a concentration of 5 mM resulted in a 24–30% decrease in electron paramagnetic resonance (EPR) signal intensity. The reaction of CLs and the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) revealed a more complex interaction than simple radical scavenging. Peptides (CLG and CLG") that contained both tryptophan and methionine showed stronger radical scavenging activity than did CLs containing methionine or methionine sulfoxide but not tryptophan (CLB and CLC). Irradiation of the reaction mixture of DPPH• and peptide with UV light also affected the radical scavenging behaviour. Scavenging activities of DPPH• by CLB, CLC and CLA were enhanced by light, whereas scavenging of DPPH• by the tryptophan containing peptides CLG and CLG″ was not affected. High-performance liquid chromatography with mass spectrometry (HPLC-MS) analysis of the reaction mixtures after a radical scavenging reaction was used to determine the impact of radical scavenging on the peptides. These reactions revealed new masses that were identified and characterized. It was established that DPPH• reacted with the methionine of CLB and with tryptophan in CLG and CLG, by formation of a new covalently-bonded species. Covalent linkages between these amino acids (alone or in peptides) and DPPH• have not been reported previously.

Microwave heating of food has increased rapidly as a food processing technique. This increases the concern that chemicals could migrate from food packaging to food. The specific effect of microwave heating in contrast to conventional heating on overall and specific migration from common plastic food storage boxes was studied in this work. The purpose was especially to determine the interaction effects of different plastics in contact with different types of foods during microwave heating. The study focused on polycarbonate (PC), poly(ethylene terephthalate) (PET), polypropylene homo-polymer (PP), co-polymer (PP-C) and random co-polymer (PP-R) packages. The migration determinations were evaluated at controlled times and temperatures, using a MAE device. The migrants were analyzed by GC-MS and HPLC. ESI-MS was evaluated as a new tool for migration determinations. Food/food simulant absorption and changes in degree of crystallinity during heating were also followed. Significant degradation of antioxidants Irgafos 168 and Irganox 1010 in PP packages occurred during microwave heating of the packages in food simulants containing ethanol, resulting in the formation of antioxidant degradation products. Degradation of PC by Fries chain rearrangement reaction leading to formation of 9,9-dimethylxanthene, and transesterification of PET leading to formation of diethyl terephthalate, were also observed after microwave heating the packages in ethanol and 90/10 isooctane/ethanol. These reactions were not observed during conventional heating of the packages at the same temperature, or after microwave heating of the packages in liquid food (coconut milk). The microwave heating also significantly increased the migration of cyclic oligomers from PET into ethanol and isooctane at 80 °C. Migration of compounds into coconut milk was slightly lower than calculated amounts using the EU mathematical model to predict migration of additives into foodstuffs. The results thus show that the use of ethanol as a fat food simulant during microwave heating can lead to a significant overestimation of migration as well as degradation of polymer or the incorporated additives. Some other detected migrants were dimethylbenzaldehyde, 4-ethoxy-ethyl benzoate, benzophenone, m-tert-butyl phenol and 1-methylnaphthalene. All identified migrants with associated specific migration limit (SML) values migrated in significantly lower amounts than the SML values during 1 h of microwave heating at 80 °C. The antioxidant diffusion coefficients in PP and PP co-polymers showed larger relative differences than the corresponding degrees of crystallinity in the same polymers and PP-R showed by far the fastest migration of antioxidants. / Mikrovågsuppvärmning av mat har ökat markant under de senaste åren. Detta ökar risken för att ämnen i plast migrerar från matförpackningar till mat. Den specifika effekten av mikrovågsvärmning i kontrast till konventionell värmning på total och specifik migrering från vanliga matförvaringslådor av plast studerades i denna avhandling. Syftet var i huvudsak att bestämma interaktionseffekter mellan olika typer av plaster och olika typer av mat under mikrovågsvärmning. Studien fokuserades på förpackningar av polykarbonat (PC), polyetentereftalat (PET), polypropylen homopolymer (PP), copolymer (PP-C) och random copolymer (PP-R). Migreringstesterna utfördes under kontrollerade tider och temperaturer genom att använda MAE. Migranterna analyserades med hjälp av GC-MS och HPLC. ESI-MS-analys utvärderades också som ny analysmetod för migreringstester. Absorption av mat- och matsimulanter samt förändringar i kristallinitetsgrad följdes också. Signifikant nedbrytning av antioxidanterna Irgafos 168 och Irganox 1010 i PP-förpackningar inträffade under mikrovågsvärmning av förpackningarna i etanol-innehållande matsimulanter, vilket resulterade i bildning av nedbrytningsprodukter från antioxidanterna. Nedbrytning av PC genom en Fries omfördelningsreaktion, vilket orsakade bildning av 9,9-dimetylxanten, samt transesterifikation av PET, vilket orsakade bildning av dietyltereftalat, observerades också efter mikrovågsvärmning av förpackningarna i etanol och 90/10 isooktan/etanol. Dessa reaktioner observerades ej efter konventionell värmning av förpackningarna under samma temperatur och ej heller efter mikrovågsvärmning av förpackningarna i riktig mat (kokosmjölk). Mikrovågsvärmningen ökade också betydelsefullt migrering av cykliska oligomerer från PET till etanol och isooktan under 80 °C. Specifika ämnens migrering till kokosmjölk var alla något lägre än migreringsvärden beräknade m. h. a. EU's officiella matematiska modell för förutsägelse av migrering från matförpackningar till mat. Dessa resultat visar att användandet av etanol som matsimulant för fet mat under mikrovågsvärmning kan leda till betydande överestimering av migrering, samt nedbrytning av polymer och additiv i polymeren. Andra detekterade migranter var till exempel dimetylbenzaldehyd, 4-etoxy-etylbenzoat, benzofenon, m-tertbutylfenol och 1-metylnaftalen. Alla identifierade migranter med tillhörande ‘specific migration limit’ (SML)-värden migrerade i betydelsefullt mindre mängder än ämnenas tillhörande SML-värden under 1 h mikrovågsvärmning under 80°C. Diffusionskoefficienterna för antioxidanterna i PP-förpackningarna visade större relativa skillnader än förpackningarnas motsvarande kristallinitetsgrader och migrering av antioxidanter var snabbast från PP-R. / <p>QC 20120530</p>

Flavonoids are of great interest for their antioxidant and health-promoting activities. Ascorbate (vitamin C) has antioxidant activities but also sometimes displays pro-oxidant activities <i>in vitro</i> and reportedly <i>in vivo</i>. This research investigated to what extent flavonoids moderate oxidative stress from vitamin C <i>in vivo</i>.<p>Dietary experiments were conducted in two phases using adult male Wistar rats. First, all animals were maintained for two weeks on a control flavonoid-free diet with the dietary requirement (27 IU) of vitamin E/kg diet. In the subsequent four weeks, the animals were treated in four groups (8 rats/group), being fed the following diets: flavonoid-free control (C), ascorbate-supplemented (7.55 mmol/kg diet) (A), flavonoid-supplemented (2.67 mmol/kg diet) (F) and flavonoids (2.67 mmol/kg diet) plus ascorbate (7.55 mmol/kg diet)-supplemented (T). Measurements were done on in vivo biomarkers of oxidative stress, tissue antioxidants and on tissue in vitro susceptibility to oxidative stress.<p>In the combined feeding of ascorbate plus flavonoids, endogenous thiobarbituric acid reactive substances (TBARS) increased in liver by 114%. No effects of dietary ascorbate or flavonoids were seen on endogenous TBARS in brain or heart, or on plasma thiols or erythrocyte fragility.<p><i>In vitro</i>, the susceptibility to TBARS formation of liver homogenate (incubated for 60 min at 37ºC in air) showed a significant 60% increase in ascorbate-fed animals compared to control, but no increase in animals fed ascorbate plus flavonoids, suggesting that the additional feeding of flavonoids helped to prevent the increase produced by ascorbate-feeding. Incubation of liver mitochondria with 300 µM ascorbate in vitro produced a large (2-7 fold) increase in TBARS, but there was no difference among mitochondria from the different feeding groups.<p>The ability of flavonoid-feeding in protecting against oxidative stress from ascorbate in vivo could not be demonstrated in this study, even showing pro-oxidant effects of flavonoids in combination with ascorbate in liver. However, in vitro tests in liver suggest a protective effect of flavonoid-feeding against susceptibility to oxidative stress from ascorbate. Further investigations are needed in order to resolve the differences observed in vitro and in vivo and to determine the endogenous effects of specific flavonoids under ascorbate-induced oxidative stress.

Oxidation plays an important role in the basic processes of life, such as the production of energy and phagocytosis employed by the immune system. However, when an imbalance between oxidants and antioxidants exists in vivo, oxidation can become uncontrolled and result in diseases such as arthritis, cancer, artherosclerosis, and Alzheimer’s Disease. Dietary antioxidants including polyphenolic compounds, proteins, and peptides have been identified as being physiologically functional foods capable of contributing to the restoration of this oxidant-antioxidant balance. The objective of this study was to explore the production of antioxidant soy peptides from a commercially available soy protein isolate (SPI) by enzymatic hydrolysis in a process similar to that occurring in the human digestive tract. In this study Archer-Daniels Midland SPI PRO-FAM 974 was used as a raw material for the production of antioxidant soy peptides. The digestion consisted of enzymatic digestion of the SPI (3.12 wt %) with pepsin (37ºC, pH 1.5) and/or pancreatin (40ºC, pH 7.8) either individually or sequentially. The enzyme concentration and digestion time for each enzyme was optimized using a 2^4 factorial experimental design to produce the greatest concentration of peptides quantified in PheGly equivalents by the OPA assay. A maximum peptide concentration of approximately 65 mM PheGly equivalents was achieved in the follow-up digests resulting from this factorial design model, using pepsin (0.15 g/L, 15 minutes) and pancreatin (4.5 g/L, 120 minutes) sequentially to digest the SPI. Fractionation of the peptides by sequential dead-end membrane ultrafiltration with molecular weight cut-offs (MWCO) of 3 kDa and 1 kDa was performed to produce peptide fractions with increased antioxidant capacity. The permeate flux as a function of time was fit to empirical models, revealing that the membrane fouling resulting in the permeate flux decline is largely reversible and most likely the result of cake filtration. Antioxidant capacity was quantified by the DPPH, FCR, and ORAC assays to determine the electron-donating and proton-donating capacities of the soy peptides. The electron-donating DPPH assay was not suitable to quantify the antioxidant capacity of the soy peptides due to poor peptide solubility in the assay media and sensitivity. The electron-donating FCR assay and the proton-donating ORAC assay were used to distinguish between the ultrafiltration and digestion conditions employed to produce the soy peptides and the antioxidant capacity was quantified in equivalence to the standard antioxidant Trolox. The soy peptide fraction with the greatest antioxidant capacity was produced by enzymatic digestion with pancreatin (4.5 g/L, 120 minutes) alone and had a molecular weight cut-off of between 3 kDa and 1 kDa. This fraction had an equivalent antioxidant capacity of approximately 190 mg Trolox/g sample in the ORAC assay and approximately 180 mg Trolox/g sample in the FCR assay. A preliminary linear model for the optimum digestion and ultrafiltration conditions for the production of antioxidant peptides with the greatest ORAC antioxidant capacity was also developed. The model includes a positive pancreatin digestion time term and a negative pepsin digestion time term. No ultrafiltration terms were found to be significant in this preliminary model, but a large constant term persisted. In conclusion, the enzymatic digestion of commercially available SPI with pancreatin and fractionated by ultrafiltration successfully produced a soy peptide fraction with increased antioxidant capacity.

Flavonoids are of great interest for their antioxidant and health-promoting activities. Ascorbate (vitamin C) has antioxidant activities but also sometimes displays pro-oxidant activities <i>in vitro</i> and reportedly <i>in vivo</i>. This research investigated to what extent flavonoids moderate oxidative stress from vitamin C <i>in vivo</i>.<p>Dietary experiments were conducted in two phases using adult male Wistar rats. First, all animals were maintained for two weeks on a control flavonoid-free diet with the dietary requirement (27 IU) of vitamin E/kg diet. In the subsequent four weeks, the animals were treated in four groups (8 rats/group), being fed the following diets: flavonoid-free control (C), ascorbate-supplemented (7.55 mmol/kg diet) (A), flavonoid-supplemented (2.67 mmol/kg diet) (F) and flavonoids (2.67 mmol/kg diet) plus ascorbate (7.55 mmol/kg diet)-supplemented (T). Measurements were done on in vivo biomarkers of oxidative stress, tissue antioxidants and on tissue in vitro susceptibility to oxidative stress.<p>In the combined feeding of ascorbate plus flavonoids, endogenous thiobarbituric acid reactive substances (TBARS) increased in liver by 114%. No effects of dietary ascorbate or flavonoids were seen on endogenous TBARS in brain or heart, or on plasma thiols or erythrocyte fragility.<p><i>In vitro</i>, the susceptibility to TBARS formation of liver homogenate (incubated for 60 min at 37ºC in air) showed a significant 60% increase in ascorbate-fed animals compared to control, but no increase in animals fed ascorbate plus flavonoids, suggesting that the additional feeding of flavonoids helped to prevent the increase produced by ascorbate-feeding. Incubation of liver mitochondria with 300 µM ascorbate in vitro produced a large (2-7 fold) increase in TBARS, but there was no difference among mitochondria from the different feeding groups.<p>The ability of flavonoid-feeding in protecting against oxidative stress from ascorbate in vivo could not be demonstrated in this study, even showing pro-oxidant effects of flavonoids in combination with ascorbate in liver. However, in vitro tests in liver suggest a protective effect of flavonoid-feeding against susceptibility to oxidative stress from ascorbate. Further investigations are needed in order to resolve the differences observed in vitro and in vivo and to determine the endogenous effects of specific flavonoids under ascorbate-induced oxidative stress.