Quercetin (Q), a water-soluble flavonoid that is ubiquitous to foods of plant origin is postulated to protect against colon cancer due to its antioxidant activity. In contrast, we have shown that a dietary combination of fish oil (FO; n-3 fatty acids) and pectin may protect against colon cancer by decreasing endogenous antioxidant enzyme activities leading to increased reactive oxygen species (ROS), an inducer of apoptosis. We hypothesized that adding an antioxidant to a FO diet may negate the beneficial effects of FO by counteracting FO effects on colonocyte redox status. To test this, we provided 40 rats with FO or CO (fiber = pectin) diets with Q being 0 or 0.45% of the diet for 10 wk. All rats were injected with azoxymethane (AOM) on d 21 and 28. Measurements included: aberrant crypt (AC) enumeration (colon cancer marker); apoptosis (TUNEL assay); catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities; reduced and oxidized glutathione concentrations (GSH/GSSG); and oxidative DNA damage (8-OHdG adducts). AC numbers were lower in FO vs CO rats (p<0.0001), but tended to increase for FO diets containing Q (P<0.098). The apoptotic index was higher (p<0.0001) when Q was added to the FO and CO diets. Total SOD (lipid main effect, p=0.0136) and GPX activity (p=0.0025) was elevated in CO rats. CAT activity was higher (p=0.0204) in FO rats, however Q diminished this effect. GSH was not affected by diet; yet, GSSG accumulated (p=0.0554) in CO rats with Q as compared to CO rats without Q. The GSH/GSSG ratio was lower (p=0.0314) in CO rats than in FO rats. There was no difference in 8-OHdG adduct levels in FO vs CO rats, however, Q decreased 8-OHdG adducts in CO rats (p=0.0428). Despite increasing apoptosis, Q did not significantly lower AC formation. These data suggest that the distinct effects of the CO/Q and FO/Q combinations are functioning through different mechanisms to induce apoptosis. The long-term consequences of adding antioxidants such as Q to a diet thought to exert its anticancer effect through a pro-oxidant mechanism are unknown and deserve further study.

Past research conducted by our lab demonstrated that potatoes contain significant levels of phytochemicals important to human health. However, since potatoes are not consumed raw, it is important to determine the effects of processing on these levels. Therefore, the changes in carotenoid content, antioxidant activity, and phenolic content were investigated using combinations of cultivars, cooking methods, storage treatments, and low-dose ionizing irradiation. Carotenoid content was measured via absorbance at 445 nm, 450 nm, and HPLC identification. Antioxidant activity was measured initially and at stabilization via the DPPH method and phenolic content was measured via the Folin method and HPLC identification. Microwaved, baked, fried, and raw potato samples contained more carotenoids than boiled samples. The samples microwaved, baked, and fried contained higher antioxidant activity and phenolics than the boiled or raw samples. However, the compound quercetin dihydrate appeared to decrease with cooking. Carotenoids, antioxidant activity, and phenolics appeared to decrease with storage; however, high storage temperatures and long storage times were believed to cause a dehydration and concentration of compounds, which caused levels to be equal to or greater than before storage. However, this decreasing trend was not linear and there were multiple significant interactions. The compound chlorogenic acid appeared to be quite sensitive to high temperature storage. Irradiation dose appeared to have only a minor, if any, effect on carotenoid levels. The interaction between storage time and irradiation dose was very influential on antioxidant activity. In early stages of storage, higher doses of irradiation had greater antioxidant activity, while, with continued storage, low doses had higher antioxidant activity. Exposure to irradiation appeared to cause an increase in phenolic content, determined by the Folin method. There may be a stimulation, induction, or release of some compounds due to processing; however, its magnitude is not believed to be as great as genetic control. The effects of processing can not be denied and should continue to be investigated. Future studies investigating the health properties of fruits and vegetables, particularly potatoes should include processing effects.

Four in vitro models were used to measure the antioxidant activity of 11 citrus phytochemicals. The citrus limonoids and bergapten showed very weak antioxidant activity. The flavonoids demonstrated mild, to moderate, to strong antioxidant activity. In addition to some other commonly accepted structural features our data indicated that the hydroxyl group in position 6 of ring A could also increase the antioxidant activity of flavonoids. Compared with the active flavonoids, limonoids are highly oxygenated triterpenoids, with fewer hydroxyl groups to stabilize unpaired electrons (or scavenge free radicals). Bergapten lacks a hydroxyl group. This is the first report on the antioxidant activity of limonoids and neoeriocitrin. A feeding study using Syrian hamsters was followed to determine the effect of citrus limonoids and flavonoids on plasma cholesterol. Hamsters fed with limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp significantly inhibited the increase of LDL/HDL-cholesterol (36.6%, 52.9% and 57% respectively) compared with the basal control (65.8%) and the pectin control (70%). Furthermore, hamsters fed with limonin had significantly larger LDL particle size (21.21 nm) compared with the control group (19.96 nm). Further studies demonstrated that LDLs from hamsters fed with limonin and limonin 17-Beta-D-glucopyranoside were less susceptible to oxidation. These data suggest that limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp have potential inhibitory effects against atherogenesis. Supercritical CO2 (SC-CO2) was attempted to extract limonoids from grapefruit seeds and molasses. Limonin aglycone was successfully extracted with SC-CO2 directly from grapefruit seeds with the yield of 6.3 mg/g seeds at 48.3 MPa, 50&#730;C and 60 min with CO2 top feeding; and the limonin glucoside was extracted using SC-CO2 and ethanol as co-solvent from the defatted seeds with the yield of 0.73 mg/g seeds at 42 MPa, 52&#730;C, 45% ethanol (XEth=0.45) and 40 min with CO2 top feeding; and limonin glucoside also was extracted using SC-CO2 and ethanol with the yield of 0.61mg/g grapefruit molasses at 48.3 MPa, 50&#730;C and 10% ethanol (XEth=0.1), 40 min with CO2 top feeding. CO2 flow rate was around~5 l/min in experiments. The results demonstrated SC-CO2 extraction of limonoids from citrus juice industry byproducts has practical significance for future commercial production.

The purpose of this study was to investigate the effects of 6 weeks of hydroxytyrosol (HT) supplementation on markers of muscle damage in healthy, recreationally active men before and throughout acute aerobic exercise bouts. Using a randomized, double-blind, repeated-measures, placebo-controlled design, sixty-one (n = 61) subjects (21.46 ± 0.22 yrs, 179.46 ± 0.79 cm, 78.91 ± 1.19 kg) consumed either a high dose (HI) HT supplement (150 mg HT), a low dose (LO) HT supplement (50 mg HT), or a placebo (PLA) every day for 6 weeks. Throughout the course of the study, the subjects performed four time trial rides (TT1-TT4) on a cycle ergometer. TT1 occurred before supplementation, TT2 halfway through the supplementation period, and TT3 and TT4 occurred in the sixth week and final two days of supplementation. Blood was drawn prior to (pre) and just before termination (end) of each time trial to measure markers of muscle damage during exercise. We observed that endurance exercise increased indicators of muscle damage, CPK and myoglobin, but an association between HT treatment and reduced muscle damage indicators during exercise were not demonstrated. However, the HT supplementation for 6 weeks in recreationally-active males improved time trial performance in the HT treatment groups over the course of the study and this improvement was accompanied by a lower increase in myoglobin concentration in blood in the HI treatment group than in the LO treatment group. Also, performance was improved after 6 weeks in the PLA group. This improvement was associated with an increase in rating of perceived exertion (RPE). RPE was not increased in either the LO and HI treatment groups, although time trial performance was significantly improved. It is possible that HT can improve performance by altering perception of effort. We conclude that chronic and acute HT supplementation did not reduce markers of muscle damage in this population at rest, during, or following exercise, but improved aerobic performance. / text

Consumers’ demands for ω-3 polyunsaturated fatty acids (PUFAs) are at all-time high. Algae, a common source of PUFAs, and antioxidants are both used as supplements in livestock feeds, are known to affect the overall quality of meat. To implement PUFA deposits into broiler meat, this study evaluated combining antioxidants and algae in broiler feed to enhance the breast meat quality. Broilers were fed diets supplemented with 50 IU Vitamin E or 200 g/ton EconomasE (EcoE, an antioxidant pack) plus 10 IU Vitamin E, with or without 0.5% algae extract (SP-1). The feed oil was partially oxidized soybean oil (POV: 86 mEq of O2/kg). The feed supplementation with combined SP-1 and EcoE increased meat lipid oxidation but had no effect on protein. This combination supplement substantially reduced (P < 0.05) meat exudation during refrigerated storage while no evident differences were seen on cooking loss or tenderness between diets. Meat from SP-1 supplemented diets was found less acceptable than meat from other diets due to detected off-flavors. The results indicate that EcoE at a supplementation level other than 200 g/ton may be required to overcome off-flavors of broiler meat due to feed incorporation of 0.5% SP-1 with oxidized oil.

The fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum) impacts production and yield in one of Canada’s number one crops, canola (Brassica napus). Unfortunately, few cultivars show any tolerance to this devastating fungal pathogen. Thus, understanding how the plant responds to this aggressive fungus at the cellular level will facilitate the identification of genes and gene products responsible for improved plant performance. While our understanding of the host pathogen interaction is becoming clearer, there is remarkably little information available for Sclerotinia, especially its pathogenicity in canola. Moreover, we know nothing about how this interaction is specified at the cellular, physiological or molecular level directly at the site of infection in mature leaves following petal inoculation. Thus, we compared differences in plant structure, antioxidant response, and genes involved in the salicylic acid, jasmonic acid and ethylene defense pathways in a susceptible cultivar, Westar, and a previously described tolerant cultivar, Zhongyou821 (ZY821). Our data showed that at the cellular level, ZY821 was able to suppress the Sclerotinia penetration. The ascorbate-glutathione pathway and resistant signaling pathways were all associated with the canola defense response to S. sclerotiorum, while stronger antioxidant and signaling pathways responses were observed in ZY821 leaves at the site of infection. Also, transcriptional regulators not previously associated with plant defense in the Arabidopsis- S. sclerotiorum pathosystem were identified through bioinformatics approaches. By comparing plant susceptibility to S. sclerotiorum between Arabidopsis wild type and seven loss-of-function mutants, I found transcription factor JAM2 might be involved in plant tolerance to S. sclerotiorum. / October 2014

To maintain intracellular redox homeostasis, genes encoding many endogenous antioxidants and phase II detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis- antioxidant responsive elements (AREs) in their promoter regions. Nrf2 has emerged as the pivatol transcription factor responsible for ARE-dependent transcription, and has been shown to play critical roles in hepatotoxicity, chemical carcinogenesis, pulmonary inflammatory diseases, neurodegenerative diseases and aging. Therefore, understanding the molecular mechanism of the Nrf2-dependent cytoprotective system is important for development of drugs for therapeutic intervention.Nrf2 is targeted by Keap1 for ubiquitin-mediated degradation under basal conditions. Upon oxidative stress, distinct cysteine residues of Keap1 are alkylated, leading to inhibition of Keap1 and activation of Nrf2. However, it was not clear how Nrf2 is re-entered into the repression status when redox homeostasis is re-achieved. In this dissertation, we establish that the post-induction repression of Nrf2 is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination/ degradation machinery. We show that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2 signaling; ubiquitination of Nrf2 is carried out in the cytosol; Keap1 nuclear translocation is independent of Nrf2; and the Nrf2-Keap1 complex does not bind the ARE. Collectively, these results suggest that Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE and mediates nuclear export of Nrf2 followed by ubiquitination and degradation of Nrf2 in the cytoplasm.In addition to Keap1-mediated negative regulation, we identified a novel positive regulatory mechanism of Nrf2 mediated by transcription co-activator p300/CBP. We show that p300/CBP directly binds and acetylates Nrf2 in response to oxidative stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner both in vitro and in vivo. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

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.

The total phenolic content (TPC) of common vegetables grown in Ontario was determined by the Folin-Ciocalteu method, and it was found that the broccoli inflorescence had an exceptionally high TPC on average value, followed by cabbage, onion, potato and carrot. The TPC values of darkpurple potatoes and carrots were higher than the common potatoes and carrots. Positive correlations between the TPC and TAA were observed with varied degrees in all vegetables. Choice of cultivar and production practices can be used to increase TPC and TAA in a wide range of vegetables. Insecticide application did not influence the TPC and TAA of broccoli leaves and flowers. Higher N rate decreased the TPC and TAA of cabbage cultivar ‘Huron’ and of carrot. Fungicide and biofungicide applications did not influence TAA in carrots. Fertilizer applications did not influence the TAA of onions, but there was a decrease in TPC. The rate of MAP (mono ammonium phosphate 52% P2O5) affected the TAA of onions, but the influence was inconsistent between two antioxidant assays. High temperature with possibly high rainfall capacity occurred in the year increased the TPC and TAA of most studied vegetable crops. / Ontario Ministry of Agriculture, Food and Rural Affairs/University of Guelph Sustainable Production Systems Program