Consumers view natural antioxidants as a safe means to reduce spoilage in foods. In addition, these compounds have been reported to be responsible for human health benefits. Identification of these compounds in peanut skins may enhance consumer interest, improve sales, and increase the value of peanuts. This study evaluated analytical methods which have not been previously incorporated for the analysis of peanut skins. Toyopearl size-exclusion chromatography (SEC) was used for separating phenolic size-classes in raw methanolic extract from skins of Gregory peanuts. This allowed for an enhanced analysis of phenolic content and antioxidant activity based on compound classes, and provided a viable preparatory separation technique for further identification. Toyopearl SEC of raw methanolic peanut skin extract produced nine fractions based on molecular size. Analysis of total phenolics in these fractions indicated Gregory peanut skins contain high concentrations of phenolic compounds. Further studies revealed the fractions contained compounds which exhibited antioxidant activities that were significantly higher than that of butylated hydroxyanisole (BHA), a common synthetic antioxidant used in the food industry. This indicates peanut skin extracts are a viable antioxidant source, and that synthetic antioxidants can be replaced with those naturally-derived from peanut by-products. Structures contained in each fraction were identified using high performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion trap mass spectrometry (MSn). Prior to this study, approximately 20 compounds have been identified in peanut skins. The combination of Toyopearl SEC with ESI-HPLC-MSn allowed for the identification of 314 phenolic-based compounds, most of which are newly discovered compounds in peanut skins. Many compounds identified are known to have powerful antioxidant effects, and also have been reported to exhibit numerous beneficial chemical and biological activities, including the treatment of various human health-related conditions. It is evident that peanut skins may be a potential untapped source for the extraction of natural food antioxidants, nutracueticals, and even pharmaceuticals. Because peanut skins are largely a wasted resource to peanut processors, the novel polyphenols identified in this research could have a significant financial impact on the peanut industry. / Ph. D.

Molecular dynamics simulations were performed to study the interactions of bioactive catechins and theaflavins commonly found in tea with lipid bilayers, as a model for cell membranes. Previously, multiple experimental studies rationalized the anticarcinogenic, antibacterial, and other beneficial effects of these compounds in terms of physicochemical molecular interactions with cell membranes. To contribute toward understanding the molecular role of tea polyphenols on the structure of cell membranes, simulation results are presented for seven catechins and three theaflavins in lipid bilayer systems which are both pure (POPC) and representative of HepG2 cancer cells (POPC and POPE). Our simulations show that the catechins and theaflavins evaluated have a strong affinity for the lipid bilayer \textit{via} hydrogen bonding to the bilayer surface, with many of the catechins able to penetrate beneath the surface. Epigallocatechin-gallate (EGCG) and Theaflavin-3,3'-digallate showed the strongest interaction with the lipid bilayers based on the number of hydrogen bonds formed with lipid headgroups. The simulations also provide insight into the functional characteristics of the tea compounds that distinguish them as effective compounds to potentially alter the lipid bilayer properties. The results on the hydrogen-bonding effects may contribute to a better understanding of proposed multiple molecular mechanisms of the action of catechins and theaflavins in microorganisms, cancer cells, and tissues. / Ph. D.

The prevalence of obesity and diabetes is increasing in the United States and abroad and strategies are needed to prevent the progression from an at-risk state to the clinically diagnosed diseases. Flavanols in cocoa powder have been shown to reduce blood glucose concentrations, improve insulin sensitivity, and decrease gut permeability in animals and humans, but it is unknown if this occurs in adults with prediabetes. Therefore, we first hypothesized that an acute dose of cocoa would reduce postprandial glucose and enhance insulin and incretin hormone responses to a mixed meal challenge compared to a placebo. Second, we hypothesized that 15 g cocoa/day for 4-weeks would reduce gut permeability, attenuate endotoxin response to a high fat meal, improve insulin sensitivity, and improve measures of skeletal muscle substrate flexibility in a randomized, double blinded, placebo controlled parallel group design. To test the first hypothesis, 30 overweight or obese volunteers who were at-risk for diabetes completed two meal challenges using a randomized crossover design. Blood samples were collected hourly for 4 hours and were analyzed for glucose, insulin, C-peptide, glucagon-like peptide 1 (GLP-1), and gastric inhibitory peptide (GIP). Cocoa did not influence these measures. However, participants with the lowest fasting blood glucose concentrations were more likely to respond to the cocoa as hypothesized. To test our second hypothesis, 15 overweight or obese adults at risk for developing diabetes consumed either the cocoa or placebo treatments along with a controlled diet for one month. Overall, cocoa did not seem to influence insulin sensitivity, gut permeability, or endotoxin levels, although cocoa may influence skeletal muscle substrate metabolism. In conclusion, the data for both studies suggests that cocoa did not exert substantial effects on the evaluated outcomes. However, the experiments did provide valuable information about incretin hormone levels in adults with impaired glucose tolerance. More research is needed to understand how cocoa can affect glucose homeostasis for overweight or obese adults. / Ph. D.

Ce travaille de thèse concerne la synthèse de dérivés de polyphénols de type flavanol, ellagitannins C-glucosidique et procyanidine, modifiés avec un espaceur comportant une biotine terminale. Cette biotine terminale a permis d’immobiliser ces polyphénols modifiés sur des surfaces SPR, permettant ainsi l’étude d’interactions polyphénol-protéine en temps réel. Ainsi, la topoisomerase II alpha et l'actine fibrilaire ont montré une plus grande affinité pour les polyphenols de type ellagitannins que pour ceux de type flavanol. Nous avons également pu montrer que d’autres protéines (BSA, myoglobine, actine globulaire, streptavidine, collagen type I) n’avaient pas d’interaction avec les flavanols et les ellagitannins. / This work concerns the synthesis of derivatives of polyphenols of the type flavanol, C-glucosidic ellagitannin and procyanidin, which are modified to bear a spacer ending with a biotin unit. This biotin ending unit allowed to immobilize these modified polyphenols on to SPR surfaces, which allowed the study of polyphenol-protein interactions in real time. The proteins topoisomerase II alpha and fibrilar actin showed a higher affinity for the polyphenols of the type ellagitannins than for those of the type flavanol. It was also showed that other proteins (BSA, myoglobin, globular actin, streptavidin, collagen type I) did not interact with either the flavanols or the ellagitannins

Ce travail concerne l'extraction d'enzymes de la famille des polyphénol oxydases à partir de champignons, leur caractérisation biochimique et leur immobilisation dans des matrices solides. Ces enzymes ont tout d'abord été extraites du champignon de Paris (Agaricus bisporus) puis partiellement purifiées. Une étude de leur activité enzymatique, de leur domaine de stabilité et de leur comportement thermique a été effectuée, ainsi que l'identification d'inhibiteurs. Cette approche a été étendue à la polyphénol oxydase de la truffe de désert (Terfezia leonis Tul.). Ces deux enzymes ont ensuite été piégées dans des gels de silice pour le dosage de la dopamine par un biocapteur optique et dans un gel d'alginate pour la dégradation du phénol. / This work is devoted to the extraction of enzymes belonging to the polyphenol oxidase family from mushrooms, their biochemical characterization and their immobilization in solid hosts. These enzymes were first extracted from Paris mushrooms (Agaricus bisporus) and partially purified. A study of their enzymatic activity, stability conditions and thermal behavior was performed, together with the identification of inhibitors. A similar approach was applied to polyphenol oxidase extracted from desert truffle (Terfezia leonis Tul.). These enzymes were then trapped in silica gels for dopamine determination using an optical biosensor and in an alginate gel for phenol degradation.

Strawberries are a rich source of polyphenols which contribute to berry color and plant disease resistance, and have been shown to lower the risk of many chronic when consumed. While a considerable body of work exists on the polyphenolic composition of commercial strawberry (Fragaria x ananassa Duch.), less information is available concerning polyphenols in Fragaria vesca, or Alpine strawberry, considered a model system for the Rosaceae family of crop species. The study of natural and genetically-engineered F. vesca mutants with white fruit can provide unique insight into regulation of metabolic flux through the complex branched phenylpropanoid/flavonoid biosynthetic pathway. Thus, the identity and quantity of major phenolic-derived anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acids, and ellagic acid (EA)-derived compounds, of red-fruited versus white-fruited genotypes of F. vesca and F. x ananassa were compared by high performance liquid chromatography-mass spectrometry. Due to the unknown origin of all but one white-fruited mutant of F. vesca, it was assumed that each resulted from independent mutation events and would exhibit different flavonoid profiles. A total of 27 phenolic-derived compounds were identified. The white genotypes of both species had very low anthocyanin levels. Total content of free EA and its conjugated forms were generally higher in white than in red F. vesca, but were the opposite in F. x ananassa, more in red than in white berries. Differences in content of individual flavonoids and in group totals among the white F. vesca genotypes suggested that they may represent different mutations affecting flavonoid production. Polyphenol profiles of a red and a white cultivar of F. vesca during four fruit developmental stages were determined along with transcriptional analyses of key structural and regulatory genes of the phenylpropanoid/ flavonoid biosynthesis. The final concentration of polyphenolic groups in red versus white F. vesca was due to the differential expression patterns of key pathway genes, especially dihydroflavonol-4-reductase, anthocyanidin synthase, and UDP-glucose-flavonoid-3-O-glucosyltransferase. The efficacy of phenolic compounds were evaluated in an in vitro study for inhibiting growth of Colletotrichum spp. associated with anthracnose fruit rot of strawberry. Only trans-cinnamic, p-coumaric, and ferulic acid inhibited isolates of the pathogen.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / This study describes: 1. The preparation of chloroform, methanol and aqueous extracts of unfermented and fermented rooibos (Aspalathus linearis). 2. The chromatographic fractionation of aqueous rooibos extracts and an investigation into the polyphenol content and antioxidant activity of the fractions. 3. The preparation of ovine adrenal microsomes containing active steroidogenic P450 enzymes, including cytochrome P450 17a-hydroxylase, CYP17, and cytochrome P450 steroid 21-hydroxylase, CYP21. 4. An investigation into the influence of chloroform and methanol extracts of rooibos on the binding of steroid substrates, progesterone and 17-hydroxyprogesterone, to CYP17 and CYP21.

Copper metallothionein (Cu-MT) was extracted from the induced biomass of Aspergillus niger. The crude extract (FI), obtained by cell homogenization, was partially purified by heat treatment (FII) and ultrafiltration (FIII). Further purification of the Cu-MT extract by affinity chromatography resulted in three major fractions, FIVa, FIVb and FIVc, of which fraction FIVc was considered to be the Cu-MT extract fraction. Fraction FIVc was re-chromatography on affinity chromatography and the eluted fraction showed a single peak (FIVc'). Spectrophotometric analysis of fraction FIVc' demonstrated a maximum absorption peak at 268 nm. Native and denatured electrophoretic analysis of fraction FIVc ' showed the presence of a single band with an estimated molecular weight of 9.5 and 10.0 kDa, respectively. Inhibition of mushroom tyrosinase (PPO) by the Cu-MT extracts was investigated, using selected phenolic substrates, including catechin, chlorogenic acid, catechol, 4-methylcatechol, caffeic acid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3-( p-hydroxyphenyl) propionic acid, p-hydroxyphenylpyruvic acid, p- and m-cresol. The results showed that the inhibitory effect of the Cu-MT extract increased with the degree of purification. The results revealed that the Cu-MT extracts were effective inhibitors of PPO activity and the best inhibitory effect was demonstrated with catechin as substrate; however, PPO activity was not inhibited by the Cu-MT extract when p-hydroxyphenylpyruvic acid and p- and m-cresol were used as substrates. The results also showed that the Cu-MT extracts exhibited different types of inhibition, including mixed, competitive and uncompetitive on PPO activity. In addition, the experimental findings indicated that the nature and degree of enzymatic inhibitions by the Cu-MT extracts were dependent upon the structural nature of the substrates as well as the methods including, spectrophotometer and polarograph, used for the detection of enzyme

Two natural enzymatic browning inhibitors, copper-metallothionein (Cu-MT) and polyphenol esterase (PPE), were obtained from A. niger and investigated. Reflectance measurements, expressed as L (lightness variable) and a (red to green degree of color) were used to compare, over extended periods of time, the relative inhibitory effectiveness of Cu-MT and PPE to those observed with the use of selected chemicals including ascorbic acid (AA), citric acid (CA), ethylenediaminetetraacetic acid (EDTA), sodium bisulfite (NaHSO3) and 4-hexylresorcinol (4HR), in the prevention of browning on the cut surfaces of selected food products such as apple and potato slices as well as freshly prepared apple juice. Treatment of each food product required an optimum concentration of the selected inhibitor for the inhibition of browning. (Abstract shortened by UMI.)

It was hypothesized that ester-linked gallic acid glycosides could be absorbed and metabolized in vivo due to the instability of an ester-linked glycosides at neutral pH. To evaluate in vivo absorption of Mangifera Indica, L. var. Keitt polyphenolics, it was first necessary to chemically characterize the compounds present using HPLC-MSn analysis. Mango pulp and extracts were also incubated with a pectinase, cellulase, pectinase with ß-glucosidase activity, and a pure ß-glucosidase to learn the extent of hydrolysis with potential application to enhancing bioavailability as a result of incubation to increase mango juice yield. After which the same extracts were assessed in differentiated Caco-2 cells to discern stability at physiological pH and to characterize metabolite formation in vitro. Finally, human urinary metabolites were characterized after 10 day consumption of 400 g in 11 individuals. Mass spectroscopic characterization and HPLC quantification of mango pulp revealed for the first time two monogalloyl glucosides (MGGs) with distinct differences in their glycoside linkages, with the ester form dominating, as well as the presence of five other phenolic acid glycosides; hydroxybenzoic acid glucoside, courmaric glucoside, ferulic acid glucoside, and sinapic acid mono and di-glucosides. Six oxygenated carotenoid derivatives were identified for the first time in a phytochemical extract, namely, a phytohormone, abscisic acid and its glycoside, two catabolism products of abscisic acid, dihydrophasic acids, and two hydroxy-dimethyl decadiene-dioic acid glucopiranosylesters. Gallotannins ranging from tetra-galloyl glucosides to nona-galloyl glucosides were also identified in the pulp, but not quantified. Clearzyme 200XL and Rapidase AR2000 were the most effective at increasing juice yield of mango pulp due to their pectinase action. Cz reduced the amount of ester-linked MGG by 70% after 4 hours of incubation while Rap hydrolyzed the ether linked MGG. The instability of ester-linked galloyl-glycosides at pH 7.4 was characterized by HPLC-MS and after only four hours of incubation a shift from HWM tannins (>8GG) to LMW (<8GG) occurred and resulted in 25 mg/L free gallic acid. Caco-2 cells metabolized gallic acid, MGG from a mango extract, and a gallotannin extract into O-methyl gallic acid indicating catechol-O-methyl transferase (COMT) as a major metabolizing enzyme for gallic acid. Urinary metabolites were identified by HPLC-MSn in dependant scans. O-methylgallic acid-O-sulfate was identified as the major metabolite 0-6 hours post consumption, followed by O-methylgallic acid at a lower concentration. The presence of gallic acid metabolites in the urine indicates absorption of ester-linked glycosides. Colonic metabolites were detected beginning 3-6 hours after consumption of mango, and were identified as pyrogalloyl derivatives. They are hypothesized to be the products of microbial breakdown of gallotannins. Daily consumption of mango for 10 days increased the concentration of O-methylgallic acid-O-sulfate, but was not significant.