Analysis of antioxidant activity (AOA) of entries in the 2002 Regional Southernpea Cooperative Trial revealed not only significant differences among entries, but that entries with pigmented (black and red) seed coats were clustered among the highest, cream types were the lowest, while pinkeye and blackeye types were intermediate. Red colored peas were higher in antioxidant activity than black types. These findings provided strong evidence that compounds responsible for pigmentation were involved in AOA. The objectives of the present investigation were to investigate the inheritance of AOA in cowpea and further study the relationship between AOA and seed coat color. Four advanced selections, ARK95-356 (black), ARK98-348 (red), ARK96-918 (cream), and LA92-180 (cream), were crossed in a complete diallel mating design, generating F1, F1', F2, F2', BC1, and BC2 populations. Individual seeds were ground and samples were extracted in methanol and analyzed for AOA using the free radical 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) method. Combining ability tests using Griffing??s Method I Model I indicated presence of highly significant general combining ability (GCA), specific combining ability (SCA), and reciprocal (REC) and maternal (MAT) effects, with pigmented lines exhibiting positive GCA and MAT, while non-pigmented lines exhibited negative GCA and MAT. AOA in the F1 was not significantly different from the maternal parent, with seed coat color also resembling the maternal parent. Segregation for seed coat color was observed in the F2 and F2'. Additive, dominance, and epistatic effects were significant. The broad sense heritability estimate was 0.87. Minimum number of genes responsible for AOA was estimated at about five. Factors governing high AOA appeared to be the same as those responsible for seed coat color, with apparent pleiotropic effects. In conclusion, breeding for high AOA is possible using highly pigmented parental lines.

The effect of currently used antioxidants and sorghum bran in pre-cooked beef patties was evaluated at two different fat levels (10 and 27%, w/w). Pre-formulated ground beef was purchased at a retail store on three different processing days. Within each fat level, ground beef portions were weighed and randomly assigned to control, butylated hydroxanisole (BHA) and butylated hydroxytoluene (BHT) (0.001%), rosemary (0.25%) or sorghum bran (0.25, 0.5 or 1.0%). After mixing in the appropriate antioxidant, 200-g patties were formed, and pH and objective color measurements for each raw patty were performed. Patties were cooked to an internal temperature of 73oC. Cooked patties were packaged and stored at 4oC. Two patties per treatment were sampled after 0, 1, 3 and 5 d of storage and analyzed for 2-thiobarbituric acid reactive substances (TBARS), non-heme iron, pH, instrumental color and trained flavor and texture descriptive attributes. The addition of BHA/BHT and rosemary extract to patties reduced non-heme iron, TBARS values, and cooked beef fat flavor attributes, but increased beef/brothy flavor attributes relative to control patties (P<0.05). As sorghum bran level increased, cooked beef patties were darker (P<0.05), less yellow (P<0.05), had higher non-heme iron (P<0.05), lower TBARS (P<0.05) and higher sandy/gritty (P<0.05) sensory texture. Cooked patties containing antioxidants did not differ in other sensory attributes (P>0.05). Fat mouthfeel of control patties were higher than treated patties (P<0.05). Sorghum bran delayed lipid oxidation by reducing TBARS values and cooked beef fat flavors, and when used at 0.25 and 0.5%, minimal effects on color and sensory attributes were observed. Our results suggested that sorghum bran can be a desirable natural antioxidant in pre-cooked ground beef.

Abstract C-Phycocyanin (C-PC), a water-soluble protein pigment, is one of the major constituents of Spirulina platenisi,. Which is a blue green algae and used in many countries as dietary supplements.The C-PC used in present study is a phycobiliprotein, and consist of two distinguishable protein subunits designated as £\ and £] subunits. The aim of this study was to screen pharmacological effects including analgesic, anti-inflammatory and hepatoprotective effects of C-PC in an animal model. Concomitantly, the antioxidant effect of C-PC was measured to confirm its free radical scavenger activity. The analgesic effect of C-PC measured by tail-flick test shown the latency (sec) of control mice is approximately 4-5 sec. After administration different doses of C-PC (0.1, 1.0, 10 mg/kg), the latency resulted in a dose-dependent phenomenon and prolong from 4 sec to 4.5, 5.6, and 6.9 sec, respectively. The anti-inflammatory effect of C-PC was analyzed in the £f ¡Vcarrageenan injected rat. The hind paw edema percentage change by injection of different doses (0.1~1.0 mg/kg) of C-PC, our results have indicated that C-PC possessed a dose-dependent inhibitory effect on carrageenan-induced edema formation. Intraperitoneal (i.p.) injection of different dose (0.01,0.1 1, 1.0 mg/kg) of C-PC on CCl4 (0.3 ml/kg) challenged rats have indicated that C-PC possessed a significant hepatotoprotective effect and significant decrease the serum levels of glutamate pyruvate transaminase (SGPT) and serum glutamate oxyloacetic transaminase (SGOT). Antioxidant effect of C-PC was measured by both xanthine oxidase method, and cytochrome c method. The IC50 of C-PC measured by xanthine oxidase method is 7.23 ¡Ó0.21 mg/ml. The IC50 value of C-PC estimated by cytochrome c method is approximately 6.1¡Ó0.74 mg/ml. Moreover, the direct active oxygen (¡PO2-) and hydrogen oxide (¡POH) radical scavenging (SOD-like) activity was also confirmed by using advance electron spin resonance (ESR) method. We used the spin-trapping technique to evaluate free radical scavenging ability of C-PC. The IC50 (¡PO2- ) and (¡POH) scavenging activities are 1.08 ¡Ñ 103 unit/g and 7.11 unit/g. Our results have shown the superoxide radical (¡POOH) scavenging activity of C-PC is approximately 3.28 mg/ml. By contrast, IC50 of the hydroxyl radical (¡POH) scavenging activity of C-PC is 4.75 mg/ml. Human hepatoma cells (HepG2 and 2.2.15 cells) and rat glioma C6 cells were cocultured with different concentration of C-PC (1.0, 10 and 20 £gg/ml)for 72 hours, the cell cycle regulation of C-PC was measured by flow cytometer. We found the C-PC possessed a G0/G1 phase-arrest effect on all of the three kind cancer cell line. Whereas, the cell cycle modulations of C-PC are more significant in human hepatoma cells. In conclusion, both our pharmacological screening tests and antioxidant bioactivity assay have indicated that C-PC is a potential antitumor, anti-inflammatory and analgesic agent.

Analysis of antioxidant activity (AOA) of entries in the 2002 Regional Southernpea Cooperative Trial revealed not only significant differences among entries, but that entries with pigmented (black and red) seed coats were clustered among the highest, cream types were the lowest, while pinkeye and blackeye types were intermediate. Red colored peas were higher in antioxidant activity than black types. These findings provided strong evidence that compounds responsible for pigmentation were involved in AOA. The objectives of the present investigation were to investigate the inheritance of AOA in cowpea and further study the relationship between AOA and seed coat color. Four advanced selections, ARK95-356 (black), ARK98-348 (red), ARK96-918 (cream), and LA92-180 (cream), were crossed in a complete diallel mating design, generating F1, F1', F2, F2', BC1, and BC2 populations. Individual seeds were ground and samples were extracted in methanol and analyzed for AOA using the free radical 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) method. Combining ability tests using Griffing??s Method I Model I indicated presence of highly significant general combining ability (GCA), specific combining ability (SCA), and reciprocal (REC) and maternal (MAT) effects, with pigmented lines exhibiting positive GCA and MAT, while non-pigmented lines exhibited negative GCA and MAT. AOA in the F1 was not significantly different from the maternal parent, with seed coat color also resembling the maternal parent. Segregation for seed coat color was observed in the F2 and F2'. Additive, dominance, and epistatic effects were significant. The broad sense heritability estimate was 0.87. Minimum number of genes responsible for AOA was estimated at about five. Factors governing high AOA appeared to be the same as those responsible for seed coat color, with apparent pleiotropic effects. In conclusion, breeding for high AOA is possible using highly pigmented parental lines.

Thesis (DTech(Biomedical Technology ))-- Cape Peninsula University of Technology, 2012 / Antioxidants are an important class of compounds that quench reactive free radical intermediates formed during oxidative reactions. They prevent oxidative reactions in food and protect biological tissues against oxidative damage. Plant phenols and phenolic acids are increasingly becoming a subject of intensive research due to their bioactive properties such as antioxidant, anti-mutagenic, anti-viral and anti-inflammatory activity. Modification of the structure of natural phenolic compounds can be achieved through the use of enzymes in biocatalysis reactions with the potential to enhance the antioxidant capacity of these natural phenolic compounds. The work reported here employed the oxidative enzyme, laccase from Trametes pubescens, in the modification of the antioxidant phenolic molecules, ferulic acid and 2,6-dimethoxyphenol (2,6-DMP) as a way of enhancing their antioxidant capacity. In addition, various phenolic compounds were focused upon for coupling reactions, with the aim to increase the antioxidant capacity of the compounds. The T. pubescens strain was cultured in a 4L airlift reactor and extracellular laccase production was monitored using the standard ABTS assay. The enzyme was isolated and purified from the culture filtrate once optimal enzyme production was detected. The enzyme was purified using standard ammonium sulphate precipitation and dialysis.

Radical mediated autoxidation is a pervasive phenomenon in commercial (i.e. rubbers, fuels, lubricants, etc.) and biological contexts (i.e. lipid peroxidation associated with neurodegeneration, cancer, and aging), which can be strategically managed with radical-trapping antioxidants (RTAs). While various phenol and polyphenol RTAs have enjoyed much academic fanfare, particularly in biochemical circles, there are other RTA scaffolds that tend to be overlooked despite possessing rather promising activity. Among these seldom studied RTA scaffolds is that of the tricyclic aromatic amine phenoxazine. The inherent reactivity of phenoxazines as RTAs was explored using a quantitative linear free energy approach. A library of phenoxazines was synthesized and Hammett correlations developed between BDE/(log kinhPhCl)/Eo and the electrophilicity substituent parameters (Σ σp+) served to unambiguously demonstrate that phenoxazine RTAs collectively represent the most reactive family of RTAs yet reported. The high inherent reactivity of phenoxazine RTAs towards peroxyl radicals necessitated the co- development of an approach to enable the accurate prediction of kinhPhCl based on the inhibition rate constants measured in hydrogen-bond accepting solvent mixtures containing chlorobenzene, 1,4-dioxane, and/or dimethylsulfoxide. The catalytic antioxidant activity of phenoxazines was established in hexadecane autoxidations at elevated temperatures, where they demonstrated superior activity to industry standard alkylated diphenylamine (ADPA), but also an inferiority to phenothiazine. These results combined with amine/nitroxide monitoring experiments serve to re-emphasize some current design considerations for high-temperature RTAs while challenging others. Lastly the activity of these as inhibitors of lipid peroxidation was assessed in liposomes and mammalian cell culture. The kinetics of peroxyl radical-trapping of the various phenoxazines in phosphatidylcholine liposomes enabled the quantitation of H- bonding interactions to the phosphate head groups of the phospholipids on RTA activity – which diminish the kinetics by up to two orders of magnitude relative to non-H- bonding hydrocarbons. The potency of these phenoxazines to subvert ferroptosis corresponds well with their inhibition activity in liposomes which further affirms the role that non-enzymatic autoxidation plays in this form of cell death.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2016 / In this study, antioxidant activity methodologies were evaluated in terms of analytical performances. The total antioxidant activity from Athrixia phylicoides leaves (Bush tea) determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH•) method, cupric ion reducing power (CUPRAC) method and cyclic voltammetry (CV). Folin-Ciocalteu method was used to quantify total phenolic content (TPC) in Athrixia phylicoides leaves. The influence of chemical and physical parameters on the total phenolic content and antioxidant activity determination were investigated. Results from direct sample and crude sample were compared. Antioxidant activity and phenolic content from Athrixia phylicoides leaves were compared with those from commercialised green tea, black tea and rooibos tea using two chosen antioxidant capacity method with acceptable characteristics. Results from the evaluation of the methods demonstrated excellent recoveries (99 to 103%) consistently, good linearity within the calibration concentration range (R2 = 0.997) and repeatable low coefficient of variation < 5% were indicative of good precision except for CV method. The average total antioxidant activity of various extracts of Athrixia phylicoides leaves ranged from 0.039 to 0.122 mg/mL (EC50), 0.031 to 0.233 mg/mL (EC50) and 339 to 429 mV (anodic potential) for DPPH method, CUPRAC method and CV method, respectively. The total antioxidant activity values for each Athrixia phylicoides samples determined by CUPRAC method were higher than the values produced by DPPH and CV methods. The highest antioxidant activities in the DPPH and CUPRAC methods were found in water extracts (direct sample). However, concentrated samples for DPPH method and CV gave a different trend with the methanol extract (crude sample) displaying the highest antioxidant capacity. Increasing the infusion time only increased total antioxidant activity determined by CUPRAC method, whilst DPPH and CV methods had the highest antioxidant activity in the lowest infusion time (3 min). Even though the results are inconclusive with regard to the effect of solid to solvent ratio effect on the total antioxidant activity, 1:150 ratio and 1:100 ratio extracts for both CUPRAC and DPPH methods and for CV gave the highest antioxidant capacities, respectively. The total antioxidant activities in pure antioxidant standards and in the teas were ranked in the following order by both CUPRAC and DPPH methods: Quercetin > catechin > Trolox and Chinese green tea > Joko black tea > Athrixia phylicoides leaves > Laager rooibos tea, respectively. Comparative study showed the necessity of employing more than one method, as each method for the same sample yielded different results. CUPRAC and DPPH methods displayed higher sensitivity and repeatability as compared to the CV method with poor precision.

Thesis (MSc. (Chemistry)) -- University of Limpopo, 2016 / In this study, antioxidant activity methodologies were evaluated in terms of analytical performances. The total antioxidant activity from Athrixia phylicoides leaves (Bush tea) determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH•) method, cupric ion reducing power (CUPRAC) method and cyclic voltammetry (CV). Folin-Ciocalteu method was used to quantify total phenolic content (TPC) in Athrixia phylicoides leaves. The influence of chemical and physical parameters on the total phenolic content and antioxidant activity determination were investigated. Results from direct sample and crude sample were compared. Antioxidant activity and phenolic content from Athrixia phylicoides leaves were compared with those from commercialised green tea, black tea and rooibos tea using two chosen antioxidant capacity method with acceptable characteristics. Results from the evaluation of the methods demonstrated excellent recoveries (99 to 103%) consistently, good linearity within the calibration concentration range (R2 = 0.997) and repeatable low coefficient of variation < 5% were indicative of good precision except for CV method. The average total antioxidant activity of various extracts of Athrixia phylicoides leaves ranged from 0.039 to 0.122 mg/mL (EC50), 0.031 to 0.233 mg/mL (EC50) and 339 to 429 mV (anodic potential) for DPPH method, CUPRAC method and CV method, respectively. The total antioxidant activity values for each Athrixia phylicoides samples determined by CUPRAC method were higher than the values produced by DPPH and CV methods. The highest antioxidant activities in the DPPH and CUPRAC methods were found in water extracts (direct sample). However, concentrated samples for DPPH method and CV gave a different trend with the methanol extract (crude sample) displaying the highest antioxidant capacity. Increasing the infusion time only increased total antioxidant activity determined by CUPRAC method, whilst DPPH and CV methods had the highest antioxidant activity in the lowest infusion time (3 min). Even though the results are inconclusive with regard to the effect of solid to solvent ratio effect on the total antioxidant activity, 1:150 ratio and 1:100 ratio extracts for both CUPRAC and DPPH methods and for CV gave the highest antioxidant capacities, respectively. The total antioxidant activities in pure antioxidant standards and in the teas were ranked in the following order by both CUPRAC and DPPH methods: Quercetin > catechin > Trolox and Chinese green tea > Joko black tea > Athrixia phylicoides leaves > Laager rooibos tea, respectively. Comparative study showed the necessity of employing more than one method, as each method for the same sample yielded different results. CUPRAC and DPPH methods displayed higher sensitivity and repeatability as compared to the CV method with poor precision.

Autoxidation, a radical chain reaction, is largely responsible for the degradation of most man-made and biological materials. These include chemical products such as lubricants, plastics and rubber; as well as biological molecules and membranes within our bodies. The development of means to hinder this process has been a major focus of the petroleum, chemical, pharmaceutical and biotechnology industry over the past century. The two most common strategies to emerge from these efforts have been the use of compounds that either prevent the initiation of autoxidation or trap the propagating radicals, so-called radical-trapping antioxidants (RTAs). Herein, we describe our efforts towards the design and development of extremely potent heterocyclic diarylamine RTAs, and their activity in a variety of applications ranging from isotropic organic solution to mammalian cells. We have elucidated the important structural motifs and mechanistic considerations necessary for the development of next-generation arylamine RTAs. Some of the substituted heterocyclic diarylamines analogs we disclose are among the best inhibitors of high temperature autoxidations described to date. Alongside, we developed novel analytical tools to facilitate the studies and acquisition of results for characterizing RTA activity in organic solutions and lipid bilayers. These fluorescent probes are highly relevant and allow for the determination of hydroperoxide and acid concentrations rapidly, as well as screen (or counter-screen) RTAs in liposomal membranes. Our methodologies address numerous drawbacks from frequently used ‘plug-and-play’ assays and we anticipate they will fill a current unmet need in both industrial and academic laboratories worldwide. Moreover, the recent characterization of ferroptosis – a novel regulated necrotic-like cell-death pathway associated with the accumulation of lipid hydroperoxides – has paved a way forward for studying oxidation induced damage in a biological context. Utilizing our expertise in lipid peroxidation and inhibition, we elucidated the prominent role of autoxidation in the execution of ferroptotic cell death. Alongside, our analytical tools and RTAs have also enabled the identification and characterization of novel ferroptosis inhibitors. Furthermore, this has prompted the development of a correlation to predict anti-ferroptosis activity of small-molecules using simple spectrophotometric assays.

The objective of this work was to produce bioactive peptides from the enzymatic hydrolysis of chicken skin proteins that could be used to treat hypertension, oxidative stress and associated health conditions using a metabolomics approach. Enzymatic hydrolysis of chicken thigh and breast muscle skin proteins was carried out using alcalase or a combination of pepsin/pancreatin (PP) at 1–4% enzyme concentrations. Chicken skin protein hydrolysates (CSPH) were each fractionated by membrane ultrafiltration into different molecular weight peptides (<1, 1–3, 3–5 and 5–10 kDa). Investigation of their in vitro antihypertensive and antioxidant activities showed that alcalase hydrolysates had significantly (p < 0.05) higher ACE-inhibitory activity compared to PP hydrolysates. ACE inhibition was inversely related to size of ultrafiltration membrane peptides. Renin-inhibitory activity varied from 15–36%, and was dependent on the type of protease; PP hydrolysates showed significantly (p < 0.05) higher inhibition than alcalase hydrolysates. CSPHs also significantly (p < 0.05) scavenged antioxidant radicals, increasing with enzyme concentration but decreased as peptide size increased. Kinetics studies revealed that peptide-dependent enzyme inhibition pattern was mostly of the mixed-type for both ACE and renin. Short-term (24 hr) oral administration of 100 mg peptides/kg body weight to spontaneously hypertensive rats (SHRs) led to maximum systolic blood pressure (SBP) reduction of –32.67 and –31.33 mmHg after 6 h for chicken thigh skin hydrolysate and chicken breast skin hydrolysate, respectively. During a 6-week feeding trial, CSPH at 1.0 and 0.5% feed substitutions had significant (p<0.05) antihypertensive effects in SHRs (-36 and -31 SBP reductions, respectively). SBP reduction was directly related to lower plasma ACE but not renin activity. Plasma total antioxidant capacity of the rats was also high. Metabolomics analysis revealed several metabolites with significant changes (≥ 2-fold changes, p < 0.05) in urine and plasma of SHRs fed CSPH, such as Symmetric Dimethylarginine (SDMA), N2-acetyl-L-ornithine, buthionine sulfoximine, uric acid, Vitamin E succinate, L-isoleucine and phospholipids which may be considered important biomarkers/pathways for hypertension and oxidative stress. We conclude that CSPHs may be used as ingredients to formulate functional foods and nutraceuticals for the management of oxidative stress and hypertension-related diseases. / October 2015