Evaluation of Antioxidant Capacity (ABTS and CUPRAC) and Total Phenolic Content (Folin-Ciocalteu) Assays of Selected Fruit, Vegetables, and Spices.

Antioxidant (AOX) capacity assays are important analytical tools, used worldwide to measure the AOX capacities of various food commodities. Although numerous protocols have been published to ascertain AOX capacities, there are increasing concerns about the reliability of many of these assays. Poor correlation of results between various assays, as well as problems with reproducibility, consistency, and accuracy, is to blame. Published AOX assays also differ markedly from each other by employing different reaction conditions, using different extracting solvents, and applying dissimilar quantification methods. In this study, AOX capacities of a range of fruit, vegetables, and spices, commonly consumed and of commercial importance in Australia and worldwide, were measured in both hydrophilic and lipophilic solvents by using two different assay systems. As the polyphenolic compounds present in any sample matrix are the main contributors to its AOX properties, the commodities were also analysed for total phenolic content (TPC), again using both solvent systems. Analysis of the results from the current study with values from the published literature exposed the challenges that make direct comparison of any quantitative results difficult. However, a strong mutual correlation of our assay results facilitated a meaningful comparison of the data within the laboratory. Concurrent use of lipophilic and hydrophilic solvents made the results more reliable and understandable. Findings from this study will aid to address the existing challenges and bring a more rational basis to the AOX capacities. This unique analytical approach also provided a platform to build an internal reference database for the commonly consumed and commercially important food commodities with the potential to broaden the scope into a database for similar food matrices.

Organic acids in Kakadu plum (Terminalia ferdinandiana): The good (ellagic), the bad (oxalic) and the uncertain (ascorbic).

The phenolic ellagic acid (EA) is receiving increasing attention for its nutritional and pharmacological potential as an antioxidant and antimicrobial agent. The Australian native Kakadu plum (Terminalia ferdinandiana) fruit is an abundant source of this phytochemical. The fruit also contains large amounts of vitamin C (mainly as ascorbic acid, AA) and possibly the undesirable oxalic acid (OA). Regular consumption of high oxalate foods poses a variety of health risks in humans including interference with calcium absorption and kidney stone formation. Oxalate is also the end-product of AA metabolism so that consumption of fruit with heightened AA content has the potential to elevate urinary oxalate levels. The aims of this study were to investigate the distribution of EA and the presence of other bioactives in other Kakadu plum tissues. Chemical analysis of Kakadu plum fruit and leaves for EA (free and total), OA (water-soluble and total), calcium (Ca) and AA indicated that EA and AA concentrations were high in the fruit while the leaves had significantly higher EA levels but little or no detectable AA. OA content in fruit and leaves was substantial with the fruit being placed in the high-Oxalate category. These findings suggest that there is potential to elevate oxalate levels in the urine of susceptible people and intake of fruit-derived products should be closely monitored. By measuring tissues collected from specific trees, high EA-producing or low OA-containing individuals were identified.

Increase in ß-ionone, a carotenoid-derived volatile in zeaxanthin-biofortified sweet corn.

Carotenoids are responsible for the yellow color of sweet corn (Zea mays var. saccharata), but are also potentially the source of flavor compounds from the cleavage of carotenoid molecules. The carotenoid-derived volatile, ß-ionone, was identified in both standard yellow sweet corn ('Hybrix5') and a zeaxanthin-enhanced experimental variety ('HZ') designed for sufferers of macular degeneration. As ß-ionone is highly perceivable at extremely low concentration by humans, it was important to confirm if alterations in carotenoid profile may also affect flavor volatiles. The concentration of ß-ionone was most strongly correlated (R(2) > 0.94) with the ß-arm carotenoids, ß-carotene, ß-cryptoxanthin, and zeaxanthin, and to a lesser degree (R(2) = 0.90) with the α-arm carotenoid, zeinoxanthin. No correlation existed with either lutein (R(2) = 0.06) or antheraxanthin (R(2) = 0.10). Delaying harvest of cobs resulted in a significant increase of both carotenoid and ß-ionone concentrations, producing a 6-fold increase of ß-ionone in 'HZ' and a 2-fold increase in 'Hybrix5', reaching a maximum of 62 µg/kg FW and 24 µg/kg FW, respectively.

Key role of Fe(2+) in epithiospecifier protein activity.

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe(2+) is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe(2+) requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.

Screening sweetcorn for enhanced zeaxanthin concentration.

BACKGROUND: New varieties of fruits and vegetables, with higher carotenoid levels, are being developed to improve the potential health benefits to consumers. To assist the development of a new variety of high zeaxanthin sweetcorn, an analytical screening method was developed, including chromameter measurement of hue angle and optimized extraction for HPLC, and applied to 385 lines of a breeding population and six commercial varieties. RESULTS: Saponification had no effect on carotenoid extraction. In the breeding population, carotenoid levels had a wide range with the highest levels of zeaxanthin being 11.9 mg kg(-1) fresh weight, which was at least six times greater than the tested commercial varieties. The regression of hue angle versus zeaxanthin was described by the equation, hue angle = 76.16 + 4.50 x exp(-0.24 x zeaxanthin) + 11.73 x exp(-0.24 x zeaxanthin), r(2) of 0.59. The top 6% of lines, with regards to zeaxanthin (zeaxanthin + beta-cryptoxanthin + beta-carotene) and total carotenoids, all had hue angles

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds.

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 degrees C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 degrees C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.

Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulphoraphane nitrile.

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.