Blaž Cigić

DPPH assay of vegetable oils and model antioxidants in protic and aprotic solvents

The rate of reaction of phenolic antioxidants with DPPH depends on solvent composition. The rate constants can differ by more than two orders of magnitude for the same phenolic compound. Reactions are faster in alcohols than in ethyl acetate that is used routinely for the analysis of antioxidant potential (AOP) of nonpolar samples such as vegetable oils. Incorporation of an acid base pair into the assay solvent buffers the system against acid impurities such as free fatty acids and CO2 from the air. This is shown to increase the rate of oxidation and number of electrons of phenolic compounds exchanged with DPPH. Typically, DPPH assays are performed for predetermined time intervals at which phenolic compounds are not fully oxidized and therefore higher reaction rates result in higher values of AOP. More than twofold AOP was obtained for oleuropein, sesamol, sinapic acid, caffeic acid and protocatechuic acid in buffered alcohols than in ethyl acetate. The AOP of sesame, pumpkin seed and extra virgin olive oil is accordingly higher when determined in buffered alcohols. DPPH assays in ethyl acetate result in underestimation of AOP of unrefined vegetable oils.

Stability and transformation of products formed from dimeric dehydroascorbic acid at low pH

Dehydroascorbic acid dimer (DDHA) is the commercially available form of oxidised vitaminC. When DDHA enters an aqueous environment, it will interact with water, but the details of this process are not well defined. Hydration of DDHA was analyzed as a function of concentration, pH and temperature. Above pH 5 only hydrated dehydroascorbic acid monomer (DHA) and its degradation product 2,3-diketogulonic acid (DKG) were observed. At pH lower than4 and in MilliQ water, relatively stable hydrated dimeric species were additionally detected. They are intermediates formed from DDHA and transformed into DKG, either directly or through DHA. A lower temperature of dissolution favours the formation of hydrated dimeric products, while the concentration of DDHA has no effect on their yield. All products were separated chromatographically and analyzed by three detectors: diode array, refractive index and electrospray ionisation mass spectrometry. The stability and transformations of products were studied in the mixture and in the isolated fractions. Two initially formed monohydrated products are transformed to a dihydrated product that is degraded to DHA and DKG. Once formed, the dihydrated product and one of the monohydrated products are sufficiently stable to be identified at neutral pH. All three hydrated products are interconvertible and can be also formed after dissolution of dried DHA.

LC–MS analysis of phenolic compounds and antioxidant activity of buckwheat at different stages of malting

The impact of malting on the profile of the phenolic compounds and the antioxidant properties of two buckwheat varieties was investigated. The highest relative increases in phenolic compounds were observed for isoorientin, orientin, and isovitexin, which are consequently major inducible phenolic compounds during malting. Only a minor relative increase was observed for the most abundant phenolic compound, rutin. The radical-scavenging activity of buckwheat seeds was evaluated using ABTS and DPPH assays. A considerable increase in total phenolic compounds and higher antioxidant activity were observed after 64h of germination, whereas kilning resulted in decreased total phenolic compounds and antioxidant activity. Higher antioxidant activities for extracts were found for buffered solvents than for pure methanol and water. Changes in the composition of the phenolic compounds and increased antioxidant content were confirmed by several methods, indicating that buckwheat malt can be used as a food rich in antioxidants.

Contribution of SO2 to antioxidant potential of white wine

The reactivity of SO2 with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and in Folin Ciocalteu (FC) assays was analysed under different experimental conditions. There was significantly higher reactivity between SO2 and DPPH in buffered methanol than in methanol alone. When DPPH and FC assays were performed in a mixture of caftaric acid and SO2, there were synergistic effects that were more pronounced with the FC assay. Phenolics are an important parameter of wine quality, and their accurate characterisation in wine is essential. Analysis of white wines with DPPH and FC assays overestimates the contribution of phenolics to the antioxidant potential (AOP). SO2 contributes (from 20% to 45%) to the AOP of the white wines analysed. As SO2 reactivity depends highly on buffer composition, pH, time of incubation and other compounds, e.g. phenolics and aldehydes, different experimental protocols can produce large variations in AOPs, and therefore control of experimental conditions is extremely important.

Influence of Solvent Composition on Antioxidant Potential of Model Polyphenols and Red Wines Determined with 2,2-Diphenyl-1-picrylhydrazyl

Solvent composition has a large influence on measured antioxidant potential (AOP) of model polyphenols and red wines with 2,2-diphenyl-1-picrylhydrazyl (DPPH). We have shown that incorporation of aqueous buffer in the assay medium results in higher reactivity of catechin and caftaric acid, which are among major polyphenolic constituents of wines. Consequently, AOPs of red wines determined after 60 min of incubation at 25 °C in buffered methanol are 1.5-1.6-fold of values determined in methanol. Even in buffered methanol, wine polyphenols are not fully oxidized after 60 min. Only about half of Trolox equivalents were determined in comparison to the Folin-Ciocalteu assay. Buffer composition, pH, time of incubation, temperature, and concentration of antioxidants and DPPH all contribute to the resulting value of the AOP being standardized or at least strictly reported because minor differences in experimental procedures can account for large variations in determined AOP with DPPH for the same samples.

A quantitative technique for determining proteases and their substrate specificities and pH optima in crude enzyme extracts.

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala-AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg-AMC, several enzymes active against Leu-AMC, and (for the first time in plants) several enzymes active against Phe-AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.

Rapid Estimation of Tocopherol Content in Linseed and Sunflower Oils-Reactivity and Assay.

The reactivity of tocopherols with 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied in model systems in order to establish a method for quantifying vitamin E in plant oils. The method was optimized with respect to solvent composition of the assay medium, which has a large influence on the course of reaction of tocopherols with DPPH. The rate of reaction of α-tocopherol with DPPH is higher than that of γ-tocopherol in both protic and aprotic solvents. In ethyl acetate, routinely applied for the analysis of antioxidant potential (AOP) of plant oils, reactions of tocopherols with DPPH are slower and concentration of tocopherols in the assay has a large influence on their molar reactivity. In 2-propanol, however, two electrons are exchanged for both α- and γ-tocopherols, independent of their concentration. 2-propanol is not toxic and is fully compatible with polypropylene labware. The chromatographically determined content of tocopherols and their molar reactivity in the DPPH assay reveal that only tocopherols contribute to the AOP of sunflower oil, whereas the contribution of tocopherols to the AOP of linseed oil is 75%. The DPPH assay in 2-propanol can be applied for rapid and cheap estimation of vitamin E content in plant oils where tocopherols are major antioxidants.

Mechanical Stress Results in Immediate Accumulation of Glucosinolates in Fresh-Cut Cabbage

The intensity of mechanical stress and the temperature significantly affect the levels of individual and total glucosinolates in shredded white cabbage (cv. Galaxy). Mild processing (shredding to 2 mm thickness) at 8°C resulted in the accumulation of glucosinolates (40% increase) in comparison with unshredded cabbage, which was already seen 5 min after the mechanical stress. Severe processing (shredding to 0.5 mm thickness) at 20°C, however, resulted in an initial 50% decrease in glucosinolates. The glucosinolates accumulated in all of the cabbage samples 30 min from processing, resulting in higher levels than in unshredded cabbage, except for the severe processing at 20°C where the increase was not sufficient to compensate for the initial loss. Glucobrassicin and neoglucobrassicin were the major glucosinolates identified in the cabbage samples. Mechanical stress resulted in an increase in the relative proportion of glucobrassicin and in a decrease in neoglucobrassicin.