Dilek Ozyurt

Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay

It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET)- and hydrogen atom transfer (HAT)-based assays. The majority of HAT assays are kinetics-based, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II)-neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity) method. This method offers distinct advantages over other ET-based assays, namely the selection of working pH at physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively), applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH), completion of the redox reactions for most common flavonoids (unlike FRAP), selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay -SH bearing antioxidants (unlike FRAP). Other similar ET-based antioxidant assays that we have developed or modified for phenolics are the Fe(III)- and Ce(IV)-reducing capacity methods.

Modified Folin-Ciocalteu antioxidant capacity assay for measuring lipophilic antioxidants.

The Folin-Ciocalteu (FC) method of performing a total phenolics assay, originally developed for protein determination, has recently evolved as a total antioxidant capacity assay but was found to be incapable of measuring lipophilic antioxidants due to the high affinity of the FC chromophore, that is, multivalent-charged phospho-tungsto-molybdate(V), toward water. Thus, the FC method was modified and standardized so as to enable simultaneous measurement of lipophilic and hydrophilic antioxidants in NaOH-added isobutanol-water medium. Optimal conditions were as follows: dilution ratio of aqueous FC reagent with iso-BuOH (1:2, v/v), final NaOH concentration of 3.5 × 10(-2) M, reaction time of 20 min, and maximum absorption wavelength of 665 nm. The modified procedure was successfully applied to the total antioxidant capacity assay of trolox, quercetin, ascorbic acid, gallic acid, catechin, caffeic acid, ferulic acid, rosmarinic acid, glutathione, and cysteine, as well as of lipophilic antioxidants such as α-tocopherol (vitamin E), butylated hydroxyanisole, butylated hydroxytoluene, tertiary butylhydroquinone, lauryl gallate, and β-carotene. The modified FC method reliably quantified ascorbic acid, whereas the conventional method could not. The modified method was reproducible and additive in terms of total antioxidant capacity values of constituents of complex mixtures such as olive oil extract and herbal tea infusion. The trolox equivalent antioxidant capacities of the tested antioxidant compounds correlated well with those found by the Cupric Reducing Antioxidant Capacity reference method.

Effect of Oven and Microwave Heating on the Total Antioxidant Capacity of Dietary Onions Grown in Turkey

The active components of largely consumed dietary onions (Allium species) in Turkey were extracted with water and ethanol, separately heated in a drying oven (at 50°C, for 1–4 h) and in a microwave oven (at 90 W, for 1–4 min), and their total antioxidant capacity was determined with different electron transfer-based assays. Five different onion species/aerial parts, namely yellow, red, white, fresh green leaf, and fresh green root, were measured for total antioxidant capacity with different methods, the hierarchic order in aqueous extracts being: CERAC: yellow-skinned > red-skinned > spring-root > spring-leaf > white-skinned onion; CUPRAC: spring-leaf > red-skinned > spring-root > yellow-skinned > white-skinned onion; Folin-Ciocalteau method: spring-leaf > spring-root > red-skinned > yellow-skinned > white-skinned onion. Using all three methods, white onion, showed the lowest total antioxidant capacity, while with respect to two methods (i.e., CUPRAC and Folin-Ciocalteau), spring onion-leaf showed the highest value. In ethanolic extracts, white onion exhibited the lowest total antioxidant capacity using two methods (CUPRAC and Folin-Ciocalteau). Of the heat-processed onions, the highest CERAC and Folin-Ciocalteau total antioxidant capacity values were obtained for red-skinned onions, while the highest CUPRAC value was for spring onion leaves. All three assays marked white-skinned onion as the lowest total antioxidant capacity content of heat-processed products. The change in total antioxidant capacity caused by both heating processes was not drastic; spring onion leaves essentially maintained its total antioxidant capacity level after 4 min microwave or 4 h drying oven heating. Onion processing by heat treatment did not cause a drastic loss in antioxidant values, favourable for traditional cooking practices.

Solid-Phase Extraction Spectrophotometric Determination of Total Antioxidant Capacity in Antioxidant-poor Samples by Using the Ferric-Ferrozine Method

Ferrozine (FZ) preferentially stabilizes Fe(II) over Fe(III) to raise the ferric reduction potential and oxidize antioxidants. The advantages of the ferric-ferrozine method over other iron-based total antioxidant capacity assays were: (i) higher molar absorptivity and enhanced sensitivity, (ii) lower interference from foreign ions, (iii) wide pH tolerance (iv) additivity of the absorbances for mixtures. Solid-phase extraction (SPE) could be combined with spectrophotometry, because the magenta-colored anionic Fe(II)-FZ complex was quantitatively sorbed on Sephadex QAE A-25 resin. The sensitivity enhancement using the resin enabled us to conduct total antioxidant capacity (TAC) measurements of antioxidant-poor samples. The apparent molar absorptivity, linear concentration range and trolox equivalent antioxidant capacities (TEAC) of certain antioxidants were found. The calibration curves (lines) of trolox, rutin, and rosmarinic acid individually and in herbal infusions-by using the method of standard additions-were parallel, confirming that the added antioxidants did not interact with herbal constituents to cause chemical deviations from Beers law.

HPLC–UV method development and validation for the determination of low level formaldehyde grown culture mushrooms in Turkey

B utilizing Localized Surface Plasmon Resonance (LSPR) offers relatively inexpensive, label-free, facile detection that is amenable to on-chip devices. Such devices can provide exquisite sensitivity at a low cost and should prove extremely useful in resource limited environments. However, several challenges remain, such as: sensitivity to small molecule binding, specificity in complex biological solutions, detection of membrane-associated species and integration into on-chip devices. This presentation will highlight recent advances in LSPR-based biosensing devices developed in the Sagle group to overcome these limitations. One study we have done to increase sensitivity is an assay in which gold nanostars are aggregated upon addition of an analyte. Due to increased surface area of contact, a large decrease in Kd and limit of detection in the attomolar range was observed with this simple aggregation assay. In addressing the second challenge, we have incorporated shape complementarity on the nanoparticle surface to carry out size-selective biosensing with improved selectivity. The third challenge is tackled through the development of a novel plasmonic platform containing a solid supported lipid bilayer so that label-free measurements of membrane associated species can be carried out. This device is shown to have improved sensitivity over existing platforms. Lastly, large-scale patterning of the nanoparticle arrays enabling the interfacing of these arrays with microfluidic on-chip devices are also presented.