Burcu Bekdeşer

tert-Butylhydroquinone as a Spectroscopic Probe for the Superoxide Radical Scavenging Activity Assay of Biological Samples

As a more convenient and less costly alternative to electron spin resonance (ESR) and nonspecific nitroblue tetrazolium (NBT) and cytochrome c assays of superoxide radical (SR, O(2)(•-)) detection, a novel probe, tert-butylhydroquinone (TBHQ), is introduced for SR nonenzymatically generated in the phenazine methosulfate-β-nicotinamide adenine dinucleotide (PMS-NADH) system. SR attacks both TBHQ and SR scavengers incubated in solution for 30 min where scavengers compete with TBHQ for the O(2)(•-) produced. TBHQ, but not its O(2)(•-) oxidation product, tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of Cu(II)-neocuproine reagent by the remaining TBHQ was higher in the presence of O(2)(•-) scavengers (due to less conversion to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct reduction of NBT by tested scavengers were overcome.

Optimization of microwave-assisted extraction of polyphenols from herbal teas and evaluation of their in vitro hypochlorous acid scavenging activity.

Hypochlorous acid (HOCl) is an important reactive oxygen species (ROS) and non-radical and is taking part in physiological processes concerned with the defense of the organism, but there has been limited information regarding its scavenging by polyphenols. This study was designed to examine the HOCl scavenging activity of several polyphenols and microwave-assisted extracts of herbal teas. HOCl scavenging activity has usually been determined spectrophotometrically by a KI/taurine assay at 350 nm. Because some polyphenols (i.e., apigenin and chrysin) have a strong ultraviolet (UV) absorption in this range, their HOCl scavenging activity was alternatively determined without interference using resorcinol (1,3-dihydroxybenzene) as a fluorogenic probe. In the present assay, HOCl induces the chlorination of resorcinol into its non-fluorescent products. Polyphenols as HOCl scavengers inhibit the chlorination of the probe by this species. Thus, the 25% inhibitive concentration (IC25) value of polyphenols was determined using the relative increase in fluorescence intensity of the resorcinol probe. The HOCl scavenging activities of the test compounds decreased in the order: epigallocatechin gallate > quercetin > gallic acid > rutin > catechin > kaempferol. The present study revealed that epigallocatechin gallate (IC25 = 0.1 μM) was the most effective scavenging agent. In addition to polyphenols, four herbal teas were evaluated for their HOCl activity using the resorcinol method. The proposed spectrofluorometric method was practical, rapid, and less open to interferences by absorbing substances in the range of 200-420 nm. The results hint to the possibility of polyphenols having beneficial effects in diseases, such as atherosclerosis, in which HOCl plays a pathogenic role.

Novel spectroscopic sensor for the hydroxyl radical scavenging activity measurement of biological samples

A novel spectroscopic sensor was developed and validated for hydroxyl radical scavenging (HRS) activity estimation using terephthalate (TP) as probe. This sensor was designed by electrostatic immobilization of the chromogenic oxidizing agent of the CUPric Reducing Antioxidant Capacity (CUPRAC) method, Cu(II)-Neocuproine (Cu(II)-Nc) complex, on a Nafion cation-exchange membrane, and the spectrophotometric assay developed in aqueous-alcoholic solutions was integrated to the CUPRAC sensor. Hydroxyl radicals ((•)OH) generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide attacked both the probe and the (•)OH scavengers in 37 °C-incubated solutions for 1/2h. The HRS activity was measured using the decrease in CUPRAC absorbance at 450 nm - arising from the reduction of Cu(II)-Nc reagent to the Cu(I)-neocuproine chelate - of the hydroxylated probe (TP) undergoing radical attack in the presence of (•)OH scavengers. The HRS activity was evaluated as the second-order rate constants of biologically active compounds for (•)OH scavenging and also as the percentage scavenging of a measured compound or sample relative to a reference compound. Using this reaction, a kinetic approach was adopted to assess the HRS activity of amino acids, plasma- and thiol-antioxidants. This assay, applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for serine and albumin for which the widely used TBARS (thiobarbituric acid-reactive substances) test is nonresponsive. Under optimal conditions, about half of the probe (TP) was converted into 2-hydroxyterephthalate (hTP), and this monohydroxylated derivative, being the only product of hydroxylation, was a more specific marker of (•)OH than the non-specific malondialdehyde end-product of the TBARS test. The sensor gave a linear response to scavenger concentration in the competition kinetic equation.

Identification and Determination of Phenolics in Lamiaceae Species by UPLC-DAD-ESI-MS/MS

This study reports the phenolic profile screening of aromatic Lamiaceae species such as marjoram (Origanum majorana L.), lavender (Lavandula officinalis) and pennyroyal (Mentha pulegium L.) using a novel and validated ultra performance liquid chromatography method coupled with DAD diode array detector and tandem mass spectrometry (MS/MS) in negative mode of electrospray ionization. Identification and quantification of phenolics in these plant extracts has been realized within 12 min. This method showed good precision (percentage relative standard deviation; RSD% 0.54-2.72 for intra-day, 1.71-4.64 for inter-day), reproducibility (percentage recovery, REC% 92.0-109.0) and linearity (r = 0.9988-0.9999). Limits of detection ranged from 0.02 to 18.2 ng/mL. The extraction of plants was performed using microwave-assisted extraction technique and 60% (v/v) aqueous methanol solvent medium was selected as suitable solvent because of maximum extraction efficiency. Total antioxidant capacity, total phenolic content and free radical scavenging activity of these plant extracts were tested and the results correlated well among each other. According to the Folin assay, phenolic contents of Origanum majorana L., Mentha pulegium L. and Lavandula officinalis were calculated as 119 ± 3.4, 85.1 ± 2.8 and 57.8 ± 2.1 mg GAE/g dry matter, respectively.

The CUPRAC Methods of Antioxidant Measurement for Beverages

In this chapter, total antioxidant capacity (TAC), ascorbic acid content, and reactive oxygen species (ROS) scavenging activity of beverages (orange, pomegranate, red grape, apple, cherry, and apricot juices) were investigated. TAC was spectrophotometrically determined by using the main and sensor-based cupric reducing antioxidant capacity (CUPRAC) methods. The modified CUPRAC methods were used to determine the ROS (hydrogen peroxide, hydroxyl and superoxide radical) scavenging activity. The ascorbic acid (AA) content of beverages was measured by La-CUPRAC method. This study showed that the antioxidant capacities of beverages, unlike their ROS scavenging activities, are correlated with their ascorbic acid contents. The TAC values and AA contents of freshly squeezed fruit juices were higher than those of ready-to-drink juices. In addition, the scavenging activities of ready-to-drink juices showed irregular variations with respect to the type of ROS scavenged. This report aims to summarize the measurement of TAC, AA, and ROS scavenging activity of various fruit juices, together with correlation of data and possible effects of processing.

Novel Spectroscopic and Electrochemical Sensors and Nanoprobes for the Characterization of Food and Biological Antioxidants

Since an unbalanced excess of reactive oxygen/nitrogen species (ROS/RNS) causes various diseases, determination of antioxidants that can counter oxidative stress is important in food and biological analyses. Optical/electrochemical nanosensors have attracted attention in antioxidant activity (AOA) assessment because of their increased sensitivity and selectivity. Optical sensors offer advantages such as low cost, flexibility, remote control, speed, miniaturization and on-site/in situ analysis. Electrochemical sensors using noble metal nanoparticles on modified electrodes better catalyze bioelectrochemical reactions. We summarize the design principles of colorimetric sensors and nanoprobes for food antioxidants (including electron-transfer based and ROS/RNS scavenging assays) and important milestones contributed by our laboratory. We present novel sensors and nanoprobes together with their mechanisms and analytical performances. Our colorimetric sensors for AOA measurement made use of cupric-neocuproine and ferric-phenanthroline complexes immobilized on a Nafion membrane. We recently designed an optical oxidant/antioxidant sensor using N,N-dimethyl-p-phenylene diamine (DMPD) as probe, from which ROS produced colored DMPD-quinone cationic radicals electrostatically retained on a Nafion membrane. The attenuation of initial color by antioxidants enabled indirect AOA estimation. The surface plasmon resonance absorption of silver nanoparticles as a result of enlargement of citrate-reduced seed particles by antioxidant addition enabled a linear response of AOA. We determined biothiols with Ellman reagent−derivatized gold nanoparticles.

Modified Radical Scavenging and Antioxidant Activity Measurement of β-Carotene with β-Cyclodextrins Complexation in Aqueous Medium

In order to evaluate the antioxidant capacity/activity of β-carotene (BC) in aqueous media, we investigated the inclusion complexes of BC with methyl-β-cyclodextrin (Me-β-CD), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and 2-hydroxyethyl-β-cyclodextrin (HE-β-CD) that enhance water solubility and chemical stability. The inclusion complexes (monitored by FTIR) exhibited higher solubility than free BC, and phase solubility studies showed a linear increase in the solubility with the Me-β-CD concentration. Cupric ion-reducing antioxidant capacity (CUPRAC), ABTS-persulfate, peroxyl and hydroxyl radical scavenging assays were applied. CD-complexed β-carotene exhibited less effective antioxidative and radical scavenging than free BC dissolved in acetone. β-Carotene showed the highest antioxidant capacity in the presence of HE-β-CD, and the lowest with Me-β-CD, probably due to the deeper and more hydrophobic cavity of the latter. We believe that this is the first report on devising simple spectrophotometric methods for the wholistic assessment of antioxidant activity/capacity, hydroxyl and peroxyl radical scavenging activity of β-carotene in aqueous solution with CDs.