Novel tributyl phosphate-based deep eutectic solvent: Application in microwave assisted extraction of carotenoids.

A contribution to the use of deep eutectic solvents (DES) and microwave-assisted extraction (MAE) was made for bioactive compounds recovery, especially those with lipophilic character, from tomato and carrot samples rich in carotenoids. For the first time, a novel deep eutectic solvent was synthesized, comprising tributyl phosphate (TBP) as a hydrogen bond acceptor and acetic acid (AcOH) as a hydrogen bond donor. The total antioxidant capacity (TAC) of tomato and carrot extracts obtained by MAE, in which optimization of operational parameters and modeling were made with the use of Box-Behnken design of the response surface methodology (RSM), was evaluated using the Cupric Reducing Antioxidant Capacity (CUPRAC) method. For the highest TAC, operational parameters that best suit the MAE procedure were set at 80 °C, 35 min, and 25 mL/2.0 g. The TAC values of extracts obtained by MAE using TBP:AcOH, 1:2 (mol/mol) were examined against those of extracts acquired by classical solvent extraction using a mixture of hexane, ethanol and acetone (H:E:A, 2:1:1 (v/v/v)) mixture. TAC of extracts in DES varied between 5.10 and 0.71 lycopene equivalents (mmol LYC kg-1). The highest extraction yield comparable to conventional organic solvents was obtained with TBP:AcOH (1:2). It was observed that, in addition to lipophilic antioxidants, some hydrophilic antioxidant compounds were partially extracted with the proposed DES. Moreover, the extracted antioxidant compounds were identified and quantified by HPLC analysis. The proposed DES and MAE process will find potential application for hydrophobic antioxidant extraction from tomatoes and carrots on an industrial scale after further studies.

Colorimetric Sensing of Antioxidant Capacity via Auric Acid Reduction Coupled to ABTS Oxidation.

In this study, a simple and sensitive colorimetric assay has been developed for total antioxidant capacity measurement. The assay is based on the absorption measurement of the bluish-green oxidized product (ABTS·+) formed as a result of the oxidation reaction of the chromogenic reagent ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) with gold(III). However, in the presence of antioxidants, the ABTS oxidation process is effectively suppressed due to the reduction of gold(III) ions to the zerovalent state forming gold nanoparticles (AuNPs). Relatively lighter colors and a significant decrease in absorbance are observed depending on the total antioxidant capacity. Taking advantage of this situation, qualitative and quantitative total antioxidant capacity (TAC) measurements, with the naked eye and UV-vis spectroscopy, respectively, could be successfully performed. The assay is named "auric reducing antioxidant capacity" (AuRAC) because the gold(III) ion-reducing ability of antioxidants is measured. The AuRAC assay was applied to dietary polyphenols, vitamin C, thiol-type antioxidants, and their synthetic mixtures. Trolox equivalent antioxidant capacity (TEAC) values obtained with the AuRAC assay were found to be compatible with those of the reference CUPRAC (cupric reducing antioxidant capacity) assay. The AuRAC assay was validated through linearity, additivity, precision, and recovery, demonstrating that the assay is reliable and robust. Compared to the simple TAC assays in the literature based on AuNP formation with subsequent surface plasmon resonance (SPR) absorbance measurement, this indirect assay has a smoother linear range starting from lower antioxidant concentrations. This method displays much higher molar absorption coefficients for antioxidant compounds than other conventional single electron transfer (SET) assays because 3-e- reduction of trivalent gold (i.e., Au(III) → Au(0)) produces three chromophore cation radicals (ABTS·+) of the assay reagent. The sensor has been successfully applied to complex matrices, such as tea infusions and pharmaceutical samples. The AuRAC assay stands out with its high molar absorptivity connected to enhanced sensitivity as well as its potential to convert into a paper-based colorimetric sensor.

Peroxyl radical scavenging activity measurement of antioxidants using histidine-stabilized and glutathione-capped fluorescent gold nanoclusters.

A fluorescent gold nanocluster was used for determining peroxyl radical scavenging activity of antioxidants. Histidine was used as a green reducing and protective agent, and glutathione (GSH) enhanced the fluorescence intensity of histidine-stabilized gold nanoclusters (AuNCs) by ligand exchange process. When AAPH-induced oxidation of GSH occurred, the initial fluorescence intensity of GSH-capped AuNCs (λex = 450 nm λem = 502 nm) was decreased with static quenching. The decline of fluorescence intensity of the GSH-capped AuNCs upon peroxyl radical attack is diminished with the addition of antioxidants to the reaction medium, the difference in fluorescence intensity being related to peroxyl radical scavenging activity of antioxidants. The 50 % inhibitive concentration of related antioxidant compounds were determined and compared to those of crocin bleaching assay. Inhibition % of sage (Salvia officinalis L.) and green tea (Camellia sinensis) infusions against peroxyl radicals were investigated. The proposed assay can be used for simple and selective estimation of the peroxyl radical scavenging activity in complex matrices, as histidine-stabilized GSH-capped AuNCs were selective toward peroxyl radicals, not affected by other ROS at the studied concentrations.

Microwave-assisted extraction of antioxidant compounds from by-products of Turkish hazelnut (Corylus avellana L.) using natural deep eutectic solvents: Modeling, optimization and phenolic characterization.

An environmentally friendly method using natural deep eutectic solvents (NADES) and microwave-assisted extraction (MAE) for the recovery of bioactive compounds from hazelnut pomace (a hazelnut oil process by-product) was developed to contribute to their sustainable valorization. Eight different NADES were prepared for the extraction of antioxidant constituents from hazelnut pomace, and choline chloride:1,2-propylene glycol (CC-PG) was determined as the most suitable NADES, considering their extraction efficiency and physicochemical properties. After selecting suitable NADES, operational parameters for the MAE process of antioxidants from hazelnut pomace were optimized and modeled using response surface methodology. For the highest recovery of antioxidants, the operational parameters of the MAE process were found to be 24% water, 38 min, 92 °C and 18 mL/0.1 g-DS. Under optimized conditions, extracts of both pomace as a by-product and unprocessed hazelnut flours of three different hazelnut samples (Tombul, Çakildak, and Palaz) were prepared, and their antioxidant capacities were evaluated by spectrophotometric methods. Antioxidant capacities of CC-PG extracts of all hazelnut samples were 2-3 times higher than those of ethanolic extracts. In addition, phenolic characterization of the prepared extracts was carried out using the UPLC-PDA-ESI-MS/MS system. The results of this study suggest that hazelnut by-products can potentially be considered an important and readily available source of natural antioxidants. Furthermore, the modeled MAE procedure has the potential to create an effective and sustainable alternative for pharmaceutical and food industries.

Redox-based colorimetric sensing of H2O2 after removal of antioxidants with ABTS radical oxidation.

Monitoring and determining H2O2 in many industries, treatment plants and biochemical media is important because of its harmful effects even at low concentrations. This work proposes a redox-based colorimetric sensor for the determination of hydrogen peroxide in the presence of antioxidants which are known interferents causing positive errors. On the other hand, the widely used peroxidase-based methods are interfered by enzyme inhibitors. The proposed method consists of two stages, namely antioxidant removal and H2O2 determination. In the first step, antioxidants were removed simply using ABTS radical (ABTS+) oxidant produced by persulfate. After antioxidant elimination, H2O2 in samples was determined by using the CUPRAC colorimetric sensor. The CUPRAC reagent, copper (II)-neocuproine (Cu(II)-Nc), immobilized on a Nafion persulfonate membrane was used for sensor preparation. The light blue Cu(II)-Nc was reduced by H2O2 to the yellow-orange colored Cu(I)-Nc chelate on the sensor, and the absorbance increase at 450 nm was recorded. The LOD and the LOQ values obtained for H2O2 were 0.33 and 1.10 µM, respectively. The proposed assay was validated in terms of linearity, additivity, precision and recovery. The H2O2 contents of spiked food extracts, synthetic serum and certain commercial products (i.e. food sterilization solution, whitening toothpaste and hair bleaching solution) were found to be comparable to the results of peroxidase-ABTS and titanyl sulfate reference assays. In addition, peroxide-type explosive triacetone triperoxide (TATP) was successfully determined in the presence of amine-type antioxidants. The proposed simple and low-cost assay is not inhibited by environmental agents (heavy metals, pesticides, sulfhydryl agents, etc.) adversely affecting enzymatic methods. It is additionally insensitive to turbidity and colored components of complex samples.

ABTS radical-based single reagent assay for simultaneous determination of biologically important thiols and disulfides.

Both the total amount of biothiols and thiol/disulfide ratio are wellness indicators of oxidative balance that play an important role in antioxidant defense system. Oxidized biothiols in disulfide form cannot be determined by conventional ABTS assay due to the biphasic kinetic pattern of the reaction between biothiols and ABTS radical cation (ABTS•+), necessitating the initial reduction of disulfides to thiols prior to measurement. In this study, direct simultaneous determination of biothiols (RSH) and their disulfides (RSSR) by using a single reagent of ABTS•+ was achieved without preliminary chemical reduction. Thus, conventional problems of preliminary operations arising from direct borohydride reduction of disulfides to thiols, followed by formaldehyde removal of borohydride excess and complications caused by formaldehyde-thiol reactions were effectively overcome with the use of a single reagent (ABTS•+). Box-Behnken statistical experimental design was employed to specify the optimal incubation temperature and time as 60 °C and 60 min, respectively. The detection limits (LOD) of the proposed assay for biothiols were compared to those of the widely used DTNB (Ellman) reference assay known to be nonresponsive to disulfides, and were found to be much lower (4-70 times). The proposed biothiol assay was successfully applied to some pharmaceutical samples and synthetic serum without preliminary treatment, and the results were highly compatible with the HPLC findings. The proposed assay was demonstrated to have superior features such as simplicity, rapidity and higher sensitivity over the widely applied Ellman thiols assay.

A novel colorimetric sensor for measuring hydroperoxide content and peroxyl radical scavenging activity using starch-stabilized gold nanoparticles.

A novel colorimetric nanosensor was developed for evaluating peroxyl radical scavenging activity of phenolic antioxidants and for the detection of hydroperoxides formed during AAPH-induced oxidation of linoleic acid emulsions. Starch was used as a green reduction/stabilization agent for gold nanoparticles (AuNPs) synthesis in alkaline medium. When tert-butyl hydroperoxide (tert-BHP) was incubated with an excess of iodide ions in a 37 °C water bath for 90 min, triodide (I3-) was formed in an amount equivalent to tert-BHP concentration. Upon the addition of starch-stabilized gold nanoparticles (ss-AuNPs) solution to the incubation mixture, triiodide ions were rapidly adsorbed on the surface of AuNPs and caused their aggregation. A concomitant red shift (from 525 nm to 563 nm) of surface plasmon resonance (SPR) absorption of the nanoparticles was observed, absorbance linearly increasing with aqueous tert-BHP concentration. The method provided an LOD of 39 µM for tert-BHP, and was validated through linearity, precision and accuracy. The concentration of hydroperoxides estimated in linoleic acid peroxidation correlated well with those found by the reference ferric thiocyanate assay. Peroxyl radical scavenger antioxidants decreased the red-shifted SPR absorption of aggregated ss-AuNPs, thereby enabling an indirect estimation of antioxidant activity. This AuNPs-based colorimetric sensor is the first of its kind to directly determine peroxyl radical scavenging activity of polyphenols. The half-maximal inhibitive concentrations (IC50) of selected antioxidant compounds were calculated by utilizing the decrease in absorbance with increasing concentration of scavengers, and compared to those of classical oxygen radical absorbance capacity (ORAC) assay. The proposed nanosensor was superior over FL-based ORAC in determining the peroxyl radical scavenging activity of the lipophilic antioxidant α-tocopherol. The percentage scavenging of real samples such as green tea infusion and synthetic serum were determined. The proposed assay can be used for estimating the peroxyl scavenging of various food and biological samples in terms of its low cost, ease of use and compatibility.

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 ß-Carotene with ß-Cyclodextrins Complexation in Aqueous Medium.

In order to evaluate the antioxidant capacity/activity of ß-carotene (BC) in aqueous media, we investigated the inclusion complexes of BC with methyl-ß-cyclodextrin (Me-ß-CD), 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and 2-hydroxyethyl-ß-cyclodextrin (HE-ß-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-ß-CD concentration. Cupric ion-reducing antioxidant capacity (CUPRAC), ABTS-persulfate, peroxyl and hydroxyl radical scavenging assays were applied. CD-complexed ß-carotene exhibited less effective antioxidative and radical scavenging than free BC dissolved in acetone. ß-Carotene showed the highest antioxidant capacity in the presence of HE-ß-CD, and the lowest with Me-ß-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 ß-carotene in aqueous solution with CDs.

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.

A Novel Spectrofluorometric Probe for the Determination of Peroxynitrite Anion Scavenging Activity of Biothiols and Amino Acids.

In this study, a novel fluorometric method for the determination of peroxynitrite anion (ONOO-) scavenging (PAS) activity of amino acids and biothiols, which can mostly trap peroxynitrite in vivo, is described. This assay is based on the conversion of a gentisic acid probe to its non-fluorescent oxidation products with ONOO-. The attenuation of the fluorescence intensity (FI) of the probe upon peroxynitrite attack is diminished with antioxidants, the difference in FI being related to the PAS activity of the antioxidants. The IC50 (50% inhibitive concentration) values of biothiols, amino acids and tissue homogenates were estimated, in comparison with the reference Pyrogallol Red (PR) bleaching method. PR is the most suitable and frequently used dye to determine PAS activity, but is relatively insensitive. The developed fluorometric assay is highly sensitive to allow determinations of the PAS activity of amino acids.

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.

Development of a new catalase activity assay for biological samples using optical CUPRAC sensor.

A novel catalase activity assay was developed for biological samples (liver and kidney tissue homogenates) using a rapid and low-cost optical sensor-based 'cupric reducing antioxidant capacity' (CUPRAC) method. The reagent, copper(II)-neocuproine (Cu(II)-Nc) complex, was immobilized onto a cation-exchanger film of Nafion, and the absorbance changes associated with the formation of the highly-colored Cu(I)-Nc chelate as a result of reaction with hydrogen peroxide (H2O2) was measured at 450 nm. When catalase was absent, H2O2 produced the CUPRAC chromophore, whereas catalase, being an effective H2O2 scavenger, completely annihilated the CUPRAC signal due to H2O2. Thus, the CUPRAC absorbance due to H2O2 oxidation concomitant with Cu(I)-Nc formation decreased proportionally with catalase. The developed sensor gave a linear response over a wide concentration range of H2O2 (0.68-78.6 µM). This optical sensor-based method applicable to tissue homogenates proved to be efficient for low hydrogen peroxide concentrations (physiological and nontoxic levels) to which the widely used UV method is not accurately responsive. Thus, conventional problems of the UV method arising from relatively low sensitivity and selectivity, and absorbance disturbance due to gaseous oxygen evolution were overcome. The catalase findings of the proposed method for tissue homogenates were statistically alike with those of HPLC.

Resorcinol as a spectrofluorometric probe for the hypochlorous acid scavenging activity assay of biological samples.

A novel spectrofluorometric method was developed and validated for hypochlorous acid (HOCl) scavenging activity estimation using resorcinol, which is a highly sensitive and chemically stable fluorogenic probe. This assay is based on the chlorination of resorcinol to its nonfluorescent products in the presence of HOCl. HOCl reacts with both resorcinol and HOCl scavengers incubated in solution for 10 min, where scavengers compete with resorcinol for the HOCl. Thus, the relative increase in fluorescence intensity of intact resorcinol is proportional to the antioxidative activity of HOCl scavengers. Using this reaction, a kinetic approach was adopted to assess the HOCl scavenging activity of amino acids, vitamins, and plasma and thiol antioxidants. This assay, which is applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for thiol-type antioxidants for which the widely used 5-thio-2-nitrobenzoic acid (TNB) test is not accurately responsive. Thus, conventional problems of the TNB assay arising from the reactivity of thiol-type scavengers to produce extra TNB by direct reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were overcome. Moreover, unlike enzymatic assays (e.g., elastase), there is no confusion as to whether the putative scavenger actually reacts with HOCl or inhibits the enzyme.

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.

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-ß-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.