Enrichment of Hazelnut Oil with Several Polyphenols: An Alternative Approach to A New Functional Food.

Hazelnut oil has been examined according to its oxidative stability and antioxidant activity. The oil sample has been treated with gallic acid, ascorbic acid, catechin, vanillic acid, p-coumaric acid and rutin. Stability of the pure and treated oils against the oxidation has been assessed via Rancimat by detecting the protection factor. The quality parameters of the oil samples were compared depending on their antioxidant activity. D-Optimal design of Response Surface Method has been applied to optimize the enrichment conditions of hazelnut oil with several polyphenols. Principal component analysis has been applied to comprehend the relationship between the groups and their quality parameters. Depending on the analysis of variance test, the most important parameter (at p < 0.0001) affecting the relevant system has been found polyphenol type with respect to stability and antioxidant capacity. Gallic acid has enhanced the stability of hazelnut oil against oxidation ~3 times over that of pure sample. The maximum yields of protection factor, antioxidant activity and dissolved polyphenol level have been 2.738, 46.14% and 259.424 ppm under the optimum conditions (300 ppm gallic acid).

Optimizing the extraction of polyphenols from Sideritis montana L. using response surface methodology.

Sideritis montana L. endemic of Turkey was screened for its polyphenols content and antioxidant activity. Factor analysis and experimental design have been applied to understand the structure of the separation process, to determine the effective parameters, and to accomplish the performance improvement. Face-centred composite design (FCD) of response surface methodology (RSM) was applied to evaluate the influences of solvent concentration, solvent amount, extraction time, and stirring speed of homogenizer-assisted extraction (HAE) as well as to model and to optimize the HAE. Quadratic models were highly significant (p < 0.0001) for the responses studied with high coefficients of determination (R2) of 0.9440, 0.9415 and 0.9521. The result of the study suggests that 15.02 mL of 22.69% EtOH solution (v/v), 70.16 s, and 9524.52 rpm of mixing speed are the optimal conditions to obtain the highest yield of total polyphenols (TPC) and flavonoids (TFC), and the best antioxidant activity (AA). Rosmarinic acid was identified as the most abundant component.

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.

Antioxidant Activity/Capacity Measurement. 2. Hydrogen Atom Transfer (HAT)-Based, Mixed-Mode (Electron Transfer (ET)/HAT), and Lipid Peroxidation Assays.

Measuring the antioxidant activity/capacity levels of food extracts and biological fluids is useful for determining the nutritional value of foodstuffs and for the diagnosis, treatment, and follow-up of numerous oxidative stress-related diseases. Biologically, antioxidants play their health-beneficial roles via transferring a hydrogen (H) atom or an electron (e(-)) to reactive species, thereby deactivating them. Antioxidant activity assays imitate this action; that is, antioxidants are measured by their H atom transfer (HAT) or e(-) transfer (ET) to probe molecules. Antioxidant activity/capacity can be monitored by a wide variety of assays with different mechanisms, including HAT, ET, and mixed-mode (ET/HAT) assays, generally without distinct boundaries between them. Understanding the principal mechanisms, advantages, and disadvantages of the measurement assays is important for proper selection of method for valid evaluation of antioxidant properties in desired applications. This work provides a general and up-to-date overview of HAT-based, mixed-mode (ET/HAT), and lipid peroxidation assays available for measuring antioxidant activity/capacity and the chemistry behind them, including a critical evaluation of their advantages and drawbacks.

Antioxidant Activity/Capacity Measurement. 1. Classification, Physicochemical Principles, Mechanisms, and Electron Transfer (ET)-Based Assays.

Because there is no widely adopted "total antioxidant parameter" as a nutritional index for labeling food and biological fluids, it is desirable to establish and standardize methods that can measure the total antioxidant capacity (TAC) level directly from plant-based food extracts and biological fluids. In this review, we (i) present and classify the widely used analytical approaches (e.g., in vitro and in vivo, enzymatic and nonenzymatic, electron transfer (ET)- and hydrogen atom transfer (HAT)-based, direct and indirect assays) for evaluating antioxidant capacity/activity; (ii) discuss total antioxidant capacity/activity assays in terms of chemical kinetics and thermodynamics, reaction mechanisms, and analytical performance characteristics, together with advantages and drawbacks; and (iii) critically evaluate ET-based methods for analytical, food chemical, biomedical/clinical, and environmental scientific communities so that they can effectively use these assays in the correct places to meet their needs.

Antioxidant Activity/Capacity Measurement. 3. Reactive Oxygen and Nitrogen Species (ROS/RNS) Scavenging Assays, Oxidative Stress Biomarkers, and Chromatographic/Chemometric Assays.

There are many studies in which the antioxidant potential of different foods have been analyzed. However, there are still conflicting results and lack of information as a result of unstandardized assay techniques and differences between the principles of the methods applied. The measurement of antioxidant activity, especially in the case of mixtures, multifunctional or complex multiphase systems, cannot be evaluated satisfactorily using a simple antioxidant test due to the many variables influencing the results. In the literature, there are many antioxidant assays that are used to measure the total antioxidant activity/capacity of food materials. In this review, reactive oxygen and nitrogen species (ROS/RNS) scavenging assays are evaluated with respect to their mechanism, advantages, disadvantages, and potential use in food systems. On the other hand, in vivo antioxidant activity (AOA) assays including oxidative stress biomarkers and cellular-based assays are covered within the scope of this review. Finally, chromatographic and chemometric assays are reviewed, focusing on their benefits especially with respect to their time saving, cost-effective, and sensitive nature.

Design, Synthesis, Biological Evaluation, and Antioxidant and Cytotoxic Activity of Heteroatom-Substituted 1,4-Naphtho- and Benzoquinones.

In the present paper, we report the synthesis, characterization, and biological evaluation as antifungal, antibacterial, antioxidant, and cytotoxic/anticancer agents of N-, S-, O-substituted-1,4-naphtho- and 2,5-bis(amino-substituted)-1,4-benzoquinone derivatives. In the synthesized compounds, antimicrobial activity at low concentrations against Escherichia coli B-906, Staphylococcus aureus 209-P, and Mycobacterium luteum B-917 bacteria and Candida tenuis VKM Y-70 and Aspergillus niger F-1119 fungi in comparison with controls was identified. 2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-naphthoquinone 9a was the most potent, with a minimum inhibitory concentration value of 3.9 µg/mL against test culture M. luteum. The synthesized compounds were screened for their antioxidant capacity using the cupric-reducing antioxidant capacity (CUPRAC) method. 2,2'-[1-(2-Aminoethyl)piperazin-1-yl]-3,3'-dichloro-bis(1,4-naphthoquinone) 10 showed the highest antioxidant capacity, with a 0.455 CUPRAC-trolox equivalent antioxidant capacity (TEAC) coefficient. Other parameters of antioxidant activity (scavenging effects on OH(·), O2(·ï¼), and H2O2) of these compounds were also determined. The cytotoxic activity of the compounds was investigated by employing the sulforhodamine B cell viability assay against A549 (lung), MCF-7 (breast), DU145 (prostate), and HT-29 (colon) cancer cell lines. Compound 10 exhibited the most powerful cytotoxic activity at a concentration of 20 µM against all cell lines. In addition to the strongest antioxidant activity of compound 10, it also had lowest IC50 values (<3 µM), warranting further in vivo studies due to its anticancer activity.

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.

A novel differential pulse voltammetric (DPV) method for measuring the antioxidant capacity of polyphenols-reducing cupric neocuproine complex.

A novel differential pulse voltammetric (DPV) method is presented, using a chromogenic oxidizing reagent, cupric neocuproine complex (Cu(Nc)2(2+)), for the assessment of antioxidant capacity of polyphenolic compounds (i.e., flavonoids, simple phenolic acids, and hydroxycinnamic acids), ascorbic acid, and real samples for the first time. The electrochemical behavior of the Cu(Nc)2(2+) complex was studied by cyclic voltammetry at a glassy carbon (GC) electrode. The electroanalytical method was based on the reduction of Cu(Nc)2(2+) to Cu(Nc)2(+) by antioxidants and electrochemical detection of the remaining Cu(II)-Nc (unreacted complex), the difference being correlated to antioxidant capacity of the analytes. The calibration curves of individual compounds comprising polyphenolics and vitamin C were constructed, and their response sensitivities and linear concentration ranges were determined. The reagent on the GC electrode retained its reactivity toward antioxidants, and the measured trolox equivalent antioxidant capacity (TEAC) values of various antioxidants suggested that the reactivity of the Cu(II)-Nc reagent is comparable to that of the solution-based spectrophotometric cupric ion reducing antioxidant capacity (CUPRAC) assay. This electroanalytical method better tolerated sample turbidity and provided higher sensitivity (i.e., lower detection limits) in antioxidant determination than the spectrophotometric assay. The proposed method was successfully applied to the measurement of total antioxidant capacity (TAC) in some herbal tea samples such as green tea, sage, marjoram, and alchemilla. Results demonstrated that the proposed voltammetric method has precision and accuracy comparable to those of the spectrophotometric CUPRAC assay.

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.

Antioxidant/antiradical properties of microwave-assisted extracts of three wild edible mushrooms.

A microwave-assisted extraction (MAE) process for polyphenols from three wild edible mushrooms was studied. The optimal extraction conditions were found to be methanol concentration of 80%, extraction temperature of 80 °C, and extraction time of 5 min. Different antioxidant assays (i.e., total antioxidant capacity (TAC) and total phenolic content (TPC)) were utilized to evaluate the antioxidant capacity of the methanolic extracts of Terfezia boudieri Chatin, Boletus edulis, and Lactarius volemus. The reactive species scavenging activities of these extracts were also investigated in vitro. High contents of phenolic and flavonoid compounds may be the major contributors to the observed high antioxidant activities of these extracts. B. edulis showed the higher TAC and TPC; highest inhibitory effect on DPPH and on other studied reactive oxygen species (ROS). MAE showed obvious advantages of high extraction efficiency with lower solvent consumption in terms of high antioxidant capacity/activity of extracts achieved within the shortest time.

Development of a fluorescent probe for measurement of peroxyl radical scavenging activity in biological samples.

In antioxidant activity testing, it has been argued that assays capable of measuring the inhibitive action against the biologically relevant peroxyl radicals (ROO(•)) from a controllable source are preferable in terms of simulating physiological conditions because ROO(•) is the predominant free radical found in lipid oxidation in foods and biological systems. A new fluorescent probe, p-aminobenzoic acid (PABA), was developed for selective measurement of peroxyl radical scavenging (PRS) activity of biological samples, in view of the fact that the existing PRS assays are quite laborious and require the application of strictly optimized conditions. The earlier probe, ß-phycoerythrin, of a similar PRS assay of wide use, oxygen radical absorbance capacity (ORAC), varies from lot to lot of production, undergoes photobleaching, and interacts with polyphenols via non-specific protein binding, while the current probe, fluorescein, undergoes undesired fluorescence (FL) quenching and side reactions. The developed technique is based on the fluorescence decrease of the PABA probe (within an optimal time of 30 min) because of its oxidation by ROO(•), generated from the thermal dissociation of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). In the absence of the scavenger, ROO(•) reacted with the probe, generating non-fluorescent products, and caused a decrease in PABA fluorescence, whereas the ROO(•) scavenger resulted in a fluorescence increase because of the inhibition of the probe oxidation by ROO(•). Thus, the fluorescence increment of intact PABA is proportional to the ROO(•) scavenging activity of samples. The linear range of relative fluorescence intensity versus the PABA concentration was in the interval of 0.5-5.0 µM. Assay precision and accuracy were assessed by analyzing two spiked homogenates of liver and kidney at clinically relevant concentrations with 97-105% recovery and 2.3% interday reproducibility. The proposed method was successfully applied to assay the ROO(•) scavenging activity of some amino acids, plasma and thiol-type antioxidants, and albumin, with the latter showing the strongest activity with respect to both ORAC and developed PABA methods. On the other hand, the original ORAC method suffers a limitation from protein thiols in total radical-trapping antioxidant parameter (TRAP) calculations, and inconsistent results have been reported by various researchers for ORAC values of thiols, such as vastly differing values for glutathione and zero value for cysteine.

Novel oxime based flavanone, naringin-oxime: synthesis, characterization and screening for antioxidant activity.

Recent interest in polyphenolic antioxidants due to their involvement in health benefits has led to the investigation of new polyphenolic compounds with enhanced antioxidant activity. Naringin (4',5,7-trihydroxyflavanone-7-ß-l-rhamnoglucoside-(1,2)-α-d-glucopyranoside) is one of the major flavanones in citrus and grapefruit. The present study aimed to synthesize naringin oxime from naringin and to evaluate its antioxidant and anticancer potential using in vitro assay system. The structure of the synthesized compound, naringin oxime, was elucidated by FT-IR, (1)H NMR, elemental analysis and UV-vis spectroscopy. Antioxidant capacity of naringin oxime, as measured by the cupric reducing antioxidant capacity (CUPRAC) method, was found to be higher than that of the parent compound naringin. Other parameters of antioxidant activity (scavenging effects on OH, O2(-), and H2O2) of naringin and naringin oxime were also determined.

Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method.

INTRODUCTION: Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). OBJECTIVE: To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. METHODS: The separation and UV detection of polyphenols were performed on a C18 -column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. RESULTS: The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (µg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. CONCLUSION: The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower.

Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method.

In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid > epicatechin > quercetin ≈ catechin > α-tocopherol > rosmarinic acid > trolox > caffeic acid > ascorbic acid.

Selective optical sensing of biothiols with Ellman's reagent: 5,5'-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles.

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB(2-)) anion as a result of reaction with biothiols was measured at 410nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57µM and acceptable linearity ranging from 0.4 to 29.0µM (r=0.998).

Novel optical fiber reflectometric CUPRAC sensor for total antioxidant capacity measurement of food extracts and biological samples.

A novel fiber optic sensor was developed for screening the total antioxidant capacity (TAC) based on the use of cupric-neocuproine (Cu(II)-Nc) immobilized onto a Nafion cation-exchange membrane with reflectance spectrometric measurement. The reflectance change associated with the formation of the highly colored Cu(I)-Nc chelate on the membrane as a result of reaction with antioxidants was measured at 530 nm by using a miniature reflectance spectrometer. The calibration graph of trolox (TR) was linear with a slope of 3.40 × 10(3) L mol(-1) mm(-1). The limit of detection (LOD) and limit of quantification (LOQ) for TR in the reflectometric cupric reducing antioxidant capacity (CUPRAC) method were found as 0.53 and 1.76 µM, respectively. The trolox equivalent antioxidant capacities (TEAC) of various antioxidant compounds using the proposed method were comparable to those of the main CUPRAC assay. This assay was validated through linearity, additivity, precision, and recovery. The developed reflectance sensor was used to screen the TAC of some commercial fruit juices and mice tissue homogenates without preliminary treatment. The method is rapid, inexpensive, versatile, and nonlaborious, uses stable reagents on the sensor, and enables the in situ estimation of antioxidant capacity of food extracts and biological samples.

Direct measurement of total antioxidant capacity of cereals: QUENCHER-CUPRAC method.

Polyphenols in cereal samples are distributed as free, soluble-esterified, and insoluble-bound forms either esterified or etherified to cell wall constituents. In the evaluation of total antioxidant capacity (TAC) of cereals, rather difficult and time-consuming acid, alkaline and enzymatic treatments of residue have been applied to complete the extraction of bound phenolic compounds. Thus, this work is aimed to measure the TAC of cereals (i.e. barley, wheat, rye, oat) by the 'QUENCHER procedure' (involving forced solubilization of bound phenolics by oxidizing TAC reagents) with the direct use of copper(II)-neocuproine (Cu(II)-Nc) reagent of the CUPric Reducing Antioxidant Capacity (CUPRAC) assay. In this novel 'QUENCHER-CUPRAC' method, reaction time and solvent composition parameters were optimized, and the method was applied to cereal samples with CUPRAC reagent dissolved in 1:1 (v/v) ethanol-water mixture. The antioxidant capacities of soluble and insoluble fractions were simultaneously measured to give a hierarchic TAC order of cereals as: barley>rye>oat>wheat. The TAC values of cereals measured by QUENCHER-CUPRAC were higher than those of original QUENCHER method using ABTS(•+) and DPPH reagents. Polyphenolic mixtures in a cellulose matrix gave additive TAC values with QUENCHER-CUPRAC. The proposed method correlated linearly with QUENCHER-ABTS(•+) (r=0.956) and QUENCHER-DPPH (r=0.976).

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.

Determination of biothiols by a novel on-line HPLC-DTNB assay with post-column detection.

A novel on-line HPLC-DTNB method was developed for the selective determination of biologically important thiols (biothiols) such as L-cysteine (Cys), glutathione (GSH), homocysteine (HCys), N-acetylcysteine (NAC), and 1,4-dithioerythritol (DTE) in pharmaceuticals and tissue homogenates. The biothiols were separated on C18 column using gradient elution, reacted with the postcolumn reagent, DTNB in 0.5% M-ß-CD (w/v) solution at pH 8, to form yellow-colored 5-thio-2-nitrobenzoic acid (TNB), and monitored with a PDA detector (λ=410 nm). With the optimized conditions for chromatography and the post-column derivatization, 40 nM of NAC, 40 nM of Cys, and 50 nM of GSH can be determined. The relative standard deviations of the recommended method were in the range of 3.2-5.4% for 50 µM biothiols. The negative peaks of biothiol constituents were monitored by measuring the increase in absorbance due to TNB chromophore. The detection limits of biothiols at 410 nm (in the range of 0.04-0.58 µM) after post-column derivatization with DTNB+M-ß-CD were much lower than those at 205 nm UV-detection without derivatization, and were distinctly lower than those with post-column DTNB alone. The method is rapid, inexpensive, versatile, nonlaborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of biothiol constituents of biological fluids and pharmaceuticals.