The Protective Role of Hippophae rhamnoides L. on Rat Brain and Liver Tissues Exposed to Cold Plus Immobilization Stress Model.

AIM: To investigate the protective role of Hippophae rhamnoides L. (Sea buckthorn, SBT) in cold plus immobilization stress-induced oxidative and nitrosative stress in rats. MATERIAL AND METHODS: Thirty-two Wistar albino rats were randomly divided into four groups: control (i.p. physiological saline), SBT (i.p. 200 mg/kg/48h SBT extract), stress (i.p. physiological saline; 6-h cold plus immobilization stress) and SBT+stress (i.p. 200 mg/kg/48h SBT; 6-h cold plus immobilization stress).* In liver and brain tissues 3-nitrotyrosine levels were determined by ELISA while total antioxidant capacity, total thiol, total glutathione, total nitrite+nitrate levels, superoxide dismutase and glutathione peroxidase activities were measured using colorimetric methods. RESULTS: In the SBT+stress group, the total glutathione levels and glutathione peroxidase activities were significantly higher in both tissues, whereas the total nitrite+nitrate levels and superoxide dismutase activities decreased compared with the stress group. The 3-nitrotyrosine levels as oxidative and nitrosative stress markers were found to be significantly higher in SBT+stress group in both tissues than in the control. No significant differences were found between the stress and SBT+stress groups in the liver. CONCLUSION: The results show that SBT has antioxidant properties against cold plus immobilization stress-induced oxidative and nitrosative stress and that it can be recommended as a natural antioxidant and nutritional supplement.

HPLC Detection and Antioxidant Capacity Determination of Brown, Red and Green Algal Pigments in Seaweed Extracts.

This study was carried out to determine the main pigments in some different selected seaweeds and to reveal their antioxidant potential regarding the ever-increasing demand for utilization of marine pigments in human health and nutrition. The individual amounts of algal pigments were found by reverse phase high-performance liquid chromatography (HPLC) and their total antioxidant capacities (TAC) by two spectrophotometric TAC assays, namely: CUPRAC (CUPric ion Reducing Antioxidant Capacity) and ABTS/TEAC (2,2'-azinobis [3-ethyl benzo thiazoline-6-sulfonate])/(trolox equivalent antioxidant capacity). These two tests gave the same rank order for TAC. The TAC of HPLC-quantified compounds accounted for a relatively much lower percentage of the observed CUPRAC capacities of seaweed extracts, namely ranging from 11 to 68% for brown, from 4 to 41% for red and from 3 to 100% for green species, i.e., some TAC originated from chromatographically unidentified compounds. Fucoxanthin, chlorophyll a, and pheophytin a compounds were major pigments in brown algae. The relative carotenoid contents in red marine algae were generally lower than those of chlorophylls. Overall total quantities were quite low compared with those of brown species. In general, chlorophyll a and chlorophyll b were chiefly present in green algae, but ß-carotene, violaxanthin and siphonaxanthin were also detected substantially higher in some species of green algae such as Caulerpa racemosa var. cylindracea and Codium fragile.

Colorimetric sensors and nanoprobes for characterizing antioxidant and energetic substances.

The development of analytical techniques for antioxidant compounds is important, because antioxidants that can inactivate reactive species and radicals are health-beneficial compounds, also used in the preservation of food and protection of almost every kind of organic substance from oxidation. Energetic substances include explosives, pyrotechnics, propellants and fuels, and their determination at bulk/trace levels is important for the safety and well-being of modern societies exposed to various security threats. Most of the time, in field/on site detection of these important analytes necessitates the use of colorimetric sensors and probes enabling naked-eye detection, or low-cost and easy-to-use fluorometric sensors. The use of nanosensors brings important advantages to this field of analytical chemistry due to their various physico-chemical advantages of increased surface area, surface plasmon resonance absorption of noble metal nanoparticles, and superior enzyme-mimic catalytic properties. Thus, this critical review focuses on the design strategies for colorimetric sensors and nanoprobes in characterizing antioxidant and energetic substances. In this regard, the main themes and properties in optical sensor design are defined and classified. Nanomaterial-based optical sensors/probes are discussed with respect to their mechanisms of operation, namely formation and growth of noble metal nanoparticles, their aggregation and disaggregation, displacement of active constituents by complexation or electrostatic interaction, miscellaneous mechanisms, and the choice of metallic oxide nanoparticles taking part in such formulations.

Use of modified CUPRAC and dinitrophenylhydrazine colorimetric methods for simultaneous measurement of oxidative protein damage and antioxidant defense against oxidation.

A modified CUPRAC (cupric reducing antioxidant capacity) method was developed for the simultaneous estimation of protein oxidation and counteracting antioxidant defense, and the results were compared with those of a modified 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay. The alkaline carbonyl method was cleared off interferences by solvent extraction using a cationic surfactant. Both solution and Nafion membrane sensor CUPRAC methods were used to measure the oxidative hazard in protein solutions. Bovine serum albumin, fetal bovine serum and egg white were used as protein probes, exposed to oxidation by Fe(II)-induced Fenton reaction in the absence and presence of selected antioxidants (ascorbic acid, cysteine, gallic acid, glutathione, and N-acetyl cysteine). Protein probes were initially unreactive toward the CUPRAC and DNPH reagents, but produced colored products upon Fenton oxidation which were bleached by antioxidants, enabling an indirect measurement of antioxidant activity (AOA) by difference. Spearman's rank test for antioxidants demonstrated that there was a strong correlation (+0.7 to +0.9) between the modified CUPRAC and carbonyl assays. There was also a strong correlation between the results of the solution phase and optical sensing CUPRAC methods (R2 > 0.95). As opposed to conventional antioxidant assays not using biologically relevant probes, this work utilizes protein probes for AOA assessment.

CUPRAC colorimetric and electroanalytical methods determining antioxidant activity based on prevention of oxidative DNA damage.

An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the 'comet' assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC-responsive. The DNA-protective effects of water-soluble antioxidants were used to devise a novel antioxidant activity assay, considered to be physiologically more realistic than those using artificial probes. Our method, based on the measurement of DNA oxidative products with CUPRAC colorimetry proved to be 2 orders-of-magnitude more sensitive than the widely used TBARS (thiobarbituric acid-reactive substances) colorimetric assay used as reference. Additionally, the DNA damage was electrochemically investigated using pencil graphite electrodes (PGEs) as DNA sensor platform in combination with differential pulse voltammetry (DPV). The interaction of the radical species with DNA in the absence/presence of antioxidants was detected according to the changes in guanine oxidation signal.

A colourimetric sensor for the simultaneous determination of oxidative status and antioxidant activity on the same membrane: N,N-dimethyl-p-phenylene diamine (DMPD) on Nafion.

A colourimetric sensor capable of simultaneously measuring oxidative status (OS) in terms of the hazard produced by reactive oxygen species (ROS) and antioxidant activity (AOA) in regard to ROS-scavenging ability of antioxidant compounds was developed. The coloured cationic semi-quinone derivatives, caused by ROS oxidative degradation of N,N-dimethyl-p-phenylene diamine hydrochloride (DMPD) in pH 5.7 acetate-buffered medium, were formed in solution and immobilized on a perfluorosulfonate-based Nafion membrane. ROS, namely hydroxyl (·OH) and superoxide (O2(·-)) radicals, were produced by Fenton/UV and xanthine/xanthine oxidase methods, respectively. The pink-coloured, (+)-charged chromophore (referred to as DMPD-quinone or DMPDQ), resulting from the reaction between DMPD and ROS, could be completely retained on the solid membrane sensor by electrostatic interaction with the anionic sulfonate groups of Nafion. After equilibration, the Nafion membrane surface was homogeneously coloured enabling an absorbance measurement at 514 nm, while the aqueous phase completely lacked colour. Antioxidants, when present, caused an absorbance decrease on the membrane due to their ROS scavenging action, giving rise to less DMPDQ production. The absorbance decrease on the sensor was linearly dependent on antioxidant concentration over a reasonable concentration range, enabling the simultaneous determination of OS and AOA-against ROS. The proposed antioxidant sensing method was tested in synthetic and real antioxidant mixtures, and validated against standard antioxidant capacity assays (i.e. ABTS and CUPRAC) for a variety of polyphenolic and antioxidant compounds. The dynamic linear ranges of antioxidants with the DMPD sensor in protection against hydroxyl and superoxide radicals generally varied within the micromolar to a few tens of micromolar concentration interval over one order-of-magnitude. Choosing three representative compounds in the high (epigallocatechin gallate), medium (quercetin) and low (p-coumaric acid) molar absorptivity range, the detection limits ranged within the concentration intervals of 0.2-0.9 µM, 0.3-0.8 µM, and 4-14 µM, respectively, depending on the radical scavenged.

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions.

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like ß-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

Correlation of total antioxidant capacity with reactive oxygen species (ROS) consumption measured by oxidative conversion.

Although both antioxidant capacity and oxidative conversion (hazard) are important in food and bioanalytical chemistry, there is considerable confusion in the literature between the results of these two types of assays. After the generation of ROS in the medium via Fe(III)-H2O2 reaction, attenuation of total oxidative conversion (TOC; as measured by thiobarbituric acid-reactive substances (TBARS) and N,N-dimethyl-p-phenylenediamine (DMPD) assays) was tested for possible correlation with the total antioxidant capacity (TAC; as measured by cupric reducing antioxidant capacity (CUPRAC) and trolox equivalent antioxidant capacity (ABTS/TEAC) assays) of the introduced antioxidant sample. The inverse relationship between oxidative conversion and antioxidant capacity was processed to establish a curvilinear relationship between the absolute values of TAC increments and TOC decrements as a function of added antioxidant concentration. This simple relationship may form a bridge between the two diverse disciplines of medical biochemistry and food analytical chemistry mainly using TOC and TAC results, respectively.

Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods.

Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC(FRAP) value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-L-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.

Modified cupric reducing antioxidant capacity (CUPRAC) assay for measuring the antioxidant capacities of thiol-containing proteins in admixture with polyphenols.

Proteins are not considered as true antioxidants but are known to protect antioxidants from oxidation in various antioxidant activity assays. This study aims to investigate the contribution of proteins, especially thiol-containing proteins, to the observed overall antioxidant capacity measured by known methods. To determine the antioxidant properties of thiol-containing proteins, the CUPRAC method of antioxidant assay using the oxidizing reagent Cu(II)-neocuproine previously used for simultaneous analysis of cystine and cysteine was adopted. While the CUPRAC method is capable of determining all antioxidant compounds including thiols in complex sample matrices, the Ellman method of thiol quantitation basically does not respond to other antioxidants. The antioxidant quantities in the selected samples were assayed with the ABTS and FRAP methods as well as with the CUPRAC method. In all applied methods, the dilutions were made with a standard pH 8 buffer used in the Ellman method by substituting the Na(2)EDTA component of the buffer with sodium citrate. On the other hand, the standard CUPRAC protocol was modified by substituting the pH 7 ammonium acetate buffer (at 1M concentration) with 8M urea buffer adjusted to pH 7 by neutralizing with 6M HCl. Urea helps to partly solubilize and denaturate proteins so that their buried thiols be oxidized more easily. All methods used in the estimation of antioxidant properties of proteins (i.e., CUPRAC, Ellman, ABTS, and FRAP) were first standardized with a simple thiol compound, cysteine, by constructing the calibration curves. The molar absorptivities of these methods for cysteine were: epsilon(CUPRAC)=7.71x10(3), epsilon(Ellman)=1.37x10(4), epsilon(ABTS)=2.06x10(4), and epsilon(FRAP)=2.98x10(3)L mol(-1)cm(-1). Then these methods were applied to various samples containing thiols, such as glutathione (reduced form:GSH), egg white, whey proteins, and gelatin. Additionally, known quantities of selected antioxidants were added to these samples to show the additivity of responses.

Spectrophotometric determination of paracetamol in urine with tetrahydroxycalix[4]arene as a coupling reagent and preconcentration with triton X-114 using cloud point extraction.

In the present paper, conventional spectrophotometry in conjunction with cloud point extraction-preconcentration were investigated as alternative methods for paracetamol (PCT) assay in urine samples. Cloud point extraction (CPE) was employed for the preconcentration of p-aminophenol (PAP) prior to spectrophotometric determination using the non-ionic surfactant Triton X-114 (TX-114) as an extractant. The developed methods were based on acidic hydrolysis of PCT to PAP, which reacted at room temperature with 25,26,27,28-tetrahydroxycalix[4]arene (CAL4) in the presence of an oxidant (KIO(4)) to form an blue colored product. The PAP-CAL4 blue dye formed was subsequently entrapped in the surfactant micelles of Triton X-114. Cloud point phase separation with the aid of Triton X-114 induced by addition of Na(2)SO(4) solution was performed at room temperature as an advantage over other CPE assays requiring elevated temperatures. The 580 nm-absorbance maximum of the formed product was shifted bathochromically to 590 nm with CPE. The working range of 1.5-12 microg ml(-1) achieved by conventional spectrophotometry was reduced down to 0.14-1.5 microg ml(-1) with cloud point extraction, which was lower than those of most literature flow-through assays that also suffer from nonspecific absorption in the UV region. By preconcentrating 10 ml sample solution, a detection limit as low as 40.0 ng ml(-1) was obtained after a single-step extraction, achieving a preconcentration factor of 10. The stoichiometric composition of the dye was found to be 1 : 4 (PAP : CAL4). The impact of a number of parameters such as concentrations of CAL4, KIO(4), Triton X-100 (TX-100), and TX-114, extraction temperature, time periods for incubation and centrifugation, and sample volume were investigated in detail. The determination of PAP in the presence of paracetamol in micellar systems under these conditions is limited. The established procedures were successfully adopted for the determination of PCT in urine samples. Since the drug is rapidly absorbed and excreted largely in urine and its high doses have been associated with lethal hepatic necrosis and renal failure, development of a rapid, sensitive and selective assay of PCT is of vital importance for fast urinary screening and antidote administration before applying more sophisticated, but costly and laborious hyphenated instrumental techniques of HPLC-SPE-NMR-MS.

Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat (veal and turkey), sardine, various milk products, and egg white were analyzed with the proposed and Lowry methods, and the results correlated appreciably (r=0.98). The method was validated by Kjeldahl analyses of the tested samples; the data sets of complex samples assayed by Cu(II)-Nc and Lowry correlated to the findings of Kjeldahl yielded correlation coefficients r=0.96 and 0.97, respectively, with slopes being close to 1. Interferences of glucose and thiol compounds at relatively low concentrations could be compensated for by selecting a lower alkaline pH (i.e., pH 10) at a cost of slightly reduced sensitivity and adding an identical amount of interferent to the reagent blank, respectively, since the absorbances due to BSA and interferent were additive. Thus a novel spectrophotometric method for total protein assay using a stable reagent and chromophore, which was simple, rapid, sensitive, flexible, and relatively selective, was developed, and applied to a variety of food products.