Chemiluminescence of synephrine based on the cerium(IV)-rhodamine B system.

A new chemiluminescence (CL) method is described for the determination of synephrine. It is based on the reaction between synephrine and Ce(IV) in a nitric acid medium and measurement of the CL intensity produced by rhodamine B used as a luminophore, similar to luminol or lucigenin in basic media, instead of as a sensitizer. In the optimum conditions, the increase of CL intensity was correlated with synephrine concentration over the range 5.0 x 10(-9)-1.0 x 10(-6) g/mL with a detection limit of 1.0 x 10(-9) g/mL. The relative standard deviation (RSD) was 2.9% for 1.0 x 10(-7) g/mL synephrine (n = 11). The method was applied to the determination of a drug in herbal products, citrus fruit and biological samples, with satisfactory results. The results given by the proposed method are in good agreement with those given by HPLC-UV and UV spectrophotometry.     

A study of the antioxidant and membranotropic activities of equinochrome a using different model systems

Echinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone) isolated from the body of sand dollar Scaphechinus mirabilis is an active substance of cardioprotective medication Histochrome and exerts a wide spectrum of anti-inflammatory activities. In the present paper, we conducted a comparative study of the antioxidant (radical-scavengering) properties of echinochrome A in 2,2′-azobis(2-methylpropionamidine) dihydrochloride?luminol and hemoglobin?hydrogen peroxide?luminol systems and assessed its impact on permeability of planar bilayer lipid membranes. Trolox was used as a reference antioxidant and ascorbic acid and dihydroquercetin are taken as standards. Echinochrome A shows moderate antioxidant activity, possessing higher antioxidant capacity than Trolox and ascorbic acid, but exhibiting lower antioxidant potential compared with dihydroquercetin in tests for antioxidant activity in both investigated systems. The test substances can be arranged in the following order according to the effectiveness of the antioxidant effect: dihydroquercetin > echinochrome A > Trolox > ascorbic acid. Echinochrome A does not lead to significant changes in the permeability of planar bilayer membranes in a dose range of 1.5 to 30 μМ. Our data indicate that echinochrome A has a rather high level of radical-scavengering activity without a primary membranotropic effect. It is thought that the high levels of the cardioprotective and anti-inflammatory activities of echinochrome A may be due not only to the ability of this substance to neutralize reactive oxygen species, but also to its capacity to generate physiological concentrations of hydrogen peroxide molecules in biological systems as signaling messengers of various metabolic processes and biochemical pathways. The suspected mechanisms of the biological activity of echinochrome A are discussed.     

A fluorometric method for measurement of oxygen radical-scavenging activity of water-soluble antioxidants

The relative activities of the antioxidants Trolox, ascorbic acid, uric acid, quercetin, and rutin, and the activities of total antioxidants in serum samples were determined using a fluorometric assay based on the dye 6-carboxyfluoroscein (6C-Fl) as a fluorescent indicator; 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) as a peroxyl radical generator; 6-hydroxy-2,5,7, 8-tetramethyl-1-chroman-2-carboxylic acid (Trolox) as a calibrator; and phosphate buffer (pH 7.0) as a solvent. Incubation of 6C-Fl in 0. 075 M phosphate buffer, in the presence of AAPH at 37 degrees C, resulted in loss of its fluorescence signal at 520 nm with excitation at 495 nm. The antioxidants Trolox, ascorbic acid, and uric acid provided protection of the fluorescence of 6C-Fl, and the relative antioxidant activities, determined by the net protection area under curve technique, were found to be 1:0.4:1, respectively. Trolox and ascorbic acid were used to validate this assay. A linear correlation of the net protection value with the concentration of serum, Trolox, ascorbic acid, and uric acid was demonstrated. Quercetin and rutin were shown to have strong antioxidant activities, nearly 10 times those of vitamin C. This assay is simple, reliable, and suitable for automation to handle many samples and requires few microliters of serum samples.     

Mild hypothermia protects against postischemic hepatic endothelial injury and decreases the formation of reactive oxygen species

The ability of mild hypothermia (MH; 34 degrees C) to protect against postischemic endothelial injury and decrease reactive oxygen species' (ROS) formation was studied using lucigenin and luminol enhanced chemiluminescence (CL). Lucigenin CL is largely specific for superoxide, while luminol reacts with many ROS. Isolated rat livers perfused under constant flow in a non-recirculating system were exposed to 2.5 h of ischemia after 0.5 h perfusion with Krebs-Henseleit buffer at either normothermia (38 degrees C) or mild hypothermia (34 degrees C) (n = 5, all groups). CL (cps), vascular resistance (Woods units), O2 consumption, and potassium efflux were measured at the end of perfusion, and at 0 min reperfusion, and every 30 min during reperfusion. For both the lucigenin and luminol groups, CL and vascular resistance increased significantly (repeat measures ANOVA, P <0.05) for normothermia (NT, 38 degrees C) but not mild hypothermia. Potassium efflux did not change significantly for the mild hypothermia groups. In the luminol enhanced group, oxygen consumption was greater in the mildly hypothermic group at 1 h and 1.5 h of reperfusion. Mild hypothermia decreased postischemic ROS production. Increased vascular resistance in the normothermia group may indicate an endothelial injury. Mild hypothermia appears to protect against this injury.     

Chelating and antiradical effect of rutin during peroxidation of lipids from microsomes and liposomes

The antioxidative effect of rutin (vitamin P) on Fe2+-induced lipid peroxidation (LPO) in bovine heart microsomes and lecithin liposomes was studied. It was shown that the LPO-induced inhibition of microsomes and liposomes in the presence of rutin occurs via two mechanisms, i.e., association of Fe2+ ions to form an inactive complex and a direct interaction between rutin and free radicals. The contribution of these mechanisms depends on the composition of the reaction mixture. In bovine heart microsomes and liposomes, ascorbic acid has a dual activity towards LPO. At high concentrations of Fe2+ necessary for LPO induction (approximately 1 x 10(-3) M), ascorbic acid blocks LPO, whereas at low Fe2+ concentrations (less than 1 x 10(-4) M) it has a prooxidative effect. A combined use of ascorbic acid and rutin results in an additive antioxidative effect at high Fe2+ concentrations (approximately 1.10(-3) M). However, at low Fe2+ concentrations rutin acts as an antagonist of the prooxidative effect of ascorbic acid.     

Luminol Luminescence Induced by 2,2'-Azo-Bis(2-Amidinopropane) Thermolysis

2-2'-Azo-bis(2-amidinopropane) thermolysis induces luminol luminescence. The luminescence intensity is quenched by SOD, catalase, Trolox and human blood serum. However, the time course of the light intensity profile is different for the different additives. In particular, the quenching efficiency of Trolox and human blood serum decreases with time after addition. Double quenching experiments show that SOD and Trolox are not competitive quenchers, while a simple competition can be established between Trolox and human blood serum in trapping a common intermediate. From the kinetic analysis of the data it is concluded that, at least at low additive concentrations, Trolox scavenges a luminol derived radical. Higher concentrations of Trolox or human blood serum produce induction times that are proportional to the additives concentrations. The possibility of employing luminol luminescence in the evaluation of TRAP levels and the capacity of biological samples to scavenge free radicals is discussed.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.     

Determination of tetracycline, chlortetracycline and oxytetracycline by flow injection with inhibitory chemiluminescence detection using copper(II) as a probe ion.

This paper reports an indirect flow-injection (FI) method for the determination of the tetracycline drugs (TCs), tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC), using copper(II) as a probe ion. The method was based on the inhibition caused by these TCs to the copper(II)-catalysed chemiluminescence (CL) reaction between luminol and H(2)O(2). The CL reaction was induced on-line and injection of the sample produced negative peaks as a result of the copper(II) complexation or displacement by the analytes. The height of the peaks was proportional to the drug concentration in the sample. The choice of the catalyst ion, the concentration of luminol, H(2)O(2) and copper(II) are discussed. The linear range was 3.6 x 10(-8)-1.0 x 10(-5), 1.1 x 10(-7)-1.0 x 10(-5) and 1.9 x 10(-7)-1.0 x 10(-5) mol/L for TC, CTC and OTC, respectively. The detection limit was 5.0 x 10(-9) mol/L for TC, 1.0 x 10(-8) mol/L for CTC and 2.0 x 10(-8) mol/L for OTC (3sigma), respectively. The method was applied to the determination of TCs in pharmaceutical preparations and human urine with recoveries in the range 95-105%.     

Semi‐micro flow injection analysis method for evaluation of quenching effect of health foods or food additive antioxidants on peroxynitrite

A semi‐micro flow injection analysis (SMFIA) method for evaluation of quenching effect of food additive antioxidants or health foods on peroxynitrite (ONOO^‐) is described. The injected sample was carried with phosphate buffer containing NaNO₂, mixed with a trigger solution to generate ONOO^‐ and then detected CL generated after mixing with luminol solution. Selective chemiluminescence caused by ONOO^‐ in this generation system was confirmed by catalase treatment. Ascorbic acid (ASA), Trolox and ascorbyl palmitate (ASP) were used as food additive antioxidants. EC₅₀ values of ASA, Trolox and ASP were 2.6, 6.4 and 43 µg/mL, respectively. The amount of reagents required for an assay by this SMFIA system could reduce the time by a third compared with the conventional method previously reported. Furthermore, as an application of the proposed method, the quenching effect of commercially available Noni (Morinda citrifolia) juices was evaluated. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.