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Excessive amounts of reactive oxygen species (ROS), unless counterbalanced by antioxidants, can cause cellular damage under oxidative stress conditions; therefore, antioxidative defenses against ROS must be measured. With the development of nanotechnology, nanoparticles have found numerous applications in science, health, and industries. Magnetite nanoparticles (Fe3O4:MNPs) have attracted attention because of their peroxidase-like activity. In this study, hydroxyl radicals (·OH) generated by MNPs-catalyzed degradation of H2O2 converted the N,N-dimethyl-p-phenylenediamine (DMPD) probe into its colored DMPD·+ radical cation, which gave an absorbance maximum at ? = 553 nm. In the presence of antioxidants, ·OH was partly scavenged by antioxidants and produced less DMPD·+, causing a decrease in the 553 nm-absorbance. Antioxidant concentrations were calculated with the aid of absorbance differences between the reference and sample solutions. The linear working ranges and trolox equivalent antioxidant capacity coefficients of different classes of antioxidants were determined by applying the developed method. In addition, binary and ternary mixtures of antioxidants were tested to observe the additivity of absorbances of mixture constituents. The method was applied to real samples such as orange juice and green tea. Student t-test, F tests, and the Spearman's rank correlation coefficient were used for statistical comparisons. © 2020 TUBITAK. All rights reserved. 28963 This study was funded by İstanbul University-Cerrahpaşa with the project number 28963. We express our gratitude to İstanbul University-Cerrahpaşa Application and Research Center for the Measurement of Food Antioxidants (GAAM) for assay development. This study was funded by Istanbul University-Cerrahpa?a with the project number 28963. We express our gratitude to Istanbul University-Cerrahpa?a Application and Research Center for the Measurement of Food Antioxidants (GAAM) for assay development.

Summary In the food industry, in the process of creating new agricultural plant products, and in the testing of anti-cancer drugs there is often a need to assay multiple samples of low molecular weight antioxidants, plant samples and foods rich in antioxidants, with minimal additional costs and low degrees of uncertainty. With these demands in mind, we decided to study the fully automated assay of antioxidants using not only automated sample measurements but also automated processing of samples and application of reagents. The automated pipetting system epMotion 5075 and the automated spectrophotometer BS 400 were chosen for the assay purposes. Five methods were introduced for the automation: 2-diphenyl-1-picrylhydrazyl (DPPH) test, ferric reducing antioxidant power (FRAP) method, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) based test, N,N-dimethyl-1,4-diaminobenzene (DMPD) based test and the free radicals method. Samples containing one of the four antioxidants (standard rutin, quercitrin, ferulic and gallic acid) in a range 1–1000 μg/ml were used throughout. All of the tested methods were found suitable for implementation in an automated assay. However, some of them, such as the ABTS test failed to assay all tested antioxidants. The coefficients of determination were also unequal. From the analytical point of view, FRAP methods provided the most reliable results in the automated assay; because of the capacity of the method, approximately 240 samples per hour (one sample per 15 seconds) can be assayed using the automated protocol. We were encouraged by the data received and we expect further interest in the practical performance of such automation. As a mean of testing the robustness of our method, in the next step of our study, oxidative status was assessed in model cell lines derived from prostate cancer (PC-3, PNT1A and 22RV1) that were cultured on ellipticine (0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15 μmol/l) supplemented agar. Antioxidant activity was assessed (DPPH, ABTS, FRAP, DMPD, FR) and calculated on the phenolic antioxidant level (rutin, quercitrin, ferulic and gallic acid), and thus an estimation was formulated of the oxidative stress as a result of the impact of anti-cancer drugs. It can be demonstrated that the new method has wide applicability.

6 páginas, 4 figuras, 2 tablas, Ascorbic acid (AA) and dehydroascorbic acid (DHAA) were analysed in fish muscle by high performance liquid chromatography (HPLC) coupled with fluorescence determination using 4,5-dimethyl-ophenilenediamine (DMPD) as a derivative agent. The use of the DMPD in combination with isobutanol extraction eliminated many matrix interferences and increased significantly the specificity and sensitivity of the method. A strong pH dependence in the derivatization reaction was also demonstrated. Satisfactory results in terms of sensitivity, stability and reproducibility were obtained. The analyses demonstrated that the relative ratio of AA and DHAA in post-mortem fish muscle differs among fish species and seems to be related with the susceptibility to oxidation., This work was performed within the research project PPQ2003-06602-C04-03 and the Integrated Research Project SEAFOODplus, contract no. FOOD-CT-2004-506359. The financing of this work by the Spanish Ministry of Science and Technology and the European Union is gratefully acknowledged. J. Iglesias acknowledges his doctoral grant from CSIC (Consejo Superior de Investigaciones Científicas).