J Appl Biomed 10:155-167, 2012 | DOI: 10.2478/v10136-012-0006-y

Automated assay of the potency of natural antioxidants using pipetting robot and spectrophotometry

Miroslav Pohanka¹, Jiří Sochor^2,3, Branislav Ruttkay-Nedecký^2,3, Natalia Cernei^2,3, Vojtěch Adam^2,3, Jaromír Hubálek^2,3, Marie Stiborová⁴, Tomáš Eckschlager⁵, René Kizek^2,3,*

¹ Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic

² Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic

³ Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

⁴ Department of Biochemistry, Charles University, Faculty Sci., Praha, Czech Republic

⁵ Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine Charles University, Praha, Czech Republic

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.

Keywords: DPPH; FRAP; ABTS; DMPD; free radicals; antioxidant

Received: October 12, 2011; Revised: December 13, 2011; Published: July 31, 2012  Show citation