Rapid automated assay of anti-oxidation/radical-scavenging activity of natural substances by sequential injection technique (SIA) using spectrophotometric detection

Abstract

A PC-controlled sequential injection analysis (SIA) system equipped with a spectrophotometric diode-array detector is used for rapid monitoring and evaluation of antioxidation/radical scavenging activity of biological samples. The automated method is based on the known reaction of stable 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) with antioxidants in organic or aqueous-organic media resulting in bleaching of DPPH due to its "quenching" by the interaction with the analytes. The decrease of the absorbance of DPPH (compared to blank experiment carried out with water-ethanol 1:1 instead of the test solution) measured at 525 nm is related to concentration of an antioxidant in the test solution. With the optimised SIA procedure it is possible to detect down to micromolar concentrations of model antioxidants such as ascorbic acid, caffeic acid, (+)-catechin, (-)-epicatechin and rutin and to evaluate the concentration of these antioxidants in the micromolar to millimolar range. The sample throughput is 45 h(-1). Thanks to its rapidity and sensitivity, the proposed SIA method is suitable for performing routine screening tests for the presence of various antioxidants in large series of lyophilised herbal or mushroom extracts (the amount of sample needed for the analysis is several milligrams).

Genesis and development of DPPH method of antioxidant assay

“…Inclusion of automation Several automation in the original DPPH assay, based on flow injection analysis (FIA) (Ukeda et al 2002;Ukeda 2004), sequential injection analysis (SIA) (Polasek et al 2004) and HPLC-FIA (Yamaguchi et al 1998) have been implemented in recent years. These methods were applied for screening and evaluation of scavenging capacity of several pure compounds and complex matrices, such as plant extracts and beverages.…”

“…4) (Polasek et al 2004). The method is based on the reaction of DPPH with antioxidants in organic or aqueous-organic media resulting in bleaching of DPPH due to its "quenching" by the interaction with the analytes and measured at 525 nm.…”

“…With the optimised SIA procedure, micromolar concentrations of 45 antioxidant samples could be determined in one hour (Vít et al 2007) which may not be possible in the batch version of the DPPH quenching method, since it is rather time consuming, the standard reaction time being 16-30 min (Guo et al 2001). The development of an automated DPPH method, based on SIA technique was supposed to be suitable for rapid testing of antioxidation activity in large series of plant extracts (Polasek et al 2004).…”

“…The proposed method is suitable for rapid screening of large series of natural samples for their anti-oxidation/radical scavenging activity (Polasek et al 2004). c. Biamperometric determination of antioxidant activity A novel electrochemical method for selective determination of antioxidant activity based on DPPH ·/DPPH redox couple and biamperometric technique is proposed by Milardovic et al (Fig.…”

Determinação da capacidade antioxidante de produtos naturais in vitro pelo método do DPPH•: estudo de revisão

“…(Huang et al, 2005;Wu;Schaich, 2005). O método DPPH• não é apropriado para medir a capacidade antioxidante de plasma e soro humano, porque as proteínas podem precipitar no meio da reação em solução alcoólica (Karadag et al, 2009).…”