Optimisation and Validation of HPLC-ABTS Assay for Screening of Radical Scavengers

Abstract

Large quantities of free radicals are produced by exogenous sources, such as ionizing radiation, tobacco smoke, pesticides, air pollutants, pharmaceuticals; also quite a big part of free radicals is continuously produced as by-products in cells of numerous intracell systems: cytoplasm molecules, membranes ferment systems, peroxisomes, mitochondria electron transmission systems, and microsome electron transmission systems [1]. When the protective antioxidant systems of the organism are insufficient, or if there is a lack of intrinsic anti-oxidants, free radicals may cause oxidative stress [2]. Oxidative stress in a human body causes initiation and development of most of the neurodegenerative, cancerous diseases. Research of raw herbal materials is the first step in the search of natural antioxidants [3]. Biologically active compounds of the herbal raw materials are distinct for different strength of the effect and different activity mechanisms [4]. Phenols of herbal origin are known for their radical scavenging activity [5]. The objective of this work is to develop and validate the on-line HPLC-ABTS assay; to apply the latter in detection of compounds possessing the radical scavenging ability in complex mixtures; to evaluate and compare the antioxidant activity of separate compounds. Currently the on-line methods, which combine HPLC distribution and post-column reaction, are designed for the search of individual antioxidants. Free radical ABTS•+ is most commonly used in post-column reaction sustaining the widely spread principle of spectrophotometric method. The greatest advantages of on-line methods are their selectivity, informatory capability and high sensitivity for precise determination of antioxidant-active compound and evaluation of its activity in complex compounds. When validating on-line HPLC-ABTS assay, [...].