3.1.1 Antioxidant Activity

Interest in natural antioxidants for both health and improved food stabilization has intensified dramatically since the last decade of the twentieth century. Health appli­cations have been stimulated by observations that free radicals and oxidation are involved in many physiological functions and cause pathological conditions. Natural antioxidants offer food, pharmaceutical, nutraceutical, and cosmetic manufacturers a “green” label, minimal regulatory interference with use, and the possibility of multiple actions that improve and extend food and pharmaceutical stabilization [168]. Determining antioxidant capacity has become a very active research topic, and a plethora of antioxidant assay methods are currently in use. Despite of it, there are no standard methods due to the sheer volume of claims and the frequent contra­dictory results of “antioxidant activities” of several products.

Reactive oxygen species which include superoxide anion (O2- . a free radical), the hydroxyl radical COH) and hydrogen peroxide (H2O2) are produced by ultravio­let light, ionizing radiation, chemical reaction, and metabolic processes. These reac­tive species may contribute to cytotoxicity and metabolic changes, such as chromosome aberrations, protein oxidation, muscle injury, and morphologic and central nervous system changes in animals and humans [34]. Effective antioxidants must be able to react with all these radicals in addition to lipids, so, consideration of multiple radical reactivity, in antioxidant testing, is critical.

In general terms, three big groups can be distinguished: chain reaction methods, direct competition methods, and indirect methods [154].

1. Among the chain reaction methods two approaches have been used: measuring the lipid peroxidation reactions or the kinetics of substrate oxidation.

There are two modes of lipid peroxidation that may be used for testing. The first one is autoxidation, in which the process is progressing spontaneously, with self-acceleration due to accumulation of lipid hydroperoxide (LOOH). The kinet­ics of autoxidation is highly sensitive to admixtures of transition metals and to the initial concentration of LOOH. As a result, the repeatability of experiments based on the autoxidation is still a problem. The second, much more promising approach, is based on the use of the kinetic model of the controlled chain reaction. This mode offers to obtain reliable, easily interpretable, and repeatable data. This approach has been applied, among others, to test natural water-soluble antioxi­dants, microheterogeneous systems, micelles, liposomes, lipoproteins (basically low-density lipoprotein [LDL]), biological membranes, and blood plasma [154].

When choosing a substrate of oxidation, preference should be given to individual compounds. Among individual lipids, methyl linoleate, and linoleic acid seem to be the most convenient. These compounds are relatively cheap and their oxidation is quite representative of the most essential features of biologi­cally relevant lipid peroxidation. The main disadvantage, when using them in biological materials, is that the extract must be free of the elected compound, as it is impossible to provide the identity of substrate. Besides, biologically originated substrates usually contain endogenous chain-breaking antioxidants (vitamin E, etc.), which can intervene in the testing procedure.

2. The direct competition methods are kinetic models, where natural antioxidants compete for the peroxyl radical with a reference-free radical scavenger:

• b-Carotene bleaching: competitive bleaching b-carotene during the autoxidation of linoleic acid in aqueous emulsion monitored as decay of absorbance in the visible region. The addition of an antioxidant results in retarding b-carotene decay [114].

• Free-radical induced decay of fluorescence of R-phycoerythrin: The intensity of fluorescence of phycoerythrin decreases with time under the flux of the peroxyl radical formed at the thermolysis of APPH (2,2′ — azobis-2-methyl — propanimidamide) in aqueous buffer. In the presence of a tested sample containing chain-breaking antioxidants, the decay of PE fluorescence is retarded [147].

• Crocin bleaching test: Crocin (strongly absorbent in the visible range) under­goes bleaching under attack of the peroxyl radical. The addition of a sample containing chain-breaking antioxidants results in the decrease in the rate of crocin decay [12].

• Potassium iodide test: KI reacts with the AAPH-derived peroxyl radical with the formation of molecular iodine. The latter is determined using an auto­matic potentiometric titrator with sodium thiosulfate. In the presence of antioxidant-containing samples, the rate of iodine release decreases [154].

3. When the indirect approach method is applied, the ability of an antioxidant to scavenge some free radicals is tested, which is not associated to the real oxidative degradation, or effects of transient metals. For instance, some stable colored free radicals are popular due to their intensive absorbance in the visible region [154]. There are two ways for presenting results, as equivalents of a known antioxidant compound (i. e., Trolox Equivalent Antioxidant Capacity, TEAC) or as the con­centration needed to reduce concentration of free radicals by 50% (EC50).

• DPPH test: It is based on the capability of stable-free radical 2,2-diphenyl-1- picrylhydrazyl (DPPH) to react with H-donors. As DPPH’ shows a very intensive absorption in the visible region (514-518 nm), it can be easily deter­mined by the UV-Vis spectroscopy [13]. This method has been applied online with TLC [65] and HPLC [5] to determine antioxidant activity in different algae extracts.

• ABTS test: The decay of the radical cation ABTS+^ (2,2′-azinobis(3-ethylben — zothiaziline-6-sulfonate) radical cation) produced by the oxidation of ABTS+^ caused by the addition of an antioxidant-containing sample is measured. ABTS+^ has a strong absorption in the range of 600-750 nm and can be easily determined spectrophotometrically. In the absence of antioxidants, ABTS+^ is rather stable, but it reacts energetically with an H-atom donor, such as pheno — lics, being converted into a noncolored form of ABTS [115].

• Ferric reducing antioxidant power (FRAP): The FRAP assay is based on the ability of antioxidants to reduce Fe3+ to Fe2+ [154]; if the reaction is coupled to the presence of some colored Fe2+ chelating compound like 2,4,6-trypyridyl — s-triazine, it can be measured spectrophotometrically.

• Cyclic voltammetry: The general principle of this method is as follows: the electrochemical oxidation of a certain compound on an inert carbon glassy electrode is accompanied by the appearance of the current at a certain poten­tial; while the potential at which a cyclic voltammetry peak appears is deter­mined by the redox properties of the tested compound, the value of the current is proportional to the quantity of this compound, in the presence of an antioxi­dant compound the signal will be lower [155].