Types, Properties And Activities Of Antioxidants
An antioxidant is a substance that when present at relatively low concentration(s), compared with those of the oxidisable substrate, significantly delays or inhibits oxidation of that substrate (Apak et al. , 2013) Antioxidants are divided into two classes; primary (or chain breaking) antioxidants and secondary (or protective antioxidants). Primary antioxidants occur naturally, but they can be produced synthetically for example butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) and the gallants (Antolovich, Prenzler, Patsalides, McDonald, & Robards, 2002; Lima, Vianello, Corrêa, Campos, & Borguini, 2014) in the food industry.
Naturally occurring antioxidants have been promoted because of concerns regarding the safety of synthetic antioxidants (Antolovich et al. , 2002). Primary antioxidants when in trace amounts may either delay or inhibit the initiation step of oxygen reactive species by reacting with a lipid radical or inhibit the propagation step by reacting with peroxyl or alkoxyl radicals (Antolovich et al. , 2002). The secondary or preventive antioxidants retard the rate of oxidation. In this way the antioxidant is able to eliminate free radicals. The secondary antioxidants include the polyphenols, flavonoids, vitamin C and E (Antolovich et al. , 2002). The biosynthesis and concentration of phenolic compounds in plants depends on genetic (Dilshad et al. , 2016; Gramazio et al. , 2014; Guo, Guo, Wang, Wang, & Ni, 2017; Martínez‐García et al. , 2016) and environmental factors (Apolot, 2018). Fruit and vegetable consumption has attracted growing interest because many epidemiological and biochemical studies have consistently demonstrated a clear and significant positive association between intake of these natural food products, consumed regularly as part of the diet, and reduced rates of heart disease, common cancers, diabetes (Testa, Bonfigli, Genovese, De Nigris, & Ceriello, 2016) and other degenerative diseases, as well as aging (Garcia-Salas, Morales-Soto, Segura-Carretero, & Fernández-Gutiérrez, 2010).
The protection that fruits and vegetables provide against these maladies is attributed to the presence of several antioxidants, especially to antioxidative vitamins, including ascorbic acid (vitamin C), α-tocopherol (vitamin E) and β-carotene (provitamin A) (Garcia-Salas et al. , 2010; Valko et al. , 2007). Phenolic substances are the main phytochemicals with antioxidant properties found in higher plants (Garcia-Salas et al. , 2010). Epidemiological studies reveal that the intake of phenolic compounds is inversely correlated with the risk of coronary heart disease(Lima et al. , 2014; Valko et al. , 2007) and in the human body, these phytochemicals are thought to provide health benefits by several mechanisms, including; free-radical scavenging; protection and regeneration of other dietary antioxidants (vitamin E); and chelating of pro-oxidant metal ions(Garcia-Salas et al. , 2010). The concentration of these phytochemicals is affected by environmental factors and processing (Cartea, Francisco, Soengas, & Velasco, 2010; Lima et al. , 2014). The other factors that affect the amounts of these antioxidants include light, temperature, mineral nutrition, pathogens, mechanical damage, plant-growth regulators (Garcia-Salas et al. , 2010).
The effect of processing and postharvest handling on phenolics, flavonoids and total antioxidant activities is different in different vegetable products and this in turn can depend on the post-harvest handling method (Cartea et al. , 2010). Natural antioxidants are difficult to isolate, detect and quantify individually from biological matrix due to their chemical diversity (Apak et al. , 2013). There are several methods used to determine total antioxidant activity of fresh vegetables. These methods are generally classified into two. (i) The electron transfer assay (ET-based assay) in which the antioxidant reacts with a fluorescent or coloured probe. (ii) The hydrogen atom transfer based assay (HAT-based assay) which measures the capability of an antioxidant to quench free radicals by donating hydrogen atom to a radical for example a peroxyl radical (ROO•) forming the aryloxy radical (ArO•) which is stabilized by resonance (Apak et al. , 2007). ROO• + ArOH → ROOH + ArO•The HAT-based assays include oxygen radical absorbance capacity (ORAC) assay, TRAP assay with R-phycoerythrin as the fluorescent probe, crocin bleaching assay with 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) as the radical generator, and β-carotene bleaching assay. In these assays the total antioxidant activity (TAA) is determined from competition kinetics by measuring the fluorescence decay curve of the probe in the absence and presence of antioxidants and the TAA is got by integrating the area under the curves (Apak et al. , 2007). In the ET-based assay the antioxidants react with a fluorescent or coloured oxidizing agent instead of the peroxyl radicals. For the spectrophotometric ET-based assays the capacity of an antioxidant to reduce an oxidant radical in reagent is measured basing on the change in colour when oxidant is reduced. The degree of colour change (either an increase or decrease of absorbance at a given wavelength) correlates with the concentration of antioxidants in the sample (Apak et al 2007 & Pisoschi, Cheregi, & Danet, 2009).
The ET-based assays include (i) 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/Trolox-equivalent antioxidant capacity (TEAC) and diphenylpicrylhydrazyl (DPPH) in which absorbance at a specified wavelength decreases. (ii) Folin total phenols assay, ferric reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC) in which there is an increase in absorbance at a particular specified wavelength as the antioxidant in sample reacts with the chromogenic reagent. In the FRAP assay the Fe(II) forms charge transfer complexes with the ligand for example 2,4,6-tripyridyl-s-triazine ligand (TPTZ). (λmax = 595 nm) and reacts with antioxidant in the sample as shown in equation below. Fe(TPTZ)23+ + AROH Fe(TPTZ)22+ + ArO• + H+ The DPPH• radical is deep violent as it stabilized by delocalization and this gives it absorption at band at about 520nm. The absorbance decreases when DPPH• solution is mixed with the sample with antioxidant which donates hydrogen atom to the DPPH• radical forming the reduced form (DPPH) with loss of the violet colour (Pisoschi, Cheregi, & Danet, 2009). DPPH• + ArOH → DPPH + ArO• + H+The discolouration of DPPH• solution increases with increasing amount of antioxidant in the sample.
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