Antioxidative activity measurement

Asharani et al. (2010) compared the antioxidant activity (measured as a-tocopherol units per gram) of methanolic extracts from different varieties of finger millet ( . coracana), little millet (P. sumatrense), foxtail millet (S. italica), and proso millet (P. miliaceum). Extracts from ragi averaged 15.3 0.6 while those of little millet, foxtail millet, and proso millet were 4.7 1.1,5.0 0.4, and 5.1 0.8, respectively. The total tocopherols in these millets were 4.1 0.2,1.3 0.2,1.2 0.008, and 3.6 0.1 mg/lOOg flour. 

Thyroid hormones and their structural analogs showed lower DPPH-scavenging activity in comparison with butylated hydroxytoluene (BHT) as a standard compound. 3,5,3, 5 -tetraiodothyroacetic acid, 3,3,5 -triiodo-L-thyronine, and thyroxine showed the highest antioxidant activity measured by DPPH reduction, 3,5,3 5 -tetraiodothyroacetic acid having over 20% of the activity of BHT (05). 

A9. Alho, H., and Leinonen, J., Total antioxidant activity measured by chemiluminescence methods. Meth. Enzyntol. 299, 3-14 (1999). 

The results obtained by ABTS and DPPH tests show that the antioxidant activity order for these different plant parts was approximately similar in both assays. However, the antioxidant capacity using DPPH compared to the one obtained by ABTS essay was underestimated about 33%. Arnao, (2000) and Delgado-Andrade et al, (2005) report the same occurrence and they explain that the DPPH is only dissolved in alcoholic media. In contrast, the ABTS radicals being solubilised in aqueous and in organic media the antioxidant activity measured is due to the hydrophilic and lipophilic nature of the compoimds. In addition, at 515 nm near the visible region where the antioxidant activity is measured, interferences occur with the DPPH coloration. 

Antioxidant Activity Measurement by HPLC Seagent(s) x - Chromogen Radical ... 

Mambro et al. (2003) reported a batch CL inhibition method for the antioxidant activity measurements of different forms of vitamin E in pharmaceutical formulations and compared their CL inhibition on a luminol-hydrogen peroxide-horseradish peroxidase enzyme system for 10 min at 25°C in 10 J,L samples. a-Tocopherol, mixed tocopherols... 

The antioxidant activities of carotenoids and other phytochemicals in the human body can be measured, or at least estimated, by a variety of techniques, in vitro, in vivo or ex vivo (Krinsky, 2001). Many studies describe the use of ex vivo methods to measure the oxidisability of low-density lipoprotein (LDL) particles after dietary intervention with carotene-rich foods. However, the difficulty with this approach is that complex plant foods usually also contain other carotenoids, ascorbate, flavonoids, and other compounds that have antioxidant activity, and it is difficult to attribute the results to any particular class of compounds. One study, in which subjects were given additional fruits and vegetables, demonstrated an increase in the resistance of LDL to oxidation (Hininger et al., 1997), but two other showed no effect (Chopra et al, 1996 van het Hof et al., 1999). These differing outcomes may have been due to systematic differences in the experimental protocols or in the populations studied (Krinsky, 2001), but the results do indicate the complexity of the problem, and the hazards of generalising too readily about the putative benefits of dietary antioxidants. 

The terminology describing the action of antioxidants is unfortunately not clear. Terms such as antioxidant power , antioxidant effectiveness , antioxidant ability , antioxidant activity , and antioxidant capacity are often used interchangeably and without discrimination. Here we use the term antioxidant activity as meaning a measure of the rate of antioxidant action, and the term antioxidant capacity as meaning a measure of the extent of antioxidant action, i.e. the amount of radicals or intermediates and products produced during oxidation that are quenched by a given antioxidant. Thus antioxidant activity is related to the kinetics of the antioxidant action and antioxidant capacity to the stoichiometry. 

DPPH- has an intense absorption maximum around 520 run (Yordanov and Christova, 1997), and antioxidant capacity and activity measured by the reduction of DPPH- are easily quantified by VIS-spectroscopy (Brand-Williams et al, 1995 Bondet et al, 1997, Espin et al, 2000). The stable radicals Fremy s salt (potassium nitrosodisulphonate) and galvinoxyl (2,6-di-tert-butyl-a-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-l-ylidene)-p-tolyloxy radical) have been used in a similar manner but with ESR detection, which can be used with samples that are not optically transparent (Gardner et al, 1998). 

An interesting development is the combination of HPLC and on-line measurement of reducing capacity or antioxidative activity. This approach allows both direct identification of antioxidative species in complex foods and quantification of the contribution to the overall antioxidative capacity in the absence of synergistic and antagonistic effects. Major advantages are less sample handling and the ability to rim large series of samples in an automated process. 

The on-line measurement of reducing capacity can be performed with either a single or a series of electrochemical detectors, and linear correlations have been demonstrated between total antioxidative activities determined by the electrochemical detection and those determined by DPPH- reduction or by the ORAC assay (Guo et al, 1997 Peyrat-Maillard et al, 2000). The reducing capacity must also be quantified by post-column reactions, either with DPPH- or by the reduction of phosphomolybdenum complexes followed by UV-VIS-detection (Bandoniene and Murkovic, 2002 Cardenosa et al, 2002). A combination of HPLC and semi-automatic ORAC analysis has also been described (Caldwell, 2001). 

BENZIE i FFand STRAIN J J (1999) Ferric reducing/antioxidant power assay Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, Meth Enzymol, 299, 15-27. 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

Rgure 2.3 The antioxidant activity of butyiated hydroxytoluene in the presence of exogenous iipid hydroperoxides. The oxidation of LDL was monitored by measuring the increase in absorbance at 234 nm as described in Fig. 2.2 and the lag phase (time before the phase of maximum rate of oxidation) estimated as described by Esterbauer et at. (1989). Samples of LDL were supplemented with the cortcentrations of 13-hydroperoxyoctadecanoic acid (13-HPODE) indicated and in the presence of 3 fM BHT. The lag phase in the absence of BHT for this preparation of LDL was 48 min. 

Mooradian (1993) has studied the antioxidant properties of 14 steroids in a non-membranous system in which the fluorescence of the protein phycoerythrin was measured in the presence of a lipid peroxyl radical generator (ABAP). Oxidation of the protein produces a fluorescent species. Quenching of fluorescence by a test compound indicates antioxidant activity. Oestrone, testosterone, progesterone, androstenedione, dehydroepian-drosterone, cortisol, tetrahydrocortisone, deoxycorti-... 

Probucol, another di-r-butyl phenol, is an anti-atherosclerotic agent that can suppress the oxidation of low-density lipoprotein (LDL) in addition to lowering cholesterol levels. The antioxidant activity of probucol was measured, using EPR, with oxidation of methyl linoleate that was encapsulated in liposomal membranes or dissolved in hexane. Probucol suppressed ffee-radical-mediated oxidation. Its antioxidant activity was 17-fold less than that of tocopherol. This difference was less in liposomes than in hexane solution. Probucol suppressed the oxidation of LDL as efficiently as tocopherol. This work implies that physical factors as well as chemical reactivity are important in determining overall lipid peroxidation inhibition activity (Gotoh et al., 1992). 

Table 3.10 shows the recovery from PP of Irgafos 168 and its oxidised and hydrolysed by-products by various extraction procedures. As may be observed, One-Step Microwave-Assisted Extraction (OSM) and US lead both to negligible hydrolytic additive degradation. The measured additive decay (by oxidation) is essentially due to the antioxidant activity during the processing (extrusion) step of the polymer and not to the US or microwave heating treatment. 

Sun T and Powers JR. 2007. Antioxidants and antioxidant activities of vegetables. In Shahidi F, Ho C-T, editors. Antioxidant Measurement and Applications. Washington, DC American Chemical Society, pp. 160-183. 

The thiocyanate method involves measurement of the peroxide value using linoleic acid as substrate and has also been widely used to measure the antioxidant activity in plant-based foods such as ginger extracts (Kikuzaki and Nakatani 1993), fruit peels (Larrauri and others 1996 1997), extracts from vegetable by-products (Larrosa and others 2002 Llorach and others 2003 Abas and others 2006 Peschel and others 2006), blueberry juice, wines, and vinegars (Su and Chien 2007). 

Decomposition of the primary products of lipid oxidation generates a complex mixture including saturated and unsaturated aldehydes such as hexanal. Hexanal is the most commonly measured end product of lipid oxidation, and both sensory and physicochemical methods are used for its determination. Where other antioxidant activity tests may be nonspecific, physicochemical measurement of hexanal offers the advantage of analyzing a single, well-defined end product. 

The scavenging ability toward O2 can also be measured by using electron spin resonance (ESR) spectrometry. The 02 anion is trapped with 5,5-dimethyl-1-pyrroline TV-oxidc (DMPO), and the resultant DMPO-OH adduct is detected by ESR using manganese oxide as internal standard. Noda and others (1997) used this technique to evaluate antioxidant activities of pomegranate fruit extract and its anthocyanidins (delphinidin, cyanidin, and pelargonidin). 

The photochemiluminiscence (PCL) assay was initially used by Popov and others (1987). Popov and Lewin (1994 1996) have extensively studied this technique to determine water-soluble and lipid-soluble antioxidants. The PCL assay measures the antioxidant capacity, toward the 02 radical, in lipidic and water phase. This method allows the quantification of both the antioxidant capacity of hydrophilic and/or lipophilic substances, either as pure compounds or complex matrices from different origin synthetic, vegetable, animal, human, etc. The PCL method is based on an approximately 1,000-fold acceleration of the oxidative reactions in vitro by the presence of an appropriate photosensitizer. The PCL is a very quick and sensitive method. Chua and others (2008) used this assay to determine the antioxidant potential of Cin-namomum osmophloeum, whereas Kaneh and Wang and others (2006) determined the antioxidant capacity of marigold flowers. The antioxidant activity of tree nut oil extracts was also assessed by this method (Miraliakbari and Shahidi 2008). 

In this method, ai-antiproteinase inhibits the hydrolytic enzyme elastase, and the remaining elastase activity is measured by monitoring increases in absorbance at410 nm. Martmez-Tome and others (2001) used a method based on this reaction to measure the antioxidant activity of broccoli amino acids and of Mediterranean spices. 

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed by Wolfe and Liu (2007). The method measures the ability of compounds to prevent the formation of dichlorofluorescein (DCF) by ABAP-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The method... 

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