Methods for Measuring Antioxidant Activity

Fogliano V, Verde V, Randazzo G and Ritieni A. 1999. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J Agric Food Chem 47(3) 1035-1040. 

Miller NJ, Rice-Evans C and Davies MJ. 1993. A new method for measuring antioxidant activity. Biochem Soc Trans 21(2) 95S... 

Current methods used to measure antioxidant activity are complicated, expensive, time-consuming, and as a rule, cannot be used for continuous monitoring. Potentiometry with the use of a mediator system and nanoparticles containing electrodes provides a very simple express-method for measuring antioxidant activity of biological liquids, nutrients, drugs and foodstuffs. 

A sensitive and simple chemiluminescent (CL) method for measuring antioxidant activity was developed. The determination of TEAC (Trolox equivalent antioxidant capacity) is based on the inhibition of CL intensity of luminol by an antioxidant. Antimicrobial activity was tested as a minimal inhibitory concentration (MIC) by broth microdilution method on gram-positive Enterococcus faecalis. Staphylococcus aureus) and gram-negative Pseudomonas aeruginosa, Escherichia coli) bacterial strains. 

A variety of natural and synthetic antioxidants are used in fat-containing foods in order to inhibit lipid oxidation with a wide range of efficiencies, depending on their properties, concentrations, and processing conditions. The need to measure antioxidant activity is well documented. Although numerous methods have been proposed for measurement of antioxidant activity, the essential features of any test are a suitable substrate, an oxidation initiator, and an appropriate measure of endpoint (9). Therefore, certain aspects should be taken into consideration when selecting a test for measuring antioxidant activity. These include the model food system used for... 

Other measurements of antioxidant activity include FRAP (ferric reducing-antioxidant power) (123-126), TRAP (total radical-trapping antioxidant parameter) (123, 127), phycoerythrin assay (128, 129), and test of metal chelating capacity (130, 131), among others. Reviews on methods for testing antioxidant activity have been published (9, 12). 

Methods of category (3) tract the capacity of the test compound to capture radicals or to inhibit radical formation rather than monitoring the actual oxidation product formation or substrate oxidation. Several new methods are developed based on this concept, and a variety of new parameters for expressing results are used. It is expected that a high correlation exists between these two types of measurements. It should be noted here that there are no standard units for reporting the antioxidant activity because such activity (assay, capacity, efficiency, effectiveness, etc.) is independent of the test procedure. Table 4 summarizes the methods available for measuring antioxidant activity and how the results of such determinations are expressed. 

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... 

N2. Naguib, Y. M., A fluorometric method for measurement of oxygen radical-scavenging activity of water-soluble antioxidants. Anal. Biochem. 284, 93-98 (2000). 

The CL method has been proven to be a convenient, reliable, rapid, and sensitive method for measuring oat antioxidant capacity compared with the P-carotene bleaching method. The latter has been used to measure the antioxidant activity of oats and is considered to be convenient and rapid compared with conventional accelerated-storage trials. Repeated measurements of an oat extract by using the CL method gave consistent results with low variations. The method is two orders of magnitude more sensitive than the P-carotene method. It requires only 15 min of automated sampling per extract compared with 2 h for continuous spectrophotometric measurement of several samples. 

Natural antioxidants can be simply defined as a group of natural compounds that exhibit antioxidant activity. This chapter will discuss the reasons for incorporating natural antioxidants into functional foods, the factors that affect antioxidant concentrations in extracts or oils, and applications of these materials in food systems. Methods for measuring natural antioxidants are not presented, but additional information is available in the referenced literature. 

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. 

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. 

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... 

In another study conducted on marine zooplankton (Byun et ah, 2009b), antioxidant activity was measured for the DPPH radical of hydrolysates produced by Alcalase, a-chymotrypsin, Neutrase, papain, pepsin, and trypsin. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (MW 1076Da) and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (MW 1033Da) by Q-TOF ESI mass spectroscopy. IC50 values of purified peptides were... 

Food and herbs are main natural sources of antioxidants. In this connection, antioxidant activity (AOA) of these products is essential for planning right diet as well as for sound therapy. Screen-printed platinum sensor and potentiometric method of measuring AOA of food and herbal extracts are presented below. 

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