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Several methods are available of the determination of the antioxidant activity of individual compounds and total antioxidant activity of food extracts and beverages. Using appropriate examples, describe how these in vitro methods operate (50%), their advantages and disadvantages (25%) and their appropriateness for determining oxidative stress in vivo (25%).
As the knowledge of healthy benefits of antioxidants are wide spread nowadays, started to become of great interest for food and supplement manufacturers in response of great demand from consumers. There are several methods being used to assess antioxidant activity of food extracts and beverages in vitro, which a few of them will be discussed critically and their suitability in determining oxidative stress in vivo. However, in order to have a better understanding of the methods a brief review about free radicals, antioxidants and the oxidative stress is explained below.
Free radicals may be defined as chemical species that has an odd or unpaired electron. They are neutral, short lived, unstable and highly reactive to pair up the odd electron to finally achieve stable configuration. Moreover, the two major types of free radicals can be derivative from (1) reactive oxygen species (ROS) and (2) Reactive nitrogen species (RNS), Types of ROS include the hydroxyl radical, the super oxide anion radical, hydrogen peroxide, singlet oxygen, hypochlorite radical, and various lipid peroxides, as for RNS depending on the environment nitric oxide (NO) can be converted to nitrosonium cation (NO+), nitroxyl anion (NO) or peroxynitrite (ONOO).1
To avoid the cells and organ damage of the body from the action of ROS and RNS substances, humans possess a complex antioxidant protection system, that functions interactively and synergistically to neutralize free radicals. Moreover, antioxidants acting stabilizing or deactivating, free radicals before they attack cells2. Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and well-being as well as food integrity for long period.
Moreover, naturally there is a dynamic balance between the amount of free radicals produced in the body and antioxidants to scavenge or quench them to protect the body against deleterious effects. An oxidative stress may result if antioxidants fall under normal physiological level as the body decrease the ability to get rid of these radicals from the metabolism. Consequences are damage of cell membrane, nucleic acids, proteins and enzymes and other small molecules, resulting in cellular damage and loss of function, that contribute to aging3 and degenerative diseases of aging such as cancer4, cardiovascular disease, immune system decline, diabetes mellitus, inflammation, renal failure and others.
There are several methods used nowadays to determine antioxidant activity of food extracts and beverages some of them were reviewed in 2009 and will be discussed below:
Oxygen Radical Absorbance capacity method (ORAC)
The assay method reflect a hydrogen atom transfer (HAT) reaction mechanism, measuring the oxidative degradation using a fluorescence probe, either beta-phycoerythrin or fluorescein, after mixed with a peroxyl radical generated by heat , using an Azo-initiator compounds, which damages the fluorescent molecule resulting in the loss of fluorescence. This reaction is usually recorded for 35 minutes after the addition of a free radical generator.
The results, fluorescence intensity vs. time, are recorded and plotted in a graph and the area under the curve (AUC) between the two decay curves (AUCsample- AUCblank) is calculated. Later, the degree of antioxidant-mediated protection is quantified by comparing with a standard solution, a vitamin E analogue, antioxidant trolox. Different concentrations of trolox are used to make a standard curve. Results for test samples (foods) have been published as "trolox equivalents" or TE.
One benefit of using the ORAC method to evaluate substances antioxidant capacity is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain multiple ingredients with various slow and fast acting antioxidants, as well as ingredients with combined effects that cannot be pre-calculated.
On the other hand, only antioxidant activity against mainly peroxyl and hydroxyl radicals is measured, also the nature of the damaging reaction is not characterized and the fluorescence probe used are not lipid soluble, which decreases in intensity in non-polar organic solvent. Moreover, the use of AMVN , a synthetic lipid - soluble azo compound, to generate free radical can be used but the method lose about 100 times its sensitivity due to low efficiency of the radical generator.5
The Ferric Reducing Ability of Plasma (FRAP)
This method is based on the ability of antioxidants to reduce ferric iron (Fe3+), by comparing the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions (Fe2+) in known concentration. Absorbance changes are linear over a wide concentration range with antioxidant mixtures, including plasma, and with solutions containing one antioxidant in purified form.
The advantages of the FRAP assay are: a quick, inexpensive, reagents are simple to prepare, results are highly reproducible and the procedure is straightforward. The FRAP assay offers an accepted index of antioxidant, or reducing, potential of biological fluids and can be used by automate, semiautomatic and manual methods.
Disadvantages are that not all reductants with the ability to reduce Fe3+ are antioxidants, therefore any electron donating substance can interfere in the FRAP results giving overestimated results. Another one is that some chelators in food can react with Fe3+, which may react with antioxidants. Finally FRAP cannot detect compounds such thiols and proteins and this may cause underestimations in the results when using serum as a substrate.
One similarity between TEAC and FRAP is that there is no link between the numbers of electrons that an antioxidant may donate during reactions. Also, depending on the sample used can take a long time and false values can be read before the reaction has ended in these methods.
Total Radical - Trapping Antioxidant Parameter (TRAP)
This method monitors the ability of antioxidant compounds to counteract the effect of a peroxyl radical generated again by the use of azo initiator compound 2, 2â€²-azo-bis (2-amidinopropane) dihydrochloride (AAPH) as the free radical source. A fluorescent probe is used, R-phycoerythrin, and the method can be monitored using spectrophotometry at a determined range (495nm).
The method has an advantage to be a sensitive to all known chain breaking antioxidants and can be used to quantify and compare antioxidant capacity.
However, this method can present limitations when the sample does not present a lag phase. The method is complex not easily reproducible as require high expertise and is time consuming. Moreover, quantifying antioxidant capacity may be difficult when using coloured radical reagents to read lag time response, since not every antioxidant has an obvious lag phase.
This method is characterized as a stable free radical due to the delocalization of the spare electron over the molecule. Thus, the molecule cannot dimerise, as would happen with other free radicals. The delocalization gives rise to a deep violet colour characterized by an absorption band at about 520 nm. When a DPPH+ solution is mixed with a substance which can donate a hydrogen atom, the reduced form is generated, accompanied by the loss of the violet colour.8 furthermore, the spectrophotometric method used to assess the total antioxidant activity is based on the absorbance decrease monitoring of the DPPH· radical (2,2-diphenyl-1-picrylhydrazyl) in the presence of antioxidants, the value of the absorbance diminishes as the antioxidant concentration increases because more DPPH+ is quenched by Trolox®.
Advantages of the method are that is simple and rapid as it needs only UV-Vis spectrophotometer and also can analyse a large number of samples using microplates.
The disadvantages are that (1) the DPPH can be only used in organic media, but not aqueous media. Also, when measuring foodstuffs and phenolic compounds careful must be taken when reading the absorbance as the DPPH radical at 517nm pos reaction win an antioxidant is decreased by light11, oxygen and types of solvent. (2) many antioxidants that may react quickly with peroxyl radicals in vivo may react slowly or no reaction at all to DPPH. (3) false results can be read with samples that contains eugenol and other phenols in the structure.
Trolox Equivalent Antioxidant Capacity (TEAC)
The method is based on electron transfer (ET) ability of antioxidant molecules to reduce the long-lived ABTS·+, a blue-green chromophore with characteristic absorption at 734 nm, compared with that of Trolox, a water-soluble vitamin E analogue. The addition of antioxidants to the preformed radical cation reduces it to ABTS, determining a decolourization.
In favour of the method the peroxydase substract used, ABTS, can be solubilised either in hydrophilic or lipophilic solutions without affecting the ionic strength. Another advantage is that the method permits to study over an extensive range of pH and is wide used and is simple to be operated.
The drawback is that the method the result found is not the inhibition of the oxidative process but the capability of the sample to interact with ABTS+. Moreover, depending on the sample used can take a long time and false TEAC values can be read before the reaction has ended.
Cellular antioxidant activity (CAA)
A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements, moreover, the method measures the ability of compounds to prevent the formation of dichlorofluorescein (DCF) by 2, 2'-azobis (2-amidinopropane) dihydrochloride (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 antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay.
The positive point is that the assay is more relevant than the chemical assays as they take into consideration some other aspects like cell uptake, distribution, and metabolism of antioxidant compounds.6 In addition, the method was already used successfully in phytochemicals and fruit extracts as well as pure compounds such quercetin, myricetin and others. However, the result of this method has not yet been correlated with In-vivo methods to be validated.
On the basis of the inactivation mechanism involved, major antioxidant capacity methods have been generally divided into two categories though: (1) hydrogen atom transfer (HAT) reaction and (2) electron transfer (ET).
HAT-based methods measure the classical ability of an antioxidant to scavenge free radicals by hydrogen donation to form stable compounds. As for ET methods, relative reactivity is based on deprotonation and ionization potential of the reactive functional group, so ET reactions are pH dependent. In general, ionization potential values decrease with increasing pH, reflecting increased electron-donating capacity with deprotonation.7
Despite the uses of a wide range of these chemical antioxidant activity assays, their appropriateness and ability to predict an in-vivo activity still not being demonstrated as they not reflect cellular physiological conditions and do not consider the bioavailability, uptake, and metabolism of the antioxidants, nevertheless in-vitro tests are useful when comparing a range of food for its total antioxidant capacity (TAC).
Biological systems are much more complex than the simple chemical mixtures employed besides antioxidant compounds may operate via multiple mechanisms and the food extracts have different antioxidant capacities for each particular assay, therefore the same substance has a different result depending on the assay used, which contributes to a large variability within the same food item due to the lack of standardization of the essays.8
It is obvious that the use of animal models and human studies, are the best methods yet to measure the efficacy of the antioxidants however, they require a high level of mobilization of resources, both financially, expertise and time. In addition, is not suitable for initial screening of foods and dietary supplements.