**5. Discussion — Conclusion**

reagent is much more stable than other radicals such as DPPH, ABTS. The redox reaction giving rise to a coloured chelate of Cu(I)-Nc is relatively insensitive to a number of parameters such as air, sunlight, humidity, and pH. The CUPRAC reagent can be adsorbed on a membrane to

A variant of CUPRAC assay is Bioxytech using bathocuproine instead of neocuproine [18].

The HAT-based methods involve a synthetic radical generator, oxidisable molecular probe and an antioxidant compound. This method measures the ability of an antioxidant to quench free radicals by hydrogen donation as in equation 2. Assays that are based on HAT mechanisms

Antioxidant with hydroxyl component OH donates an H atom to an unstable free radical to give a more stable radical. HAT reactions are solvent and pH independent and are usually quite rapid, typically completed in seconds to minutes. The presence of reducing agents, including metals, is a complication in HAT assays and can lead to erroneously high apparent

The ORAC assay has been used widely in measuring the net resultant antioxidant capacity (or

The ORAC assay was developed by [28] for the determination of reactive oxygen species in biological systems. [29] modified the method using fluorescein (FL) as a more stable and reproducible fluorescent probe. This method exists under several adaptations but the principle always remains the same: using a fluorescent probe and AAPH (2,2'-azobis(2-amidinopro‐ pane) dihydrochloride) to generate peroxyl radicals. A HAT reaction occurs between antiox‐ idant samples (or standard) and the peroxyl radicals generated by thermal degradation of

The final results (ORAC values) were calculated using the differences between blank and sample areas under the quenching curves of fluorescein, and were expressed as micromoles

The ORAC method is superior to similar methods because it combines inhibition time and inhibition degree of free radicals. The ORAC using fluorescein is specific for antioxidants and is sensitive, precise and robust. This assay can model reactions of antioxidants with lipids in both food and physiological systems and it can be adapted to detect both hydrophilic and hydrophobic antioxidants with minor modifications. However, the need of a fluorometer, which may be not routinely available, is considered as a disadvantage of this method. The long

This assay was developed by [30] and modified by other researchers. This assay is based on the generation of a stable β –carotene radical from β –carotene peroxyl radical; the latter

analysis time has also been a major criticism even if this assay can be automated.

peroxyl radical absorbance capacity) of botanical and other biological samples.

AAPH. These reactions lead to a loss of fluorescence measured at 515 nm.

build an optical antioxidant sensor.

measure competitive kinetics [22].

**• Oxygen radical absorbance capacity (ORAC)**

**4.2. Systems based on HAT**

reactivity [18].

86 Biotechnology

of Trolox equivalents (TE).

**• β -carotene bleaching test**

Many results on the determination of the antioxidant activity of purified molecules and / or food raw material have been published over the last few years. However, the obtained data present a broad variability even for a given method or a given molecule. To overcome these problems, some authors have proposed other alternatives by developing new methods, or new ways to process data and express the results. [34] proposed the « quencher method », where the antioxidant activity is directly measured from the solid sample without the extraction step. Free radicals are mixed with the food sample and a spectrophotometric method (ABTS, DPPH) was used. [35] developed the global antioxidant response (GAR) method which uses an in vitro approach with enzymatic digestion, designed to mimic digestion through the gastrointestinal tract aimed at releasing antioxidants in foods. [36] suggested a general method of standardi‐ zation of estimations of total antioxidant activity (TAA) by extrapolating parameters to zero sample concentration based on a pseudo-first-order kinetics model. Accurate results were obtained in comparison with the ABTS method. Moreover, several papers deal with the standardization of the extraction procedures and the results analysis for a given method in order to minimize the observed variability [37]. However, it appears difficult to find a universal method knowing that many kinds of antioxidants and radicals are present. Four general sources of antioxidant have been identified: (i) enzymes (superoxide dismutase, gluthatione peroxidase), (ii) large molecules (albumin, ferritin), (iii) small molecules (phenols, ascorbic acid, carotenoids) and (iv) and some hormones (estrogen, melatonin). Many kinds of free radicals can be found, for example O2▪-, HO▪, NO▪, RO(O)▪, LO(O)▪. Moreover, the stability, the selectivity of the radicals and the reaction mechanisms can be also different. Thus, it is possible that no single method may be able to express the antioxidant capacity of different antioxidants taken independently or in a mixture [18]. Previous studies demonstrated that it is not appropriate to use one-dimensional methods to evaluate the antioxidant activity of multifunctional food such as fruits and vegetables, since they contain a large diversity of natural antioxidants.

The determination of antioxidant activity in the food matrix needs a sample preparation to extract the active molecules and then an accurate method of measurement and an expression of the results. (i) During sample preparation, precautions must be taken to avoid the loss of antioxidants due to the drastic conditions of extraction. A determination of all food constitu‐ ents is necessary because a certain interference with antioxidants can occur. Antioxidant capacity values should only be compared where the method, the solvent and the analytical conditions are similar [38]. Indeed, some authors underlined that there is an effect of the solvent used for the extraction or used to solubilize antioxidants on the result of the antioxidant activity evaluation [39-42]. This is due to interference of the reaction mechanism and the solvent [38]. (ii) The method to measure the antioxidant activity must be chosen according to the nature of the active molecules present in the samples. Some methods described in part 3 are more appropriate for some kinds of antioxidants. For example the DPPH method is more adequate to lipophilic systems. Moreover, several assays must be carried out to determine a value of antioxidant activity. (iii) Results of antioxidant activity measurement can be expressed as EC50 (quantity of antioxidant necessary to assure 50% depletion of free radicals), tEC50 (time to reach 50% depletion of free radicals), tEC50 (time to reach 50% depletion of free radical) or AE (antiradical efficiency defined as the inverse of the product between EC50 and tEC50). Thus, taking these 3 parameters into account can be relevant to have a more comprehensive evalu‐ ation of antioxidant activity [38].

The determination of the antioxidant capacity by in vivo methods is not always feasible but it appears interesting because it simulates an environment closer to that really happening in biological systems. Methods using HAT reactions will be preferred to SET reactions because peroxyl radicals used in HAT assays are the predominant free radicals found in lipid oxidation and biological systems. To elucidate a full profile of antioxidant capacity against various ROS, the development of different methods specific for each ROS/ RNS may be needed. [18] proposed a comparison of different in vitro methods; conclusions given that ORAC, TRAP and LDL are considered to be the most biologically relevant assays [18] because the antioxidant capacity measured reflects closer the in vivo action of the antioxidants. So, it appears clearly that the antioxidant activity determination needs a standardization of the procedure used and a combination of at least two or three methods. The use of only one method does not reflect the antioxidant activity of food raw material due the variability of the molecules that act as antioxidant.
