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    • The current literature clearly states that there are

    2024-03-25

    The current literature clearly states that there are no widely adopted standardized methods to determine the total antioxidant capacity of foods or biological samples. One should consider the antioxidant reaction mechanism in system to determine the most appropriate methods for the measurement. It has commonly been assumed that hydrogen Spironolactone transfer (HAT) based assays (such as ORAC, TRAP) are physiological relevance due to the depending on the peroxyl radicals as most found radicals in biological systems and involving kinetics based monitoring (Prior et al., 2005). However, these methods are not suitable to utilize as standardized methods since they have number of limitations as mentioned before. Electron transfer (ET) based methods (ABTS, DPPH, FRAP, CUPRAC, FC) have distinct advantages over HAT-based assays, namely simplicity, availability and stability of reagents, reproducibility over a wide concentration range; therefore, these methods have been more preferred to determine the antioxidant capacity of foods or biological samples. Even so, it is also important to bear in mind that current ET-based methods ignore the reaction kinetics and the antioxidant reaction period for different antioxidants. It has been known that some antioxidants show an immediate very fast reaction against selected radicals, while others show a combination of fast and slower stages, and some show only slow continuous reactions. However, these methods have been questioned how much react rather than how fast those analyzed antioxidants and this leads to incorrect assessment of antioxidant activity, both quantitatively and qualitatively. Therefore, monitoring the kinetics of antioxidant reactions may be better to further investigation of antioxidant activity. Each of classical antioxidant capacity measurement assays based on either hydrogen atom transfer mechanism or electron transfer mechanism, are not sufficient alone to determine the total antioxidant capacity as each antioxidant shows different antioxidant activity against different radical forms. Therefore, researchers have considered more than one method to evaluate the real antioxidant capacity of a sample and elucidate the mechanism of antioxidant activity. They have investigated the correlations among antioxidant activities of samples measured by different methods. Generally, ET- or HAT- based methods correlate well among themselves due to the similar antioxidant mechanism. On the other hand, it was reported that there was no or low correlation between ET- and HAT- based assays. Floegel, Kim, Chung, Koo, and Chun (2011) evaluated the antioxidant capacity of most common antioxidant-rich foods using DPPH and ABTS assays as ET-based methods, and compared those results with HAT-based ORAC results from USDA reports. They concluded that ABTS assay was strongly positively correlated to that by DPPH assay as well as it showed a stronger correlation with ORAC from the USDA database compared with DPPH assay. Ou, Huang, Hampsch-Woodill, Flanagan, and Deemer (2002) conducted a large scale vegetable analysis using two different in vitro assays, FRAP and ORAC, and obtained weak correlation between antioxidant capacities determined by FRAP and ORAC assays. The reason of this weak correlation could be explained as ORAC measured the peroxyl radical scavenging activity, whereas FRAP estimated only the Fe (III) reducing activity. In addition, in most studies, there was a low correlation between assays based on inhibition of lipid peroxidation and HAT- or ET-based assays, which means classical HAT- or ET-based assays could not represent the lipid oxidation and its inhibition (Apak, Özyürek, Güçlü, & Çapanoğlu, 2016). Moreover, it was shown that the correlation between the assays also depended on the structure of antioxidant compounds. For example, it was revealed that EGCG and EC content in tea showed high correlation with DPPH and ORAC, respectively. Normally, it was believed that the increased number of hydroxyl groups in the flavonoid structure resulted in high antioxidant capacity that was coherent with DPPH assay but not with ORAC assay (Roy et al., 2010). The reason of this contradiction might be the prooxidation of EGCG or formation of conjugates with EGCG itself during peroxyl radical scavenging activity. In view of all that has been mentioned so far, one may suppose that multiple assays should be used to evaluate overall antioxidant capacity and elucidate antioxidant profile of samples according to several mechanisms such as scavenging of different types of radicals, radical and metal reducing activity.