## 5 Hydrogen Atom Transfer-Based Assays  
### Oxygen Radical Absorbance Capacity (ORAC)  
#### 5.1.1 Method Principle  
The oxygen radical absorbance capacity (ORAC) method, developed by [PERSON], [PERSON], and [PERSON] [126], is based on measuring the decrease in the fluorescence of fluorescein (3',6'-dihydroxyroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one). Moreover, the ORAC assay enables the assessment of peroxyl radical scavenging, with radicals generated by 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). This compound generates peroxyl radicals that oxidize fluorescein; however, in the presence of antioxidants, degradation is delayed, and fluorescence is maintained. In this system, AAPH acts as a peroxyl radical initiator, fluorescein serves as the molecular probe, and the antioxidant functions as the radical scavenger. Phosphate buffer is used as a blank sample [127]. The ORAC reaction mechanism is illustrated in Figure 4. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Tested Material** & **Standard** & **Substrates** & **Type and Time** & **Wavelength** & **Source** \\ \hline Soybean, green & Gallic acid & 0.25 mL of extract, 0.25 mL FCR and 4.5 mL 20\% Na\({}_{2}\)CO\({}_{3}\) & 25 min, room  temperature & 730 nm & [115] \\ \hline Strawberries & Gallic acid & 0.05 mL of extract, 0.43 mL MQ, 0.02 mL FCR, 0.05 mL 20\% Na\({}_{2}\)CO\({}_{3}\) & 60 min, room  temperature & 725 nm & [116] \\ \hline Black tea & Gallic acid & 1 mL of diluted extract, 1 mL of 3-fold-diluted FCR, 2 mL of 35\% Na\({}_{2}\)CO\({}_{3}\) & 30 min, room  temperature & 700 nm & [117] \\ \hline Vegetable juices & Gallic acid & 0.2 mL of extract, 1.5 mL of 10-fold-diluted FCR, 1.5 mL of 6\% Na\({}_{2}\)CO\({}_{3}\) & 90 min, room  temperature & 725 nm & [118] \\ \hline Berries & Gallic acid & 0.5 mL of extract, 2.5 mL of FCR, 2 mL 7.5\% Na\({}_{2}\)CO\({}_{3}\) & 60 min, room  temperature & 765 nm & [119] \\ \hline Tomato and & Quercetin & 0.5 mL of extract, 2.4 mL MQ, 0.1 mL FCR, 2 mL 2\% Na\({}_{2}\)CO\({}_{3}\) & 60 min, room  temperature & 750 nm & [120] \\ \hline Fruit juices & Gallic acid & 0.1 mL of extract, 0.5 mL MQ, 0.1 mL & 90 min & 760 nm & [121] \\ \hline Pickled Chinese  cabbage & Gallic acid & 0.5 mL of extract, 2.5 mL FCR, 2 mL  of 7.5\% Na\({}_{2}\)CO\({}_{3}\) & 60 min,  darkness & 760 nm & [122] \\ \hline Cabbage: white,  Chinese, and red & Gallic acid & 0.05 mL of extract, 0.1 FCR, 2 mL 1\% Na\({}_{2}\)CO\({}_{3}\), 1\% Na\({}_{2}\)CO\({}_{3}\), 1\% Na\({}_{2}\)CO\({}_{3}\), 1\% Na\({}_{2}\)CO\({}_{3}\) & 90 min, room  temperature & 750 nm & [123] \\ \hline _Dovylalis caffra_ & Gallic acid & 0.3 mL of extract, 0.5 mL of FCR, 5.8 mL of MQ, 1.5 mL of 20\% Na\({}_{2}\)CO\({}_{3}\) & 2 h, room  temperature & 760 nm & [124] \\ \hline Tea extracts & Ascorbic acid & 0.1-0.3 mL of tea sample, 0.5 mL of  FCR, 0.5 mL of 30\% NaOH temperature & 630 nm & [63] \\ \hline Buckwheat & Gallic acid & 0.3 mL of sample, 1.5 mL of FCR, 1.2 mL of 7.5\% Na\({}_{2}\)CO\({}_{3}\) & 30 min, room  temperature & 765 nm & [125] \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Comparison of previous approaches in FC assay. The ORAC assay measures both hydrophilic and lipophilic antioxidant capacities against biologically relevant peroxyl radicals. This feature makes ORAC superior to other spectrophotometric assays for assessing antioxidant potential, as the radicals used in the test are more representative of those found in biological systems [128,129]. The ORAC scale is used by the United States Department of Agriculture (USDA) to compare the antioxidant activity of various foods [130]. Notably, other oxidants present in the body, such as singlet oxygen, are not accounted for in the ORAC assay [25]. However, the assay may be affected by secondary reactions related to the radical chain-breaking process or the presence of metal ions [131]. Elements like Cu, Fe, zinc (Zn), and aluminum (Al) can reduce the antioxidant capacity measured by the ORAC assay. This effect is attributed to the formation of phenol-metal complexes.