## 6 Summary and Future Challenges  
This article critically reviewed widely applied spectrophotometric methods for evaluating antioxidant potential. A key challenge in this area is the lack of standardized protocols, leading to considerable variability in standards, solvents, extraction procedures, incubation times, and assay conditions [142; 143]. These methodological differences can influence reaction kinetics, potentially leading to inconsistent and inaccurate results. Furthermore, the use of varying measurement units within the same assay hinders the direct comparison of results. This is especially noticeable in the DPPH method, where a wide range of units are in use. To date, no consensus has been reached on a universal unit for reporting antioxidant capacity. In addition, extrapolating in vitro findings to in vivo conditions remains challenging due to significant differences in radical species and pH levels between experimental assays and the human body [26]. Nevertheless, some methods show greater biological relevance. The CUPRAC assay, for instance, operates at a near-physiological pH, while the ORAC method targets peroxyl radicals commonly present in the human body. These characteristics may improve the applicability of in vitro results to in vivo conditions. The popularity of spectrophotometric techniques stems largely from their simplicity, low cost, and ease of use. According to the Association of Official Analytical Collaboration (AOAC), existing guidelines mainly focus on validation procedures [144]. This may explain \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Tested Material** & **Standard** & **Substrates** & **Type and Time of Incubation** & **Wavelength** & **Source** \\ \hline Various plants & & 0.3 mL of extract, 75 mM phosphate & & & \\ extracts & & buffer (pH 7.4), 1.8 mL of fluorescein (70 nM), 0.9 mL of AAPH (12 mM) & & & \\ \hline Polyphenols & & 20 \(\mu\)L of sample (75 mM phosphate & & & \\  & & buffer (pH 7.0) mixed with 10 mg of & & & \\  & & test sample), 0.2 mL of fluorescein (94.4 nM), 75 \(\mu\)L of AAPH (31.7 mM) & & & \\ \hline Oranges & & 50 \(\mu\)L of extract, 75 mM phosphate & & & \\  & & buffer (pH 7.0), 50 \(\mu\)L of fluorescein (78 nM), 25 \(\mu\)L of AAPH (221 mM) & & & \\ \hline Berries & & 100 \(\mu\)L of extract, 75 mM phosphate & & & \\  & & buffer (pH 7.4), 60 \(\mu\)L of fluorescein (200 nM), 40 \(\mu\)L of AAPH (60 mM) & & & \\ \hline Maple syrup & & 120 \(\mu\)L of extract, 75 mM phosphate & & & \\  & & buffer (pH 7.4), 30 \(\mu\)L of fluorescein (400 nM), 50 \(\mu\)L of AAPH (200 mM) & & & \\ \hline _Chlorella vulgaris_ & & 100 \(\mu\)L of extract, 75 mM phosphate & & & \\  & & buffer (pH 7.4), 800 \(\mu\)L of fluorescein (40 nM), 100 \(\mu\)L of AAPH (400 mM) & & & \\ \hline Acai pulp & & 100 \(\mu\)L of extract, 75 mM phosphate & & & \\  & & buffer (pH 7.0), 2.6 mL of fluorescein (40 nM), 300 \(\mu\)L of AAPH (200 mM) & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Comparison of previous approaches in the ORAC assay. why most researchers tend to optimize nearly all parameters of a given method, including incubation time, temperature, solvent type, and wavelength. Undoubtedly, the evaluation of antioxidant activity should not be performed by a single assay. The use of multiple techniques, each representing different reaction mechanisms of bioactive compounds, increases the likelihood of accurately assessing their antioxidant capacity. A positive correlation between results from different methods strengthens the validity of the overall outcomes. Nevertheless, significant differences in methodology still pose a major problem to comparing data across studies. Variations in reagent selection, experimental protocols, and reaction conditions remain key factors contributing to this lack of consistency (Table 7). Among the methods discussed, CUPRAC has shown the highest consistency.