## 3 Results and Discussion
### Reactive Oxygen Species Scavenging Capacity Assays  
Reactive species are the principal cause of oxidative stress, being divided in reactive oxygen and nitrogen species (ROS and RNS, respectively). These molecules, produced during the body aerobic metabolism, may cause oxidative damage of amino acids, DNA, lipids, and proteins [82; 83; 84]. The radical scavenging activity results are summarized in Table 2.  
Among ROS, superoxide radical (O\({}_{2}\)\({}^{\bullet-}\)) has particular importance as it is one of the most aggressive oxygen species in the human organism, being enrolled in the development of aging and chronic diseases, such as atherosclerosis, ischemic heart disease, diabetes mellitus, cancer, neurodegenerative diseases, immunosuppression and others [85; 86; 87; 88]. Phenolic compounds have demonstrated a strong scavenging capacity of superoxide anion [89]. Concerning this species quenching assay (Table 2), gallic acid and catechin were the best scavengers, with IC\({}_{50}\) values of 24.55 ug/mL and 84.40 ug/mL, respectively, followed by MAE condition 2 (IC\({}_{50}\) = 134.59 ug/mL), UAE condition 2 (IC\({}_{50}\) = 178.68 ug/mL) and UAE condition 1 (IC\({}_{50}\) = 221.88 ug/mL). It is important to emphasize that there are no significant differences (\(p>0.05\)) between MAE condition 2 extract and catechin used as positive control. Among the ROS studied, the best results were achieved by hypochlorous acid (HOCI). For this species, the UAE condition 1 (IC\({}_{50}\) = 1.88 ug/mL) and MAE condition 1 (IC\({}_{50}\) = 2.29 ug/mL) extracts achieved the best results, being not significantly different from gallic acid (IC\({}_{50}\) = 3.27 ug/mL) used as positive control. These results are followed by MAE condition 2 (IC\({}_{50}\) = 6.32 ug/mL) and UAE condition 2 (IC\({}_{50}\) = 13.50 ug/mL) extracts. Regarding the oxygen radical absorbance capacity, the values of _C. pepo_ extracts varied between 0.04 ug TE/mg DW (MAE condition 2) and 1.13 ug TE/mg DW (UAE condition 1), with no significant difference between the extracts and the positive controls employed. To the best of our knowledge, this is the first study that screened the radical scavenging activity of _C. pepo_ extracts. Nevertheless, comparing with other seed matrixes, the results are very interesting. For example, using tea seed oil extracted by cold pressing, [PERSON] et al. obtained a IC\({}_{50}\) = 1.73 mg/mL for the O\({}_{2}\)\({}^{\bullet-}\) radical [90], which indicates that this extract has a lower radical scavenging capacity than the _C. pepo_ extracts tested. Annatto seed \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & & \multicolumn{4}{c}{**Reactive Oxygen Species**} \\ \hline
**Extraction** & **Conditions** & \begin{tabular}{c} **O\({}_{2}\)\({}^{\bullet-}\)** \\ **IC\({}_{50}\) (μg/mL)** \\ \end{tabular} & \
\begin{tabular}{c} **HOCI** \\ **IC\({}_{50}\) (μg/mL)** \\ \end{tabular} &
\begin{tabular}{c} **ORAC** \\ **\# TE/mg DW** \\ \end{tabular} \\ \hline MAE & Condition 1 & - & 2.29 \(\pm\) 0.11 \({}^{\text{c}}\) & 0.28 \(\pm\) 0.025 \\  & Condition 2 & 134.59 \(\pm\) 29.31 \({}^{\text{b,c}}\) & 6.32 \(\pm\) 0.10 \({}^{\text{b}}\) & 0.04 \(\pm\) 0.01 \\ UAE & Condition 1 & 221.88 \(\pm\) 0.00 \({}^{\text{a}}\) & 1.88 \(\pm\) 0.23 \({}^{\text{c}}\) & 1.13 \(\pm\) 0.28 \\  & Condition 2 & 178.68 \(\pm\) 42.60 \({}^{\text{a,b}}\) & 13.50 \(\pm\) 0.75 \({}^{\text{a}}\) & 0.67 \(\pm\) 0.06 \\ _Positive controls_ & & & & \\  & Catechin & 84.40 \(\pm\) 10.33 \({}^{\text{c,d}}\) & 0.31 \(\pm\) 0.05 \({}^{\text{d}}\) & 6.60 \(\pm\) 9.29 \\  & Gallic acid & 24.55 \(\pm\) 3.49 \({}^{\text{d}}\) & 3.27 \(\pm\) 0.29 \({}^{\text{c}}\) & 7.31 \(\pm\) 2.63 \\ \hline \hline \end{tabular} Different letters (a, b, c, d) in the same column indicate significant differences between extracts (\(p<0.05\)). IC\({}_{50}\) = 1n vitro concentration required to decrease in 50% the reactivity of the studied reactive species in the tested media. ORAC: Peroxyl radical scavenging assay; TE: trolox equivalents; DW: dry weight.  
\end{table}