## Results and Discussion
### Antioxidant Activity of F3 and F4 Fractions In Vivo  
The antioxidant potential of F3 and F4 fractions was assessed in vivo in zebrafish _Danio rerio_ exposed to H\({}_{2}\)O\({}_{2}\)-induced oxidative stress. F4 induced a concentration-dependent increase in mortality (half maximal lethal concentration (LC\({}_{50}\)) = 17.13 \(\pm\) 1.27 ug/mL) and developmental abnormalities (half maximal effective concentration (EC\({}_{50}\)) = 29.52 \(\pm\) 4.27 ug/mL), as presented in Figure 8a. Although no mortality was observed during the exposure to F3, the highest tested concentration (62 ug/mL) increased the incidence of developmental abnormalities (16.67 \(\pm\) 11.55% of specimens; data not shown). When compared to F3 fraction, F4 fraction contained a higher amount of fatty acid amides such as oleamide, palmitamide, and erucamide that might be responsible for elevated toxicity. This was also the case in our recent study where _Ericaria crinita_ and _Ericaria amentacea_ fractions enriched with mentioned fatty acid amides negatively affected the survival of zebrafish embryos [11]. Considering the observed toxicity, the determination of in vivo antioxidant potential was decided to be conducted on 2.5, 5.0, and 10.0 ug/mL of F4 and 11.3, 22.5, and 45.0 ug/mL of F3. As shown in Figure 8b, the survival rate of the zebrafish larvae was significantly reduced upon exposure to H\({}_{2}\)O\({}_{2}\). However, pre-treatment with 45 ug/mL of F3 significantly protected zebrafish from H\({}_{2}\)O\({}_{2}\)-induced mortality (survival rate increased by 35.3% compared to the treatment group on H\({}_{2}\)O\({}_{2}\); \(p<0.05\)). H\({}_{2}\)O\({}_{2}\) increased ROS production in zebrafish up to 255.8 \(\pm\) 6.99% compared to the control treatment group on artificial water (100%; Figure 8c), which revealed a high fluorescence of the positive control (Figure 8d). Following the treatment with 45 ug/mL of F3, the ROS levels reduced to 164 \(\pm\) 6.2% (\(p<0.05\); Figure 8c,d).  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** &
\begin{tabular}{c} **IC\({}_{50}\) Value** \\ **(mg/mL)** \\ \end{tabular} & **Confidence Interval** & **Hillslope** & **R\({}^{2}\) Value** \\ \hline F3 & 0.498 & 0.419–0.595 & 2.48 & 0.981 \\ F4 & 0.798 & 0.696–0.937 & 1.97 & 0.992 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Dose–inhibition results for _D. vermicularis_ nonpolar fractions using ABTS in vitro assay (\(n=4\)) to obtain the half-maximal inhibitory concentration (IC\({}_{50}\)) with presented confidence intervals, Hills slope, and R\({}^{2}\) value.  
Figure 7: Concentration–inhibition response curves for F3 and F4 fractions from _D. vermicularis_ used for the calculation of their antioxidant activity by using the reduction in the radical cation (ABTS assay).  
These findings are in correlation with the results obtained with UHPLC-ESI(+)-HRMS analysis. Both fractions contain the compounds that have antioxidative properties such as porphyrin-based compounds [40] and xanthophylls [41], as well as sphingolipids [42]. Fucoxanthin and zeaxanthin/lute (Table 3), molecules well-known for their antioxidant potential, have a greater abundance in F3, which might be the reason of the stronger antioxidant activity of F3 in relation to F4. Nevertheless, one should notice that the determination of the antioxidant potential of F4 in vivo was conducted within the lower concentration range (2.5-10.0 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) compared to F3 (11.3-45.0 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). Taken together, the obtained results indicate the pharmaceutical potential of _D. vermicularis_.