## 3 Results and Discussion
### Efficacy of Nanocarriers  
The AIA of extract was tested both in vitro and in vivo. In vitro pro-AIA based on NO inhibition was registered in Table 2. According to these results, the extract-loaded PLGA NPs led to a lower value of NO inhibition when compared to the extract-loaded PCL NPs. Higher concentrations of both NPs carriers cannot be tested due to limitations of the method, i.e., increasing NPs' concentration, turbidometry of the suspension also increased and no reliable values were obtained by spectrophotometry. In any case, these results suggest that the encapsulation appears to be an advantage, especially for extract-loaded in PLGA and PCL NPs, as demonstrated by statistical differences in comparison to the free extract. A comparative in vivo study using two different types of NPs for methanolic elderflowers extracts was reported for the first time. The results, concerning the in vivo AIA of the extract in the different formulations compared with controls, are presented in Table 3. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Samples** & **Concentration** & **NO Inhibition** \\  & & **(\%)** \\ \hline Free Extract & 35 \(\mu\)g/mL & 23.6 \(\pm\) 1.8 \\ Extract-loaded PLGA NPs & 35 \(\mu\)g/mL & 43.5 \(\pm\) 4.0 *** \\ Extract-loaded PCL NPs & 35 \(\mu\)g/mL & 55.3 \(\pm\) 1.8 *** \\ Negative control (DMSO) & 0.5\% & 0.0 \(\pm\) 0.0 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: In vitro AIA: NO inhibition values relative to LPS-stimulated RAW264.7 macrophages incubated with free extract and with extract-loaded NPs (mean \(\pm\) S.E.M., \(n\) = 6, *** \(p\) \(<\) 0.001, when compared to the free extract). Figure 13: Cell viability (%) of (**a**) HaCaT and (**b**) HFF cells after 48 h incubation (%, mean \(\pm\) S.D. ; \(n\) > 4): free extract (orange columns), empty PLGA NPs (white columns), extract-loaded PLGA NPs (black columns), empty PCL NPs (light grey columns) and extract-loaded PCL NPs (dark grey columns). The model used/applied created an oedema in rats' paws. The positive control has an inhibition of oedema. The highest inhibition of extract-loaded NPs was observed for PLGA NPs, presenting more 23% of inhibition, when compared to the free extract. Moreover, the encapsulation into PLGA NPs presented a better result than the same extract into PCL NPs. Histologically, corresponding results in similar magnitude in the dorsal face of the paws were observed. Extract-loaded PLGA NPs paws showed lower oedema and fewer inflammatory infiltrates (Figure 14d, arrows) in comparison with the other groups. The effect of the free extract (Figure 14c) was slight milder than in the negative control (Figure 14a). Considering the biochemical analysis, the results are displayed in Table 4. It is important to note that the values obtained for the different parameters evaluated in the plasma of the rats for each group are in line with the oedema inhibition and histological results. Lower TNF-\(\alpha\) and IL-6 values were observed for rats treated with diclofenac (positive control), followed by extract-loaded PLGA NPs, extract-loaded PCL NPs and, finally, the free extract. Regarding IL-10, higher values were observed for the positive control group, followed by extract-loaded PLGA NPs and, finally, by the extract-loaded PCL NPs. However, the free extract presented a decrease of the IL-10 concentration. Figure 14: Representative histological images of rat’s paws (2.5\(\times\), H&E) after topical administration. (**a**) Negative Control; (**b**) Positive Control; (**c**) Free extract; (**d**) Extract-loaded PLGA NPs; (**e**) Extract-loaded PCL NPs. Arrows show the inflammation area.