## 3 Results and Discussion
### Efficacy of Nanocarriers
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Group** & **Formulations** & **Dose** & **Inhibition** \\  & & **(mg/kg)** & **(\%)** \\ \hline Negative Control & Carbopol 940® gel & – & \(0.0\pm 0.0\) \\ Positive Control 1  & Diclofenac in gel & \(1.0\) & \(133.5\pm 12.6\) 2  \\ Free Extract 2  & Extract in gel & \(1.0\) & \(41.2\pm 15.3\) \\ Extract-loaded PLGA NPs & Extract-loaded PLGA NPs in gel & \(1.0\) & \(60.7\pm 9.0\) 2  \\ Extract-loaded PCL NPs & Extract-loaded PCL NPs in gel & \(1.0\) & \(21.7\pm 12.7\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: AIA of topically applied extract formulations on the carrageenanan-induced rats’ hind paw oedema model (mean \(\pm\) S.E.M, \(n=5\), unless otherwise indicated; a—\(n=6\); b—\(n=8\), * \(p<0.05\) and *** \(p<0.001\), when compared to negative control). The in vitro AIA revealed an increase of NO reduction after extract nanoencapsulation, mainly into the PCL NPs, whereas in the in vivo AIA studies, the extract's administration revealed higher AIA with PLGA NPs. This event can be associated to the highest release of rutin from PLGA NPs than from PCL NPs at PBS pH 5.5, the pH of the skin, which can contribute to a higher permeation of rutin with a consequent increase of paw oedema inhibition. This last observation was also supported by histological analyses, where the cytokines levels of extract-loaded PLGA NPs led to similar results of IL-6, IL-10 and TNF-\(\alpha\) in comparison to the positive control (dclofenac). According to the literature, there are some compounds that can inhibit the expression or activity of inducible nitric oxide synthase (iNOS) and they have AIA properties [53]. Some potential compounds are flavonoids and other polyphenols, which are involved in suppression of the cytokine's production [54]. They also seem to present a protective effect against inflammation, reducing the severity of the inflammatory process and regulating the expression of pro-inflammatory cytokines [54]. The extract of elderflowers used in this work presented a rich composition of phytocompounds including the flavonoids rutin and naringenin, as mentioned above. Rutin and naringenin probably might be responsible for the suppression of iNOS protein through the inhibition of nuclear factor-kB (NF-kB) [55; 53]. Naringenin was also reported as having mRNA expression and NO production in activated macrophages in a dose-dependent manner [53]. Flavonoids present AIA, acting in the reduction of eicosanoids production through inhibition of the phospholipase A2 (PLA2), cyclooxygenase (COX), and lipoxygenases (LOX) activities, as well as inhibiting phosphodiesterase, protein kinases, histamine releasing, and modulators of the transcription of genes, or due to their AA [54]. As the exact mechanism is still under research, the authors hypothesized that the reported biological activities probably result from a synergistic effect of several extract components with an impact on AIA. This hypotesis is also supported by previous references. [PERSON] et al. reported that rutin inhibits the TNF-\(\alpha\) production in human peripheral blood neutrophils [56; 57]. Rutin is also associated to a reduction of IL-6 expression, a reduction of TNF-\(\alpha\)[56] and an increase of IL-10 concentration [58]. Remarkably, in the current study, AIA was increased after extract encapsulation in PLGA NPs, which was corroborated by IL-6 and TNF-\(\alpha\) levels. Compared to positive control, dclofenac is generally associated to a decrease of IL-6 concentration, a decrease of TNF-\(\alpha\), and to an increase of IL-10 concentration [59; 60; 61]. In addition, this study also suggests that the mechanism responsible for the AIA of the nanoencapsulated methanolic _S. nigra_ extract is not only related to the pro-inflammatory cytokines [6], but also based on the anti-inflammatory cytokines' production.