## 3 Results and Discussion
### Characterization of Nanoparticles
The rutin-polymer complexes demonstrated similar energy stabilizing profile involving non-bonding dihedral and bonding van der Waals (vdW) components. The dihedral energy component confirmed the anti-inflammatory molecule as a good fit for the polymer matrix. To the best of our knowledge, a comparative release study of the methanolic elderflowers' extract from two different types of NPs was herein performed for the first time. Two different pH were used in order to simulate the biological conditions: pH 5.5 similar to the pH of the skin [32] and pH 7.4 that corresponds to the biological pH of the blood and interstitial's fluid [42]. The graphic representation is shown only until 4 h, since the percentage of rutin released was constant up to 24 h (Figure 9). Rutin of the free extract was immediately dissolved in both buffers. But, after encapsulation, a different release of rutin was observed in both NPs (Figure 9a,b). In comparison to PLGA, the rutin release from PCL NPs was faster, especially in PBS at pH 7.4 (Figure 9b). The results show a higher rutin release from PLGA NPs after 30 min in PBS pH 5.5, whereas in pH 7.4, PCL NPs presents a complete rutin release. The results presented the same behavior for the same type of NPs when berberine was encapsulated, PLGA NPs presented a higher release when exposed to the PBS pH 5.5 than to the pH 7.4 [43]. Another study with PCL NPs and curcumin presented similar results with the ones obtained in the present study, with lower release from PCL NPs in PBS pH 5.5 than in pH 7.4 [44]. Figure 8: Representation of (**a**,**c**) geometrical preferences (**b**,**d**) electrostatic molecular graph after molecular mechanics simulations in vacuum (**a**,**b**) PCL-Rutin (**c**,**d**) PLGA-Rutin [Colour code for elements: C = cyan; H = white; O = red; N = blue]. Rutin: tube rendering; PCL: stick rendering; PLGA: stick rendering. Figure 9: Percentage of the major compound (rutin) release over time in (**a**) PBS at pH 5.5 and (**b**) PBS at pH 7.4: free extract (orange line), extract-loaded PLGA NPs (black line) and PCL NPs (grey line) (mean \(\pm\) S.D.). Thus, according to our previous results, the encapsulation may be an additional advantage due to the lower release in the first time points when compared with the free extract. Furthermore, these data also are in agreement with the results obtained for rutin-polymers complexes, where a higher energy variation for rutin-PLGA NPs was observed. Moreover, the presence of H-bonding in the rutin-PLGA complex may have led to the retention of the rutin molecule within the PLGA matrix at physiological pH. Another important factor that can be associated with the rutin release is the fact that PCL presents higher hydrophilicity than PLGA ([PERSON] et al., 2018). The antioxidant activity (AA) was assessed by three different methods: DPPH, ORAC and HOSC. Concerning DPPH, and according to previous studies performed by our group, this extract presents a high AA, similar to positive controls (Figure 10a). Empty NPs were also tested, revealing no AA. The free extract presents an AA of \(79.5\pm 1.6\%\), while the ascorbic acid and the quercetin present values of \(83.0\pm 3.7\%\) and \(95.1\pm 0.2\%\), respectively. Differences were only observed between the extract and the quercetin (\(p<0.001\)). The AA results obtained by DPPH revealed that the extract-loaded NPs presented a significant decrease of the AA in comparison with the free extract. The decrease of AA after encapsulation probably means that the compounds responsible for the AA might not be completely exposed or released, as suggested by the interactions between the complex rutin-polymers (PLGA or PCL), due to the reduced reaction time of this assay (very fast assay \(\approx\)30 min). To perform the DPPH method, these radicals have to be easily accessible, which was not verified for the extract-loaded NPs. Afterwards, these systems were analyzed by using the same method, 24 h after incubation in PBS buffer pH 5.5 and 7.4 (Figure 10b).