## 2 Materials and Methods
### Methods
#### 2.2.11 Antioxidant Activity  
Different methods to evaluate the antioxidant activity (AA) of extract-loaded NPs were used: 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and hydroxyl radical scavenging capacity (HOSC). * 2,2-Diphenyl-1-picrylhydrazyl Assay (DPPH)For this purpose, 10 mg of extract was dissolved in 1 mL of MeOH. Aliquots of 10 \(\upmu\)L of each solution (free extract, positive controls and extract-loaded NPs) were diluted in 990 \(\upmu\)L of a DPPH solution in ethanol 70% (_v/v_). The reaction mixture was incubated at RT for 30 min in the dark. After incubation, the absorbance (_Abs_) was measured at 517 nm (Thermo Scientific Evolution 300 UV-vis, Loughborough, UK). The positive controls consisted of a 10 mg/mL of quercetin solution in ethanol and 10 mg/mL of ascorbic acid solution in water. An absorbance control (_Abs\({}_{control}\)_) containing 10 \(\upmu\)L of ethanol, and 990 \(\upmu\)L of DPPH was prepared. The rutin concentration was 0.75 mg/mL in ethanol. This study was performed in triplicate (_n_ = 3) and the free radical scavenging effect was then calculated according to Equation (2):  
\[\textit{Searveing activity}\ (\%)=\frac{Abs_{control}-Abs_{sample}}{Abs_{control}} \times 100 \tag{2}\]  
\(\bullet\) Oxygen Radical Absorbance Capacity Assay (ORAC)  
The ORAC's method evaluates the samples' antioxidant capacity to peroxyl radicals, based on previous works [19], but modified in order to use fluorescein (FL)\(\times\) 800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) [20]. In this method, the measurements of AA were made against peroxyl radical induced by 2,2\({}^{\prime}\)-azobis(2-amidinopropane) dihydrochloride (AAPH) at 37 \({}^{\circ}\)C [21]. This assay consists of a measure of the samples' ability (antioxidant species) through the inhibition of the FL's (\(3\times 10^{-4}\) mM) oxidation catalysed by AAPH [20]. The consumption of FL associated to its incubation with AAPH was estimated by fluorescence (F) measurements at 538 nm on excitation at 480 nm (Genios Spectrofluorimeter, Helvetic Tecan Group Ltd., Mannedorf, Zurich, Switzerland) for a period of 40 min [22]. The values of instantaneous fluorescence/initial fluorescence (F/F\({}_{0}\)) were plotted in function of the time. The integration of area under curve (AUC) was performed for each time that F/F0 reached a value of 0.2 or 0.4 [23]. A reaction mixture containing AAPH (41.4 mg/mL), FL (\(4\times 10^{-3}\) mM) and the tested samples (free extract, extract-loaded PLGA NPs and extract-loaded PCL NPs) was incubated in phosphate buffer solution (PBS, 75 mM, pH 7.4) (USP41) at 37 \({}^{\circ}\)C [23]. PBS was used as a blank and 5, 10, 20, 30, and 40 \(\upmu\)M Trolox solutions were used as control standards. All the samples, including the blank and the controls, were analyzed in triplicate. The final ORAC's values were calculated by using a regression equation between the Trolox concentration and the net area under the FL decay curve [22]--Equation (3):  
\[ORAC-FL=\frac{AUC_{sample}-AUC_{0}}{AUC_{Trolox}-AUC_{0}}\times f[Trolox] \tag{3}\]  
where \(AUC_{Sample}\) is the \(AUC\) in the presence of the tested sample, integrated between time zero, which corresponds to 60% or 80% of the probe consumption; \(AUC_{0}\) is the \(AUC\) for the control (target molecule plus AAPH solution); \(AUC_{Trolox}\) is the \(AUC\) for Trolox; f is the dilution factor, equal to the ratio between the total volume of the working solution (target molecule plus AAPH and the sample aliquot) and the added sample volume; [Trolox] is the Trolox molar concentration. Data were expressed as micromoles of Trolox equivalents antioxidant capacity (TEAC) per g of sample (extract, rutin or per NPs) [18; 22]. The degraded extract-loaded NPs were also analyzed. The experiments were performed in triplicate [22].