## 2 Materials and Methods
### Methods
#### 2.2.14 In Vitro and In Vivo Anti-Inflammatory Activity  
The anti-inflammatory activity (AIA) was assessed in vitro and in vivo according to the following procedures:  
* In Vitro Anti-Inflammatory Activity  
In vitro AIA was determined through reduction of nitric oxide (NO) production in RAW264.7 cells. The RAW264.7 cells (ATCC(r) SC-6003(tm)) were used in DMEM culture medium supplemented with 10% of heat-inactivated FBS, 1% of L-glutamine (2 mM), and 1% of Pen (50 U/mL)/Strep (50 mg/mL), at 37 \({}^{\circ}\)C in a humidified atmosphere and 5% CO\({}_{2}\). The cells were seeded in 96-well tissue plates until 80% of confluence. Then, different samples, namely free extract (35 mg/mL) and extract-loaded NPs (35 mg/mL), were incubated with cells (density \(2.5\times 10^{5}\) cells/well) in a 96-well-plate containing 100 mg/mL of lipopolysaccharidesaccharide (LPS), for 24 h. An equivalent concentration of empty NPs was also tested. Cellular viability was determined by the MTT assay, in relation to a control with the vehicle (0.5%, DMSO), and according to Equation (7) as described elsewhere [13]. Free nitrites in culture medium were measured by spectroscopy at 562 nm using the _Griess_ method. The amount of nitrite was calculated from a NaNO\({}_{2}\) standard curve (concentration range 0-100 uM). The equation was y = 0.0114x + 0.0578 with R\({}^{2}\) of 0.9962. The _NO production_ (%) of the tested samples [NO\({}^{2-}\)] in relation to the negative control [NO\({}^{2-}\)] was calculated using Equation (9):  
\[NO\;Production\;(\%)=\;\frac{\left[\text{NO}^{2-}\right]\text{t}}{\left[\text{ NO}^{2-}\right]\text{c}}\times 100 \tag{9}\]  
The _NO inhibition_ (%) was then determined by using Equation (10):  
\[NO\;Inhibition\;(\%)=\;100-\;NO\;Production \tag{10}\]  
* In Vivo Anti-Inflammatory Activity  
The carrageenanan paw oedema model in rats is one of the most used tools to study the acute local inflammation [13; 27]. In this assay, animals were randomly allocated into different groups (each group with \(n=8\), except positive control \(n=6\)) and were anesthetized by intraperitoneal injection with a mixture of ketamine (75 mg/kg) and medetomidine (0.50 mg/kg). The AIA of the extract in the free form or loaded in NPs was evaluated after topical administration. Each formulation was applied in the right hind paw of each animal (200 uL). The extract concentration applied (free extract and extract-loaded NPs) was 1.0 mg/kg body weight. In the negative control group, a placebo Carbopol(r) 940 gel was applied. In the case of the positive control group, a dose of 1.0 mg/kg body weight commercial diclofenac sodium-containing formulation was used. Based on the literature, the Carbopol(r) 940 gel was prepared with some modifications [28]. In brief, 500 mg of Carbopol(r) 940 were dispersed in water under magnetic stirring (400 rpm). After the polymer was fully solubilized, 0.2% of methyl 4-hydroxybenzoate and 0.02% of propyl-4-hydroxybenzoate were added under constant stirring. Finally, 0.2 g of NaOH was added under magnetic stirring, promoting the gelation of Carbopol(r) 940. Both NPs were incorporated into the gels, as well as in the free extract. Incorporation of extract-loaded PLGA NPs was performed by adding 2.1 mL per mL of gel; the incorporation of extract-loaded PCL NPs was performed by adding 1.65 mL per mL of gel. Finally, 1.5 mg per mL of gel were added in the free extract. The concentrations of free extract and extract-loaded NPs administered were based on the EE obtained for each NP and on the maximum concentration of the extract previously obtained [6]. The experimental design was performed according to Figure 1. Paw oedema was induced by a single subplantar injection of 0.1 mL of a \(\lambda\)-carrageenanan (1%, _w/v_) sterile saline solution 30 min after topical treatment in the rats' right hind paw. The paws' volume was measured before the sample topical application and carrageenanan injection (V\({}_{0}\) or basal volume) and 6 h later (V\({}_{6}\)) using a plethysmometer ([PERSON], Gemonio (VA), Italy).