## 2 Materials and Methods
### Physicochemical Characterization
#### 2.3.3 Determination of EO Content in CE EO and NLC EO  
EO Isolation from CE EO and NLC EO  
The EO was isolated from both CE EO and NLC EO by liquid-liquid extraction. An aliquot of 1 mL from each formulation was transferred to a test tube and then diluted by adding 2.5 mL of deionized water. Extraction was performed by three consecutive extractions of each solution with 2.5 mL of hexane. After vigorous shaking, the organic phase was collected and transferred to a clean test tube. Then, the total organic phase was washed with saturated NaCl solution, and the organic phase was collected and dried by the addition of anhydrous sodium sulfate. The organic phase was then concentrated on a vacuum rotary evaporator. The essential oil isolated from each emulsion was stored at \(-20\)\({}^{\circ}\)C in hermetically sealed glass vials.  
Analysis of the EO by Gas Chromatography-Mass Chromatography (GC-MS)  
The initial EO and the EOs isolated from CE EO and NLC EO were analyzed by GC-MS on a non-polar HP-5 MS (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\mu\)m film thickness) column usinga 6890N GC interfaced with a 5975B mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) in electron impact (70 eV) ionization mode. In an aliquot of 2 \(\upmu\)L of EO, 20 \(\upmu\)L of octane (internal standard) was added, and the mixture was diluted with 98 \(\upmu\)L of ethyl acetate. Helium was used as the carrier gas at 1 mL/min. The volume injected into an oven at 325 \({}^{\circ}\)C was 1 \(\upmu\)L. The column temperature was initially set at 50 \({}^{\circ}\)C. It was then raised to 80 \({}^{\circ}\)C, where it remained for 2 min. The temperature was gradually increased to 110 \({}^{\circ}\)C at a rate of 1.5 \({}^{\circ}\)C/min, then to 130 \({}^{\circ}\)C at a rate of 4 \({}^{\circ}\)C/min, then to 150 \({}^{\circ}\)C at a rate of 4 \({}^{\circ}\)C/min, and finally to 260 \({}^{\circ}\)C at a rate of 8 \({}^{\circ}\)C/min, where it remained for 2 min. Immediately after, the temperature decreased gradually to 50 \({}^{\circ}\)C. The duration of the procedure was 62.75 min. The mass scan range was \(m/z\): 30-1050.  
The identification of each compound was achieved by the comparison of mass spectra and retention indices (Als) from the van der Dool and Kratz equation relative to n-alkanes (C8-C20) with those of spectral libraries [19; 20]. The software used for the analysis was AMDIS (Automated Mass spectral Deconvolution & Identification System v.2.73, NIST Institute, Gaithersburg, MD, USA) and WSearch VS2020 (Wsearch Software by [PERSON]).  
For the quantification of the components, eucalypttol (1,8-cineole) was chosen as an external standard since it was the major component of the EO. Standard solutions of several concentrations of eucalypt in ethyl acetate were prepared and analyzed by GC-MS. A calibration curve (y = 4.1928x \(-\) 0.0575, R\({}^{2}\) = 0.9942) was constructed using the ratio of the peak area of eucalypttol (external standard) to the peak area of octane (internal standard) and the concentration of the standard compound (0.01-1.00 mg/mL). Thus, the amount of essential oil in the emulsions (NLC EO and CE EO) was determined, expressed in mg of eucalypt equivalents/mL of emulsion. The entrapment of the essential oil components (the ratio of the content of the respective component in the EO that has been incorporated into the NLC EO or CE EO to the content of the same component in the EO before it was incorporated) was also calculated.