## 2 Materials and Methods
### Methods
#### 2.3.5 Chromatographic Analysis  
The HPLC-MS method was used to analyze and quantify polyphenols, methoxylated flavones, and phytosterols. The HPLC equipment used consisted of an Agilent 1100 HPLC Series system (Agilent, Santa Clara, CA, USA), which included a G1322A degasser, G13311A binary gradient pump, column thermostat, G1313A autosampler, and G1316A UV detector. This system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap VL). The data were acquired and processed using the Chem Station and Data Analysis software from Agilent (version 01.11) [47; 48; 49; 50; 51; 52; 53; 54].  
The HPLC-MS analysis of phenols was performed by injecting 5 \(\upmu\)L of the RG extracts into a reverse-phase analytical column Zorbax SB-C18 (100 \(\times\) 3.0 mm i.d., 3.5 \(\upmu\)m particles). The separation of compounds was achieved using a mobile phase consisting of a mixture of methanol and 0.1% (\(p/v\)) acetic acid. All polyphenols were eluted in less than 40 min under the following conditions: flow rate of 1 mL/min and working temperature of 48 \({}^{\circ}\)C. The binary gradient used for elution started with 5% methanol in a linear mode and ended with 42% methanol over 35 min. For the last 3 min, an isocratic elution was performed with 42% methanol. Prior to the next injection of the sample, the column was re-equilibrated with 5% methanol for 7 min. The detection of the compounds was performed using UV at different wavelengths, specifically at 330 nm until 17.5 min and at 370 nm until the end of the analysis. The MS system was also used to detect compounds, and it was operated in the negative mode using an electrospray ionization source [47; 48; 50; 51; 52; 53; 54].  
3.6 Preparation and Characterization of Chitosan-Sodium Alginate-_Ruta gravelens_ L. Extract Microspheres (CARG-Ms)  
To prepare the microspheres, two colloidal aqueous phases were selected: a chitosan solution of three concentrations (0.1%, 0.2%, and 0.4%) obtained by dispersing it in a 2% acetic acid aqueous solution and a sodium alginate solution with a single concentration (0.75%) obtained by dispersing it in distilled water. The RG extract was encapsulated using two different procedures to determine which method was more effective. A 4% calcium chloride solution was added to the chitosan solutions to prevent the precipitation phenomenon that may occur when working at pH values close to the acidity constants of chitosan and sodium alginate. If the precipitation phenomenon were to occur, the resulting microspheres would be unstable, formed from an irregular matrix that would result in the loss of the trapped substances [55].  
The complex coacervation method involves dropping a solution of sodium alginate into a mixture of chitosan-RG extract (method 1) or a mixture of sodium alginate-RG extract into a solution of chitosan at a rate of 0.5 mL/min and under continuous stirring at 100 rpm for 60 min. The resulting microspheres, which were weakly greenish or deep green in color, were then filtered, washed, and dried in the dark at room temperature until a constant weight was achieved, approximately 24 h, and kept in tightly closed containers until further use [39; 56]. Schematic representations of the procedures for obtaining the microspheres following the two methods are shown in Figure 1, and the formulas used to prepare the microspheres are shown in Table 2.  
For comparison with microspheres prepared by the two methods, empty microspheres (E1, E2, and E3) were also prepared that did not contain the RG extract, as shown in Table 2. These were used in subsequent determinations.