## 2 Materials and Methods
### Methods
#### 2.3.9 Confocal Laser Scanning Microscopy (CLSM) Analysis  
The roughness of the microspheres was determined using a Confocal 3D Laser Scanning Microscope (Text OLS 4000, Olympus, Tokyo, Japan) with a 405 nm laser light source [58]. Figure 1: Schematic representation of the two methods for the preparation of microspheres with RG extract. 2.3.10. Scanning Electron Microscope (SEM) Analysis  
The appearance of each microsphere sample was analyzed using scanning electron microscopy (SEM) on an FEI Quanta FEG 250 microscope (FEI, Hillsboro, OR, USA). To avoid affecting the samples, the images were processed in a low-vacuum system [29]. 2.3.11. Optical Scanning Analysis  
The optical scanning process of the microspheres was conducted under reflective light (i.e., the classical method) and under transmittance light (i.e., as for a color-positive photographic film). The scanning resolution was 9600 dpi, which generated a pixel resolution of 0.002645 mm. The optical scanner was from Canon, a Canon Scan 9000 F, which has a 12-line sensor array with FARE technology (dual LED D65 equivalent illumination) and color calibration before each image was scanned. The transmittance scanning illumination source was F11. For the reflective scanning procedure, a black-mate sheet was used to cover the microspheres. 2.3.12. Analysis of the In Vitro Extract Release  
The in vitro release test of the RG extract from the obtained microspheres was performed according to a method published in another study [56] and the method described in the United States Pharmacopoeia (USP 43) [59] using the dissolution test apparatus equipped with rotating blades, an Electrolab TDT-08L Dissolution Tester, and 8 baskets of 400 mL capacity with a rotation speed of 30 rpm [60]. The quantity of microspheres with RG used was 0.25 g/sample, and the pH medium used was a phosphate buffer solution with a pH of 7.4, and the temperature was 37 \({}^{\circ}\)C. The samples were collected at 0.25, 0.50, 0.75, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h, and the release of the extract was determined spectrophotometrically at a wavelength of 362 nm. The experiment was performed in triplicate, and the results are expressed as the mean value \(\pm\) SD. 2.3.13. Statistical Analysis  
The Design of Experiment (DOE) data were analyzed using univariate and multivariate statistical methods. Bifactorial ANOVA (Analysis of Variance) was used to compare the average values of EE% concerning two statistical factors: Samples and Method. The SWL% averages were compared using tetra factorial ANOVA with four statistical factors: Samples, pH, Time, and Method. The in vitro release capacity of the phytocomplex from microspheres was compared using trifactorial ANOVA on three statistical factors: Samples, Time, and Method. GraphPad Prism 5.3 software (GraphPad Software, 2365 Northside Dr. Suite 560, San Diego, CA, USA) was used for univariate analysis. Multivariate analysis, consisting of PCA (principal components analysis), multivariate analysis of variance (MANOVA), and HCA (hierarchical cluster analysis), was performed to determine the most efficient selected materials to ensure the highest percentage of in vitro release of the phytocomplex in the polymeric matrix system. All data for the analyzed parameters were collected in triplicate, and the results obtained were presented as the average \(\pm\) SD. Statistical analysis and graphical representations were performed using standardized statistical methods in MATLAB Software, version R2013a (MatWorks Inc., 1 Apple Hill Drive, Natick, MA, USA). 3. Results and Discussions  
3.1.