## 2 Materials and Methods
### Analysis of the In Vitro Extract Release  
The in vitro release test of the microspheres loaded with RG extract was performed in phosphate buffer solutions with a pH of 7.4. The release capacity of the microspheres synthesized in simulated intestinal juice was determined at a temperature of 37 \({}^{\circ}\)C. As can be seen in Table 5 and Figure S13, the release of polyphenols from the microspheres was complete after 24 h.  
Univariate statistical analysis demonstrated that, after 24 h, microspheres prepared using method 2 released a higher concentration of the entrapped extract (97.94%) compared with those prepared using method 1, which released a smaller concentration of extract (91.7%). Another observation was that microspheres with a higher concentration of chitosan (0.4%) released a smaller concentration of extract (51.19%) compared with those with a lower concentration of chitosan (0.1%), which released 57.55% extract. The rate of release of the extract from microspheres prepared using the two methods is represented in Figure S14a,b, and, as can be seen, was different and occurred in two stages: the first stage showed a rapid  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multicolumn{8}{c}{**Time (h)**} \\ \cline{2-13}  & **0.25** & **0.5** & **0.75** & **1** & **2** & **3** & **4** & **5** & **6** & **8** & **12** & **24** \\ \hline m1\_Ch01 & 0.06 \(\pm\) & 7.75 \(\pm\) & 16.24 \(\pm\) & 27.46 \(\pm\) & 42.12 \(\pm\) & 54.96 \(\pm\) & 65.09 \(\pm\) & 74.92 \(\pm\) & 80.54 \(\pm\) & 86.22 \(\pm\) & 90.02 \(\pm\) & 92.71 \(\pm\) \\  & 2.25 & 1.37 & 0.71 & 5.28 & 4.13 & 9.36 & 7.41 & 6.28 & 0.46 & 1.59 & 3.27 & 6.91 \\ m1\_Ch02 & 0.02 \(\pm\) & 6.50 \(\pm\) & 14.12 \(\pm\) & 25.93 \(\pm\) & 37.46 \(\pm\) & 50.18 \(\pm\) & 61.68 \(\pm\) & 70.42 \(\pm\) & 79.08 \(\pm\) & 84.57 \(\pm\) & 89.63 \(\pm\) & 91.82 \(\pm\) \\  & 0.88 & 1.44 & 2.46 & 5.28 & 4.13 & 7.69 & 6.28 & 1.01 & 2.23 & 3.67 & 4.02 & 7.69 \\ m1\_Ch04 & 0.01 \(\pm\) & 3.64 \(\pm\) & 12.97 \(\pm\) & 21.59 \(\pm\) & 33.52 \(\pm\) & 47.41 \(\pm\) & 58.76 \(\pm\) & 67.28 \(\pm\) & 75.32 \(\pm\) & 81.39 \(\pm\) & 87.32 \(\pm\) & 90.57 \(\pm\) \\  & 5.50 & 1.23 & 0.12 & 4.18 & 2.39 & 9.56 & 7.45 & 1.08 & 3.67 & 5.48 & 8.46 & 0.22 \\ m2\_Ch01 & 0.09 \(\pm\) & 13.62 \(\pm\) & 26.64 \(\pm\) & 42.13 \(\pm\) & 55.09 \(\pm\) & 66.46 \(\pm\) & 74.92 \(\pm\) & 82.71 \(\pm\) & 89.73 \(\pm\) & 95.25 \(\pm\) & 97.84 \(\pm\) & 98.63 \(\pm\) \\  & 2.69 & 0.58 & 0.62 & 3.64 & 3.45 & 0.15 & 1.05 & 6.39 & 8.15 & 2.05 & 4.15 & 7.02 \\ m2\_Ch02 & 0.08 \(\pm\) & 10.55 \(\pm\) & 21.24 \(\pm\) & 35.07 \(\pm\) & 49.28 \(\pm\) & 59.91 \(\pm\) & 70.23 \(\pm\) & 80.34 \(\pm\) & 87.12 release of the extract from the microspheres, followed by the second stage, where a slow release of the extract occurred by the end of the 24 h.  
The dissolution medium used affected both the chitosan and sodium alginate molecules, causing the ionization of the carboxyl groups of sodium alginate. Additionally, the deprotonation of sodium alginate caused the swelling of the complex network of microspheres, leading to the disintegration of microspheres and the complete release of the extract [84; 85]. Therefore, at pH 7.4, microspheres could swell to their maximum capacity, disintegrate, and release the entire amount of entrapped extract within 24 h. Simultaneously, a uniform and rapid hydration of microspheres occurred, forming a gel barrier in a uniform layer where the diffusion process of the extract took place [85]. According to previous studies, _Acalypha indica_ L. extract release from chitosan-casein microparticles was more effective at a pH of 5 compared with those at pH levels of 7 and 10. This is because microparticles have a rigid and hydrophobic core in an alkaline environment [79]. In another study, _Cassia fistula_ L. ethanolic extract from chitosan microspheres was tested in media with two different pH levels: acid, at 2.0, and alkaline, at 7.4.