## 2 Materials and Methods
### Swelling Index (SVL%) Analysis  
Microspheres were tested for their swelling index in media with five different pH levels (1.2, 3.0, 5.0, 6.8, and 7.4) for 8 h, according to Figures S4-S7 and Tables 2 and S1-S5. According to Table 3, microspheres prepared by method 2 swelled the least. The largest swelling was observed in the empty samples (E1, E2, and E3) and in the samples prepared by method 1 (m1_Ch01, m1_Ch02, and m1_Ch04). Another observation was that the variation in the chitosan concentration influenced the swelling index values. The samples that included 0.1% chitosan had a swelling index of 42,512.44-147,682.7% and those with 0.4% chitosan had an index of 24,487.56-105,008.6% in a pH medium of 7.4.  
Following this study, it was observed that the swelling index decreased with increasing chitosan concentration in the microspheres, regardless of the pH medium applied, also decreasing the solubilization tendency of the microspheres. In acidic media, smaller swelling is observed than that in alkaline media due to ionic repulsion, minimal osmotic pressure, and because the amino groups in the chitosan structure were not protonated. In the Supplementary Material (Tables S1-S5), the swelling index for all microspheres at different pH values is presented. Due to the fact that the swelling index was highest at pH 7.4 when, after 8 h, the microspheres could no longer be weighed because they began to disintegrate, the in vitro release study was performed at this pH, considering it to be the most appropriate.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Samples**} & \multicolumn{5}{c}{**SVL\%**} \\ \cline{2-6}  & **pH 1.2** & **pH 3** & **pH 5** & **pH 6.8** & **pH 7.4** \\ \hline m1\_Ch01 & 1663.05 \(\pm\) 0.36 & 5518.6 \(\pm\) 0.67 & 10,037.18 \(\pm\) 0.85 & 36,548.76 \(\pm\) 0.12 & 79,841.36 \(\pm\) 0.21 \\ m1\_Ch02 & 1041.91 \(\pm\) 0.26 & 3638.24 \(\pm\) 0.41 & 7276.32 \(\pm\) 0.75 & 26,605.56 \(\pm\) 0.88 & 73,338.97 \(\pm\) 0.93 \\ m1\_Ch04 & 980.31 \(\pm\) 0.11 & 2305.47 \(\pm\) 0.74 & 6450.86 \(\pm\) 0.53 & 19,643.47 \(\pm\) 0.45 & 67,504.3 \(\pm\) 0.19 \\ m2\_Ch01 & 431.59 \(\pm\) 0.02 & 1063.01 \(\pm\) 0.28 & 4125.96 \(\pm\) 0.13 & 13,708.03 \(\pm\) 0.24 & 42,512.44 \(\pm\) 0.37 \\ m2\_Ch02 & 226.01 \(\pm\) 0.48 & 851.93 \(\pm\) 0.67 & 2103.82 \(\pm\) 0.42 & 10,314.47 \(\pm\) 0.33 & 35,246.45 \(\pm\) 1.02 \\ m2\_Ch04 & 110.17 \(\pm\) 0.73 & 520.30 \(\pm\) 0.52 & 1044.61 \(\pm\) 0.51 & 7121.89 \(\pm\) 0.54 & 24,487.56 \(\pm\) 0.72 \\ E1 & 2826.03 \(\pm\) 0.51 & 8637.18 \(\pm\) 0.12 & 26,074.33 \(\pm\) 0.55 & 71,097.53 \(\pm\) 0.18 & 147,682.7 \(\pm\) 0.62 \\ E2 & 2303.86 \(\pm\) 0.63 & 6476.38 \(\pm\) 1.05 & 17,952.78 \(\pm\) 0.31 & 57,811.12 \(\pm\) 0.71 & 128,678 \(\pm\) 0.44 \\ E3 & 2040.62 \(\pm\) 0.11 & 4410.86 \(\pm\) 0.37 & 9081.73 \(\pm\) 0.62 & 45,286.95 \(\pm\) 1.01 & 105,008.6 \(\pm\) 0.55 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Swelling index (SVL%) of the RG extract in microspheres.  
Previous research has explored the swelling abilities of microspheres loaded with plant extracts in various pH environments. One study found that _Thymus serpyllum_ L. aqueous extract microencapsulated in chitosan microspheres using glutaraldehyde as a reticular agent had a higher percentage of swelling in a simulated gastric environment (pH 2.2) compared with a simulated intestinal environment (pH 6.8). However, the intestinal environment had a longer swelling period, and a high polymer concentration slowed the swelling process [81]. Another study on _Cassia fistula_ L. ethanolic extract microencapsulated in chitosan microspheres found that they had a higher swelling index in acidic (pH 2.0) versus alkaline (pH 7.4) environments due to chitosan's solubility in acidic conditions [82].