## 4 Results and Discussion
### Preparation and Physicochemical Characterizations  
An appropriate carrier is necessary for delivering \(\alpha\)-M to the site of action. In this study, ChAlg/\(\alpha\)-M HF was prepared as a novel RAS therapy. To prepare ChAlg/\(\alpha\)-M HF, firstly, the ChAlg solution was inserted into a propylene box (\(5\times 5\times 4\) cm\({}^{3}\)) and dried at 60 \({}^{\circ}\)C for 24 h to obtain ChAlg HF. Then, \(\alpha\)-M (25 mg) in methanol was casted into ChAlg HF [18]. ChAlg HF was successfully prepared using the solvent evaporation method (Figure 2A). The Ch and Alg molecules effectively formed the HF by evaporating the water solvent in a drying process at 60 \({}^{\circ}\)C. In addition, ChAlg/\(\alpha\)-M HF was successfully prepared with macroscopically smooth.  
Figure 1: In silico studies of chitosan (Ch), alginate (Alg), and \(\alpha\)-Mangostin (\(\alpha\)-M). Discovery studio visualizer and AutoDock 1.6 applications were applied for molecular docking studies.  
We next analyzed ChAlg/\(\alpha\)-M HF using SEM to confirm that \(\alpha\)-M existed in the ChAlg HF. The surface of HFs was observed by SEM at 5000 magnification. The results of SEM clearly showed that \(\alpha\)-M existed on the surface of ChAlg HF (Figure 2B) with the halo pattern of Alg-Ch, whether the Alg HF was less homogenous than ChAlg HF. This polyelectrolyte complex film surfaces showed the irregular and porous properties similar to the results in the previous studies [29; 30; 31; 32].  
XRD and DSC studies had been done to identify the crystallinity of \(\alpha\)-M in HFs. XRD patterns of HFs without \(\alpha\)-M (Alg HF, Ch HF, and ChAlg HF) showed a halo pattern, indicating an amorphous form (Figure 3A). In contrast, pure \(\alpha\)-Mand ChAlg/\(\alpha\)-M HF showed high peaks at around 5\({}^{\circ}\), suggesting that Ch Alg/\(\alpha\)-M HF contained \(\alpha\)-M. Interestingly, the crystallinity of \(\alpha\)-M was decreased in ChAlg/\(\alpha\)-M HF as shown by peaks reduction of \(\alpha\)-M from around 7.5 to 12.5\({}^{\circ}\). These results corroborate the in silico study which showed a probability to form an interaction with a hydrogen bonding between Ch, Alg, and \(\alpha\)-M.  
Figure 3: Characterization of ChAlg/\(\alpha\)-M HF. (**A**) XRD patterns; (**B**) DSC thermographs. The experiments were performed three times, and representative data are shown.  
Figure 2: Macroscopic and microscopic appearances of hydrogel films (HFs). (**A**) Macroscopic appearances of ChAlg/\(\alpha\)-M HF; (**B**) SEM Analysis of ChAlg/\(\alpha\)-M HF. The experiments were performed three times, and representative data are shown.  
The DSC thermograms presented the endothermic peaks of Ch HF, Alg HF, ChAlg HF, and ChAlg/\(\alpha\)-M HF at around 100 \({}^{\circ}\)C, representing water evaporation (Figure 3B). This phenomenon maybe related to the amount of free water in HFs. The water molecules in HFs, can be bounded through three polar groups of polymers: A mine, carboxyl, and hydroxyl [33]. The degradation of carboxylic groups in Alg appeared at the temperature range of 200-230 \({}^{\circ}\)C. As shown in the DSC thermogram of Ch HF, ChAlg HF, and ChAlg/\(\alpha\)-M HF at a temperature range of 270-280 \({}^{\circ}\)C. the thermal stability of Alg increased due to complexation between Alg and Ch, representing deacetylation and partial depolymerization of the Ch chains [31]. In addition, a strong endothermic peak of \(\alpha\)-M appeared at 179 \({}^{\circ}\)C, representing the melting point of \(\alpha\)-M. However, it slightly decreased and moved to 180 \({}^{\circ}\)C in ChAlg/\(\alpha\)-M HF, specifying a lower crystalline of \(\alpha\)-M in ChAlg HF compared to pure \(\alpha\)-M. The DSC studies imply that \(\alpha\)-M interacted with chitosan and alginate in the hydrogel film and these phenomena were supported by previous molecular docking and XRD results.