## 3 Results and Discussions
### Formulation of Liposomes with Mulberry Extract and Their Characterization  
Formulation of Liposomes Using the Lipid Film Hydration Method and Microscopic Characterization of the Formed Liposomes  
In addition to identifying the optimal concentration of mulberry extract with healing effect (MnE 25 g/mL), the main aim was to incorporate this extract into liposomal formulas, characterize them, and evaluate the healing potential of it. To confirm that the lipid vesicles were formed spontaneously following the hydration of the lipid film according to the authors [PERSON] ([PERSON]) et al., 2021 [24] and [PERSON] et al., 2020 [59], microscopic analysis was used.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Samples** & **TPc** & **TFc** & **Ascorbic acid** & **FRAP** \\
**(mg GAE/100 g dw)** & **(mg QE/100 g dw)** & **(mg AA/100 g fw)** & **(mmol TE/100 g dw)** \\ \hline MnE & 1460.37 \(\pm\) 61.42 & 296.48 \(\pm\) 79.53 & 43.71 \(\pm\) 1.481 & 80.23 \(\pm\) 0.56 \\ \hline \end{tabular}  
* The results are presented as mean \(\pm\) SD (\(n=3\)). TFc—Total phenols content; Gallic acid equivalent (GAE); TFc—Total flavonoids content; QE—Quercetin; AA—Ascorbic acid; FRAP—Ferric-Reducing Antioxidant Power; TF—Trolov equivalent; MnE—_Morus nigra_ extract.  
\end{table}
Table 2: Total phenols, flavonoids, ascorbic acid content and antioxidant capacity determined by the FRAP method of MnE *.  
Thus, following the microscopic analysis, it can be seen in Figure 2 that the empty liposomes (L) and mulberry-loaded liposomes (MnL) have a spherical or round appearance, and are not agglomerated, repelling each other. Our results are according with those of other authors [24; 59] who obtained liposomes with the similar characteristics.  
[PERSON] et al., 2018 [20], report that phospholipids and sterols such as cholesterol are the most common ingredients used in liposome formulations, and that hydrophilic phase buffer systems are most commonly used. The ratio of phosphatidylcholine to cholesterol of 3:1 \(w/w\) that was used in the formulation of our liposomes (MnL) is one that confers stability to the formulated vesicles according to [PERSON] et al., 2019 [60].  
Depending on the compound/extract to be entrapped or included, liposomes are prepared differently. For example, for the inclusion of plant extracts, volatile oils, etc. it is considered that obtaining multilayered and giant (with size up to 500 nm) liposomes is favorable [22; 61]. Thus, the main method of obtaining this type of liposomes is the method that uses organic solvents for the formation of a lipid layer to be hydrated, the method called the lipid film hydration method. Hydration according to the authors [PERSON] et al., 2021 [62] is important to be done with hydrophilic compounds that give the electrical charge to the formed system (for example the phosphate buffer) to increase the stability of the emulsion.  
Conventional liposome formulation methods according to [PERSON] et al., 2010 [21] include four important stages: the formulation of the lipid phase to be hydrated, its hydration, purification and analysis of the formulated vesicles.  
The effect of black mulberry fruit extract encapsulated in spray-dried liposomal powders was studied compared to those coated with chitosan, both included in chocolate, at different pH and temperatures (40, 60 and 80 \({}^{\circ}\)C). The results of the study showed that liposomal powders coated with chitosan provided better protection of anthocyanin content, both at increasing temperature and pH. In addition, encapsulation of mulberry fruit in liposomes improved the in vitro bioaccessibility of anthocyanins [9].