# Development of a Mouthwash Using Freeze-Drying Technique: An Optimization Study
## 3 Results and Discussion
### Development and Evaluation of the Cetraria islandica Extract Mouthwash
#### 3.2.7 Optimization of the _Cetraria islandica_ Mouthwash Formulation  
To obtain an optimal formula in terms of the desired lyophilized characteristics, an optimization process was performed. For that, a set of constraints was applied to the developed models of certain responses and only the formulations that fulfill those conditions with a certain degree of probability were considered. The conditions envisaged the maximization of fracturability, minimization of weight loss percentage, minimization of reconstitution time and maximization of viscosity. Such a formulation would ensure easy manipulation, with no material loss when extracted from the package, fast reconstitution, and a pleasant mouthfeel with good adherence to the oral cavity surface. The composition of the optimal formula generated by the software is presented in Table 5.  
For the next step, we prepared the sample according to the generated formula, freeze-dried it, and then analyzed it as the other samples before. The experimental values were  
Figure 6: The influences of formulation factors (X1, X2, X3) on the viscosity of the reconstituted mouthwash (Y10).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Factor** & **Value** & **Ingredient** & **Quantity (\(w\)/\(w\)\%)** \\ \hline Filler Type (X1) & Mannitol & **Mannitol** & **7.00** \\ \% filler (X2) & 7 & Poloxamer 407 & 5.00 \\ \% MC (X3) & 0.5 & **Methylcellulose** & **0.50** \\  & & Allantoin & 5.00 \\  & & Hyaluronic acid & 0.125 \\  & _Cetraria islandica_ extract & 5.00 \\  & & Flavoring agent & 0.50 \\ \hline \hline \end{tabular} The bold data underlines the factors from the DoE.  
\end{table}
Table 5: The quantitative and qualitative composition of the optimal mouthwash formulation.  
collected using Modde software and, in the end, the predicted values were compared to the experimental ones, as residual values. As shown in Table 6, the values obtained experimentally were close to the values predicted by the software. Therefore, the experimental design showed high reliability and may be used in further studies regarding freeze-dried mouthwashes. This is an innovative product, with no correspondents on the market to be compared with. However, among pharmaceutical products, the oral lyophilates are quite similar in terms of the production process, product characteristics and use. Literature reports give a hardness minimum limit of 4N for oral lyophilates and a disintegration time of maximum 180 s, which are both in accordance with the developed freeze-dried mouthwash.  
Up to this point, the study demonstrated that freeze-dried mouthwashes with appropriate characteristics in both solid state and as reconstituted solutions are feasible and that the DoE strategy enabled the optimization of products. _Cetraria islandica_ extract was successfully incorporated for its mentioned applications such as mouth and throat anti-irritant, antimicrobial, or moisturizing and soothing effects [29]. Besides the previously described roles of _Cetraria islandica_ extracts, recent studies reported the ability of its polysaccharides to induce keratinocyte differentiation with important implications in wound healing [30]. Our results revealed the lack of toxicity of extracts and an antibacterial activity on _S. aureus_ and _E. faecalis_ which supported the hypothesis of oral care product preparation. As freeze-drying is a state-of-the-art method to dry or concentrate extracts without altering their biological properties, and its use was reported for lichen species, we assumed that the lyophilized mouthwash formulations would preserve the effects of extracts [16]. Future studies envisage the biological evaluation of freeze-dried formulations and in vivo studies in healthy volunteers.