## 3 Results and Discussion
### Reduction of Phosphodiesterase Type 5 (PDE5) Protein and Transcript Levels  
To determine whether the formulation affects the expression of the PDE5 enzyme, human muscle epithelial cells were incubated with Icarifil and L-Citrulline in the same amount present in the mixture. Figure 4 reports the variations in PDE5 protein level and transcriptional levels of epithelial cells after treatment. Icarifil (0.5 mg/mL) was able to reduce the level of human PDE5 in a significantly higher manner than the vehicle used as a control, and L-Citrulline was dosed in an equivalent amount present in the mixture. Specifically, Icarifil showed efficacy in reducing PDE5 protein levels higher than L-Citrulline by 22% and 45% compared to the control. This difference further increased when transcriptional levels of PDE5 were evaluated, where the total mixture was more effective than L-Citrulline alone at levels of about 40%. It is clear from these data that when L-Citrulline is used in combination with the other component in a unique formulation, the efficacy on the PDE5 enzyme is more evident than when it is used by itself [28]. Figure 2: Effect on the proliferative activity of murine penile muscle cells of Icarifil (100 \(\upmu\)L of 0.5 mg/mL) and different mixtures of components, dosed in quantities equivalent to those present in the formulation compared to the medium and medium with 100 \(\upmu\)L of PBS added used as controls. Data represent mean \(\pm\) SD with significance, **_p_ \(<\) 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test. Figure 4: Treatment effect of Icarifil (0.5 mg/mL) on human phosphodiesterase were 5 activity testing human muscle epithelial cells compared with the action of L-Citrulline alone (equivalent amount present in the formulation). PDE5 protein level variation (**A**) and PDE5 relative transcriptional levels (**B**) The medium was used as a control. Data represent mean \(\pm\) SD, significance \(\mathbf{\ast}\)\(p\) < 0.05, using the One-WAY ANOVA test followed by Bonferroni test as a post-test. To verify whether these results translated into mouse penile muscle cells, the cells were incubated with Icarifil or components comprising the formulation. The results showed that Icarifil (0.5 mg/mL) was able to reduce the activity of phosphodiesterase type 5 significantly more effectively than the control and the mixture of L-Citrulline and L-Carritine alone or in combination with _Eruca vesicaria_, _Turnera diffusa_, and Tribulus Terrestris, dosed in an equivalent ratio to that present in the mixture (Figure 5A). Specifically, the total mixture showed efficacy in reducing PDE5 levels higher than 65% compared to the control and 35% compared to the mixture of L-Citrulline and L-Carritine, the major components of Icarifil. This difference was also evident in the relative transcriptional levels of PDE5 (Figure 5B), where Icarifil was substantially more effective than the control (about 57%) and the mixture consisting of L-Citrulline and L-Carritine (about 22%). These results could be  
Figure 3: Dose-dependent effect of 400, 600, and 900 \(\upmu\)L of a solution consisting of Icarifil and culture medium in 1:1 ratio on weight gain of muscle epithelial cells compared with the culture medium used as a control. Data represent mean \(\pm\) SD with significance \(\mathbf{\ast}\)\(p\) < 0.05, \(\mathbf{\ast}\)\(p\) < 0.01 using the One-WAY ANOVA test followed by Bonferroni test as a post-test. explained by the synergic effect between the components of the multicomponent mixture, demonstrating that this complex mixture could reduce PDE5 levels more effectively than the single components. After assessing the ability of Icarifil to reduce the activity of PDE5 enzyme more than the single extract, its activity was compared with a well-known PDE5 inhibitor. Therefore, mouse penile muscle cells were treated with an aqueous solution of tadalafil alone (10 umol/L and 5 umol/L) and in co-treatment with Icarifil (0.5 mg/mL).