## 3 Results
### Efficacy Assessment
#### 3.3.4 Anti-Age Effect  
The anti-aging effect was evaluated in the first instance through the evaluation of both senescence-related (beta-galactosidase) and aging-related markers (MMP-9). Subsequently, the expression levels of well-aging and trophic-related markers (collagen type 1, hyaluronic acid and elastin) were analyzed. These evaluations were conducted on BJ-cells and full-thickness skin tissues.  
Results on BJ-Cells  
As demonstrated in Figure 8, exposure to UV and pollution (CTR+) resulted in elevated MMP-9 levels and beta-galactosidase activity in comparison to the negative control (CTR-). Treatment with PLG-GRAPE, at all concentrations, resulted in a reduction of MMP-9 levels and beta-galactosidase activity.  
Furthermore, the expression of key components of the extracellular matrix was evaluated, with results from the assessment of collagen type I (Figure 9A,B), elastin (Figure 10A,B), and hyaluronic acid (Figure 11) revealing that cells exposed to UV radiation and pollution exhibited significantly lower levels of collagen type I, elastin, and hyaluronic acid to the negative control. Treatment with PLG-GRAPE led to a significant enhancement in the levels of all these key extracellular matrix components, thereby further validating the anti-aging efficacy of the product.  
Figure 8: Evaluation of anti-age activity on BJ cells through: (**A**) MMP-9 quantification; (**B**) Beta-galactosidase activity evaluation. Values are shown as mean \(\pm\) st. dev, based on three replicates (\({}^{*}p<0.05\)\(t\)-test vs. negative control).  
Figure 9: _Cont._Figure 10: Evaluation of elastin levels using the ELISA method (**A**) and immunofluorescence technique (**B**). Values are shown as mean \(\pm\) st. dev, based on three replicates (* \(p<0.05\)\(t\)-test vs. negative control).  
Figure 9: Evaluation of Collagen type three levels through ELISA method (**A**) and immunofluorescence technique (**B**). Values are shown as mean \(\pm\) st. dev, based on three replicates (* \(p<0.05\)\(t\)-test vs. negative control).  
Results on Full Thickness Skin Tissues  
The experiments were also conducted on full-thickness skin tissue. As demonstrated in Figure 12, also in this case, the expression of MMP-9 and the activity of beta-galactosidase increased in the CTR+ samples compared to the CTR\(-\) samples. However, this increase was significantly reduced in the tissues treated with PLG-GRAPE. These results confirm the anti-aging efficacy of the sample in a more complex model.  
Furthermore, the key components of the extracellular matrix were evaluated in full-thickness skin tissues. The assessment of collagen type I (Figure 13A) elastin (Figure 13B), and hyaluronic acid (Figure 13C) revealed that tissues exposed to UV radiation and pollution exhibited significantly lower levels of collagen type I, elastin, and hyaluronic acid compared to the negative control. Treatment with PLG-GRAPE led to a significant enhancement in the levels of these critical extracellular matrix components, thus, validating the anti-aging efficacy of the product in full-thickness skin tissues.  
Figure 11: Evaluation of hyaluronic acid levels using the ELISA method. Values are shown as mean \(\pm\) st. dev, based on three replicates (* \(p\) < 0.05 _t_-test vs. negative control).  
Figure 12: Evaluation of anti-age activity on full-thickness skin tissues through MMP-9 quantification (**A**) and Beta-galactosidase activity evaluation (**B**). Values are shown as mean \(\pm\) st. dev based on three replicates (* \(p\) < 0.05 _t_-test vs. negative control).