## 3 Results and Discussion
### Biological Activity Evaluation of Grape Pomace Extracts Prepared in Optimal Conditions  
In the pursuit of understanding and harnessing the full potential of grape pomace extract in NADESs, our study delved into the preliminary biological activity evaluation of extracts prepared under optimal conditions. The biological evaluation of optimal extracts holds paramount significance in both the food and cosmetic industries, as it provides a comprehensive understanding of the potential benefits and safety considerations associated with these extracts. In this context, examining the biological properties of optimal extracts emerges as a pivotal step in the development and formulation of products for the food and cosmetic sectors. To date, the Grasevina grape pomace extract in BGlc has been assessed on the HaCaT cell line, and cytotoxicity was determined through an in vitro assay specific to the HaCaT cell line (keratinocytes). The extract prepared with BGlc exhibited a stimulatory effect on HaCaT cell proliferation, showing a potential increase of up to 40% depending on the extract volume ratio. This suggests a proportional relationship between polyphenol concentration and the stimulatory effect, making them promising for application in the cosmetic industry [30]. In this study, we further explored additional methods of biological activity and compared them with the previously used ethanol extract in the research by [PERSON] et al. [30]. In this research, various biological evaluations were undertaken to assess the potential applications of extracts obtained under optimal conditions at the preparative scale in the cosmetic and food industry. The study involved the examination of collagenase activity inhibition, in vitro wound healing through a scratch test, and the simulation of polyphenol permeability from grape pomace extracts across an artificial membrane using the PAMPA (Parallel Artificial Membrane Permeability Assay) method simulating the gastrointestinal tract barrier. First, HPLC analysis of the NADEES extract prepared under optimal conditions and the ethanol extract used as a control was conducted, and the results are presented in Table 2.  
Considering compatibility and in comparison with previous work [29], no significant differences were observed in biologically active compounds. Different solvents dissolve various types of compounds, and a synergistic effect is possible, making it worthwhile to further investigate the biological activity. The potential inhibition of collagenase was examined for the fixed concentration of polyphenols in the grape extract. The results indicate that the negative control had the lowest collagenase inhibition, followed by the positive control. Among the extracts, the BGlc extract demonstrated values of statistically higher (\(p<0.05\)) inhibition compared to the EtOH extract (Figure 4). \begin{table}
\begin{tabular}{l l l} \hline \hline  & \multicolumn{2}{c}{**Extracts**} \\ \hline
**Compound** & **GPBGlc** & **GPEOH** \\ \hline Epigallocatechin & 0.235 \(\pm\) 0.010 & 0.279 \(\pm\) 0.001 \\ Catechin & 0.739 \(\pm\) 0.006 & 0.820 \(\pm\) 0.002 \\ Epicatechin & 0.750 \(\pm\) 0.005 & 0.471 \(\pm\) 0.009 \\ Gallic acid & 0.549 \(\pm\) 0.008 & - \\ Procyanidin B1 & 1.017 \(\pm\) 0.005 & 0.505 \(\pm\) 0.002 \\ Procyanidin B2 & 0.265 \(\pm\) 0.022 & - \\ Procyanidin B3 & 0.966 \(\pm\) 0.003 & 1.197 \(\pm\) 0.030 \\ Procyanidin C1 & 0.258 \(\pm\) 0.053 & - \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Polyphenolic content (mg g dw\({}^{-1}\) of pomace) in prepared extract. Specific polyphenol contents and total polyphenol content were expressed as the means (n = 3) \(\pm\) S.D. The findings presented in our study align with prior research emphasizing the significance of natural extracts in addressing skin aging processes, particularly collagenase activity [37; 38]. So far, several research articles have compared the impact of NADES on collagenase activity, with findings indicating notable results.