## 3 Results and Discussion
### Biological Activity Evaluation of Grape Pomace Extracts Prepared in Optimal Conditions
Specifically, NADES formulations based on ChCl/Urea and ChCl/glycerol exhibited significant inhibition values for collagenase (91.1% and 92.7%, respectively) [38]. However, it is important to mention that choline chloride is restricted for use in cosmetics, which means these NADES formulations might not be the best choice for the final cosmetic product [10]. Moreover, we want to investigate the impact of extracts on in vitro wound healing. Cell migration is an important step in wound healing. Tests that can assess cell migration are very useful for evaluating in vitro wound healing. A gap in the keratinocyte monolayer, mimicking wound formation, was created using the scratch test [39]. This experiment examined the impact of the extract obtained in the NADES and the solvent itself on cell migration, compared to the control (cells only in a monolayer). Figure 5 shows the results of the scratch test. Upon comparing the images on both sides of the display, with the left side representing a mimicked wound and the right side showcasing the healing process after 48 h, it is evident that cells treated with extract in NADES effectively bridged the gap. Notably, there were more cells in the treated group compared to the untreated cells (control). Even cells treated with NADES alone exhibited growth, contributing to wound healing. These observations strongly suggest that the extract in NADES (BGlc) and NADES itself play a role in promoting cell migration and wound healing, highlighting their potential application in the cosmetics industry. Similarly, [PERSON] et al. [40] tested the influence of hydrophobic DES based on menthol and saturated fatty acids. This formulation also enhanced wound healing. However, it is crucial to acknowledge a limitation in this experiment. The in vitro cell culture environment did not fully replicate an in vivo surgical wound. While in vitro results are promising, the transition to in vivo studies is crucial for a comprehensive understanding of their effectiveness and safety in wound healing of real scars [41]. Figure 4: The impact of polyphenols extracted in BGlc and EtOH in concentration of 300 mg mL\({}^{-1}\). Results are the mean \(\pm\) standard deviation (n = 3). Positive control—1 mg mL\({}^{-1}\) of gallic acid. Negative control—distilled water. GPBlc—grape pomace extract in NADES BGlc obtained under optimal conditions. GPEtOH—grape pomace extract in aqueous ethanol (70%. \(v/v\)). Furthermore, simulations to assess the permeability of polyphenols from grape pomace extracts through an artificial membrane that mimics the gastrointestinal tract were conducted. The PAMPA method was employed to investigate the passive transport of polyphenols, particularly in the natural deep eutectic solvent (NADES) BGlc and ethanol, across the small intestine epithelium and into the bloodstream. The apparent permeability coefficients (Papp) were determined before and after in vitro small intestinal simulation, revealing that the permeability of polyphenols in BGlc was 2.5 times higher than that in ethanol (Table 3). This enhanced permeability is attributed to potential interactions between the NADES and the phospholipid bilayer of the artificial membrane. Although the observed permeability falls into the category of low absorption, with values less than 1 \(\times\) 10\({}^{-6}\) cm s\({}^{-1}\)[42], the results obtained by PAMPA still go in favor of the extraction in the NADES compared to a conventional solvent. Figure 5: Migration assessment of HaCaT cells before (1 st column) and 48 h after the initial scratch and treatment with GPBGlc and BGlc (2 nd column). GPBGlc—grape pomace extract in NADES BGlc obtained under optimal conditions. \begin{table}
\begin{tabular}{l l} \hline \hline  & **Permeability (10\({}^{-11}\) cm s\({}^{-1}\))** \\ \hline GPBGlc & 5.15 \(\pm\) 0.47 \({}^{\text{a}}\) \\ GPEtOH & 1.99 \(\pm\) 0.11 \({}^{\text{b}}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Permeability coefficient (cm s\({}^{-1}\)) calculated for the different systems.