## 3 Results and Discussion
### Optimization of Preparative-Scale Parameters for Ultrasound-Assisted Extraction Process  
Following the identification of the optimal UAE method, the response surface methodology to further enhance the extraction process was employed. Notably, the chosen extraction solvent here was a natural deep eutectic solvent (NADES), betaine/glucose (BGlc), previously selected by [PERSON] et al. [30], aligning with the principles of green extraction. This selection aimed to investigate potential improvements in extraction efficiency associated with the use of NADESs in order to achieve ready-to-use stable extracts [31]. The efficiency of extraction depends on various factors, including the solvents used, extraction time, the power of ultrasound, and the mass-to-solvent ratio [32]. These four independent variables were encoded at one level, resulting in the experimental design shown in Table 1.  
Figure 2: Polyphenol extraction efficiency from grape waste (6 g) with water (0.3 L). Extraction was conducted using a batch process with different probe diameters (22 mm, 34 mm, and 50 mm). Contents of polyphenols were expressed as the means (n = 3) \(\pm\) S.D. Presented values followed by different lower-case letters (a,b) are significantly different (\(p<0.05\)) as measured by [PERSON]’s HSD test. According to the obtained results, as shown in Figure 3, the highest concentration of TP was achieved in experiment 12. A BGlc with 30% of water content (\(v/v\), %) was applied. The extraction was conducted at an amplitude of 20%, for 8.5 min, and with a mass-to-volume ratio of 0.06. The lowest polyphenol concentration was obtained in experiment 27, where a NADES with 30% water, 100% amplitude, 8.5 min of extraction, and a mass-to-volume ratio of 0.06 was used. The TP content ranged from 6.64 to 20.94 mg g\({}^{-1}\) dry weight. Additionally, the RSM model was evaluated, and ANOVA was used to determine the statistical significance of the quadratic model and the regression coefficients for each response. The results are summarized in Table S1. The determined optimal conditions for extracting polyphenols from grape pomace using NADES BGlc (1:1) are as follows: a NADES ratio of 70%, an amplitude strength of 28.13%, an extraction time of 13.21 min, and a mass-to-solvent ratio of 0.04. The obtained concentration of TP after extraction under these optimal conditions was 21.6 mg\({}_{\text{GAE}}\) g\({}^{-1}\) experimentally, while when it was theoretically predicted, the value was. 21.1 mg\({}_{\text{GAE}}\) g\({}^{-1}\). The collected experimental data were used to create three-dimensional response surface plots for the TP yield as a function of the NADES content, time, amplitude, and mass-to-solvent ratio. According to Figure 3, it is evident that the concentration of TP increased with the higher content of NADES in the solvent. The highest concentrations were achieved at a NADES content of 70%, while the lowest concentrations were obtained when extraction was performed with water, as has been previously noted [25]. The TP yield was also influenced by amplitude and the mass-to-volume ratio. The yield of TPs increased with decreasing amplitude and decreasing mass-to-volume ratio. Extraction time had a positive influence but did not have a significant  
\begin{table}
\begin{tabular}{l l l l l l} \hline \hline
**Experiment** & \begin{tabular}{l} **Factor 1** \\ **\% NADES** \\ **[\%, w/w]** \\ \end{tabular}  & \begin{tabular}{l} **Factor 2** \\ **A [\%]** \\ \end{tabular}  & \begin{tabular}{l} **Factor 3** \\ **t [min]** \\ \end{tabular}  & \ \begin{tabular}{l} **Factor 4** \\ **Mass/Solvent** \\ **[g mL\({}^{-1}\)]** \\ \end{tabular}  & \begin{tabular}{l} **Response** \\ **TP** \\ **[mg\({}_{\text{GAE}}\) g\({}^{-1}\)]** \\ \end{tabular}  \\ \hline
1 & 35.00 & 100.00 & 8.50 & 0.02 & 12.47 \\
2 & 35.00 & 100.00 & 8.50 & 0.10 & 8.88 \\
3 & 0.00 &  
impact on polyphenol extraction yield.