## 3 Results and Discussion
### Cascade Protocol for Grape Stalk Valorization
After lignin isolation, an antioxidant-rich liquid fraction remained (see Figure 2), thus suggesting the exploitation of GS as source of bioactive compounds (BACs). The antioxidant activity of this crude was evaluated by using the free radical method with DPPH by comparison to the standard Trolox and ascorbic acid. Moreover, the TPC was evaluated by the Folin-Ciocalteau assay for the liquid fraction after alkaline treatment. It should be noted that TPC cannot be performed for NaDESs extracts because of the stratification of the end products of the Folin reaction, meaning that their phenolic compounds might not be detected by UV-vis spectrophotometry [20]. Results are detailed in Table 2.  
As mentioned, alkaline delignification led to a highly structurally modified lignin, which means that a deep depolymerization occurred. This fact could explain the high scavenging activity showed for experiments at 60 min (0.2616 \(\pm\) 0.0015 IC\({}_{50}\)) (Table 2, entry 1). By contrast, for 120 min treatment (entry 2), the antioxidant power significantly decreases (_p_ < 0.05), probably due to the occurrence of recondensation reactions that reduced the presence of free phenol groups, thus diminishing the scavenging activity. Regarding TPC, the number of total polyphenols was similar for both 60 and 120 alkaline GS pretreatment (Table 2, entries 1-2). During delignification, some hemicellulose chains could be hydrolyzed to monosaccharides and give interference by reacting with the Folin-Ciocalteau reagent [46]. Furthermore, polyphenols can be degraded by exposition to US for too long, according to literature [47]. Therefore, the slight decrease at 120 min might be due to the formation of free hydroxyl radicals by the presence of high water content under UAE, which can negatively affect the antioxidant power. [48]. Considering the liquid fractions obtained after lignin extraction with NaDES, the antioxidant power was higher for longer pretreatments (120 min) compared to 60 min (Table 2, entries 3-8), with statistical significance for each system (_p_ < 0.05). This fact agrees with the results previously reported (Figure 1) in terms of delignification, except for ChLevA, which showed the lowest activity, quite surprisingly. The results for ChLAGly \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Liquid Fraction Samples** & **DPPH IC\({}_{50}\) (Related to C\({}_{0}\) *)** & **TPC (mg GAE/g DM) ** \\ \hline
1 & NaOH (10\%), 60 min & 0.2616 \(\pm\) 0.0015 \({}^{\mathrm{d},e}\) & 5.7873 \(\pm\) 0.0569 \\
2 & NaOH (10\%), 120 min & 0.4305 \(\pm\) 0.0018 \({}^{\mathrm{c}}\) & 5.3693 \(\pm\) 0.0170 \\
3 & ChLx, 60 min & 0.5848 \(\pm\) 0.0089 \({}^{\mathrm{b}}\) & n.d. *** \\
4 & ChLA, 120 min & 0.3195 \(\pm\) 0.0059 \({}^{\mathrm{d}}\) & n.d. \\
5 & ChLAGly, 60 min & 0.4490 \(\pm\) 0.0063 \({}^{\mathrm{c}}\) & n.d. \\
6 & ChLAGly, 120 min & 0.1819 \(\pm\) 0.0022 \({}^{\mathrm{e},f}\) & n.d. \\
7 & ChLevA, 60 min & 1.1867 \(\pm\) 0.0258 \({}^{\mathrm{a}}\) & n.d. \\
8 & ChLevA, 120 min & 0.6650 \(\pm\) 0.0244 \({}^{\mathrm{b}}\) & n.d. \\
9 & Ascorbic acid (standard) & 0.0869 \(\pm\) 0.0010 \(\lx@math@degree\) & - \\
10 & Trolox (standard) & 0.1081 \(\pm\) 0.0005 \({}^{\mathrm{e},g}\) & - \\ \hline \hline \end{tabular}  
* C\({}_{0}\): initial concentration for each extract. ** mg GAE/g DM: mg gallic acid equivalents/g dry matter. ** non-determined.  
\end{table} 
Table 2: Scavenging activity by DPPH assays and TPC for the liquid fractions obtained after grape stalks (GS) delignification processes via UAE (40 kHz, 200 W). Results are expressed as mean \(\pm\) SD. Values within each variable (for DPPH) with different letters are significantly different (_p_ < 0.05). at 120 min were worth noting (entry 6), being significantly higher compared to the others (\(p<0.05\)), and comparable with the standard Trolox (\(p>0.05\)). This fact could be attributed to a stronger depolymerization degree compared to the bicomponent NaDESs, which could also explain the absence of precipitated lignin with this solvent.