# Impact of Deep Eutectic Solvents on Extraction of Polyphenols from Grape Seeds and Skin
## 3 Results and Discussion
### Antioxidant Activity  
Dietary antioxidants, widely spread in fruits and vegetables, have received considerable scientific attention and investigation of their bioactivity became the main goal of many conducted studies. Regardless of the large number of validated antioxidant in in vitro tests, there is still a problem concerning the differences in chemical principles and mechanisms of the most common methods. In order to provide comprehensive information about total antioxidant capacity, and to distinguish the dominant mechanism of antioxidant activity, it was suggested to perform several tests pointing different aspects of antioxidant properties [41]. Therefore, four antioxidant methods were conducted--FRAP, CUPRAC, DPPH, and ABTS (Table S1: Antioxidant activity of grape seed extracts, Table S2: Antioxidant activity of grape skin extracts). FRAP is a typical electron transfer method based on the capability of antioxidants to reduce Fe\({}^{3+}\) tripyridyl triazine complex (colorless) to Fe\({}^{2+}\) tripyridyl triazine (blue color) in acidic medium. CUPRAC assay measures the ability of test compounds to reduce Cu\({}^{2+}\) to Cu\({}^{+}\) in aqueous-ethanolic medium (pH 7.0) in the presence of neocuproine. DPPH assay, the oldest indirect method for evaluating antioxidant capacity, measures the reduction of stable organic DPPH radical in presence of antioxidants  
Figure 2: Total polyphenol content of skin extracts obtained from ten grape varieties. Data are expressed as mg GAE g\({}^{-1}\) DW for each variety, means \(\pm\) SD, \(n=3\). Different lower case letters (a–c) indicate significant differences among solvents (\(p\leq 0.05\)) based on post hoc Tukey’s test. Abbreviations: TPC—total polyphenol content; GAE—gallic acid equivalents; DW—dry weight; SD—standard deviation; AcEtOH—acidified aqueous ethanol; ChCit—choline chloride: citric acid; ChGlc—choline chloride: glucose. to the yellow colored 2, 2-diphenyl-1-picrylhydrazyn. Another commonly used radical scavenging test is ABTS/TEAC assay, based on the ability of test compounds to reduce ABTS radical [41]. Taking into account many limitations and lacks of each test applied for evaluating antioxidant activity, results obtained from them were used for calculating unique antioxidant composite index (ACI). ACI is a simple, widely used mathematical calculation that enables deeper insight into antioxidant activity, since it combines several tests based on different chemical mechanisms. ACI values calculated for grape seed extracts are presented in Figure 3. Regarding conventional solvents, the highest antioxidant activity was determined for seeds obtained from 'Gamay' variety. Extraction with ChCit also revealed 'Gamay' as the most potent, followed by Serbian autochthonous varieties, such as 'Black Tamjanika' and 'Prokupac'. In the case of ChGlc, the highest ACI value was calculated for seed extracts of 'Prokupac' variety. When it comes to grape skin (Figure 4), ACI calculation revealed apparently red skin prominence in antioxidant actions. 'Merlot', 'Zacinak', and 'Cabernet Sauvignon' were characterized with the highest antioxidant capacity. Observed differences among varieties, regarding both seeds and skin, represent a confirmation of many research studies conducted so far [20; 42]. ACI values for both, seeds and skin (Figures 3 and 4) were significantly correlated with TPC of corresponding extracts (0.798 \(\leq\) r \(\leq\) 0.967; \(p\) < 0.01). Therefore, such strong relationship confirmed the previous disclosures about the significant contribution of polyphenol compounds to the antioxidant activity. Nevertheless, prominent antioxidant activity of ChCit extracts was revealed. Higher antioxidant activity of these extracts in comparison with ChGlc and acidified aqueous ethanol extracts could be explained by the antioxidant activity of ChCit itself, or by antioxidant activity of NADES forming compound, citric acid. This naturally occurring organic acid possess antioxidant and anti-inflammatory properties.