# Impact of Deep Eutectic Solvents on Extraction of Polyphenols from Grape Seeds and Skin
## 3 Results and Discussion
### Phenolic Composition  
Structural and physicochemical features of a solvent have a strong influence on selectivity towards biologically active compounds. In general, considering solvent extractability, the highest content of polyphenols from both, seeds and skin, was obtained with ChCit. Thus, further HPLC analysis was done to precisely characterize the phenolic profile of such ChCit extracts. Moreover, acidified aqueous ethanol extracts were also screened in order to compare NADES versus conventional solvent in terms of extraction efficiency for some of the main grape polyphenols (gallic acid and protocatechic acid within the phenolic acids group, (+)-catechin and (-)-epicatechin as flavan-3-ols representatives, quercetin 3-O-glucoside like flavonol main member). The sum of identified polyphenols by HPLC varied noticeably among varieties, for both seed and skin extracts. Polyphenol compositions evaluated by HPLC-DAD analysis of grape seed extracts are presented in Table 3. Flavan-3-ols were predominantly present, which is in accordance with previously published data [22,45]. (+)-Catechin was the most abundant followed by (-)-epicatechin. Gallic acid and protocatechic acid were present in smaller quantities. When it comes to grape skin (Table 4), distribution of phenolic compounds was significantly different amid varieties, which is in accordance with literature data [46]. Nevertheless, grape skin peaks could be assigned to the four different compounds: (+)-catechin, (-)-epicatechin, protocatechic acid, and quercetin 3-O-glucoside. Regardless of the similar polarity and acidity of extraction solvents, differences between acidified aqueous ethanol and ChCit selectivity were observed. Conventional solvent, almost without the exception, had a higher affinity than NADES for (+)-catechin, (-)-epicatechin, and gallic acid in seeds. The highest concentrations of (+)-catechin were determined in the seeds of 'Zupljanka' (15.587 and 10.197 mg g\({}^{-1}\) DW, for acidified aqueous ethanol and ChCit, respectively), followed by 'Zacinak' and 'Prokupac'. 'Prokupac' was the variety with the highest content of (-)-epicatechin, regardless the solvent. The abundance of dominant flavan-3-ols was in agreement with [PERSON] et al. [34] and [PERSON] et al. [47]. Gallic acid was predominantly present hydroxybenzoic acids. Interestingly, there was no difference between seeds obtained from white and red varieties regarding gallic acid content, although such observation was previously reported [21,48]. Among all varieties, red grape 'Prokupac'  
Figure 4: Antioxidant composite index- grape skin extracts. Data are expressed as % 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 [PERSON]’s test. Abbreviations: ACI—Antioxidant Composite Index; SD—standard deviation; AcEtOH—acidified aqueous ethanol; ChCit—choline chloride: citric acid; ChGlc—choline chloride: glucose. showed significantly higher amounts of gallic acid (2.450 and 1.850 mg g\({}^{-1}\) DW, for acidified aqueous ethanol and ChCit seed extracts, respectively). Interestingly, ChCit skin extracts contained higher amounts of (+)-catechin and (-)-epicatechin in comparison with acidified aqueous ethanol extracts. (-)-Epicatechin was dominantly present in red grape skin and the highest concentrations of this compound were determined in 'Cabernet Sauvignon' (2.612 and 5.219 mg g\({}^{-1}\) DW, for acidified aqueous ethanol and ChCit extracts, respectively). Among white varieties, isomer (+)-catechin was the most abundant.