# Impact of Deep Eutectic Solvents on Extraction of Polyphenols from Grape Seeds and Skin
## 3 Results and Discussion
### Total Polyphenol Content (TPC)
Regarding NADES, TPC values of grape seed extracts ranged from 92.36 to 182.60 and 56.17 to 156.17 mg GAE per gram of dry weight for ChCit and ChGlc extracts, respectively (Figure 1). ChCit had higher extraction efficiency than ChGlc in almost all varieties (with the exception of seeds of two red grape varieties: 'Merlot' and 'Prokupac'). Interestingly, seeds from three Serbian autochthonous varieties, 'Prokupac', 'Black Tamjanika' and 'Zupljanka', stood out with pronounced high TPC content in both NADES. By analyzing skin extracts (Figure 2) obtained using acidified aqueous ethanol, 'Merlot', 'Cabernet Sauvignon', and 'Zacinak' were outstanding with the highest TPC (16.21, 16.14, and 15.00 mg GAE g\({}^{-1}\) of DW, respectively). Concerning only white varieties, 'Zupljanka' (9.41 mg GAE g\({}^{-1}\) of DW) had the highest total phenolic content. The results of TPC in skin extracts revealed in the present study were in good agreement with literature [18; 23]. When it comes to NADES skin extracts (Figure 2), ChCit outperformed ChGlc, with some exceptions. ChCit and acidified aqueous ethanol showed similar potential considering solvent extractability; therefore, the same varieties ('Cabernet Sauvignon', 'Merlot', and 'Zacinak') had the highest total phenolic content in both extracts. ChGlc distinguished those, but also 'Frankovka', as grape varieties, which skin is abundant in phenolic compounds. The present study confirmed the observations of [PERSON] et al., who screened five different DES and proposed acid based NADES as the most promising for extraction of grape skin polyphenols [25]. In general, TPC values varied significantly among varieties (Figures 1 and 2). In addition, it is clearly observed that total phenolics of grape skin extracts were several times lower than those determined for grape seed extracts. These findings were in accordance with previous reported data for the _Vitis vinifera_ species [18]. In addition, TPC values determined for red skin were slightly higher in comparison with white skin extracts. Extracting solvent affected total polyphenol content of both seeds and skins (Figures 1 and 2). It is known that physicochemical properties play an important role in solvents selectivity towards some particular compounds. More polar solvent is expected to have a higher yield of polar molecules in comparison with non-polar ones. Thus, differences in extraction efficiency between ChCit and ChGlc could be present due to distinction in their polarity (acid based NADES are more polar). Solvent acidity is another important feature found to affect extraction selectivity. For example, anthocyanins are flavonoid phenolic compounds whose chemical form and stability depend on pH value. Namely,  
Figure 1: Total polyphenol content of seed 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. they are prevalent in the flavylium cation form, which is stable at low pH. With the increased pH value, they undergo structural transformation into forms prone to further degradation due to their instability [25; 38]. Therefore, extraction of these polyphenols is enhanced along with the growing acidity of a solvent. This could be a possible explanation for lower TPC values found in most ChGlc extracts, considering almost neutral pH of this solvent (pH values of AcEtOH and ChCit are both less than 7). ChCit showed equal or even higher extraction efficiency than referent solvent, acidified aqueous ethanol, observing both, seeds and skin. Extraordinary extraction capacity of ChCit extracts confirmed previous findings about high extraction ability of acid based NADES for some phenolic compounds [25; 38; 39; 40].