# Impact of Deep Eutectic Solvents on Extraction of Polyphenols from Grape Seeds and Skin
## 3 Results and Discussion  
### Total Polyphenol Content (TPC)  
Total polyphenol content of grape seeds and skin was investigated in order to emphasize previous disclosures about the importance of profitable and sustainable utilization of wine by-products, considering appreciable amounts of biologically active compounds remained caught within the grape pomace [34; 35]. The present study was conducted on seeds and skin from ten different grape varieties, among which, five of them represent Serbian autochthonous varieties ('Bagrina', 'Zupljanka', 'Zacinak', 'Black Tamjanika', and 'Prokupac'). With intention to compare conventional and natural deep eutectic solvents, investigation of total phenolic content was performed using three different solvents: acidified aqueous ethanol (AcEtOH), and green solvents ChCit, and ChGlc. Natural deep eutectic solvents are designer solvents, and there are more than 108 different possible combinations. Commonly, it is recommended to try several typical combinations, which considerably differ in physiochemical properties in order to select a suitable NADES. In our study, choline chloride-based NADES containing citric acid or glucose were chosen based on former reports and their physicochemical properties. For instance, ChCit is characterized with low pH value and with polarity similar to polarity of conventional solvents widely used for polyphenol extraction. Conversely, pH of ChGlc is almost neutral. High stability of phenolic compounds in sugar based NADES, as well as in ChCit, was previously published [10; 14]. Furthermore, different water content in NADES should be optimized in order to reduce viscosity that can cause problem with mass transfer and pumping if the NADES are applied in industrial level [36]. In many cases, the addition of water between 20 30% (w/w) reduced viscosity of NADES and beneficially influences extraction yield of both polar and non-polar compounds. According to our experience and previous optimized works with polyphenol extraction, 30% of water was added to the NADES ChCit and ChGlc [25]. Choosing the right NADES for polyphenol extraction is a major part of the extraction process optimization, since it directly influences pH, polarity, and viscosity of a solvent, and as such, directly influences the extraction efficiency. However, NADES choice is followed by selection of the extraction method, which also significantly contributes to the extraction efficiency. In our case, ultrasound assisted extraction (UAE) was chosen since extraction yield could be improved with cavitation phenomena resulting cell leakage and better mass transfer. Additionally, process parameters to achieve the maximum extraction efficiency within NADES should be adjusted. This means optimization of a temperature, extraction time, and power of ultrasound. According to our experience, and previously published and optimized ultrasound assisted extractions of polyphenols from the grape within NADES, extraction was carried out 30 min on 50 \({}^{\circ}\)C [25]. TPC values of grape seed extracts obtained from acidified aqueous ethanol ranged from 70.86 to 146.69 mg GAE per gram of dry weight (Figure 1). The highest total phenolic content was extracted from 'Gamay' and 'Prokupac' seeds, while lowest TPC appeared in seeds obtained from 'Merlot' and 'Cabernet Sauvignon' varieties. Our results were similar to those reported for the same varieties [18; 22] and slightly lower than seed data obtained for Mediterranean grape pomace by [PERSON] et al. [37]. despite of the similarity of the varieties included in our and in the study conducted by [PERSON] et al., minor observed differences are presumably present due to seasonal variations of weather, agronomical practices, and vineyard management [22]. Nevertheless, mentioned study also reported the highest total polyphenol content in seeds obtained from Serbian autochthonous variety 'Prokupac'.