Extraction of Polyphenols from Olive Leaves Employing Deep Eutectic Solvents: The Application of Chemometrics to a Quantitative Study on Antioxidant Compounds  
[PERSON]  
1 Food Quality & Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania,  
International Centre for Advanced Mediterranean Agronomic Studies, P.O. Box 85, 73100 Chania, Greece;  
[EMAIL_ADDRESS] (H.A.); [EMAIL_ADDRESS] (S.G.);  
[EMAIL_ADDRESS] (A.K.); [EMAIL_ADDRESS] (S.L.)  
2 Analytical Biochemistry and Biotechnologies Research Laboratory (LABAB), Mouloud Mammeri University,  
Tzi Ouzouou 15000, Algeria; [EMAIL_ADDRESS]  
3 Green Processes & Biochemistry Group, School of Agricultural Sciences, University of Thessaly,  
N. Temponere Street, 43100 Karditsa, Greece; [EMAIL_ADDRESS]  
4 Department of Chemistry, National and Kapodistrian University of Athens, 15772 Athens, Greece;  
[EMAIL_ADDRESS]  
5 Hellenic Mediterranean University Research Center, Institute of Agri-Food and Life Sciences,  
7410 Heraklion, Greece  
5 Department of Agriculture, Hellenic Mediterranean University, 74140 Heraklion, Greece  
products such as phenolic compounds, which exhibit a broad spectrum of bioactivities, including antioxidant, anti-inflammatory, antimicrobial, antihypertensive, hypoglycemic, cardioprotective activities, etc. [8]. A beneficial effect on oxidative stress-based diseases has been observed with the intake of polyphenols [9]. Several investigations have been conducted to elucidate the mechanism of lipid peroxidation and to develop antioxidants to neutralize cell oxidation in order to prevent food deterioration and biological damages [10]. Currently, a safe alternative to synthetic antioxidants is being a major interest of research [11]. As a consequence, numerous natural sources (aromatic plants, fruits, vegetables, leaves, and spices) have been tested for their antioxidant activity, and OLL is one of these resources [12].  
The yield and composition of phenols originated from different resources were found to vary depending on some factors related to the extraction solvent and the operating conditions [13] and also on some other factors associated with the genetic background of the plant [9; 14; 15]. OLL phenols are mainly extracted using conventional solvents. However, recently, a move toward green methodologies under the name of green solvents, including ionic liquids (ILs) and deep eutectic solvents (DESs) or low-transition temperature mixtures (LTTMs), has emerged [16; 17; 18]. These new solvents aim at maximizing extraction efficiency and reaching new purposes such as researcher safety, environmental protection, and the use of a simplified and cheaper extraction procedure [19]. Due to the physicochemical properties of the green solvents (viscosity, density, hydrophilicity, and solubility), the drawbacks of the conventional solvents are overcome. Furthermore, because of their lower cost, DESs are particularly more attractive solvents, compared with the ILs. The DESs are known as a new class of solvents, alternative to ILs, that consists of a mixture of organic compounds with a melting point lower than that of either individual component [20].  
The present work was carried out to optimize OLL polyphenol extraction conditions using DESs based on glycerol, which represents a cheap and biodegradable source material combined with amino acids: lysine, proline, and arginine. A response surface methodology (Box-Behnken design) was implemented in order to assess the effects of the liquid-to-solid ratio (\(R_{L/S}\)), the concentration of DES (\(C_{\text{DES}}\)), and the extraction temperature (\(T\)) on the yield in total phenols (\(\Upsilon_{TP}\)). This work also aimed at comparing extraction efficiency among the DESs tested and conventional solvents, in addition to obtaining information regarding the selectivity of the DESs tested and the polyphenolic composition of the extracts.