INTRODUCTIONDeveloping new green solvents is one of the most important tasks for industry. Lately, deep eutectic solvents have been researched due to their desirable characteristics like low toxcity and biodegrability. Natural deep eutectic solvents (NADES) are defined as a mixture of two or more components, which may be solid or liquid and that at a particular composition present a high melting point depression becoming liquids at room temperature. The compounds that constitute the NADES are primary metabolites such as aminoacids, organic acids, sugars or choline derivates. These solvents are obtaind by the complexation between a hydrogen acceptor and a hydrogen-bond donor. The charge delocalization occurring is hereafter responsible for the decrease in melting point of the mixture relative to the melting points of the raw materials (figure..). Different ratios of the components of NADES, may affect stability of NADES in terms of the mixture remaining in the liquidphase for prolonged periods. The physical properties and solubilizing capacity of NADES can be modified by the addition of small quantities of water. Previous studies have reported that most of them were not liquid at room temperature and consequently there is a limitation for their applicationto extraction or reaction media at room temperature. A small amounts of water resulted in a liquid at room temperature and even at lower temperature. However, extension dilution of NADES withwater will result in the loss of existing hydrogen bonds, and consequently, the disappearance of the special structure of NADES. Furthermore, assumption is that NADES are third medium in living organisms with existent water and lipid media. Many findings strongly suggest that water and lipids are indeed not the only solvents present in living organisms. These NADES may play a role in all kinds of cellular processes, explaining mechanisms and phenomena that are otherwise difficult to understand, such as the biosynthesis of non-watersoluble small molecules and macromolecules.

In this study, citotoxity and antioxidative capacity were investigated in eight different NADES (FIGURE). Citotoxical effects of different NADES were investigated in different human cell lines, HeLa and Hek293. Antioxidative capacity was examined by using ORAC method.

Materijali/metode

The heating methode was used for preparing natural deep eutectuc solvents. The two or more components in specific ratios with calculated amounts of water were placed in glass flask with round bottom with stirring bar. The mixture was heated on temperature between 50 and 70 C until the the clear liquid was formed (30 90 min).Oxygen radical absorbance capacity(ORAC) is a well-established and reliable method to determine the antioxidant capacity of a biological samples in vitro. Theassaymeasures the oxidative degradation of the fluorescent molecule (fluorescein) after being mixed with free radical generators such asazo-initiator compounds. It is based on the inhibition of oxyradical-induced oxidation of 2,2-azobis-(2- methylpropionamidine) dihydrochloride (AAPH) by substances with antioxidant properties. Peroxyl radicals produced in a time-dependent manner during the thermal decomposition of AAPH will quench the fluorescence signal In the presence of a substance with antioxidant properties the fluorescence reduction is inhibited, depending on the substances ORAC capacity. The dynamics of the signal inhibition, expressed as the area under the curve (AUC), are used to quantify the antioxidant capacity, expressed as the ORAC value, by comparing the sample AUC to an antioxidant standard curve generated with Trolox. Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) is a vitamin E analogue and a known antioxidant. It is used as a standard by which all unknown antioxidants are compared.HeLa cells are human cervical cancer cells (the cervix is found at the top of the vagina and the entrance to the uterus). The cells were first cultured, or grown, in a laboratory in 1951 and were the first type of human cancer cell to be cultured continuously for experiments.HEK293 is a cell line derived from human embryonic kidney cells grown in tissue culture. This particular line was initiated by the transformation and culturing of normal HEK cells with sheared adenovirus 5 DNA.-citotoksinos metoda

ConclusionThis study demonstrates that mixtures of many primary metabolites can form all kinds of NADES when mixed in adequate ratios.

NADES have unique phisical and chemical properties like their negligible volatility at room temperature, high solubilization ability and low toxicity. Due to large number of various componets, a great amount of different NADES can be formed. NADES represent green alternative for classic solvents which can be used in synthetic chemistry, electrochemistry, biochemistry and separation process.