INTRODUCTION

Developing 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 liquid
phase 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 application
to 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 with
water 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. The assay measures the oxidative
degradation of the fluorescent molecule (fluorescein) after being mixed with free radical generators
such as azo-initiator compounds. It is based on the inhibition of oxyradical-induced oxidation of 2,2azobis-(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

Conclusion
This 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.