Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor
Abstract
In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the 
OH radicals via Fenton-type reaction. The interaction of the radical species with immobilised DNA in the absence and presence of antioxidants was evaluated by means of changes in the guanine oxidation peak obtained by square wave voltammetry. The results demonstrated that the DNA-based biosensor is suitable as a rapid screening test for the evaluation of antioxidant properties of samples.
Introduction
Oxygen and its reactive species are very important in oxidative metabolism. They are produced in living by normal metabolism and by exogenous sources such as carcinogenic compounds and ionizing radiations (Vertuani et al., 2004). The study of these reactive species and its effects on the organism have been of primary interest, since they induce some damage to cells by reacting with biomolecules such as proteins, lipids and cause serious lesions to DNA.
Reactive oxygen species (ROS)-induced oxidative DNA damage producing a variety of modifications at DNA level including base and sugar lesions, strand breaks, DNA-protein cross-link and base-free sites. However, DNA of all mammalian cells contains trace amounts of modified bases that are indicative of attack by oxidising species and its are removed by excision repairing enzymes, they are known to accumulate with age being associated with disease processes (Dizdaroglu et al., 2002).
One of the most reactive radical species that induce lesions in DNA is the hydroxyl radical (OH). This species cause cell injury when they are generated in excess or the cellular antioxidant defense is impaired. When OH is generated adjacent to DNA, it attacks both the deoxyribose sugar and the purine and pyrimidine bases resulting intermediates radicals, which are the immediate precursors for DNA base damage (Jaruga and Dizdaroglu, 1996). In living systems many of the hydroxyl radicals are generated from the metal (M) ion-dependent breakdown of hydrogen peroxide (Dunford, 2002, Burkitt, 2003). In the presence of ferrous or cupric ions, hydrogen peroxide is converted into the hydroxyl radical by Fenton's reaction. The Fenton-type system is important because it has been implicated as an important mediator of oxidative damage in vivo and it is of great interest in terms of reducing the possibility of mutation and consequently the cancer (Burkitt, 2003).
In contrast to biological potential of ROS formation, the cells have developed a complex defense system, which act through the enzymatic activities and protection by the low-molecular weight antioxidants. Another form of protection is the use of synthetic and natural origin compounds that show antioxidant effect on the cell. Antioxidants act as reducing agents (free radical terminators), metal chelating and singlet oxygen quenchers (Vertuani et al., 2004). Much interest has been developed in recent years in order to find and to characterize natural origin substances that exert some antioxidant potential, because worldwide growing trend toward the usage of natural antioxidants in food industry and also their health benefits. Good examples are the natural antioxidants that have already been extracted from plant sources and are produced commercially (Schuler, 1990). Natural antioxidants are presumed to be safe, since they occur in plants and they are more desirable than their synthetic counterparts. Moreover, plants with known antioxidant property usually present some antimutagenic, anticarcinogenic and anti-inflammatory action (Arnao et al., 1999).
The evaluation of antioxidant properties is not an easy task. Many methods can be used to determine this activity and substrates, conditions, analytical methods and concentration can affect the estimated activity (Litescu et al., 2001, Nyska and Kohen, 2002). In this direction, in vitro systems are easier, faster and more cost-effective compared to traditional bioassays in vivo. Therefore, test of the direct antioxidant activity in vitro is useful, because if a substance that is poorly effective in vitro will not be better in vivo (Aruoma, 2003). It can also be evaluated about some possibility of damaging effects.
Antioxidant tests can be basically classified in two groups: those assays based on the inhibition of the human low-density lipoprotein oxidation and those assays used to measure oxygen free radical scavenging ability. Current methods in vitro for determining the antioxidant efficacy of the compounds have as basic principle the oxidation inhibition of a suitable substrate. After the substrate is oxidized under standard conditions, the extent of the oxidation (an end point) is measured at a fixed time point or over the range that is characteristic of the generated free radical by UV–vis spectrometry, chemiluminescence, fluorimetry and chromatographic methods (Sánchez-Moreno, 2002).
The methodology for evaluating natural antioxidants must be interpreted according to the system and the analytical method used to determine the extent and end point of the oxidation (Frankel and Meyer, 2000). Therefore, the main features of any test are a suitable substrate, an oxidation initiator and an appropriate measurement of the end point. Several analytical strategies become possible combining these three characteristics (Arnao et al., 1999, Aruoma, 2003).
In this context, the aim of the present study is reporting the results of an electrochemical screening system based on biosensor to investigate the antioxidant properties of several plant extracts. The composition of the system consisted in a DNA layer immobilized on a screen-printed electrode as oxidation target and a Fenton-type reaction was used as method of inducing damage by generating OH radicals. Hydroxyl radicals can interact with DNA bases as well as with deoxyribose residues. The antioxidant properties of the extracts were evaluated by changes that occur into DNA layer by means of guanine oxidation peak obtained by square wave voltammetry.
Section snippets
Cleavage of DNA by Fenton system
One of the most effective systems in generating a highly reactive oxygen species, the OH radical, is the Fenton reaction. This term refers to the reaction between hydrogen peroxide and transition metals ions to produce the hydroxyl radical capable of oxidising a wide variety of organic molecules.Mn + H2O2 → Mn+1 + OH− + OH
The reaction involves one-electron transfer from metal ion (Mn) to the weak O
O bond of H2O2 to produce the hydroxyl radical and hydroxide anion. Transition metals ions that usually
Materials
Ethylenediaminetetraacetic acid (EDTA), hydrogen peroxide, sodium acetate and sodium chloride were purchased from Merck (Darmstadt, Germany). Double stranded Calf thymus DNA D4522 (dsDNA) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were supplied from Sigma (St. Louis, USA). Ferrous sulfate was acquired from Acros (New Jersey, USA). Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) was obtained from Aldrich (Milwaukee, USA). All aqueous solutions were prepared using ultrapure water (ρ
Results and discussion
The studied system involved basically three important steps: DNA-based biosensor preparation, the interaction of the biosensor with the Fenton solution in absence and presence of antioxidant samples and the evaluation of the event that occurs at electrode surface.
The DNA-based biosensor preparation procedure (step of pretreatment of the electrode surface and DNA immobilization) as well as the general assay conditions (buffer composition, ionic strength, pH, electrochemical method and parameters
Conclusions
Methods that have been developed for the measurement of the antioxidant activity of the diversity of existing samples are all essentially inhibition methods; a free radical species is generated. There is an end point by which the presence of the radical is detected and the antioxidant activity of the added sample inhibits the end point by scavenging the free radical. An important consideration that should be made is the reliability and practicability of the method; much of the literature of
Acknowledgements
The authors thank FAPESP for the financial support and LDM is indebted to this foundation for her fellowship.
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