Antioxidant Properties and Electrochemical Behavior of some Acetyl Salicylic Acid Derivatives

Article history: Received on: 28/08/2016 Revised on: 11/09/2016 Accepted on: 26/10/2016 Available online: 29/11/2016 Antioxidants are frequently used in several dietary supplements and have been probed for their significant role in the prevention of diseases like cancer or coronary heart disease. Acetyl salicylic acid has grabbed substantial attention due to their ability in impeding oxidative stress. In the present study, nine new salicylic acid derivatives: 5-(2-hydroxyphenyl-5-substituted)-1,3,4-oxadiazole-2(3H)-thiones, N-(2,5-dimethyl-1H-pyrrol-1yl)-2-hydroxy-5-substituted benzamides, 2-hydroxy-5-substituted-N'-[1-(2-oxo-2substituted-chromen-3yl)ethylidene] benzohydrazides and Hg (II) complex of N'-[(1Z)-1-(6-bromo-2-oxo-2H-chromen-3-yl) ethylidene]-2-hydroxybenzohydrazide have been synthesized and characterized in the light of advanced spectral techniques. The antioxidant properties of these compounds were investigated using 2,2-diphenyl-1picrylhydrazyl (DPPH) spectrometric assay and compared with the results of cyclic voltammetry studies. The redox properties shown by the active molecule using cyclic voltammetry correlated well with the biochemical study.


INTRODUCTION
Reactive oxygen species (ROS) are extremely reactive molecules that contain oxygen like superoxides, hydroxyl radicals, peroxides etc. which are normally generated as a byproduct of the natural oxygen metabolism that occurs within cells (Muller, 2000;Schumacker, 2006).Oxidative stress produced due to increased levels of either endogenous or exogenous ROS on exposure to ionic radiations or alterations in metabolic pathways can lead to severe consequences like ageing, DNA damage, lipid peroxidation, oxidation of amino acids in proteins, deactivation of certain enzymes eventually resulting in conditions like inflammation, cardiovascular disorders, neurodegenerative diseases or cancer (Renschler 2004;Li et al., 2013).An antioxidant is any chemical substance that can either react with or quench ROS and thereby protects the biological systems from several lethal diseases (Trachootham et al., 2009;Tochhawng et al., 2013).In the last few decades, several antioxidant supplements have been extensively used in different fields like cosmetic formulations, pharmaceutical products etc. to inhibit/delay oxidative stress.Phenolic compounds like acetyl salicylic acid and its metabolite salicylic acid exhibit significant non-enzymatic antioxidant potential via donation of hydrogen atom from the hydroxyl moiety attached to the phenyl ring and hence prevent oxidative stress (Ghasemzadeh et al., 2012;Bal-Demirci et al., 2015).
Transition metal coordination compounds are particularly suitable as biologically active ligands as they can adopt a wide variety of oxidation states, coordination numbers and geometries (Kostova and Balkansky, 2013).Metal complexes, based on their structure and on the source of the oxidative stress, might act as antioxidants or pro-oxidants.The potential prominence of antioxidants has encouraged us to investigate the cooperative effects of metal complexes and salicylic acid derivatives for improving antioxidant activity.The current study provides the synthesis of few salicylic acid derivatives and their mercury based complexes followed by the investigation of their antioxidant potential.
Antioxidant compounds are highly capable in acting as reducing agents and their solutions get easily oxidized on the surface of inert electrodes.Centered on this fact, the relationship between electrochemical behaviors and the data obtained from the spectrometric assay of the potent antioxidant compound was also explored.
Thin layer chromatography was carried out using precoated aluminum sheets with Aluchrosep silica Gel 60/ UV 254 .A mixture of ethyl acetate and hexane (1:1) was used as the eluent and the spots were observed using a UV chamber.The melting points of the new derivatives were determined via the open capillary method and are uncorrected.
The IR and mass spectra of salicylic acid derivatives were taken using Schimadzu FTIR 8400S spectrophotometer and Agilent 6510 series mass spectrometer.The 1 H and 13 C NMR were recorded on a Bruker 400 MHz instrument.The elemental analyses were performed using a Flash thermo 1112 series CHN analyser.

DPPH radical scavenging assay
The DPPH radical scavenging technique is based on the ability of the stable free radical DPPH, to become decolorized when they are exposed to antioxidants.About 100 μL each of various concentrations of test compounds and DPPH were added to respective wells of a micro plate, in order to make up a final volume of 200 μL.An equal amount of methanol and DPPH were used as the control.Ascorbic acid was employed as the standard reference.The potential of the test agents to quench DPPH was colorimetrically assessed after 20 min incubation in the dark.The absorbance was recorded using an ELISA plate recorder at 517 nm.The experiment was performed in triplicates and the mean values were considered (Shekhar and Anju, 2014;Sharma and Bhat, 2009;Manjula et al., 2010). -×100

Electrochemical Measurements
Electrochemical measurements hold many advantages over DPPH, they are fast, cheap and the oxidation potentials can be determined with high accuracy.Cyclic voltammetry studies were carried out using a three-electrode system comprising of glassy carbon working electrode, saturated calomel reference electrode and a platinum counter electrode which is used as a reference (Sochor et al., 2013).The electrodes were cleaned and polished using 0.3 µm alumina powders and rinsed before the commencement of the experiment (Brcanovic et al., 2013).Subsequent to mechanical treatment, electrochemical pretreatment of glassy carbon was carried out by potentio-dynamic cycling through11.0-0.8V in the supporting electrolyte at a slow sweep rate 10 mV/s for approximately 15-30 min.The electrolyte was deaerated during the potential cycling process.
About 20 mL of the supporting electrode solution was taken in the electro-chemical cell.The molecule under study was dissolved in DMSO and added to it so that the total volume was maintained at 30 mL.The solution was mixed well using a magnetic stirrer for a minute and cyclic voltammetry measurements were performed from +2 V to -2 V at the glassy carbon electrode at a scan rate of 50 mV/s.

Chemistry
The IR spectra of oxadiazole thiones (2a-b) showed the characteristic C=S stretching band at 1260-1270 cm -1 and their 1 HNMR spectra displayed the NH protons at 10.73 ppm.The NH and C=O stretching vibrations were observed at 3150 and 1670-1680 cm -1 in the IR spectra and the NH proton resonated at δ 11.2-11.4 in the 1 HNMR spectra of pyrrole derivatives (3a-b).The schiff bases (5a-d) exhibited the characteristic IR stretching absorption at 1600-1610 cm -1 for the C=N group.The Schiff base ligand prepared under mild conditions reacted with HgCl 2 in DMSO at 298 K to afford the corresponding mercury (II) complex 6 in 74% yield.The IR analysis showed that in this mononuclear mercury (II) complex, the Hg 2+ ions are surrounded by one nitrogen and two oxygen atoms from the schiff base ligand and the two Cl atoms.In comparison with the spectra of the schiff base 5b, the Hg(II) complex displayed the band of (HC=N) at 1604 cm - ; showing a shift to lower wave numbers from 1610 cm -1 suggesting that, the azomethine nitrogen is coordinated to Hg(II) ion (Ngai et al., 2013;Abdel-Hamid and Newair, 2011).The OH stretching vibration at 3298 cm -1 in 5b shifted to a lower frequency to 3286 cm -1 in the complex.The band of lactone (C=O) at 1737 cm -1 in the metal complex showed an absorption shift to lower wave number from 1745 cm -1 confirmed that, the oxygen of the carbonyl group is coordinated to the metal ion.The unchanged position of NH band in the metal complex indicated that this group is not involved in coordination with Hg 2+ ion.The new bands that appeared in the region of 510 and 530 cm -1 in the spectra of the complexes are attributed to (M-N) and (M-O) bond stretchings respectively.Yet another band in the region 410 cm -1 is assigned to (M-Cl) bond vibration.Thus the IR spectrum provides strong evidence for the complex formation of 5b with Hg.The molecular weight of the synthesized molecules obtained from the mass spectra was in accordance with their respective molar masses.

Antioxidant studies
All the salicylic acid derivatives were screened for their antioxidant potential using DPPH assay.The ascorbic acid which was used as the standard exhibited an IC50 value of 3.09 µg/mL.All the molecules studied exhibited moderate to good antioxidant capabilities.Especially, Schiff base 5b exhibited a good IC50 value of 14.63 µg/mL, whereas its Hg(II) complex did not show any antioxidant capacity.The graph depicting the antioxidant nature of ascorbic acid, 5b and 6 is presented in Fig. 1.

Cyclic voltammetry
Cyclic voltammetry technique was used to assess the antioxidant activity of the most active molecule 5b Cyclic voltammetry is a frequently employed technique in electrochemistry to characterize the antioxidant potential of electro-active molecules as well as electrode surfaces, mainly attributed to its simplicity, speed, and amenability to be used directly on biological samples.In this method, the redox potential of probable antioxidants is scanned at a controlled rate, which is oxidized and the current produced during redox reactions that occur on the surface of the working electrode is recorded continuously (Hynek et al., 2012).Thus in cyclic voltammetry, the antioxidants are characterized by their current-potential relationships offered at an inert glassy carbon electrode.An antioxidant can be easily oxidized at an electrode.The more powerful the reducing agent, the lower is its positive oxidation potential (Sun-Waterhouse et al., 2008).Schiff base 5b was subjected to cyclic voltammetry in order to assess its antioxidant behavior.The cyclic voltammogram of 5b is presented in Fig. 2. The cyclic voltammogram of 5b shows an increase in current that starts at 0.9 V in the forward scan, after which the potential climbs exponentially (Fig. 2).However, the swift upsurge in current does not persist for long.A peak is observed (Ep,a) at 0.65 V followed by decrease in current.