The cyclic voltammetric behavior of 2 mM AMC in 0.05 M Tris-HCl buffer (pH 7.4) at GCE in the absence and presence of varying concentration of CB-DNA has been shown in Figure 1. The voltammogram (—) shows that AMC exhibits two prominent reduction peaks and one weak oxidation peak at the bare GCE. Peak I_c at –1.06 V corresponds to the one electron reduction of carbon 3 followed by dimer formation as shown in Scheme 1b. Peak II_c at –1.40 V can be attributed to the two electron/two proton reduction of the dimer as depicted in Scheme 1c.The weak anodic peak I_a at –0.94 V may be due to the electrooxidation of the product generated from II_c as illustrated in scheme 1(d).

Voltammograms (- - -) and (- - -) in Figure 1 further indicate considerable diminution of the peak currents and shift of cathodic peak potentials to less negative values by the addition of increasing concentration of DNA. Such observations can be attributed to the decrease in free drug concentration due to its intercalation into DNA [23]. The shift in peak potential indicates the interaction of AMC with DNA.

Further examination of the cyclic voltammograms reveals the disappearance of anodic peak, which may be due to the formation of electrochemically unoxidizable drug-DNA adduct. To show that the decrease in peak current is due to the formation of slowly diffusing AMC -DNA complex, the diffusion coefficient of the drug with and without DNA was determined by using Randles- Sevcik equation [24]: (1) I p = 2.69 × 10 5   n 3 / 2 A C o * D o 1 / 2 v 1 / 2where I_p (A) is the peak current, A (cm²) is the surface area of the electrode, C_o*(mol cm⁻³) is the bulk concentration of the electroactive species, D_o (cm²s⁻¹) is the diffusion coefficient and ν (Vs⁻¹) is the scan rate.

The linearity of the plot (Figure 2) between I_p and υ^1/2 indicates that the reduction of AMC is controlled by the diffusion step. The diffusion co-efficient of 2 mM AMC in the absence and presence of 5 μM DNA was obtained as 1.11 × 10⁻⁹ m²s⁻¹ and 2.97 × 10⁻¹⁰ m²s⁻¹ from the slope of I_p vs. υ^1/2 plot. These results indicate that the diffusion coefficient of DNA – bound drug (AMC), which is an order of magnitude lower than that of the free drug is responsible for the observed decay in peak current. Similar results have also been obtained by other investigators [25–27].

The binding constant (K) of AMC- DNA interaction was obtained by using the following equation [28–29]: (2) I p 2 = 1 K [ D N A ] ( I p o 2 − I p 2 ) + I p o 2 − [ DNA ]where I_p and I_po are the peak currents of AMC with and without DNA respectively. A value of 6.15 ×10⁵ M⁻¹ was obtained from the slope of I p 2 vs. I p o 2 − I p 2 / [DNA] plot as shown in Figure 3. The strong affinity of this drug to DNA as is evident from its high binding constant which could serve as an important addition to the traditional cancer treatment.

The experimental heterogeneous electron transfer rate constant of AMC (k^o) with a value of 3.95 × 10⁻⁶ ms⁻¹ was obtained at 100 mV/s scan rate from the slope of I_p vs. C_o plot (Figure 4) using Reinmuth’s expression [30]: (3) I p = n F A C o k owhere I_p and C_o denote the peak current and concentration of the electroactive species respectively.

The electron affinity (A) of AMC with a value of 1.71 eV was calculated from its E_1/2 value by the relationship reported as [31]: (4) A = χ − E 1 / 2 − Swhere S is the solvation energy and χ is the work function of working electrode, whose value is 4.39 eV for glassy carbon electrode [32]. The electron affinity of AMC is comparable with 1- (4′-N,N-dimethylphenyl)-2-propen-1-one (1.69 eV) [33] but slightly greater than simple aromatic hydrocarbons [31– 34– 35] due to its conjugated carbonyl group.

The solvation energy was evaluated from Born′s equation [33]: (5) S = −( z 2 e 2 / 2 r )   ( 1 − 1 / ε )   ( 1 / 4 π ε o )   1 / 1 .6 x 10 − 19

The radius of AMC was calculated from the density equation [33]: (6) 4 3 π r 3 = M N d

The UV–Vis absorption spectra of AMC–DNA complexes in 10% aqueous methanol maintained at pH 7.4 (0.05 M Tris-HCl buffer) were also recorded using 5 μM AMC with varying concentration (ranging from 0.5–20 μM) of DNA for getting further clues about the mode and strength of interaction. Figure 5 shows the absorption spectra of AMC in the absence and presence of DNA. The absorption spectra exhibited peculiar bathochromic and hypochromic shifts by the addition of increasing concentration of DNA. Such findings indicate the interaction of electronic states of AMC intercalating chromophore (α,β-unsaturated ketonic part) with the DNA bases, resulting in a stable AMC-DNA complex by π–π stacking and dipole–dipole interactions [36– 37]. The bathochromic shift of the drug from 336 nm to 359 nm and hypochromic effect of 0.34 after binding to a fixed amount of DNA are typical characteristics of an intercalative mode of binding [38– 39], in which the drug inserts itself between the adjacent base pairs causing partial unwinding of DNA resulting in the failure of its translation and transcription machinery. The insertion of the drug is expected to occur through minor and major grooves as its radius (0.46 nm) is less than the width (0.6 and 1.2 nm) of both these grooves [40]. The decrease in the drug absorption intensities were exploited for the evaluation of the binding constant using the following equation [41– 42]: (7) A o A − A o = ε u ε b − ε u + ε u ε b − ε u [ 1 K [ D N A ] ]where A and A_o represent the absorbance of bound and unbound drug with ɛ_b and ɛ_u as their respective absorption coefficients.

The binding constant with a value of 2.86 × 10⁵ M⁻¹ was obtained from intercept to slope ratio of A₀/(A–A₀) vs. 1/[DNA] plot. The value of K obtained here is in close agreement with that obtained from CV measurements and is greater then the K = 2.60× .10⁵ M⁻¹ obtained for the interaction of kaempferol with DNA due to a couple factors like the presence of electrophilic carcbon and extensive aromatic system [43].

Electrochemical techniques generally provide necessary but insufficient evidence to support an intercalative mode of binding. Viscometric measurements can offer least ambiguous clues about the binding model in solution [13]. The change in the viscosity of 50 μM DNA upon the addition of the drug ranging from 20 μM to 100 μM is shown in Figure 6. Representing intrinsic viscosity of DNA with and without drug by η and η_o, data were plotted as (η/η_o)^1/3 against the concentration of the drug. The results reveal a significant increase in the relative viscosity of DNA, indicating an intercalative mode of binding [44]. The intercalating agents for their accommodation widen the gap between the adjacent base pairs and thus cause lengthening of the double helical structure resulting in a significant increase of relative viscosity of DNA [45].

AMC was kindly provided by F. L. Ansari from her combinatorial library of 120 chalcones [20– 46]. Methanol UN-no 1230 with more than 99.5% purity was used without further purification. Tetrabutylammonium perchlorate (TBAP, Fluka, 99% purity) was further purified by recrystallization using methanol as a solvent. Doubly distilled water was used to prepare all the solutions and buffered at pH 7.4 by using 0.05 M Tris-HCl buffer. DNA was extracted from chicken blood by Falcon method [21]. Stock solution of chicken blood DNA (CB-DNA) was prepared by dissolving an appropriate amount of DNA in doubly distilled water and stored at 4°C. The concentration of the stock solution of CB-DNA (200 μM in nucleotide phosphate, NP) was determined by UV absorbance at 260 nm using the molar extinction coefficient (∈) of 6600 M⁻¹ cm⁻¹ [22].

The nucleotide to protein (N/P) ratio of 1.85 obtained from the ratio of optical density at 260 and 280 nm (A₂₆₀/A₂₈₀ = 1.85) was taken as evidence for protein free DNA [19]. Stock solution of AMC (6 mM) was prepared by dissolving AMC in 10% aqueous methanol.

Voltammetric experiments were performed using a PGSTAT 302 equipped with Autolab GPES version 4.9 (Eco Chemie, Utrecht, the Netherlands). Measurements were carried out in a conventional three electrode cell which consisted of saturated calomel electrode (SCE, Fisher Scientific Company cat no.13-639-51) as a reference electrode, a thin Pt wire of thickness 0.5 mm with an exposed end of 10 mm as the counter electrode and a bare glassy carbon electrode (GCE) with a geometric area of 0.071 cm² as the working electrode. Prior to experiments, the GCE was polished with 0.25-μm diamond paste on a nylon buffing pad. For electrochemical measurements the test solution was kept in an electrochemical cell (model K64 PARC) connected to a Lauda model K-4R circulating thermostat. Absorption spectra were measured on a Shimadzu 1601 UV-Vis Spectrometer. For the extraction of DNA, a Table Top Centrifuge, Model PLC-05 (Taiwan) was used. Density and viscosity measurements were made on an Anton Paar Stabinger Viscometer SVM 3000.

For CV experiments both the concentration and volume of AMC were kept constant while varying the concentration of DNA in solution. The voltammograms were recorded as aliquots of known volume of DNA were added. The solutions were deoxygenated via purging argon gas for 10 min before every experiment and used to keep argon atmosphere throughout the measurements. All experiments were carried out at 25 °C and blood pH (7.4). Prior to every electrochemical assay the GCE was polished for carrying out the electrochemical process on a clean electrode surface.

Spectroscopic titrations were carried out for monitoring the system by keeping both the concentration and volume of the drug constant, while varying the concentration of DNA. The solutions were allowed to equilibrate for 5 min before measurements. The spectrum was recorded after each addition of DNA solution. The data were presented as the average of three experiments in all cases.