Journal of Electroanalytical Chemistry 621 (2008) 1–6

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

Study on the electrochemical behavior of anticancer herbal drug rutin and itsinteraction with DNA

Xue Tian a, Fengju Li b, Lu Zhu a, Baoxian Ye a,*

a Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR Chinab Zhangzhou Teacher College, Zhengzhou, 450044, China

a r t i c l e i n f o

Article history:Received 14 December 2007Accepted 11 February 2008Available online 7 March 2008

Keywords:DNARutinAnticancerInteractionDPV

0022-0728/$ - see front matter � 2008 Published bydoi:10.1016/j.jelechem.2008.02.022

* Corresponding author. Tel.: +86 371 67763 220; fE-mail address: yebx@zzu.edu.cn (B. Ye).

a b s t r a c t

In this paper, the electrochemical behavior of rutin and its interaction with DNA were investigated usingvoltammetry and spectroscopy. In 0.05 mol L�1 B–R buffer solution (50% ethanol, pH 5.02), rutin exhib-ited excellent electrochemical activity. In the presence of DNA, the peak current of rutin decreased in aquantitative fashion and the peak potential shifted to a more positive potential value in both cases ofDNA in solution and modified on electrode surface by Langmuir–Blodgett technique, indicating the dom-inance of intercalative interaction. The binding of rutin with DNA, analyzed in terms of the cooperativeHill model, yields the association constant Ka = 1.58 � 105 and a Hill coefficient m = 2.09. The results serveas a reference for the study of rutin with DNA base pairs in the natural environment of living cells.

� 2008 Published by Elsevier B.V.

1. Introduction

The study of the interaction of DNA with small molecules suchas drugs, organic dyes and metals has been an intensive topic fordecades because it provides insight into the screening design ofnew and more efficient drugs targeting to DNA, which can speedup the drug discovery and development processes [1]. The recogni-tion of DNA binders involves a complex interplay of differenceinteractive forces. It includes hydrophobic interaction along theminor groove of DNA, strong electrostatic interaction arising fromthe exterior sugar-phosphate backbone and intercalative interac-tion between the stacked bases pairs of native DNA from the majorgroove [2,3].

A variety of analytical techniques have been developed for thecharacterization and identification of the interaction betweenDNA and small molecules with relative advantages and disadvan-tages [4–8]. However, most of these methods suffer from high cost,low sensitivity and procedural complication. Up to now, electro-chemical methodologies have attracted appreciable attention dueto the inherent specificity and high sensitivity. Direct monitoring,simplicity and low cost facilitate to investigate the drug-targetingcompound interactions and obtain the quantitative analysis infor-mation in pharmaceutical formulations and biological fluids [9,10].

Elsevier B.V.

ax: +86 371 67763 654.

On the other hand, the electrochemical system serves as a versatileand illuminating model of biological system in an approach to thereal action occurring in the living cells in vivo [11]. The interactionmechanism can be elucidated in three different ways, involving theuse of drug- or DNA-modified electrodes and interaction in solu-tion [12].

In recent years, appreciable attentions are focused on electro-chemical DNA biosensor in the fields of clinical diagnostics as wellas forensic and biomedical application. Langmuir–Blodgett (LB)technique has attractive features for allowing the fabrication ofmolecular assemblies with controlled thickness at moleculardimensions and a well-defined molecular orientation. ThereforeLB technique continues to make great contributions to the furtherdevelopment of DNA-modified electrode for analytical sensing.

In the development of novel and highly effective medicines forthe treatment of various diseases, there has been a marked in-crease in the demand for herbal medicines in the more affluentcountries. Herbal medicines are widely used owing to their reputedbeneficial effects, such as unmatched chemical diversity and min-imum side effects. Flavonoids are a large group of naturally occur-ring polyphenolic compounds, ubiquitously distribute in plantsand are general present in the human diet. Owing to the antioxi-dant ability, they play an important role in inhibiting DNA and cellsfrom oxidative damage, mutagenesis and carcinogenesis [13].Thereby they possess a broad range of pharmacological activities,such as anticancer, antibacterial, enzyme inhibitory, and pro-orantimutagenic properties. As flavonoids are electro-active, the

2 X. Tian et al. / Journal of Electroanalytical Chemistry 621 (2008) 1–6

electrochemical characters of most active components in flavo-noids were investigated and reported in literatures. Brett and herco-workers [14–16] investigated the electro-oxidation of querce-tin, catechin and rutin using various voltammetries and revealedthat flavonoids can be oxidized via a complex and pH dependentelectron transfer process. Volikakis and Efstathiou [17] determi-nated 12 flavonoids using adsorptive stripping voltammetry in aflow injection system using nujol-graphite and diphenylether-graphite paste electrodes.

Rutin, shown in Fig. 1, is a kind of the most abundant naturalflavonoids, also the mainly active component of many natural Chi-nese traditional medicines. Ghica and Brett [16] previously re-ported the electrochemistry of rutin, proposing that the oxidationof rutin is pH dependent adsorption process at glassy carbon elec-trode (GCE). Based on our knowledge, there have few electrochem-ical reports about the interaction of rutin with DNA. In thiscontribution, detailed investigations on the electrochemical behav-ior of the interaction between rutin and DNA were carried outusing cyclic voltammetry (CV) and differential pulse voltammetry(DPV) under the optimum conditions. Moreover, UV–vis spectrawere studied to characterize the interaction mode and the interac-tion mechanism. It is suggestive for further fruitful research to de-sign novel antitumor drugs and diagnosis disease.

2. Experimental

2.1. Apparatus and reagents

Model 650A electrochemical analyzer (CHI Instrument Com-pany, USA) served to perform all the electrochemical measurementsin a three-electrode configuration. The working electrode was a bareor DNA-modified GCE (3 mm in diameter); a Pt wire and a commer-cial saturated calomel electrode (SCE) were utilized as counter andreference electrode, respectively. All potentials given in this paperwere referred to SCE. The UV–vis spectra were recorded by ModelUV-2102 (UNICO, Shanghai, China) spectrophotometer.

Fish Sperm DNA (Shanghai Sangon Company, China) stock solu-tion (1.0 mg mL�1) was prepared with doubly distilled water. Astock solution of 1.0 � 10�3 mol L�1 rutin (Shanghai R&D centerfor standardization of Chinese Medicines, China) was prepared bydissolving it with ethanol. Octadecylamine (Tianjin Guangfu FineChemical Research Institute, China) stock solution was preparedby dissolving it with distilled chloroform and used without furtherpurified. All spreading solutions were kept under 5 �C and renewedevery 2 weeks. Other chemicals were of analytical reagent gradeand used as received. Each assay was performed at ambienttemperature.

O

O O

O

O

OH

OH

OH OH

OHH3C

HOOH

OH

OH O

HO

A

B

C

Fig. 1. The molecular structure of rutin.

2.2. Procedure

Britton–Robinson (B–R) buffer solution (0.05 mol L�1, pH 5.02)was selected as supporting electrolyte solution, containing 50%ethanol (v/v) due to the low solubility of rutin in aqueous solu-tions. Single-stranded DNA was produced by heating the double-stranded DNA solution in water bath at 100 �C for 30 min, thenpromptly cooling it in ice bath, yielding so-called denaturedssDNA. In the following investigation, all DNA were dsDNA exceptotherwise statement. Pretreatment of GC electrode was carried outaccording to normal method.

2.3. Preparation of DNA-modified electrode

Since DNA as a polyelectrolyte salt is soluble in water, it is notpossible to directly prepare LB film from DNA. The amphiphilicoctadecylamine (ODA) is very suitable for preparing LB film. Here,DNA LB film was produced from the suspension of DNA in ODA bydissolving DNA in subphase (water) and spreading the ODA chloro-form solution on the subphase surface. When the ODA–DNA sus-pension was compressed slowly, a mono-molecule layer wasfabricated, and thus DNA LB film modified electrode resulted. Mea-surements of surface pressure (p)–area (A) isotherms and prepara-tion of LB monolayer were performed with a JML-04 LB trough(Shanghai Zhongchen Co., China). The solvent was allowed to evap-orate for 30 min before compression. Z-type DNA LB film used inthis paper was gained according to a standard vertical dipping pro-cedure by the LB technique.

3. Results and discussion

3.1. The electrochemical behaviors of rutin

Cyclic voltammetry can provide general information about theelectroactivity and possible surface activity of various compounds.The CV of 1.0 � 10�4 mol L�1 rutin in 0.05 mol L�1 B–R buffer solu-tion (50% ethanol, pH 5.02) were recorded at GCE in the potentialrange of 0.0–1.2 V. The voltammograms in succession cycles areshown in Fig. 2. Rutin exhibits a pair of well-defined reversiblepeaks (P1 and P2) with E00 = 0.306 V and an irreversible oxidationpeak (P3) at Ep = 0.987 V. The peak couple has been assigned tothe redox of dihydroxyl groups at B-ring of rutin, and the anodicpeak P3 attributed to the oxidation of the OHs at A-ring [18]. How-

Fig. 2. Cyclic voltammograms of the background (a) and 1.0 � 10�4 mol L�1 rutin(b) in 0.05 mol L�1 B–R buffer solution (50% ethanol, pH 5.02) for multi-cycles withscan rate of 0.1 V s�1.

Fig. 4. DPV curves of rutin with different concentrations of DNA in solution. Curve(a): 8.0 � 10�6 mol L�1 rutin; (b): (a) + 10 lg mL�1 DNA; (c): (a) + 20 lg mL�1 DNA;(d): (a) + 30 lg mL�1 DNA. Other conditions same as in Fig. 2.

X. Tian et al. / Journal of Electroanalytical Chemistry 621 (2008) 1–6 3

ever, the peak currents remarkably decrease in succession cycles(The number in Fig. 2 represents the cycle sequence, such as Iand II), suggesting that the peak currents are adsorption driven[16]. After the third cycle sweep, the peak currents vary slightlyand finally remain constant. Therefore, the peak current of the firstcycle was recorded and used in subsequent experiments.

The electrochemical behavior of rutin was found to be pHdependent in a wide pH range (2.0–9.6). The potentials shiftednegatively and the peak currents changed significantly withincreasing pH of the supporting electrolyte solution. The linearplots of peak potential Ep versus pH were obtained for P1 and P2

with corresponding slopes 57.5 mV/pH and 62.1 mV/pH, respec-tively. From the slope values, it could be concluded that the uptakeof electron was accompanied by an equal number of proton in bothelectrode reactions. With respect to the better sensitivity and peakprofile, pH 5.02 B–R buffer solution was selected as the supportingelectrolyte.

The influence of scan rates on the CV peaks was also examined,as shown in Fig. 3. With increasing the scan rates, the anodic peak(P1 and P3) potentials shift in gradually positive direction and thecathodic peak (P2) potentials shift in negative direction; mean-while the potential separation of P1 and P2 increases, which meansthat the electrode reaction is a quasi-reversible reaction in dynam-ics. The peak currents both P1 and P2 are directly proportional tothe scan rates in the range of 30–350 mV s�1, and the regressionequations are expressed as: ip1 (lA) = 0.875 + 0.0223t (mV s�1),R = 0.997; ip2 (lA) = 0.384 + 0.0128t (mV s�1), R = 0.995. The resultsindicate that the electrode reactions are adsorption controlled pro-cesses. According to Laviron’s theory [19]: ip = n2F2tAC/4RT = nFQt/4RT, the electron transfer number 2 was calculated at average. Sothe electrode reaction processes are both two electron transferwith two protons involved. This is in accordance with that reportedin literature [20].

3.2. Study on the interaction of rutin with DNA in solution

Considering the sensitivity, we choose the oxidation peak P1 asfocus to investigate the interaction between rutin and DNA. DPVtechnique provides higher sensitivity and better peak resolutionthan CV for studying the electrochemical behavior of biologicalsystems [21], so we use DPV in the following experiments. Priorto the trial, the mixture of rutin with DNA should be stirred for10 min to reach binding equilibrium. Fig. 4 shows the DPV curvesof 8.0 � 10�6 mol L�1 rutin in the absence (curve a) and presence

Fig. 3. Influence of scan rates on the peak potentials and peak currents of 5.0 �10�5 mol L�1 rutin. Scan rate from inner to outer: 30, 50, 100, 150, 200, 250, 300,350 mV s�1. Other conditions same as in Fig. 2.

(curve b, c and d) of DNA. In the presence of DNA, the peak currentexhibits a distinct weakness in comparison with that in the ab-sence of DNA. The decrease in the peak current is attributed tothe formation of rutin–DNA complex, which results in the decreaseof equilibrium concentration of rutin in solution. However, it isworth mentioning that the decrease of peak current of8.0 � 10�6 mol L�1 rutin is proportional to the DNA concentrationsin the range of 1.6–18 lg mL�1 with a regression equation DIp

(lA) = 0.5068 + 0.1614C (lg mL�1), and a relative coefficientR = 0.997, suggesting a means for determination of DNAconcentration.

On the other hand, the addition of DNA makes the peak poten-tial shifts in a positive direction, confirming the dominance ofintercalative interaction between rutin and DNA [23]. In addition,the plot of ip versus t is linear with respect to the addition ofDNA, indicating the electron transfer process involves adsorptivesubstances with DNA.

3.3. Comparison of the interaction of rutin with dsDNA and ssDNA

In order to further demonstrate the interaction of rutin withDNA, 10 lg mL�1 dsDNA (Fig. 5 curve c) and 10 lg mL�1 ssDNA(Fig. 5 curve b) was added into 1.0 � 10�5 mol L�1 rutin solution

Fig. 5. DPV curves of rutin with dsDNA and ssDNA in solution. (a): 1.0 � 10�5 m-ol L�1 rutin; (b): (a) + 10 lg mL�1 ssDNA; (c): (a) + 10 lg mL�1 dsDNA. Other cond-itions same as in Fig. 2.

4 X. Tian et al. / Journal of Electroanalytical Chemistry 621 (2008) 1–6

(Fig. 5 curve a), respectively. Although the peak current of rutin de-creased in both cases, it decreased more sharply with dsDNA, indi-cating that the interaction of rutin with dsDNA was stronger thanthat with ssDNA. The results coincide with the above conclusion ofrutin interposing into dsDNA with double helix structure. In con-trast, the random coil-like ssDNA cannot give a suitable environ-ment for rutin intercalation. The differently positive shift in thepeak potential may reflect a difference in the adsorption energy be-tween dsDNA and ssDNA interacting with rutin via intercalativeinteraction [21]. The stronger interaction of rutin with dsDNA im-plied the potential of rutin to discriminate dsDNA and ssDNA, andin DNA biosensor as hybridization indicator.

The surface coverage (C) of electro-active rutin was also esti-mated from integration of the oxidation peak in the DPVs accord-ing to C = Q/nFA, where Q represents the charge involved in theelectrode reaction. Other symbols have their usual meanings. Thesurface coverage of rutin with dsDNA and ssDNA were 2.27 �10�7 mol m�2 and 2.85 � 10�7 mol m�2, respectively. These valuesfurther indicate that dsDNA interacts with DNA more strongly insolution.

3.4. Investigation of the interaction of rutin with DNA-modified at GCE

3.4.1. p–A isotherms of DNA LB filmsFig. 6 shows the surface pressure (p) versus area (A) isotherm of

DNA LB films. The remarkable difference for ODA can be observeddue to the different subphase. For DNA/ODA composite Langmuirmonolayer (Fig. 6a), a rapid expansion of the molecule area wasfound along with the decrease of surface pressure. The limitingarea value of 0.38 nm2 and collapsing pressure 27.1 mN m�1 wereobserved, respectively. In contrast, the ODA monolayer (Fig. 6b)had a steep rise in surface pressure and the limiting area of0.22 nm2 per molecule, indicating the formation of DNA/ODA com-posite film. The entrapment of DNA molecules from subphase intoODA Langmuir monolayer is driven by electrostatic interaction be-tween the negatively charged DNA and cationic ODA molecules[22]. According to previous literature [23], DNA/ODA conjugatemonolayer at the air–water interface is stable and behaves likeideal amphiphilic complexes. Therefore, the LB technique offers areasonable and effective way to immobilize DNA onto the elec-trode surface and DNA retain their native form as characteristicdouble–helical structure.

To provide further information about the structure and the elec-trochemical properties of DNA Langmuir film, redox couplesFeðCNÞ3�=4�

6 was used as electrochemical probes. The cyclic voltam-

Fig. 6. p–A isotherms of ODA monolayers on DNA aqueous solution (curve a) andpure water (curve b).

mograms of 5.0 � 10�4 mol L�1 K3Fe(CN)6 were recorded at bareand DNA-modified GCE, respectively. The peak currents decreasedand the peak potentials shifted slightly at DNA-modified GCE incomparison with that at bare GCE. The result indicates that thenegative probe can pass through the DNA/ODA film and access tothe electrode surface for exchanging electrons, despite the exis-tence of some electrostatic repulsion between DNA andFeðCNÞ3�=4�

6 . This observation confirmed that DNA had been immo-bilized onto the electrode surface by LB technique.

3.4.2. Interaction of rutin with DNA-modified electrodeFig. 7 shows DPV curves of 3.0 � 10�5 mol L�1 rutin in the buffer

solution at the bare GC (curve a) and DNA–GC electrode (curve b),respectively. The peak potentials appear at 340 mV and 361 mV,corresponding to the Ep (bulk) and Ep (surf), respectively. The morepositive peak potential Ep (surf) than the Ep (bulk) indicates that theintercalative and stacking interactions of rutin to the base pairsof DNA, in long term, overcome the electrostatic attractions[9,24]. In the previous studies [25], when foreign molecules bind-ing to DNA via electrostatic interactions, the potential of the bindershifted in negative direction. In addition, the surface coverage of3.0 � 10�5 mol L�1 rutin at bare GCE and DNA–GCE were calcu-lated to be 5.16 � 10�7 mol m�2 and 0.77 � 10�7 mol m�2, respec-tively. This obvious difference could be attributed to the orderedsurface structure of DNA on the surface of the electrode, makingrutin less facile to reach the electrode surface and the electrontransfer lower. The dramatic decrease of peak current could be ex-plained as very strong intercalative interaction between rutin andDNA.

3.5. UV–vis spectra studies

Fig. 8 shows the UV–vis absorption spectra of rutin in the ab-sence and presence of DNA. As is known, DNA has a specificabsorption peak at 260 nm in aqueous solution (Fig. 8a). Rutinexhibits strong absorption bands appearing at 211, 257 and360 nm, along with two very weak bands at 220 and 296 nm(Fig. 8b). With the addition of various concentrations of DNA intorutin solution, spectrum (b) was changed to spectra (c) and (d),and the absorption bands of rutin at 220 and 296 nm disappeared,indicating the strong interaction between rutin and DNA. It can befound that the absorption spectrum (c) and (d) are significantly dif-ferent from the sum of corresponding absorption spectrum ofalone DNA and rutin. The absorbance change at 211 and 257 nmmay be attributed to the formation of new p-conjugated system.

Fig. 7. DPV curves of 3.0 � 10�5 mol L�1 rutin in the buffer solution at the bare GC(a) and DNA–GC (b) electrode, respectively. Other conditions same as in Fig. 2.

Fig. 8. The UV–vis spectra of rutin and DNA � RUm complex in 0.05 mol L�1 B–R buffer solution (50% ethanol, pH 5.02). (a): 5 lg mL�1 DNA; (b): 3.0 � 10�5 mol L�1 rutin; (c):(b) + 40 lg mL�1 DNA; (D): (B) + 50 lg mL�1 DNA.

Fig. 9. The relationship between log[DI/(DImax � DI)] and log[RU]. Other conditionssame as in Fig. 2.

X. Tian et al. / Journal of Electroanalytical Chemistry 621 (2008) 1–6 5

Bathochromic shift and hypochromic effect are suggested due to astrong interaction between the electronic states of the intercalativechromophore and that of the DNA bases [26,27].

3.6. Association equilibrium constant and stoichiometric coefficient

According to the literature [28], rutin (RU) and DNA associate toa single complex DNA � RUm, with the reaction scheme

DNAþmRU ¼ DNA � RUm ð1Þ

In terms of the overall Hill cooperativity model, the fraction ofrutin bound to DNA is given by

f ¼ ½DNA � RUm�=½DNA � RUm�max ¼ ½RU�m=ð½RU�m þ Kmd Þ ð2Þ

where [DNA � RUm]max represents the maximum concentration ofcomplexed DNA, and [RU] is the concentration of free rutin. Thenthe following formula can be deduced:

Kmd ¼ ½RU�mð1� f Þ=f ð3Þ

Kd is the equilibrium dissociation constant and m the Hill coeffi-cient. Note that Kd = [RU]0.5 at f = 0.5, i.e., at half occupation. Thebinding constant Ka is given by the reciprocity: Ka = Kd

�1.Mass conservation dictates that the concentration of free DNA is

available from the binding DNA:

½DNA� ¼ ½DNA � RUm�max � ½DNA � RUm� ð4Þ

For the same reason

½RU� ¼ ½RU�o �m½DNA � RUm� ð5Þ

where [RU]o is the total concentration of rutin. Under the givenexperimental conditions, the current I is given as

I ¼ k � ½RU� ð6Þ

DI ¼ IðRUÞo � IðRUÞ ð7Þ

Insertion of Eqs. (5) and (6) into Eq. (7) yields

DI ¼ kð½RU�o � ½RU�Þ ¼ k �m � ½DNA � RUm� ð8Þ

DImax ¼ k �m � ½DNA � RUm�max ð9Þ

From Eq. (3) we obtain the following equation:

log½f=ð1� f Þ� ¼ m log Ka þm log½RU� ð10Þ

Insert Eqs. (8) and (9) into Eq. (10) to yield

logDI

DImax � DI

��¼ m log Ka þm log½RU� ð11Þ

If DNA and rutin form a single adduct, the plot of log[DI/(DImax � DI)] versus log[RU] is linear with slope of m. The experi-mental data fit (shown in Fig. 9) yields m = 2.09 and m log-Ka = 10.40, so Ka = 1.58 � 105. The stoichiometry of thecooperative rutin binding is at least 2 per base pair unit.

4. Conclusions

In this work, the electrochemical behavior of anticancer herbaldrug rutin and its interaction with DNA were studied by electro-chemical and spectroscopic methods. All the experimental resultsindicate that the principal interaction mode of rutin with DNA iscooperative intercalative interaction. The interaction can be quan-tified in terms of the Hill model of cooperative interactions. The re-sults demonstrate that the electrochemistry combined withspectrophotometry is available and provides significant promiseto study the mechanism of the interaction of DNA with targetingcompounds not only from macrocosmic but also at molecular level.

6 X. Tian et al. / Journal of Electroanalytical Chemistry 621 (2008) 1–6

Acknowledgement

The financial support provided by National Natural ScienceFoundation of China (20775073) is greatly appreciated.

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