1. Introduction Flavonoids are polyphenolic compounds and constitute the non-nutritive parts of plants. Due to their tremendous biological importance and broader range of pharmacological activities including antioxidant, anticancer, antitubercular, antibacterial, antiallergic, antimicrobial, anti-inflammatory, antiviral, antitumor, antimutagenic, antidiabetic, hepatoprotective and cardiovascular activities, falvonoids have gained much attention and become a topic of interest for researchers in the last decades [1-8]. Karanjin (Kj) and karanjachromene (Kc) are furanoflavonoid and pyranoflavonoid, respectively (Scheme 1) and extracted from Pongamia pinnata seeds’s oil [9,10]. Karanjin has been reported for

its antifungal, antibacterial, antihyperglycemic activities and as nitrification inhibitors [10-16].

Reactive oxygen species (ROS) like superoxide (O2

¯˙), hydrogen peroxide (H2O2) and, hydroxyl radical (OH¯˙), and peroxyl radical (HOO∙) are normal metabolic products of biological systems [2,3] and react with DNA and damage it. Modification in DNA structure (DNA lesion) as a result of its interaction with ROS arises as a result of oxidative stress [17,18]. Since DNA is a carrier of genetic information in all living things, its damage/modification (impropriety in the base pairs) ultimately bring changes in true genetic information and result in mutagenesis and carcinogenesis [19]. The damaging of DNA is important from both a chemical and medicinal point of view. Experimental evidence has proven that

Central European Journal of Chemistry

UV–absorption studies of interaction of karanjin and karanjachromene with ds. DNA:

Evaluation of binding and antioxidant activity

* E-mail: nasimaa2006@yahoo.com

Received 30 May 2013; Accepted 18 July 2013

Abstract:

© Versita Sp. z o.o.Keywords: Flavonoids • UV-Vis spectroscopy • Interaction with chicken blood ds.DNA • Binding constants • Antioxidant activity

1Department of Chemistry, Allama Iqbal Open University,

Islamabad 44000, Pakistan

2Department of Biochemistry, Quaid-i-Azam University,

Islamabad 45320, Pakistan

Nasima Arshad1*, Naghmana Rashid1, Sajida Absar1, Muhammad S. A. Abbasi1, Samreen Saleem2, Bushra Mirza2

Research Article

Two flavonoids, karanjin (Kj) and karanjachromene (Kc) have been investigated spectrophotometrically for their mode of interactions with double stranded (ds)-DNA at blood (7.4) and stomach (4.7) pH and at human body temperature (37ºC). Benesi-Hildebrand equation was used to evaluate the binding constants, Kb. Binding constants at both pH values and at body temperature showed stronger binding of both the flavonoids and formation of 1:1 flavonoid–DNA complex via intercalative mode. However, Kb values for karanjin were evaluated to be comparatively greater than karanjachromene at both pH values. The highest value of binding constant (1.32×105 M-1) for karanjin at blood pH (7.4) demonstrated its comparatively stronger binding and greater effectiveness at this pH. Standard Gibbs free energy changes (∆G) of flavonoid–DNA complexes were calculated as negative values and indicative of spontaneity of their binding. Both flavonoids showed significant DNA protection activity.

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Cent. Eur. J. Chem. • 11(12) • 2013 • 2040-2047DOI: 10.2478/s11532-013-0327-z

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DNA can be damaged under certain conditions including various physical and chemical factors [20]. Mutagens like oxidizing agents, alkylating agents and high-energy radiation can change the DNA sequence [21]. Great attention has been paid to recognize free radical scavengers or antioxidants as an inhibitor of DNA damaging caused due to oxidative reaction. As interest in the impact and health benefits of flavonoids continues to grow, flavonoid–DNA binding studies have become an interesting area of research and have opened the way for new findings.

Recent trends to prevent nucleic acid damage mainly emphases on the interaction of DNA with other molecules which preferentially bind to DNA reversibly (non-covalent binding). These molecules include drugs, metal-based complexes and antioxidants (flavonoids). Reversible binding is more significant than irreversible covalent binding, as chances of adverse side effects are comparatively less. Stacking between the base pairs of DNA, weaker interactions with the negative charged nucleic sugar-phosphate structure and interactions with major and minor grooves of DNA double helix are the three major modes of reversible binding of a variety of small molecules with DNA and are named as intercalation, electrostatic interactions and groove binding, respectively [22]. A variety of intercalators and minor groove binders are known for their anticancer, antiviral, antitumor, antibacterial and antifungal activities [23,24]

In the present work, the interaction and binding of two flavonoids, karanjin and karanjachromene with ds.DNA extracted from chicken blood (ck.DNA) have been investigated under physiological conditions of pH (blood pH; 7.4 and stomach pH; 4.7) and temperature (37ºC) using UV-Vis spectroscopy. Both compounds were also investigated for their DNA protection antioxidant activitiy and 1,1-Diphenyl-2- picrylhydrazyl (DPPH) radical scavenging activity.

2. Experimental procedure

2.1. Materials 2.1.1. Reagents and chemicalsAll the solutions were prepared in autoclaved water. Extraction of DNA from chicken blood was carried out in the laboratory using Falcon method [25]. Concentration of DNA (expressed as molarity of phosphate groups) was determined spectrophotometrically at 260 nm using molar extinction coefficient, ε260 = 6600 cm-1M-1 [25,26]. DNA was further checked for its purity by monitoring the ratio of the absorbance at 260 nm to that at

280 nm. The ratio (A260/A280) was evaluated greater than 1.8, which is indicative of adequate DNA purity, i.e., DNA is free from protein [27]. Karanjin and karanjachromene were extracted from Pongamia pinnata seeds in the laboratory [9]. Buffer solutions of pH 7.4 (phosphate buffer; Na2HPO4 + NaH2PO4) and 4.7 (acetate buffer; CH3COOH + CH3COONa) were prepared and experiments were conducted at both pH. Stock solutions of two flavonoids were prepared in buffered ethanol - water mixture in 1:1 ratio. For flavonoid–DNA complex formation, flavonoid concentration was kept constant while DNA was added in aliquots at both pH 7.4 and 4.7 and at 37ºC.

2.1.2. InstrumentationThe electronic absorption spectra were recorded on Shimadzu1800 spectrophotometer (±0.005 nm) equipped with a Shimadzu thermostat (±0.1°C) using 1.0 cm matched quartz cells. Hettich EBA20 Portable Centrifuge C 2002 (Max. speed: 6,000 min-1) and vortex machine was used for the extraction of DNA from chicken blood. pH meter (Janway. Model 4510) was used to measure the pH of buffers utilized in this study.

2.2. Methods2.2.1. Spectroscopic titrationsDNA Concentration was determined by UV-Visible spectrophotometer at wavelength of 260 nm and its value was calculated as 8.4×10-5 M. Spectroscopic titrations were carried out by keeping the concentration of karanjin (6.7×10-5 M) and karanjachromene (2.3×10-5 M) constant in the sample cell, while varying the concentration of ds.DNA from 1 µM to 30 µM in the sample cell at blood (7.4) and stomach pH (4.7) under normal body temperature (37ºC). The change in absorbance was measured before and after the addition of various concentrations of DNA. All the samples were equilibrated 5 min prior to every spectroscopic measurement. Also, in order to achieve the required temperature (37ºC) of the solution, sample cell was kept inside the cell cavity for few second before running the spectra.

Kj Kc

Scheme 1. Structures of karanjin (Kj) and karanjachromene (Kc).

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UV–absorption studies of interaction of karanjin and karanjachromene with ds. DNA: Evaluation of binding and antioxidant activity

2.2.2. Free radical induced oxidative DNA damage assayFree radical induced oxidative DNA damage assay is a simple assay which was used to investigate the protection or the damage effect of test compounds (karanjin and karanjachromene) on DNA in vitro. Plasmid DNA (pBR322) was diluted with the help of phosphate buffer (50 mM) to have a final concentration of 0.5 µg per 3 µL. Each reaction mixture (15 µL) had 3 µL of diluted plasmid, 5 µL of test compounds at different concentrations (10, 100 and 1000 µg mL-1), 4 µL of 2 mM FeSO4 and 3 µL of 30% H2O2. For each experiment, as a positive control (X) plasmid DNA treated with 30% H2O2 and 2 mM FeSO4 was used. FeSO4 was used as a catalyst in this experiment and Hydrogen peroxide (H2O2) to induce DNA damage by producing OH free radicals. Plasmid pBR322 DNA (untreated) was used as negative control (P) and a 1 Kb DNA ladder (L) was used as marker. Each Reaction mixture was incubated at 37ºC for 1 h in dark and then subjected to gel electrophoresis (1% agarose). Ethidium bromide was used as staining agent for DNA. Gels were visualized under UV and intensity of each bands was determined (Gel Doc, Bio Rad).

2.2.3. DPPH free radical scavenging assayThe solutions of two flavonoids (0.01M); karanjin and karanjachromene were prepared in methanol. The solution of DPPH● (10-5 M) was prepared in methanol. 0.5 mL of each sample was mixed with 2.5 mL solution of DPPH● separately. The decrease in absorption (515 nm) was monitored every one minute for 15 minutes using UV-Vis spectrophotometer. Absorption of blank sample containing the same amount of methanol and DPPH● was also measured. The solution of Gallic acid (10-5 M) was prepared and used as positive control.

3. Results and discussion

3.1. Spectral findings of flavonoid–DNA complex3.1.1. UV-spectra of flavonoidsFlavonoids are benzo-g-pyrone derivatives containing several hydroxyl groups attached to the ring structures, and the basic structure consists of two aromatic rings, A and B linked through three carbons that usually form an oxygenated heterocyclic, ring C. The reactive structures of flavonoids are a pyrogallol group, catechol group, 2,3-double bond in conjugation with 4-oxo group, 3-hydroxyl group and some additional resonance-effective substituents [28]. There are numerous evidences that the catechol-type ring-B in flavonoids is the antioxidant active moiety [29]. Yang and his colleagues have shown that the antioxidant activity of

flavonoids is due to their aromatic OH groups [28]. Most of the flavonoids have same carbon skeleton and differ only by number and position of hydroxyl substituents on ring A and B. In the UV-region, two main absorption bands are observed in flavonoids. The absorption in the range of 320–385 nm (band-I) corresponds to Ring B portion (cinnamoyl system), while absorptions in the range of 240–280 nm (band-II) correspond to Ring A portion (Benzoyl system), Scheme 2 [30].

Initially, UV-spectra of karanjin and karanjachromene in ethanol-water (1:1) mixture were recorded. Two peaks were observed in the UV-spectra of karanjin, the less intensive peak at 307 nm (band Ι) and more intensive peak at 262 nm (band ΙI). In Karanjin, the band I at higher wavelength corresponds to n ─ π* whereas the band II at comparatively low wavelength corresponds to π ─ π* chromophoric transitions [22,31,32]. Karanjachromene gave only one peak at 270 nm. The only band of karanjachromene is related to π ─ π* transition. Absence of second peak at high wavelength region (320 nm–385 nm) could be due to different orientation of ring B in karanjachromene molecule, making the absorption in this range forbidden. The low absorption intensity of band I in case of karanjin and absence of similar band in karanjachromene further predicted the possibility of n ─ π* transition. Concentration effect on UV-spectra of flavonoids is in accordance with the Beer’s law. Extinction coefficients were calculated from these spectra by plotting absorbance at λmax versus concentration, Fig. 1. Molar extinction coefficients (ε) for karanjin and karanjachromene were evaluated as 7380 M-1 cm-1 and 16600 M-1 cm-1, respectively.

3.1.2. UV-spectrophotometric studies of flavonoid–DNA interactionUV–Vis spectroscopy is considered one of the most reliable techniques to study the interactions of small molecules and the way of their binding with the DNA. The spectra of flavonoids were recorded separately after the addition of various concentrations of DNA at constant flavonoid concentration under physiological conditions

Scheme 2. Flavon Structure showing chromophores.

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of pH and temperature. Addition of DNA showed marked effects on the spectra of both flavonoids. Changes in spectral responses indicated the formation of flavonoid–DNA complex.

By the addition of various concentrations of DNA on fixed concentration karanjin, a gradual decrease in the absorption intensity of both bands (I &II) was observed along with a pronounced red shift of 3.9 nm (band I); 3.3 nm (band II) at pH 7.4 and 2.8 nm (band I); 2.2 nm (band II) at pH 4.7, respectively, Fig. 2. The increasing concentration of DNA led to hypochromic effect and bathochromic shift in the UV-absorption spectra of karanjin and inferred the interaction between electronic states of the chromophore and that of DNA base pairs, hence resulted in the formation of stable karanjin–DNA complex via intercalation [33]. The relatively longer shifting of band I at both the pH indicated the stabilization of karanjin–DNA complex due to interaction of lone pairs of karanjin with DNA. This is further attributed to the molecular structure of karanjin as its planer part may penetrate into the double helical structure of DNA and organize itself parallel to the nearby planes of the nitrogenous bases [34,35].

Addition of various concentrations of DNA on fixed concentration of karanjachromene at both pH values showed increase in the absorption peak intensity with a small blue shift of 0.7 nm at pH 7.4 and 0.3 nm at pH 4.7, respectively, Fig. 3. The observed hyperchromic effect after the addition of DNA inferred that more karanjachromene bound to DNA and may be attributed to either external interaction via electrostatic interaction/or groove binding or formation of karanjachromene–DNA complex by uncoiling DNA double helix [36,37]. However, unstacking of DNA base pairs is considered the major cause of hyperchromism as suggested by several authors [33,38,39].

3.1.3. Flavonoids–DNA binding constantIt is assumed that the changes in UV-absorption spectra after the addition of DNA to flavonoid solution is due to the binding of flavonoid to DNA as is the case with other DNA binding drugs such as Lumazine. Consequently following equilibrium may be established:

Flavonoids + DNA ↔ flavonoids – DNA (1)

Figure 1. Concentration profile of karanjin (Kj) and karanjachromene (Kc).

Figure 2. Electronic spectra of karanjin (6.7×10-5 M) in the absence (a) and the presence of 1 µM (b), 5 µM (c), 10 µM (d), 15 µML (e), 20 µM (f), 25 µM (g), 30 µM (h) DNA at pH 7.4 (A) and 4.7 (B) and at 37ºC. The direction of arrow indicates addition of DNA concentrations.

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UV–absorption studies of interaction of karanjin and karanjachromene with ds. DNA: Evaluation of binding and antioxidant activity

The formation constant or binding constant “Kb” of flavonoids–DNA complex can be determined from the change in absorbance in UV-spectra after the addition of DNA. Benesi-Hildebrand equation is used to evaluate binding constants spectrophotometerically [40]:

[ ]1o G G

o H G G H G G b

AA A K DNA

ε εε ε ε ε− −

= +− − − (2)

Where Ao and A are the absorbance’s of free and bound flavonoid, εG and εH-G are their molar extinction coefficients, respectively. A plot of Ao /(A-Ao) to 1/[DNA] have shown linearity which is suggestive of 1:1 complex formation of both flavonoids with DNA per nucleotide phosphate. The intercept to slope ratio of this plot gives the value of binding constant “Kb”, Fig. 4. Kb values were calculated at both blood and stomach pH and at body temperature (37ºC) and given in Table 1. The Kb values of both flavonoid–DNA complexes are in agreement with the binding constant values for the interaction of anthracycline molecules with DNA (K ≈ 104 – 105 M-1) already reported and depict stronger interactions of two flavonoids with the DNA at both pH values [24]. Further, Kb value (1.32×105 M-1) of karanjin at pH 7.4 and at 37ºC was found much greater than that of typical antioxidant, quercetin, whose binding constant value was reported (6.10×104 M-1) at the same pH and same temperature [31].

In general, intercalation of small molecules with DNA is related to hypochromic effect with red shift or hyperchromic effect with blue shift observed in the absorption spectra [33]. The observed changes in absorption spectra during flavonoids–DNA complex formation in present studies further demonstrated the possibility of stable complex formation via intercalation of planer parts of both flavonoids between the adjacent DNA base pairs. The binding constant (Kb) values for karanjin were evaluated comparatively greater than karanjachromene at both pH. However, the value of binding constant calculated for karanjin was obtained 10 order of magnitude higher at pH 7.4 and attributed to its comparatively stronger binding and greater effectiveness at blood pH than at stomach pH.

The values of binding constant “Kb” were further used to calculate standard Gibbs free energy “∆G” of flavonoid–DNA complex, using the following equation;

∆G = -RT ln Kb (3)

Free energy changes of karanjin and karanjachromene at both the pH were evaluated as negative values, Table 1, and showed spontaneous binding of flavonoid with DNA [22,34].

Figure 3. Electronic spectra of karanjachromene (2.3×10-5 M) in the absence (a) and the presence of 1 µM (b), 10 µM (c), 12 µM (d), 20 µM (e), 25 µM (f), 30 µM (g) DNA at pH 7.4 (A) and 4.7 (B) and at 37ºC. The direction of arrow indicates addition of DNA concentrations.

Table 1. Binding constants and free energy values for the flavonoids–ds.DNA complexes at physiological conditions.

Complex Code pH 7.4 pH 4.7Binding constant Free Energy Binding constant Free Energy

Kb/M-1 (-∆G)KJ mol-1 Kb/M

-1 (-∆G)KJ mol-1

Kj-DNA 1.32×105 29.47 3.81×104 29.29

Kc-DNA 1.68×104 25.07 2.89×104 26.47

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3.2. Free radical induced oxidative DNA damage analysisTest compounds under study (karanjin and karanjachromene) were investigated for their anti-oxidant and pro-oxidant effects in vitro by free radical induced oxidative DNA damage assay [41]. This experiment is based on Fenton reaction where Fe2+ reacts with H2O2 to produce OH radicals which have been proven to be the most dangerous to all biomolecules [42]. DNA protecting activity of test samples (karanjin and karanjachromene) was checked by in vitro free radical induced DNA damage assay at different concentrations (10, 100 and 1000 µg mL-1). Plasmid pBR322 DNA (intact) exists in super-coiled (SC) form. When the OH radical (generated from Fenton reaction) attacks on plasmid DNA, if only one strand of plasmid DNA is cleaved (single stranded nicking), the super-coiled (SC) form of plasmid DNA will be converted to open circular (OC) form (a slow moving form). If scission occurs on

both strands of plasmid pBR322 DNA (double stranded nicking), a linear form of plasmid will be generated that migrates between open circular (OC) form and supercoiled (SC) form. Thus in this assay, the effect of compounds to condense or unwind the supercoiling substrate such as plasmid DNA was investigated. Evaluation of anti-oxidant or pro-oxidant effects of test compounds on pBR322 DNA was based on the increase or loss of percentage of supercoiled form as compared with the control. In the present study, test compounds; karanjin and karanjachromene were checked for their potential aginst DNA damage induced by H2O2. In control X, when plasmid was treated with FeSO4 and H2O2, scission occurred on both strands of DNA which generated linear DNA form. Test compounds; karanjin and karanjachromene showed significant plasmid DNA protection at different concentrations (10, 100 and 1000 µg mL-1) as compared with control X as depicted in Fig. 5.

Figure 4. Plot of Benesi-Hildebrand equation for calculation of binding constant (Kb) of karanjin (a) and karanjachromene (b) at pH 7.4 and 4.7 and at 37ºC.

Figure 5. Effect of karanjachromene and karanjin on pBR322 plasmid DNA. L = ladder (1 KB), P = Plasmid (pBR322), X = pBR322 plasmid treated with FeSO4 and H2O2, Lane 1 = plasmid treated with Karanjachromene at 1000 µg mL-1, Lane 2, 3, 4 = plasmid treated with FeSO4 and H2O2 and Karanjachromene at 1000, 100 and 10 µg mL-1 respectively, Lane 5 = plasmid treated with Karanjin at 1000 µg mL-1, Lane 6, 7, 8= plasmid treated with FeSO4 and H2O2 and Karanjin at 1000, 100 and 10 µg mL-1 respectively.

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UV–absorption studies of interaction of karanjin and karanjachromene with ds. DNA: Evaluation of binding and antioxidant activity

3.3. DPPH free radical scavenging studiesAntioxidants are reported to react with DPPH (a stable free radical) via donating electron or hydrogen, as a result neutralizing it to diphenyl picrylhydrazine [43].

DPPH radicals thus reduce by antioxidant and can be determined spectrophotometrically as decrease in the absorbance. Karanjin and karanjachromene were subjected to DPPH assay to determine their antioxidant or prooxidant behavior. Decrease in the absorption peak intensity at 515 nm inferred antioxidant behavior of both the flavonoids, Fig. 6. Radical scavenging activity of an antioxidant can be calculated by the following formula;

% Inhibition = [(Ao - A)/Ao] ×100

Where: Ao = absorption of blank sample (t = 0 min);A = absorption of test compound-DPPH solution (t = 15 min).

% inhibition of karanjin and karanjachromene were evaluated 15.22% and 11.83%, respectively, which showed much less radical scavenging activities of both flavonoids from this method. As reported earlier in case of other flavonoids such as pyragallol, catechol

etc. the most important structural feature to show antioxidant activity by this method is presence and position of hydroxyl groups [44]. This opens the avenue to further investigate the effectiveness of DPPH radical scavenging method for antioxidant studies.

4. Conclusions The interactions of two flavonoids, karanjin and karanjakhromene, with ds.DNA were investigated through UV-Vis spectrophotometric method at blood (7.4) and stomach (4.7) pH and at body temperature (37ºC). The results achieved from UV-absorption spectra of both the flavonoids with DNA revealed an intercalation mode of interaction as the dominant mode. The values of standard Gibbs energy changes, (∆G), were evaluated negative inferred the spontaneity of the binding of two flavonoids with DNA. The flavonoid–DNA binding constants “Kb” were evaluated and their values showed stronger interactions of two flavonoids with the DNA to form stable 1:1 complex formation at both pH values. Binding constants calculated for karanjin were comparatively greater than kranjachromene at both pH values. However, the binding constant for karanjin was found to be 10 orders of magnitude higher at pH 7.4 than at pH 4.7, which inferred its comparatively stronger binding and greater effectiveness at blood pH. Both flavonoids showed good DNA protection activity but less DPPH● scavenging activity. This study and further investigation in this direction could be beneficial not only to determine flavonoid-DNA interactional mechanism and but also to design new promising DNA targeted drugs.

Acknowledgements This work is supported by Department of Chemistry, Faculty of Science, Allama Iqbal Open University Islamabad, Pakistan.

N. Arshad, N.K. Janjua, L.H. Skibsted, M.L. Anderson, J. Chem. Soc. Pak. 35, 544 (2013) N. Arshad, N.K. Janjua, A.Y. Khan, J.H. Zaidi, L.H. Skibsted, Monatsh. Chem.143, 377 (2012)N. Arshad, N.K. Janjua, A.Y. Khan, A. Yaqub, T. Burkholz, C. Jacob, Nat. Prod. Commun. 7, 311 (2012)N. Arshad, N.K. Janjua, S. Ahmed, A.Y. Khan, L.H.Skibsted, Electrochem. Acta 54, 6184 (2009)

K.Y. Akhilesh, T. Jayprakash, P. Om, K. Feroz, S. Dharmendra, M.G. Madan, Med. Chem. Res. 22, 2706 (2013)L. Malyane, W.K. Benguedouar, H.N. Boussenane, H. Rouibah, M. Lahouel, Pak. J. Pharm. Sci. 21, 201 (2008) A.R. Tapas, D.M. Sakarkar, R.B. Kakde, Trop. Pharm. Res. 7, 1089 (2008)R.J. Nijveldt, E.V. Nood, D.E.C.V. Hoorn,

Figure 6. Time course of absorption reduction of DPPH-Test samples (0.01 M); karanjin and karanjachromene in methanol. DPPH-Gallic Acid (10-5 M) in methanol is used as positive control.

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