IntroductionFlavonoids are phenolic compoundswhich are widely distributed in plants. They consists of two aromatic rings (Aand B) and a heterocyclic ring (C). They are abundantly found in nature in manyfruits, vegetables and beverages. They have reported to have biological as wellas pharmaceutical activity counting anti-inflammatory, antimalarial antioxidantand antitumor activity. The expected antioxidant activity is due to conjugatedouble bonds and ?-electrons which are delocalized over the both benzene rings.Flavonoid are situated in the epidermis act as UV filters as they absorb lightin the range of 280 – 315nm.

1One of the most importantnon-glycosides flavonoid is hesperetin (3′, 5, 7-trihydroxy-4?methoxyflavanone)which abundantly occurs in citrus fruits. It has been received much attentiondue to its health benefits. Recently, researchers are emphasizing thedevelopment of pharmaceutical drugs based on natural constituents. It alsoshows many properties which includes antioxidants, inhibition of thecarcinogenic cells and tumors, lowers cholesterol levels and ant allergicproperties. 2  In addition to this, growing proof on the potential therapeuticefficacy of hesperetin has been establishedin diagnosing models,including lowing plasma sterol, antiplatelet activity, inhibiting tube-shaped structure formation , preventing upset and neuroprotectiveeffects.

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Most significantly, hesperetinhas received much attentionin prevention of cancer and tumor. In consideration of the important pharmacologicactivities, a sensitive and reliable analytical methodology in determinationof hesperetin is important. A lot of work has beendone on hesperetin biologicallyand some determination techniques have been already reported, like high performance liquid chromatography (HPLC), high performance liquidchromatography with mass spectrum technique , ultra-high-performanceliquid chromatography withultraviolet detector, fast reversephase-high-performance liquid chromatography,capillary zone electrophoresis with electro kinetic supercharging, high performance liquid chromatographyand capillary electrophoresis and high performance liquid chromatographywith electrochemical detection, high performance liquid chromatographywith electrochemical detection. Thesemultisolvent extraction techniques need expensive devices and maintenance iscoupled due to low concentration and complexity of analyte.3Radicalsand AntioxidantsRadicals are atoms having freeelectrons in their configuration. Human metabolism of the body generates freeradicals and reactive oxygen species (ROS) including superoxide, hydroxylradical and peroxyl radicals.

These radicals cause oxidative damage anddegenerative diseases. These radicals are terminated by radical scavenging byantioxidants therefore antioxidants play significant role in defense mechanism4. Oxidationreactions are decisive for life; however they can also harm or kill cells. An antioxidantis a molecule that restrain oxidation reactions, therefore inhibiting the formationof free radical intermediates, and reducing its undesired effect. The radicalscavenging is obtained by being oxidized themselves and therefore antioxidantsare often reducing agents such as thioalcohols, ascorbic acid, or polyphenols.Because to their cellular protective abilities, antioxidants are widelyutilized in dietary supplements and have been investigated for the preventionof diseases like cancer, coronary heart disease and many others related tooxidative stress.

Polyphenolic compounds such as chalcones, flavonoids and manyother flavonoids are regarded as excellent antioxidants because they have shownto have  enormous radical scavenging ability.5 In flavonoidsystem, the radical scavenging is said to occur via an electron transfer (ET) process,in which the phenol is transformed into phenoxyl radical which results in theformation of the semiquinone6. Thereforeflavonoids can be considered as hydrogen donating antioxidants because of theirreducing properties of the multiple hydroxyl groups attached to their aromaticring systems, along with their ability to delocalize over the resultingphenoxyl radical within their structure7. Flavonoids and UV protectionUV radiation reaching onthe earth’s surface is divided into three types of radiation. One with lowerenergy UV-A (320-400nm), others with higher energy UV-B (280-320 nm) and UV-C(254-280 nm) regions. Ultraviolet (UV) light particularly UV-B band causes mostsevere damage and is increasing due to ozone reduction, which is the primaryshield of solar radiation.

Exposure to UV-B radiation may cause cellular damageand damage to chloroplast compartment leading to a reduction in photosynthesis.Flavonoids, lie in the epidermis of a plant, act as UV filters as they absorb lightin the280 – 315 nm regions thus preventing UV-B radiation from reaching themesophyll and protecting the plant tissue from damage and ensuring favorableplant photosynthesis8. Estimation of Antioxidant Activity using UV-Visible SpectroscopyUV-visible spectroscopytechnique is extensively used for the determination of antioxidant activity ofmany antioxidants.

In this case solution of an antioxidant is added to thesolution of free radicals which are used as model to study scavenging effectsof antioxidant. The absorbance of the UV peaks of free radicals is decreased whenthe solution of an antioxidant is added and this continues to happen onsubsequent addition of antioxidant solutions to the solutions of free radicals.This decrease in the absorbance of free radicals shows their scavenging or stabilitywith the addition of antioxidants.

Percent scavenging (%RSA) is evaluated fromthe data of UV –visible spectroscopy, which is then plotted against theconcentration of antioxidant. From the slope of this plot one can evaluate theantioxidant activity of antioxidant in terms of IC30 and IC509.Estimation of Antioxidant Activity using Cyclic VoltammetryCyclicvoltammetry (CV) is based on linear sweep potential in time. CyclicVoltammetry were used to investigate flavonoid characteristics by peakpotential, number and position of peaks at those potentials, the height ofcurrent at each peak potential for all the flavonoids. According to literature,from the first anodic peak, we get the most useful information about the antioxidantactivity of compound, it is assumed that the lower the oxidation potential ofthe first anodic peak appeared, the greater the ability of a compound to donatean electron thus behave as an antioxidant10. This wassuggested on the basis that both electrochemical (EC) oxidation and hydrogen donatingfree radical-scavenging breaks the same phenolic bonds between oxygen andhydrogen, producing the phenoxyl radical and H’, which consists of an electron(e-) and an H + ion11.Using the technique ofcyclic voltammetry, the antioxidant activity of antioxidants is evaluated.

Whenantioxidant solution is added to the solution of free radicals, the anodic peakcurrent of free radicals is decreased with addition of each aliquot. From the dataof cyclic voltammetry percent radical scavenging activity (%RSA) in terms ofIC30 and IC50 can be calculated in order to measure the effectiveness of anantioxidant9. Evaluation of pKa value Alarge number of natural as well as synthetic compounds administrates many oftheir physical, chemical and biological properties.

So these properties aredetermined by pKa at any pH12. Different methodshas been employed to find pKa value, the most common methods are potentiometrictitration where pKa is obtained from titration curve, spectrophotometrictitrations where spectra is obtained for each point in titration and change ofabsorbance is plotted against pH and capillary electrophoresis in buffersolution where pKa is evaluated from mobility of ions. Out of all thesetechniques, pKa was found from UV spectroscopy as it is a sensitive techniqueand small sample is needed. A chromosphore must be attach with a compound tofind pKa value from UV spectroscopy13.  The DVP studies and UV spectroscopy wasemployed for drug at various pH in BRB buffer14.SuperoxideAnion (O2-)Superoxide anion (O2?)is very important free radical as it is concerned with several diseases likecancer, denaturation of proteins, aging and lipid peroxidation.

This chemicalspecies is generated as a result of reduction of molecular oxygen15.Oneof the very major and key source for the natural production of superoxide anionis electron transport chain reaction that takes place in mitochondria of acell. There is a channel of electron acceptors in mitochondria which undertakeperiodic reduction-oxidation reactions, however there is possibility that freeelectrons may be transferred to molecular oxygen instead of next electronacceptor which later on generates superoxide anion.

Many iron-Sulphur clustersin a cellular respiratory machine of a body are also attributed to the generationof poisonous superoxide ions via side reactions with oxygen. Generation of superoxideAnion (O2?) free radicals from the mitochondria, is consideredas their major intracellular formation under physiological conditions.Approximately 1-2 % of daily intake of molecular oxygen when goes tomitochondria results in production of 160-325 mmol of superoxide anionapproximately in a 60 kg woman whereas in 80 kg man 215-420 mmol of superoxideis produced on daily basis16. Oxygenfree radicals have extreme effects as they’re involved with varied disorders.They disrupt the integrity of epithelium tissue that lines the system.Disruption and loss of epithelium tissue ends up in the extravasation ofintervascular fluid and blood cells into the ischemic space ends up in theelementary events of reperfusion injury.

Several mechanisms are projected tosupport the toxicity of those chemical species. Experimental evidences suggestthat xanthine oxidase enzyme is that the major supply for the assembly ofdamaging and poisonous oxygen free radicals that cause ischemia-reperfusion17.DFT and Theoretical Studies Theredox reactivity of phenolic compounds (ArOH) mostly follow two differentchemical pathways:ArOH + R• ? ArO• + RHArOH +R• ? ArOH+• + R? ?ArO• + RHReaction1 presents the homolytic dissociation of the O?H bond. This reaction can occuron each OH group present on phenolic compound (the flavone, for example) and itis governed by the BDE (Bond Dissociation Energy) of the OH groups and by theenthalpy ?E1 of reaction 1.

The BDE is an intrinsic thermodynamic parameter,whereas enthalpy of reaction 1 is dependent on the radical that is reactingwith the flavone. The lower the BDE of O?H, the easier will be the O-H bond breaking,and most significant is its role in the antioxidant reactivity18.Redoxproperties of phenolic compounds have been established theoretically, usingsemi-empirical Hartree-Fock (HF) methods19 and more recentmethod is by density functional theory (DFT)20. DFT seems liketo be the most reliable approach to achieve reliable BDE (Bond DissociationEnergy) values that are close to experimental data, while semi-empiricalmethods underestimate the ? electron delocalization and yield non-planargeometries for flavonoids and flavones (i.

e., flavonoids having a 2,3-doublebond).Recently a valid estimation of the BDE for phenolic compounds have beendone by DFT and it highlights the role of the OH groups present on B-ring21 and the 3-OHgroup in flavonoids22.Thegeometries and energies of the phenoxyl radicals were obtained after the H atomwas removed from each OH group of the optimized structure of the parentmolecule and using an unrestricted approach, which is more relevant to DFTcalculations23.Tovindicate UV/Vis spectra of atomic and molecular systems, a large panel of(time-dependent) quantum chemical methods are present.

Several years ago it wasreported on the electronic structures and spectra including molecular orbital(MO) analysis were based on Hückel and Pariser–Parr–Pople methods. Later asemi-empirical study confirmed the role of hydroxylation in the B-ring inpolyphenols. Excited states were more recently scrutinized with SE–CI (singleexcitation–configuration interaction) evaluation based on density functionaltheory (DFT) geometries. Over the past years time-dependent (TD) DFT has developedas an effective tool to evaluate UV/Vis absorption of p conjugated compounds.Recently, TD-DFT was revealed to be reliable in predicting the UV/Vis spectraof a series of polyphenol and their derivatives, whereas Hartree–Fock/CIS andsemi-empirical approaches were found insufficient to reproduce the experimentaldata results.

TD-DFT was also used to examine the optical properties of somecations. TD-DFT also allows the study of relatively large and complex systems(up to 500 atoms), which is not possible with other methods, for example ab-initiomethods thus it is a valuable tool to investigate large series of manycompounds24.Drug-DNA Binding StudiesTranscriptionand replication are important for survival of cells and proliferation.

They alsohelp body to function normally. Transcription or replication of DNA starts onlywhen it receives a signal, which is mostly in the form of a binding ofregulatory protein to a specific region of the DNA. Thus, if the specificityand strength of binding with this regulatory protein can be imitated by a smallmolecule, then DNA function can be modulated, inhibited or activatedartificially by binding this molecule as an alternative of the protein. Whenactivation or inhibition of DNA function is required then the small naturalmolecules can act as drug to treat or control a particular disease.

Chemotherapyis the technique in which these foreign molecules stop the replication of DNA suchas drugs. The molecules have anti-cancerous properties because they can interactas well as inhibit further replication of DNA. Due to their broadpharmacological activity flavonoids have got considerable interest among theselarge family of molecules25.Infact, they are best recognized for their antioxidant properties, and can act asa reducing agents, hydrogen atom donors, free radical quenchers and metal ionchelators and this may justified for the anti-tumor activities of flavonoids26.

Studieson binding mechanism of these anticancer drugs with the DNA has been recognizedas one of significant research topic during past several years. The effect ofDNA interacting drug can be perceived experimentally at molecular, cellular andclinical levels. Molecular level is the most basic level which involves directchemical interaction of drug with the giant DNA molecule. A number oftechniques have been developed to examine this level of interactions are X-rayCrystallography, Mass Spectroscopy, foot printing technique, Electrochemistry,UV-Vis and fluorescent Spectroscopy, NMR, FTIR, Surface Plasmon Resonance,Equilibrium dialysis and Molecular Modelling Studies27.DNA-bindingdrugs may be categorized into two extensive classes, one is covalently bindingdrugs and the other non-covalently binding drugs. The covalently binding drugsmake covalent bonds to nitrogenous bases forming inter-strand or intra-strandcross-linking producing more distortion to the DNA structure.

Covalent DNAbinding cannot usually be reversed28 and it canleads to complete inhibition of DNA functions, leading to cellular death. Dueto such firm and strong (as well as the non-selective) binding, covalentbinders cause lethal side effects in treated patients.Thenon-covalently binding drugs are classified only to three smaller groups28.

(1)Intercalating drugs (2) minor-groove binders and (3) drugs formingelectrostatic interactions with the negatively cha`rged sugar phosphatebackbone of DNA molecule.Intercalatingdrugs get stacked between the two neighboring base-pairs present in DNA doublehelix. They have planar heterocyclic groups and loosely bind to the nitrogenousbases of DNA through ?-? stacking interactions. Thus though they make non-covalentinteractions, the intercalators form a rather strong perturbations to DNA structure29.Asthe minor groove is narrow so the edges of bases are more accessible in majorgroove for the larger DNA-binding proteins such as transcription factors. Whilesmall-molecular drugs, favorably bind at the minor groove, which are narrowerand provides better van der Waals contact of the drug peripheral with thesugar-phosphate walls of the minor groove, weak hydrogen bond interactionsenhance the net drug-DNA binding with these interactions30.

Thespectroscopic technique was used to study the interaction of the flavanone withds.DNA. In present work the compound under investigation is UV active andexhibit a UV spectra with a peak at 287nm.

UV-Vis spectroscopy has beenemployed to study the interaction of drug with ds.DNA The spectrum of given compoundchange after the subsequent addition of DNA and from the difference inabsorbance, binding constant was calculated which gives an clue of strength ofbinding with DNA.InCyclic Voltammetry, the I-E response of compounds was observed. It was foundthat this compound undergo electrochemical oxidation. After the addition of DNAto the solution of compound prepared in PBS buffer, a change in i-E response wasobserved which shows the interaction of compound with ds.

DNA. By change in i-Eresponse the amount of compound that have been interacted can be known. Diffusioncoefficients and binding constant was also determined experimentally. Theelectrochemical data supported the spectroscopic surveys.

Moleculardocking and QSAR studies were carried out for the investigation of interactionsbetween the compound and the double stranded DNA using MOE (Molecular OperatingEnvironment) package. Different electronic descriptors and binding constant wascalculated theoretically. The higher binding constants shows the strongerbinding.

Docking with Enzyme ComplexComputer-basedapproaches are becoming progressively important and harmonizing to wetlaboratory techniques in elucidating the structure and function of manybiomolecules. Molecular docking is an often used method in structure-based rationaldrug design. IT is beyond doubt early efforts were caught up by limitedpossibilities in computational possessions, due to recent developments inextraordinary performance computing computer-generated screening methods becamemore and more proficient. These computational methods contributed to the progressof numerous drugs and drug runners that advanced to clinical field. TheoreticalMolecular Docking Studies of Drug with DNA Enzymes (DNA polymerase, Topoisomeraseand Helicase) were carried out.

These enzymes are essential for DNAreplication, repair and recombination. A strong interaction of drug was foundwith these enzymes and their binding constant with drug was calculated lateron.Present WorkInthe present study, theantioxidant activity of the compound and ascorbic acid was determined by usingUV Visible spectroscopy and Cyclic Voltammetry with DPPH radical. .Theoretically, antioxidant activity is estimated by DFT calculations bycalculating the bond dissociation enthalpy (BDE) of each O?H and Gibbs freeenergy (?G) of reaction against DPPH radical. By comparing the theoretical andexperimental results, the predictive power of DFT calculations is established.Molecularlevel interactions with DNA molecule were studied using cyclic voltammetry (CV)and UV-Vis spectroscopy at physiological conditions (pH = 7.

4 and T = 298 K).Molecular Docking was done to confirm the binding between drug and DNA.Theoretical Molecular docking studies was done to predict the interactionbetween DNA enzymes and Drug.