Introduction beverages. They have reported to have biological


Flavonoids are phenolic compounds
which are widely distributed in plants. They consists of two aromatic rings (A
and B) and a heterocyclic ring (C). They are abundantly found in nature in many
fruits, vegetables and beverages. They have reported to have biological as well
as pharmaceutical activity counting anti-inflammatory, antimalarial antioxidant
and antitumor activity. The expected antioxidant activity is due to conjugate
double bonds and ?-electrons which are delocalized over the both benzene rings.
Flavonoid are situated in the epidermis act as UV filters as they absorb light
in the range of 280 – 315nm.1

One of the most important
non-glycosides flavonoid is hesperetin (3′, 5, 7-trihydroxy-4?methoxyflavanone)
which abundantly occurs in citrus fruits. It has been received much attention
due to its health benefits. Recently, researchers are emphasizing the
development of pharmaceutical drugs based on natural constituents. It also
shows many properties which includes antioxidants, inhibition of the
carcinogenic cells and tumors, lowers cholesterol levels and ant allergic
properties. 2  In addition to this, growing proof on the potential therapeutic
efficacy of hesperetin has been established
in diagnosing models,
including lowing plasma sterol, antiplatelet activity, inhibiting tube-shaped structure formation , preventing upset and neuroprotective
effects. Most significantly, hesperetin
has received much attention
in prevention of cancer and tumor. In consideration of the important pharmacologic
activities, a sensitive and reliable analytical methodology in determination
of hesperetin is important. A lot of work has beendone on hesperetin biologically
and some determination techniques have been already reported, like high performance liquid chromatography (HPLC), high performance liquid
chromatography with mass spectrum technique , ultra-high-performance
liquid chromatography with
ultraviolet detector, fast reverse
phase-high-performance liquid chromatography,
capillary zone electrophoresis with electro kinetic supercharging, high performance liquid chromatography
and capillary electrophoresis and high performance liquid chromatography
with electrochemical detection, high performance liquid chromatography
with electrochemical detection. These
multisolvent extraction techniques need expensive devices and maintenance is
coupled due to low concentration and complexity of analyte.3

and Antioxidants

Radicals are atoms having free
electrons in their configuration. Human metabolism of the body generates free
radicals and reactive oxygen species (ROS) including superoxide, hydroxyl
radical and peroxyl radicals. These radicals cause oxidative damage and
degenerative diseases. These radicals are terminated by radical scavenging by
antioxidants therefore antioxidants play significant role in defense mechanism4. Oxidation
reactions are decisive for life; however they can also harm or kill cells. An antioxidant
is a molecule that restrain oxidation reactions, therefore inhibiting the formation
of free radical intermediates, and reducing its undesired effect. The radical
scavenging is obtained by being oxidized themselves and therefore antioxidants
are often reducing agents such as thioalcohols, ascorbic acid, or polyphenols.
Because to their cellular protective abilities, antioxidants are widely
utilized in dietary supplements and have been investigated for the prevention
of diseases like cancer, coronary heart disease and many others related to
oxidative stress. Polyphenolic compounds such as chalcones, flavonoids and many
other flavonoids are regarded as excellent antioxidants because they have shown
to have  enormous radical scavenging ability.5 In flavonoid
system, 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 the
formation of the semiquinone6. Therefore
flavonoids can be considered as hydrogen donating antioxidants because of their
reducing properties of the multiple hydroxyl groups attached to their aromatic
ring systems, along with their ability to delocalize over the resulting
phenoxyl radical within their structure7.

Flavonoids and UV protection

UV radiation reaching on
the earth’s surface is divided into three types of radiation. One with lower
energy 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 most
severe damage and is increasing due to ozone reduction, which is the primary
shield of solar radiation. Exposure to UV-B radiation may cause cellular damage
and 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 light
in the280 – 315 nm regions thus preventing UV-B radiation from reaching the
mesophyll and protecting the plant tissue from damage and ensuring favorable
plant photosynthesis8.

Estimation of Antioxidant Activity using UV-Visible Spectroscopy

UV-visible spectroscopy
technique is extensively used for the determination of antioxidant activity of
many antioxidants. In this case solution of an antioxidant is added to the
solution of free radicals which are used as model to study scavenging effects
of antioxidant. The absorbance of the UV peaks of free radicals is decreased when
the solution of an antioxidant is added and this continues to happen on
subsequent addition of antioxidant solutions to the solutions of free radicals.
This decrease in the absorbance of free radicals shows their scavenging or stability
with the addition of antioxidants. Percent scavenging (%RSA) is evaluated from
the data of UV –visible spectroscopy, which is then plotted against the
concentration of antioxidant. From the slope of this plot one can evaluate the
antioxidant activity of antioxidant in terms of IC30 and IC509.

Estimation of Antioxidant Activity using Cyclic Voltammetry

voltammetry (CV) is based on linear sweep potential in time. Cyclic
Voltammetry were used to investigate flavonoid characteristics by peak
potential, number and position of peaks at those potentials, the height of
current at each peak potential for all the flavonoids. According to literature,
from the first anodic peak, we get the most useful information about the antioxidant
activity of compound, it is assumed that the lower the oxidation potential of
the first anodic peak appeared, the greater the ability of a compound to donate
an electron thus behave as an antioxidant10. This was
suggested on the basis that both electrochemical (EC) oxidation and hydrogen donating
free radical-scavenging breaks the same phenolic bonds between oxygen and
hydrogen, producing the phenoxyl radical and H’, which consists of an electron
(e-) and an H + ion11.

Using the technique of
cyclic voltammetry, the antioxidant activity of antioxidants is evaluated. When
antioxidant solution is added to the solution of free radicals, the anodic peak
current of free radicals is decreased with addition of each aliquot. From the data
of cyclic voltammetry percent radical scavenging activity (%RSA) in terms of
IC30 and IC50 can be calculated in order to measure the effectiveness of an

Evaluation of pKa value

large number of natural as well as synthetic compounds administrates many of
their physical, chemical and biological properties. So these properties are
determined by pKa at any pH12. Different methods
has been employed to find pKa value, the most common methods are potentiometric
titration where pKa is obtained from titration curve, spectrophotometric
titrations where spectra is obtained for each point in titration and change of
absorbance is plotted against pH and capillary electrophoresis in buffer
solution where pKa is evaluated from mobility of ions. Out of all these
techniques, pKa was found from UV spectroscopy as it is a sensitive technique
and small sample is needed. A chromosphore must be attach with a compound to
find pKa value from UV spectroscopy13.  The DVP studies and UV spectroscopy was
employed for drug at various pH in BRB buffer14.

Anion (O2-)

Superoxide anion (O2?)
is very important free radical as it is concerned with several diseases like
cancer, denaturation of proteins, aging and lipid peroxidation. This chemical
species is generated as a result of reduction of molecular oxygen15.

of the very major and key source for the natural production of superoxide anion
is electron transport chain reaction that takes place in mitochondria of a
cell. There is a channel of electron acceptors in mitochondria which undertake
periodic reduction-oxidation reactions, however there is possibility that free
electrons may be transferred to molecular oxygen instead of next electron
acceptor which later on generates superoxide anion. Many iron-Sulphur clusters
in a cellular respiratory machine of a body are also attributed to the generation
of poisonous superoxide ions via side reactions with oxygen. Generation of superoxide
Anion (O2?) free radicals from the mitochondria, is considered
as their major intracellular formation under physiological conditions.
Approximately 1-2 % of daily intake of molecular oxygen when goes to
mitochondria results in production of 160-325 mmol of superoxide anion
approximately in a 60 kg woman whereas in 80 kg man 215-420 mmol of superoxide
is produced on daily basis16.


free 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 of
intervascular fluid and blood cells into the ischemic space ends up in the
elementary events of reperfusion injury. Several mechanisms are projected to
support the toxicity of those chemical species. Experimental evidences suggest
that xanthine oxidase enzyme is that the major supply for the assembly of
damaging and poisonous oxygen free radicals that cause ischemia-reperfusion17.

DFT and Theoretical Studies

redox reactivity of phenolic compounds (ArOH) mostly follow two different
chemical pathways:

ArOH + R• ? ArO• + RH

ArOH +R• ? ArOH+• + R? ?
ArO• + RH

1 presents the homolytic dissociation of the O?H bond. This reaction can occur
on each OH group present on phenolic compound (the flavone, for example) and it
is governed by the BDE (Bond Dissociation Energy) of the OH groups and by the
enthalpy ?E1 of reaction 1. The BDE is an intrinsic thermodynamic parameter,
whereas enthalpy of reaction 1 is dependent on the radical that is reacting
with 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.

properties of phenolic compounds have been established theoretically, using
semi-empirical Hartree-Fock (HF) methods19 and more recent
method is by density functional theory (DFT)20. DFT seems like
to be the most reliable approach to achieve reliable BDE (Bond Dissociation
Energy) values that are close to experimental data, while semi-empirical
methods underestimate the ? electron delocalization and yield non-planar
geometries for flavonoids and flavones (i.e., flavonoids having a 2,3-double
bond).Recently a valid estimation of the BDE for phenolic compounds have been
done by DFT and it highlights the role of the OH groups present on B-ring21 and the 3-OH
group in flavonoids22.

geometries and energies of the phenoxyl radicals were obtained after the H atom
was removed from each OH group of the optimized structure of the parent
molecule and using an unrestricted approach, which is more relevant to DFT

vindicate UV/Vis spectra of atomic and molecular systems, a large panel of
(time-dependent) quantum chemical methods are present. Several years ago it was
reported on the electronic structures and spectra including molecular orbital
(MO) analysis were based on Hückel and Pariser–Parr–Pople methods. Later a
semi-empirical study confirmed the role of hydroxylation in the B-ring in
polyphenols. Excited states were more recently scrutinized with SE–CI (single
excitation–configuration interaction) evaluation based on density functional
theory (DFT) geometries. Over the past years time-dependent (TD) DFT has developed
as 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 spectra
of a series of polyphenol and their derivatives, whereas Hartree–Fock/CIS and
semi-empirical approaches were found insufficient to reproduce the experimental
data results. TD-DFT was also used to examine the optical properties of some
cations. 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-initio
methods thus it is a valuable tool to investigate large series of many

Drug-DNA Binding Studies

and replication are important for survival of cells and proliferation. They also
help body to function normally. Transcription or replication of DNA starts only
when it receives a signal, which is mostly in the form of a binding of
regulatory protein to a specific region of the DNA. Thus, if the specificity
and strength of binding with this regulatory protein can be imitated by a small
molecule, then DNA function can be modulated, inhibited or activated
artificially by binding this molecule as an alternative of the protein. When
activation or inhibition of DNA function is required then the small natural
molecules can act as drug to treat or control a particular disease. Chemotherapy
is the technique in which these foreign molecules stop the replication of DNA such
as drugs. The molecules have anti-cancerous properties because they can interact
as well as inhibit further replication of DNA. Due to their broad
pharmacological activity flavonoids have got considerable interest among these
large family of molecules25.

fact, they are best recognized for their antioxidant properties, and can act as
a reducing agents, hydrogen atom donors, free radical quenchers and metal ion
chelators and this may justified for the anti-tumor activities of flavonoids26.

on binding mechanism of these anticancer drugs with the DNA has been recognized
as one of significant research topic during past several years. The effect of
DNA interacting drug can be perceived experimentally at molecular, cellular and
clinical levels. Molecular level is the most basic level which involves direct
chemical interaction of drug with the giant DNA molecule. A number of
techniques have been developed to examine this level of interactions are X-ray
Crystallography, Mass Spectroscopy, foot printing technique, Electrochemistry,
UV-Vis and fluorescent Spectroscopy, NMR, FTIR, Surface Plasmon Resonance,
Equilibrium dialysis and Molecular Modelling Studies27.

drugs may be categorized into two extensive classes, one is covalently binding
drugs and the other non-covalently binding drugs. The covalently binding drugs
make covalent bonds to nitrogenous bases forming inter-strand or intra-strand
cross-linking producing more distortion to the DNA structure. Covalent DNA
binding cannot usually be reversed28 and it can
leads to complete inhibition of DNA functions, leading to cellular death. Due
to such firm and strong (as well as the non-selective) binding, covalent
binders cause lethal side effects in treated patients.

non-covalently binding drugs are classified only to three smaller groups28.

Intercalating drugs (2) minor-groove binders and (3) drugs forming
electrostatic interactions with the negatively cha`rged sugar phosphate
backbone of DNA molecule.

drugs get stacked between the two neighboring base-pairs present in DNA double
helix. They have planar heterocyclic groups and loosely bind to the nitrogenous
bases of DNA through ?-? stacking interactions. Thus though they make non-covalent
interactions, the intercalators form a rather strong perturbations to DNA structure29.

the minor groove is narrow so the edges of bases are more accessible in major
groove for the larger DNA-binding proteins such as transcription factors. While
small-molecular drugs, favorably bind at the minor groove, which are narrower
and provides better van der Waals contact of the drug peripheral with the
sugar-phosphate walls of the minor groove, weak hydrogen bond interactions
enhance the net drug-DNA binding with these interactions30.

spectroscopic technique was used to study the interaction of the flavanone with
ds.DNA. In present work the compound under investigation is UV active and
exhibit a UV spectra with a peak at 287nm. UV-Vis spectroscopy has been
employed to study the interaction of drug with ds.DNA The spectrum of given compound
change after the subsequent addition of DNA and from the difference in
absorbance, binding constant was calculated which gives an clue of strength of
binding with DNA.

Cyclic Voltammetry, the I-E response of compounds was observed. It was found
that this compound undergo electrochemical oxidation. After the addition of DNA
to the solution of compound prepared in PBS buffer, a change in i-E response was
observed which shows the interaction of compound with ds.DNA. By change in i-E
response the amount of compound that have been interacted can be known. Diffusion
coefficients and binding constant was also determined experimentally. The
electrochemical data supported the spectroscopic surveys.

docking and QSAR studies were carried out for the investigation of interactions
between the compound and the double stranded DNA using MOE (Molecular Operating
Environment) package. Different electronic descriptors and binding constant was
calculated theoretically. The higher binding constants shows the stronger

Docking with Enzyme Complex

approaches are becoming progressively important and harmonizing to wet
laboratory techniques in elucidating the structure and function of many
biomolecules. Molecular docking is an often used method in structure-based rational
drug design. IT is beyond doubt early efforts were caught up by limited
possibilities in computational possessions, due to recent developments in
extraordinary performance computing computer-generated screening methods became
more and more proficient. These computational methods contributed to the progress
of numerous drugs and drug runners that advanced to clinical field. Theoretical
Molecular Docking Studies of Drug with DNA Enzymes (DNA polymerase, Topoisomerase
and Helicase) were carried out. These enzymes are essential for DNA
replication, repair and recombination. A strong interaction of drug was found
with these enzymes and their binding constant with drug was calculated later

Present Work

the present study, the
antioxidant activity of the compound and ascorbic acid was determined by using
UV Visible spectroscopy and Cyclic Voltammetry with DPPH radical. .
Theoretically, antioxidant activity is estimated by DFT calculations by
calculating the bond dissociation enthalpy (BDE) of each O?H and Gibbs free
energy (?G) of reaction against DPPH radical. By comparing the theoretical and
experimental results, the predictive power of DFT calculations is established.

level 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 interaction
between DNA enzymes and Drug.