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β-CYCLODEXTRIN INCLUSION COMPLEX OF

CURCUMIN
S. Swaroop
School of Chemical Sciences
Mahatma Gandhi University, Kottayam, Kerala

and

Beena Mishra and K. Indira Priyadarsini

Radiation & Photochemistry Division
Bhabha Atomic Research Centre

This paper was adjudged as one of the Best Posters at

the “Trombay Symposium on Radiation & Photochemistry (TSRP-2006)” held
at BARC, Mumbai, during January 5-9, 2006

Abstract

Formation of inclusion complex of curcumin with β-cyclodextrin ( β-CD) has been characterized
by absorption and fluorescence spectroscopy. From temperature-dependent fluorescence measurements,
the thermodynamic parameters ∆H and ∆S, for the complexation were estimated. Kinetics of binding was studied
by stopped flow technique, from which the binding constants for inclusion complex was estimated.
Further, influence of such inclusion complex on change in superoxide radical scavenging property
of curcumin was examined using xanthine/ xanthine oxidase assay.

Introduction
and therefore act as suitable tools for following the

Curcumin is a lipid soluble polyphenolic antioxidant
from curcuma longa, turmeric, with antioxidant, anti-
inflammatory, anti-carcinogenic properties [1]. One of
the disadvantages of curcumin, limiting its physiological
activities, is its insoluble nature in aqueous solutions. It
is possible to increase its water solubility by forming
inclusion complexes with water-soluble hosts [2]. There
are several studies on host-guest complex formation of
curcumin [2]. Formation of such host-guest systems can
be probed by following absorption and fluorescence
changes in curcumin. The photophysical properties of
curcumin vary significantly depending on the medium
inclusion behavior to different systems [3]. Cyclodextrins
are one of the most widely used synthetic model host
cavities, which provide a conical cavity for the water
insoluble guests to be encapsulated, thereby making
them water soluble [4].
Here, we made an attempt to estimate the binding
constant of 1:1 curcumin and β-cyclodextrin (β-CD)
complex by following the absorption, fluorescence
properties and also by stopped-flow studies.
The superoxide radical scavenging ability of such a
complex was compared with that of free curcumin.

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Materials and Method
Curcumin, β-CD, xanthine, xanthine oxidase and
cytochrome c, were of the highest purity available from
the local market and used as received. Nano pure water
from Millipore Q system & Spectrograde solvents from
Spectrochem India were used for the preparation of
solutions.
The absorption spectra were recorded on a JASCO
V-530 spectrophotometer and emission spectra on
Hitachi 4010 spectrofluorimeter. The stopped flow
experiments were carried out using SX.18MV stopped
flow reaction analyzer (from Applied Photo Physics Ltd.
UK) with absorption detector.
Superoxide was generated by enzymatic methods
employing xanthine and xanthine oxidase assay.
For binding studies, the stock solution of curcumin in
DMSO was diluted to 100 µM in 10 mM β-CD such
that the solution contained less than 1% DMSO solvent.
This solution was mixed with the above matrix to obtain
solutions of curcumin with varying concentrations.
Results and Discussion
Absorption spectral studies
Curcumin in the absence of β-CD shows absorption
maximum at 420 nm and β-CD does not absorb
appreciably in the wavelength region. On addition of
β-CD, the absorption maximum of curcumin showed
red shift from 420 nm to 430 nm and the absorbance at
430 nm was nearly doubled on addition of 10mM
β-CD. This spectral change is attributed to a complex
formation between curcumin and β-CD, represented by
equation (1):
(1)
The equilibrium constant for the above equilibrium is
given by equation (2)
(2)
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Here n indicates number of β-CD molecules involved in
complexation. For the above equilibrium, assuming 1:1
complex formation, binding constant was estimated by
employing the double reciprocal plot known as Benesi-
Hildebrand equation [3]. Thus linear fit of the double
reciprocal plot of absorption changes (∆A) at 430 nm
as a function of β-CD concentration (90.5 to 10 mM)
gave binding constant (K) to be 6.9 ± 0.3 x 102 M-1.
Fluorescence spectral studies
The changes in fluorescence intensity and spectra of
curcumin were also used, to evaluate the binding
constant. For this study, curcumin was excited at 350
nm, the isosbestic point for the complex formation, in
the absence and presence of different concentrations of
β-CD (0-10 mM). At pH 7, curcumin has very weak and
broad fluorescence maximum at 550 nm (Fig.1). The
fluorescence intensity was found to increase with
increasing concentration of β-CD. Also in presence of
10 mM β-CD the fluorescence maximum shifted from a
broad featureless band to a well-defined blue shifted
band λmaxem at 520 nm. The blue shift (~30 nm) in the
fluorescence spectrum confirms the binding of curcumin
to the hydrophobic cavity of β-CD. Even at the limiting
solubility of β-CD (10 mM), no saturating value for
fluorescence was observed. The fluorescence changes
are related to the association constant (K) for the 1: 1
complex as [5]
(3)
Were Iof and Icomplex are the respective fluorescence
intensities at 520 nm in the absence and presence of
different concentrations of β-CD. Inset of Fig. 1 shows
the variation of fluorescence intensity of curcumin at
520 nm as a function of β-CD concentration (0 to 10
mM).
Fitting this data to equation (3) by using non-linear
least square fitting gave binding constant (K) of
2.6 ± 0.3 x 102 M-1.
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Fig. 1: Fluorescence spectra of curcumin

(10 µM) in the absence (a) and in the presence of
different concentrations of b-ββD
(b = 0.5 mM, c= 1 mM, d = 2 mM,
e = 4 mM, f = 6 mM, g = 10 mM).
Inset shows plot of fluorescence intensity
as a function of β-CD concentration,
which is non-linear fitting to equation (4)
Fig. 2: (a) Absorbance-time profile obtained
on mixing of 10 mM curcumin with 2 mM β-CD
at 430 nm. (b) Variation in kobs as
a function of sum of equilibrium
concentration of curcumin and β-CD,
line shows fitting to equation (5).

Kinetics of binding of curcumin to β-cyclodextrin constant as a function of sum of the equilibrium

The kinetics of binding of curcumin to β-CD was followed
using stopped-flow spectrometer. For this study, aqueous
solutions of 10 µM curcumin containing 1% methanol
was mixed with different concentrations of β-CD
(0.5 – 10 mM) in the stopped flow cell and the absorption
changes at 430 nm were monitored as a function of
time. The absorption-time profile obtained as shown in
the inset (a) of Fig. 2 was fitted to an exponential
function, to obtain the observed first order rate
constant (kobs).
For the formation of 1: 1 complex, kobs is related to the
equilibrium concentration of curcumin and β-CD
according to equation (4) [3].
(4)
kf and kb are the forward and backward rate constants
for the equilibrium in equation (1), where n =1. Fig.
2b, shows linear plots for the change in first order rate
concentrations of β-CD and curcumin. From the
intercept and slope of the plot, the binding constant for
1:1 complex is found to be 5.2 ± 0.2 x 103 M-1. This
value is higher than that determined by absorption and
fluorescence method. This higher value indicates that
there are more than one kinetic steps involved in complex
formation.
Effect of temperature on the binding of curcumin
with β-CD
Thermodynamic parameters associated with binding of
curcumin with β-CD for 1:1 stoichiometry have also
been calculated by estimating the binding constant at
different temperatures, measured at 293, 308 and 323
K. The binding constant at each temperature was
determined by following the fluorescence changes in
curcumin with β-CD concentration and fitting the data
to equation (3).

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The equilibrium constant was found to decrease with
increasing temperature and according to equation (5),
the plot of logarithm of binding constant as a function
of inverse temperature gave a linear plot, whose intercept
and slope corresponds to DS/R and –DH/R respectively
[5]. From this enthalpy (DH) and entropy (DS) values
were estimated to be –8.5 kJ/mol and –0.02 kJ/mol/K
respectively.
(5)
Thus under the present conditions, the DH values for
the transfer of curcumin from water to β-CD is negative
and ∆S is also negative but to a very small extent. The
main driving force for complex formation could,
therefore, be the release of enthalpy rich water from the
cyclodextrin cavity. The water molecules located inside
the cavity cannot satisfy their hydrogen-bonding
potentials, therefore are of higher enthalpy. The energy
of this system is lowered when suitable guest
molecules, that are less polar than water, replace these
enthalpy-rich water molecules.
Effect of complexation on superoxide scavenging
ability of curcumin
After studying the photophysical properties and
estimating the thermodynamic properties, we studied
its free radical scavenging ability by following its reactions
with enzymatically-produced superoxide radicals.
Xanthine oxidase is an enzyme that converts xanthine to
uric acid, during which superoxide radical is generated
[6]. This is one of the important sources of superoxide
ions in living systems. In presence of cytochrome c,
superoxide radical reduces the Fe3+ in cyctocrome c to
Fe2+ form, which absorbs at 550 nm and is a direct
measure of superoxide radical formation in the system.
When antioxidants capable of reacting with superoxide
radical are added to the system, due to the competition
for the reaction of superoxide radical, the yield of Fe2+
form and the absorbance at 550 nm decreases [6]. The
percentage inhibition (%I) of superoxide radical inhibition
can be calculated from the following equation (6)
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(6)
Here A0 and As are the maximum absorbance values at
550 nm in the absence and in the presence of curcumin-
β-CD complex. By plotting the %I as a function of the
complex concentration, the IC50 value was estimated to
be 59 µM. Under similar conditions, the absorbance
changes were also monitored in the presence of
uncomplexed curcumin, from which the IC50 value for
curcumin was estimated to be 88 mM. This clearly shows
that on complexation with β-CD, the superoxide radical
scavenging ability of curcumin is increased.
Conclusions
Curcumin, a phenolic and lipid soluble antioxidant, has
been examined for its complexing ability with β-CD. The
binding constant of the complex was determined by
following absorption, fluorescence spectral changes and
stopped-flow studies. Stopped-flow kinetic studies which
showed binding between curcumin and β-CD, have been
estimated. From the temperature dependent studies, the
enthalpy and entropy changes on complexation with
β-CD could be estimated. From all these results it is
concluded, that β-CD complexation of curcumin takes
place in the hydrophobic cavity of β-CD. Such a complex
formation changes curcumin into a water-soluble form.
Superoxide scavenging ability of curcumin on
complexation with β-CD increased as compared to
uncomplexed curcumin.
Acknowledgements
The authors are thankful to Dr S. K. Sarkar, Head,
Radiation & Photochemistry Division for encouragement
and support.
References
1. Aggarwal, B. B., Sundaram, C., Malani, N. and
I chikawa, H. (227) Curcumin: the Indian Solid
Gold. in: The Molecular Targets and Therapeutic
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Uses of Curcumin in Health and Disease
(B.B. Aggarwal, S.Young-Joon, S. Shishodia,
Eds),Springer, NY USA, pp. 1-76.
2. Baglole, K. N., Boland, P. G. and Wagner, B. D.
(2005) J. Photochem. Photobiol. A 173
173, 230.
3. Barik, A., Priyadarsini, K. I. and Mohan,
H. (2003) Photochem. Photobiol. 77 77, 597.
About the Authors
Mr. Swaroop, S.,, received his
M.Sc. from School of Chemical
Sciences, Mahatma Gandhi
University Kottayam, Kerala in
2005. During his M.Sc. he
worked under the guidance of
Dr. K. I. Priyadarsini on the
project entitled “Photophysical
and Radiation Chemical Studies on curcumin-â-
cyclodextrin complexes”, at Radiation and
Photochemistry Division, Bhabha Atomic Research
Center, (BARC). Currently he is working at School of
Chemical Sciences, Mahatma Gandhi University,
Kottayam on electron transfer reaction of
polyphenols.
Dr B. Mishra received her M.Sc.
degree in Inorganic Chemistry
from Institute of Science,
Mumbai, in 2001. She was a DAE
research fellow working in
Radiation and Photochemistry
Division, BARC. She obtained her
Ph.D. degree in chemistry from
212
4. Li, S. and Purdy, W. C. (1992) Chem. Rev. 92 92,
1457.
5. Singh, M. K., Pal, H.,Koti, A. S. R., and Sapre,
A. V. (2004) J. Phys. Chem. A. 108
108, 1465.
6. Hodges, G. R., Young, M. J., Paul, T. and Ingold,
K. U. (2000) Free Radic. Biol. Med. 29 29, 434.
University of Mamba in 2007. She joined same
division as a K. S. Krishnan Research Associate.
Presently she is working on free radical and
biochemical studies of organoselenium
compounds.
Dr K. I. Priyadarsini joined
Bhabha Atomic Research Centre
in 1983. She is currently working
on the elucidation of mechanisms
of antioxidant action involving
natural products and herbal
extracts with potential
application as radioprotectors, employing electron
pulse radiolysis and in vitro biochemical studies. Dr
Priyadarsini has co-authored more than 100 papers
in peer reviewed international journals on radiation
chemistry, photochemistry and radiation biology.
She has been elected as the Fellow of the National
Academy of Sciences, India and is a recipient of the
Homi Bhabha Science & Technology Award.
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