Spectral investigation and structural characterization of Dibenzalacetone: β-Cyclodextrin inclusion complex

Article abstract of DOI:10.1016/j.molstruc.2014.04.004

The interaction of Dibenzalacetone (DBA) with β-Cyclodextrin (β-CD) producing an inclusion complex was carried out by co-precipitation method. The binding constant was determined using steady state and time-resolved fluorescence spectroscopy and the results suggested that the inclusion complex preferred 1:1 stoichiometry. The complex was characterized by UV-Visible, infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). The morphological characteristics of the solid complex were analyzed by Scanning electron microscope (SEM) and Atomic force microscope (AFM). The structure of 1:1 inclusion complex of DBA with β-CD is proposed. The Docking study reveals that this structure was found to be highly probable and energitically favorable model.

Full text of DOI:10.1016/j.molstruc.2014.04.004

Accepted Manuscript
Spectral investigation and structural characterization of Dibenzalacetone : β-
Cyclodextrin inclusion complex
R. Periasamy, R. Rajamohan, S. Kothainayaki, K. Sivakumar
PII:
S0022-2860(14)00357-3
DOI:
http://dx.doi.org/10.1016/j.molstruc.2014.04.004
MOLSTR 20511
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
25 October 2013
30 March 2014
3 April 2014
Please cite this article as: R. Periasamy, R. Rajamohan, S. Kothainayaki, K. Sivakumar, Spectral investigation and
structural characterization of Dibenzalacetone : β-Cyclodextrin inclusion complex, Journal of Molecular
Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.04.004
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Spectral investigation and structural characterization of
Dibenzalacetone : β-Cyclodextrin inclusion complex.
1
2
1*
3
R. Periasamy , R. Rajamohan , S. Kothainayaki and K. Sivakumar
1
Chemistry Section, FEAT, Annamalai University, Annamalainagar, Tamil Nadu, India – 608002
Department of Chemistry ,S.K.P. Institute of Technology, Tiruvannamalai- 606 611
Department of Chemistry, Faculty of Science, SCSVMV University, Enathur, Kanchipuram 631 561,Tamilnadu
2
3
Abstract
The interaction of Dibenzalacetone (DBA) with β-Cyclodextrin (β-CD) producing
an inclusion complex was carried out by co-precipitation method. The binding constant was
determined using steady state and time-resolved fluorescence spectroscopy and the results
suggested that the inclusion complex preferred 1:1 stoichiometry. The complex is characterized
by UV-Visible, infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and
X-ray diffractrometry (XRD). The morphological characteristics of the solid complex were
analyzed by Scanning electron microscope (SEM) and Atomic force microscope (AFM). The
structure of 1:1 inclusion complex of DBA with β-CD is proposed. The Docking study reveals
that this structure was found to be highly probable and potentially favorable model.
_
_____________________________________________________________________________
*Corresponding author
Tel:- +91 9486456733
Email: kothaichemish@yahoo.co.in (S. Kothainayaki)
Keyword: . 1,5-Diphenyl-1,4-pentadien-3-one, β-Cyclodextrin, Inclusion complex, Atomic force
microscopy, Fourier Transform Infra red , Thermal analysis, Molecular Docking

2
1. Introduction
Cyclodextrins (CDs) are cyclic oligosaccharides which have six, seven, and eight
D-glucopyranose units for α, β, and γ-cyclodextrin respectively. They form inclusion complexes
with guest molecules of suitable polarity and size [1-6], because of their special molecular
structure with a hydrophobic central cavity and a hydrophilic outer surface. The difference in
polarity within the molecule and the restricted space provided by cyclodextrin cavity
significantly influences photo physical and photochemical processes of guest molecule.
Chalcones are used as therapeutic agents for cardiovascular diseases [7] and exhibit
interesting biological properties [8-10]. They posses high photosensitivity, thermal stability and
are used in developing various crystalline electro-optical devices [11]. It is well known that.
Dibenzalacetone [1,5-Diphenyl-1,4-pentadien-3-one] (DBA) is a component of sun screens
because it is capable of absorbing UV light. It is also used as ligand in organometallic
chemistry. Dibenzalacetone (DBA) is less fluorescent in aqueous solution, but its fluorescence
intensity increases by the addition of β-Cyclodextrin (β-CD). Now-a-days inclusion
complexation with β-CD is widely used in various fields such as medicine [12-15], chemistry
[
16,17], agriculture [18] etc. Ramamurthy, et al., [19,20] have exploited the use of CDs as host
to examine photochemical and photo physical processes that occur in molecules complexed
within them and to compare their behavior in aqueous solution and solid state. Earlier we
reported spectro fluorometric study on inclusion complexation of 2-amino-6-fluorobenzothiazole
[21], Benzisothiazole [22] and 3-amino-5-nitro benzisothiazole [23] with β-CD. In the present
work we report inclusion complexation of DBA with β-CD in solution and solid state.

3
2
. Experimental
.1 Instruments
2
Absorption spectrum was recorded with
a
Hitachi model U-2001
spectrophotometer. Fluorescence measurements were made using
a
Jasco.FP-550
spectrofluorimeter. The isosbestic wavelengths were used as the λexi for measuring fluorescence
intensities at any analytical wavelength. pH values were measured by using an Elico LI-105
model pH meter. Fluorescence lifetimes were determined using a time-correlated Pico second
Photon counting Spectrofluorimeter (Tsunami, spectrophysics, USA). FT-IR Spectra were
obtained with Avtar-330 FT-IR Spectrophotometer using KBr pellet. The spectra were recorded
-
1
in the frequency range 4000-400 cm . Microscopic morphological (SEM) measurements were
performed with JEOI-JSM 5610 LV. Surface morphology was also recorded using AGILENT-N
9
410 A SERIES 5500 AFM. The samples were analyzed by DSC using a Mettler Toledo SR
system. The samples (5gm each) were placed into pierced aluminum container which found use
with hydrate and solvate loss as well as to relieve internal pressure in the case of ordinary
sample. The studies were performed under static air atmosphere in the temperature range of
°
°
°
2
0 C to 200 C at a heating rate of 10 C/min. The peak temperatures were determined after
calibration with standard. The powder XRD patterns of DBA, physical mixture and the complex
were recorded by using automated Philips Holland-PW 1710 scanner with filter Cu radiation
°
over the interval 5-60 /2θ. The operation data were as follows: voltage 35 KV, current 20 mA,
°
filter Cu and scanning speed 1 /min.
2.2 Reagents
Dibenzalacetone (DBA) was synthesized by Aldol condensation reaction carried
out between benzaldehyde and acetone in alkaline medium [24]. Then it was purified by
repeated recrystalisation from ethanol. β-Cyclodextrin was purchased from S.D. fine chemicals
and used as received. Triply distilled water was used for the preparation of experimental

4
3 4
solutions. pH solution (6.8) was prepared by using appropriate amounts of NaOH and H PO .
-
3
3
The concentration of experimental solutions was in the order of 3×10 mol/dm . Solutions for
UV study and fluorimetric titration were prepared just before taking measurements.
2.3 Preparation of Solid inclusion Complexes of DBA with β-CD
Solid DBA: β-CD complex was prepared using established standard
co-precipitation method [21-23]. DBA and β-CD with 1:1 molar ratio were accurately weighed
separately. Saturated β-CD solution was prepared in water. Then DBA solution in methanol was
added to β-CD solution slowly till a suspension was formed. The suspension was stirred for
3
0 min at 40 °C and kept stirring at room temperature for 24 h. After getting clear solution
(without any suspended particles) it was kept in lyophilizer for 24 hours. The precipitate
o
obtained was filtered through 0.45 µm membrane filter and dried at 40 C in an oven for 24 h.
The dried complex was ground to fine powder. Physical mixture of DBA: β-CD in 1:1 molar
ratio was prepared by homogeneously blending previously sieved (63-160 μm) individual
components in a mortar.
2.4 Job’s continuous variation method
Determination of stoichiometry of the inclusion complex was provided by the
continuous variation technique (Job’s plot) based on the difference in absorbances, ∆OD
∆OD=A -A). of DBA observed in the presence of β-CD. Equimolar solutions of the DBA and
the corresponding β-CD was prepared and mixed to standard volumes and proportions in order
that the total concentration remained constant ( [DBA] + [β-CD] = Constant ) but the ratio of
the initial concentrations varied between 0 and 1. ∆OD values in the preparation of guests were
calculated by measuring the absorbance of DBA in the absence (A ) and presence (A) of the
(
o
t
t
o
corresponding concentrations of β-CD. Also, an equimolar solution of β-CD was used as a
blank, to take into account its refractive index.

5
2.5 Molecular Docking Study
The most probable structure of the DBA: β-CD inclusion complex was
determined by molecular docking studies using the Patch Dock server [25] . The 3D structural
data of β-CD and DBA was obtained from crystallographic databases. The guest molecule
(
DBA) was docked in to the host molecule (β-CD) cavity using Patch Dock server by submitting
the 3D co-ordinate data of DBA and β-CD molecules. Docking was performed with complex
type configuration settings. Patch Dock server followed a geometry-based molecular docking
algorithm to find the docking transformations with good molecular shape complementarity.
Patch Dock algorithm separated the Connolly dot surface representation [26, 27] of the
molecules into concave, convex and flat patches. These divided complementary patches were
matched in order to generate candidate transformations and evaluated by geometric fit and
atomic desolvation energy scoring [28] function. RMSD (root mean square deviation) clustering
was applied to the docked solutions to select the non-redundant results and to discard redundant
docking structures.
.
3. Results and discussion
3.1 Interaction of DBA with β-CD in solution
The absorption maxima, and fluorescence maximum of DBA at different
concentrations of β-CD at pH 6.8 is given in Table 1. Upon increasing the concentration of
β-CD, the absorption maxima at longer wavelength are blue shifted with a gradual decrease in
-3
their absorbance (Fig.1) up to the concentration (12×10 M). This behavior has been attributed
to the enhanced dissolution of the guest molecule by inclusion complexation [22] through the
hydrophobic interaction between the guest molecule and β-CD.
The binding constant and stoichiometry of DBA: β-CD complex can be
determined from the changes in absorbance of DBA by the addition of β-CD using

6
Benesi-Hildebrand equation [BH]. BH equations for 1:1 and 1:2 complex [29] are shown by
eqns (1) and (2) respectively.
1
/(A-A
/(A-A
o
o
) = 1/∆ε + 1/K[DBA]
) = 1/∆ε + 1/K[DBA]
o
o
∆ε[β-CD]
o
o
------------(1)
2
1
∆ε[β-CD]
------------(2)
Where (A-A
o
) is the difference between the absorbance of DBA, in the presence and absence of
β-CD. ∆ε is the difference between the molar extinction co-efficient of DBA in the presence and
absence of β-CD. [DBA]
o
and [β-CD]
o
are the initial concentrations of DBA and β-CD,
2
respectively. A good linear correlation (R = 0.9919) is obtained when 1/(A-A
o
) is plotted
against 1/[β-CD] [Fig.1.inset], whereas a non-linear plot is obtained when 1/(A-A
o
) is plotted
2
against 1/[β-CD] . This shows that the complex formed between DBA and β-CD has 1:1
stoichiometry. The binding constant value ‘K’ for 1:1 inclusion complex is determined from
-
1
slope of the plot according to eqn (3) .[30, 21-23] and it is found to be 34 ±2 M at 303K
K = 1/ Slope [A'-A ] ---------------------(3)
o
Fig.2 represents the fluorescence spectra of DBA at pH 6.8 with different
concentrations in aqueous solution of β-CD. The enhancement of fluorescence intensity of DBA
(both the maximum) with increasing concentration of β-CD up to the concentration
-
3
1
2×10 M suggests the formation of inclusion complex between DBA and β-CD. An increase in
the fluorescence intensity of guest molecule with increasing the concentration of host during the
formation of inclusion complex has been reported earlier [21, 31 and 32]. The β-CD dependence
of DBA fluorescence can be analyzed by BH equations for 1:1 [21, 22] and 1:2 complex [33]
using equations (4) and (5) respectively.
1
/I-I
o
=1/I'-I
o
+ 1/K (I'-I
o
)[β-CD]------------- (4)
2
1/I-I
o
=1/I'-I
o
+ 1/K (I'-I
o
)[β-CD] ------------- (5)

7
Where ‘K’ is binding constant, I
o
is the intensity of fluorescence of DBA without
β-CD. I is the intensity of certain concentration of β-CD, and I’ is the fluorescence intensity of
o
DBA with the highest concentration of β-CD. A non - linear relation is observed when I/I is
2
plotted against 1/[β-CD] , but a linear plot is obtained for I/I
o
Vs 1/[β-CD] (Fig.2a inset). This
shows that the complex formed between DBA and β-CD has 1:1 stoichiometry. From the slope
of the 1:1 plot, the Binding constant ‘K’ calculated using the equation (6) is found to be
-
1
5
1 ±2 M at 303 K.
K
o
= 1/ Slope [I'-I ] --------------------- (6)
3.1.1 Jobs Plot
The stoichiometry of the inclusion complex DBA: β-CD is also confirmed by using
Job’s continuous variation method using the absorption data. Fig.2b shows the change in optical
density (∆OD) against mole fraction of DBA. In this plot ∆OD maximum at 0.5 which indicates
the stoichiometry of the complex between DBA and β-CD is 1:1.
3.1.2 Analysis of fluorescence decay curve
The fluorescence lifetimes are often very sensitive indicators for exploring the local
environment around the excited state fluorophore [34, 35]. With the above point of view to
acquire a deep insight into the excited state photo physics of the guest and its alterations in β-CD
confinement, we have recorded the fluorescence lifetimes of DBA in water and at different
concentration of β-CD. The excitation wavelength was fixed at 320 nm and the emission wave
length was fixed at 340 nm. The fluorescence decay behaviors of DBA both in water and in
β-CD environments are found to be quite different.
The lifetime and amplitudes of the decay of DBA with and without β-CD are given in
Table.2. In water the fluorescence decay of DBA obtained from monitoring the emission at
3
1
40 nm is a single exponential with lifetime value 7.89 ns (τ ). However in the presence of

8
various concentrations of β-CD, decay curves gave a best fit for the biexponential decay with
lifetimes of τ
1
2
in the range of 7.64 ns to 0.1 ns and τ in the range of 2.3 ns to 2.96 ns. Triple
2
exponential analysis was also attempted, but χ was not improved. On increasing the
2
concentration of β-CD, the lifetime of the complex (τ ) is higher and its amplitude is also
increased. This is due to the formation of inclusion complex between DBA and β-CD (22, 23
2
and 36]. The χ values for the single and double exponential fittings were less than 1.2. The
observed enhancement in lifetime indicates that DBA molecule experiences less polar
hydrophobic environments within the β-CD cavity resulting in the interaction of non-radioactive
decay process. Further the increase in fluorescence lifetime is a result of the significant
interaction of DBA with hydrophobic β-CD cavity.
The ratio of the pre-exponential factors (B
components by the following eqn (7)
2 1
/B ) is related to the concentration of the two
B
2
/B
1
= C2Krε
2
/C
1
1 1
Kr -----------(7)
ε
Where C
molar extinction co-efficient at the excitation wavelength respectively. The subscript 1 and 2
refer the free and complex of DBA with β-CD respectively. Since Kr is a constant and ε =ε
[37]
1
, is the concentration of DBA, Kr and ε are the radioactive rate constant and the
1
2
,
2 1 2 1
Eqn (7) is simplified as B /B = C2/C1. In excess of [β-CD] with respect to the DBA, B /B
can be written as Eqn (8).
2 1
B /B = K [β-CD] --------------(8)
2 1
Fig.2c shows the plot of B /B Vs [β-CD] . The linearity in the plot with the correlation
coefficient of 0.9947 reflects the formation of 1:1 complex between DBA and β-CD. The
-
1
binding constant K calculated for this complex is 51 ±2 M at 303 K.

9
3.1.3 Spontaneity of inclusion complexation reaction
The Gibbs free energy change (ΔG) for this inclusion process was determined from
the binding constant value using the following equation at 303K [21-23, 30]
∆
G = - RT lnK -------- (9)
Where ∆G is the Gibbs free energy change, R is the gas constant [J/mol.K] T is the temperature
-
1
in Kelvin and “K” is the binding constant in mol . When the “K” value from absorption data is
-
1
used in the equation (9), it gives the ∆G values -8.9 KJ mol and the “K” value from
-
1
fluorescence data, it is -9.8 KJ mol .
The negative values indicate that inclusion complexation reaction proceeded spontaneously
at 303 K.
3
.2 Interaction of DBA with β-CD in Solid state
.2.1 FT-IR Spectral analysis
3
The solid inclusion complex prepared by co-precipitation method is analyzed by
FT-IR spectroscopy. The FT-IR spectra of β-CD, pure DBA, physical mixture of DBA and
β-CD and solid complex of DBA with β-CD are shown in Fig.3. When the guest molecule is
encapsulated by means of inclusion complexation reaction, the absorption bands resulted from
the included part of guest molecule are generally shifted in their position or intensity may be
altered [37]. If β-CD and DBA form a solid inclusion complex, the non-covalent interactions
between β-CD and DBA such as hydrophobic interactions, van der Waals interactions and
hydrogen bonding lower the energy of the included part of DBA, thus reducing the absorption
-
1
intensities of the corresponding bands. The O-H stretching frequency appears 3361 cm in
-
1
-1
β-CD, it appears at 3369 cm in physical mixture. It is shifted to 3408 cm with reduced
intensity in the case of solid inclusion complex. Slight shifts are observed in C-H, C=O
stretching frequencies of DBA and solid inclusion complex. The aromatic ring C-C stretching

1
0
-1
-1
-1
-1
vibrations appear at 1591 cm and 1493 cm in DBA. It is shifted to 1593 cm and 1494 cm
in solid inclusion complex with considerable reduction in the intensity of the peak. Similarly
-1
-1
-1
ring C-H bending vibration of DBA appeared at 759 cm and 595 cm shifted to 760.7 cm
-
1
and 570 cm in the case of solid inclusion complex with slight reduction in peak intensity.
These changes occur when the benzene ring of DBA is entrapped into the nano hydrophobic
cavity of β-CD [30]. There is no appreciable change in the stretching frequencies of physical
mixture. These observations clearly indicate the formation of inclusion complex between DBA
and β-CD.
3.2.2 SEM image analysis
The surface morphology of β-CD, DBA, Physical mixture and solid inclusion
complex of β-CD : DBA were examined by Scanning Electron Microscopy [38]. SEM images of
β-CD, DBA, Physical Mixture and inclusion complex of DBA: β-CD are shown in Fig.4. In β-
CD, the particles are quite dense and well separated from each other whereas in DBA the
particles are aggregated and are loosely bounded to each other. The physical mixture of both the
components (Fig.4c) exhibited the characteristics of both DBA and cyclodextrin particles.
However the DBA: β-CD inclusion complex (Fig.4d) appears as a compact and homogeneous
plate like structure with a drastic change in the morphology and shape of the particles. It is no
longer possible to differentiate DBA and β-CD in the complex which confirms the interaction
between DBA and β-CD during the formation of inclusion complex [39].
3
.2.3 XRD analysis
Evidence for the inclusion complexation of DBA with β-CD is also obtained from
Powder XRD. It is a useful method for the detection of CD encapsulation and it has been used to
assess the degree of crystallinity of the given sample. Generally the crystalline nature of the
guest molecule is reduced and more number of amorphous structures are increased in the solid
inclusion complex [40,41]. Hence the solid complexes usually exhibit less numbered as well as

1
1
less intense peaks. This shows that overall crystallinity of complex is decreased and the
solubility is increased due to its amorphous nature.
DBA exhibits characteristic peaks at 2θ values of 12.93°, 18.99°, 19.31°, 19.68°,
2
0.28 , 24.75°, and 27.68° [ Fig 5b]. Most of the characteristic peaks of DBA are present in the
diffraction pattern of physical mixture without loss of intensity [Fig.5c]. The diffraction pattern
of β-CD exhibited important peaks at 2θ values of 8.95°, 10.56°, 12.45°, 18.60°, 22.4°, 31.8°,
and 35.01° [Fig.5a]. The X-ray diffraction pattern of the solid inclusion complex [Fig.5d] is
evidently different from that of DBA, βCD, and physical mixture, because the inclusion complex
formed between DBA and β-CD is semi crystalline in nature. The peak intensity of DBA at
1
2.61°, 19.47°, 20.10°, and 24.59° are reduced significantly in diffraction pattern of complex
which suggested the reduction in crystallinity [Fig.5d]. Some low intensity peaks of DBA at
7.68°, 28.16°, 29.60° and 29.92° were absent after complexation with β-CD. This different
2
pattern might be due to inclusion of DBA molecule into the cavity of β-CD. Further, appearance
of new low intense peaks at 2θ values for the patterns of DBA/ β-CD (32.19°and 33.19°)
inclusion complex indicates the change in DBA and CD environment after inclusion
complexation.
3.2.4 DSC analysis
DSC represents an effective and inexpensive analytical tool widely used
for a rapid preliminary qualitative investigation of thermal behavior of the inclusion complexes
prepared by co-precipitation method [42]. When guest molecule is included in β-CD cavity or
crystal lattice their melting and sublimation points may shift to different temperature or
disappear [43]. DSC curve of pure β-CD [fig.6a] shows an endothermic peak at 118 °C, TG of
β-CD shows a weight loss about 15 %. It could be due to the guest water molecules present in β-
CD cavity. There is no significant mass loss in TG up to 270 °C whereas above this temperature
decomposition of β-CD is started. The thermo gram of DBA [fig.6b] shows an endothermic
peak at 114 °C due to its phase transition. In the thermo gram of DBA :β-CD complex [fig.6d],
the endothermic peak is found at 108 °C, there is no weight loss in the TG curves of DBA and

1
2
DBA : β-CD solid inclusion complex. It shows clearly that they are free from hydrated water
molecules. It is also confirmed by determining the melting point of DBA and solid inclusion
complex is 113 °C and 108 °C respectively using Sigma melting point apparatus. The lower
melting point of inclusion complex is due to melting point depression by the β-CD [44], similar
observation was found when felodipine and carbamazepine were complexed with β-CD, the
endothermic temperature of inclusion complex was lowered by 3 and 5 °C respectively [44,45].
Physical mixture of β-CD and DBA, shows the weight loss nearly from 80-120 °C in TG
[
fig.6c] which is due to presence of water molecules in β-CD cavity and the endothermic peak at
13 °C is due to the melting point of DBA. The difference in thermal behavior between
inclusion complex and host (β-CD) clearly reveals encapsulation of DBA in β-CD cavity.
1
3.2.5 AFM Analysis
AFM studies are used to visualize the surface texture of the deposited films,
especially when the surface texture sizes are less than 3 micron [46]. The film is prepared by
re-suspending the compound and complex in methanol, followed by dilute dispersion on freshly
cleaved mica and then by thorough drying the particles are fixed on to the surface. AFM
analysis is quite ideal for measuring the nanometric dimensional surface roughness and for
visualizing the surface nanotexture of the deposited film. Visualization of surface topography of
the guest (DBA) and the complex using AFM analysis is given in Fig.7. The peak to valley
distance in these images is used as an indicator of the surface roughness. When the distance
between the peak and the valley is more which means that it has a rougher surface and if the
distance is less it indicates the smoothness of surface [47]. Based on this peak and valley
distance, the AFM image of pure DBA (Fig.7a) shows a rough surface and uneven distribution.
But in the case of solid complex the rough surface of DBA is changed into a smooth surface. The
changes in surface morphology and the modification of the crystals may be taken as a proof for
the interaction of DBA with β-CD (Fig.7b) (43).

1
3
3.3 Analysis using MOPAC software
The structure of the inclusion complex is established using MOPAC/AM1
software. The calculations show the total length of DBA molecule is found to be 13.34 Å
Table 3). The cavity length of β-CD is 7.8 Å, the inner diameter of β-CD cavity in the narrow
(
edge and wide edge are 5.6 Å and 6.8 Å respectively [30, 48]. According to the size matching
rule [49] only a portion of DBA molecule could be entrapped in to β-CD cavity. The low value
of Binding constant ‘K’ reveals the possibility of only partial accommodation [22] of DBA in
β-CD cavity. ‘K’ value is an important parameter to characterize the interaction between guest
and host molecule and also reflects the intensity of binding force between them [22]. Based on
these results the structure of solid inclusion complex is proposed.
Molecular docking study of inclusion process
The 3D structure of β-CD and DBA obtained from crystallographic databases are shown in
Figs. 8a and 8b. The guest molecule, DBA was docked into the cavity of β-CD using Patch Dock
server. The Patch Dock server program provides several possible docked models for the most
probable structure based on the energetic parameters such as geometric shape complementarity
score [50], approximate interface area size and atomic contact energy [28] of the DBA:β-CD
inclusion complex [Table.4]. The docked DBA: β-CD 1:1 model (Fig. 8c) with the highest
geometric shape complementarity score 3218, approximate interface area size of the complex
2
4
11.4 Å and atomic contact energy -265.9 kcal/mol was the highly probable and energetically
favorable model and it is in good correlation with results obtained through experimental
methods.

1
4
4. Conclusions
The following conclusions have arrived on the basis of the above results.
i.
Using Benesi- Hildebrand plot and Job’s continuous variation method, the stoichiometry
of the DBA: β-CD inclusion complex is found to be 1:1.
ii.
Time - resolved fluorescence spectral study in aqueous solutions shows the presence of
two species namely free DBA and DBA: β-CD inclusion complex.
iii.
The solid complex prepared by co-precipitation method is characterized by UV-Visible,
FT-IR, DSC, XRD and microscopic morphological image analysis using SEM and AFM.
Based on these findings, the interaction between DBA and β-CD is confirmed and the structure
of 1:1 inclusion complex, DBA:β-CDx is proposed. The Docking study suggests that the
proposed structure is found to be highly probable and energetically favorable model.
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Figure captions:
1
. Fig. 1. Absorption spectra of DBA with increasing concentration of β- CD
Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β- CD).
Inset : Benesi-Hildebrand absorption plot of DBA with β- CD (λabs = 230 nm)
. Fig. 2. (a) Fluorescence spectra of DBA with increasing concentration of β- CD
(1.
(
2
(1.
Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β- CDx),
(
Inset Benesi-Hildebrand fluorescence plot of DBA with β- CD (λemi = 361 nm),
(b)
Job’s plot of DBA with β- CD (λabs = 240 nm). C). Plot of [β-CD] vs B /B
2
1
3
. Fig. 3. FT-IR spectra of a) β-CD b) DBA, c) physical mixture of DBA with β- CD and
d) solid complex of DBA with β- CD.
4
. Fig. 4. . SEM images of a) β-CD ( ×5 00), b) DBA ( ×5 00), b) physical mixture of DBA
with β- CD ( ×5 00) and c) Solid complex of DBA with β- CD ( ×5 00).
5
. Fig. 5. XRD patterns of a) β- CD b) DBA, c) physical mixture of DBA with β- CD and
c) solid complex of DBA with β- CD.
6
7
Fig. 6. Thermo gram [TG-DSC] of a)β-CD b) DBA, c) physical mixture of DBA
with β-CD and d) solid complex of DBA with β- CD.
Fig. 7. Atomic force microscope 2D and 3D images of (a) DBA and (b) solid complex
of DBA with β- CD.
8
Fig 8. Ball and stick representation of (a) β-CD (b) DBA (c) 1:1 inclusion complex;
the oxygen atoms are shown as red, carbon atoms are shown as golden balls and sticks,

1
9
and hydrogen atoms are not shown.
Table. 1 The absorption and fluorescence spectral maxima of DBA in different concentration of
β- CD at pH 6.8
Concentrations
pH 6.8
of β-CD (mol
-
3
dm )
λ
max
abs
λ
max
abs
λ
flu
λ
flu
nm
nm
nm
nm
0
228.5
230.5
230.5
231.0
231.5
231.5
232.0
0.322
0.332
0.346
0.357
0.388
0.405
0.467
0.512
323.5
321.5
0.484
0.444
0.381
0.372
0.376
0.376
364
363
363
362
362
361
361
437
436
436
435
435
435
434
-
3
3
3
3
2
4
6
8
х 10
х 10
х 10
х 10
-
-
-
321.0
321.0
314.5
-3
1
0 х 10
2 х 10
315.5
-3
1
shoulder
shoulder
255.0

2
0
Table 2. Fluorescence lifetime and amplitude of DBA with increasing concentrations of β-CD
Excitation wavelength 320 nm, detection wavelength 340nm)
(
Concentration of Lifetime
β-CD (M)
Relative
amplitude
χ2
Standard
deviation
-
9
9
-9
0
7.89×10
7.64×10
100
1.103
2.367×10
-
-10
0
0
0
0
0
0
.002
60.43
39.57
49.13
50.87
56.55
43.45
33.31
66.69
26.43
73.57
19.81
80.19
1.011
1.018
1.187
0.966
0.959
1.150
2.16×10
1.25×10
3.61×10
1.35×10
8.16×10
1.35×10
3.42×10
9.50×10
1.46×10
1.30×10
8.20×10
1.21×10
-
9
-10
2.3×10
-
-
-
-
-
-
-
-
-
-
9
-10
-10
-10
-10
-10
-11
-9
.004
.006
.008
.010
.012
7.39×10
9
2
.34×10
10
9
1.26×10
.42×10
2
10
9
1.14×10
.63×10
2
10
9
1.04×10
.74×10
-9
2
10
9
-10
-10
1.00×10
.96×10
2

2
1
Table 3. Bond length between various atoms of DBA
Atom
Bond distance (Å)
H
28 - H20
13.34
7.72
28- 2
H C
C
8
- C
4
6.73
C
8
- H20
- H20
12.12
12.31
7.28
C
9
9- 4
C C
H C
19- 2
7.10
H C
29- 4
7.51

2
2
Table 4: Scores of the top 10 docked models of DBA:β-CD inclusion complex computed using
Patch Dock server.
Model
1
Geometric shape
complementarity score area size of the complex
Approximate interface Atomic contact energy
kcal/mol
2
Å
3218
411.4
- 265.9
2
3
2934
2870
355.4
343.3
-255.5
-252.8
4
5
6
7
8
9
2852
2846
2816
2808
2792
2778
2772
340.1
339.2
340.4
334.5
300.5
334.6
341.3
-243.2
-239.1
-243.5
-235.8
-213.6
-241.3
-246.0
1
0

2
3
Figures:
Fig. 1. Absorption spectra of DBA with increasing concentrations of β- CD
(
1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β- CD). ,
Inset: Benesi-Hildebrand absorption plot of DBA with β- CD (λabs = 230 nm)
(

2
4
Fig. 2. (a) Fluorescence spectra of DBA with increasing concentrations of β- CD
1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β- CD),
(
(
Inset : Benesi-Hildebrand fluorescence plot of DBA with β- CD (λemi = 361 nm),
b) Job’s plot of DBA with β- CD (λabs = 230 nm).
(

2
5
2
Fig.2c: Plot of [β-CD] vs B /B1

2
6
Fig. 3. FT-IR spectra of a) β-CD b) DBA, c) physical mixture of DBA with β- CD and d) solid
compound DBA with β- CD.

2
7
Fig. 4. . SEM images of a) β-CD ( ×5 00), b) DBA ( ×5 00), c) physical mixture of DBA with
β- CD ( ×5 00) and c) Solid complex of DBA with β- CD ( ×5 00).

2
8
Fig. 5. XRD patterns of a) β- CD b) DBA, c) physical mixture of DBA with β- CD and
d) solid complex of DBA with β- CD.

2
9
Fig. 6. Thermogram [TG-DSC] of a) β-CD b) DBA, c) physical mixture of DBA with
β- CDx and d) solid complex of DBA with β- CDx.