Salicylanilide /Cyclodextrin Inclusion Complex:
Preparation, Characterization and Molecular Docking
K Sivakumar* and M Parameswari
Department of Chemistry, Faculty of Science, Tamilnadu, India
Dr. K Sivakumar, Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa MahaVidyalaya University, Enathur,
Kanchipuram - 631 561, Tamilnadu, India. Tel: +91 9842361378; Fax: +91 044 27264285; E-mail: firstname.lastname@example.org
Received: November 20, 2015; Accepted: November 30, 2015; Published: December 05, 2015
Sivakumar K, Parameswari M (2015) Salicylanilide /Cyclodextrin Inclusion Complex: Preparation, Characterization and
Molecular Docking studies. SOJ Mater Sci Eng 3(3): 1-4. DOI: http://dx.doi.org/10.15226/sojmse.2015.00130
Interaction between Salicylanilide (SA) with β-Cyclodextrin (β-
CD) has been investigated by UV and fluorescence techniques. The
red shift in λmax enhanced absorption and emission in β-CD medium
confirms the –NH-CO-C6H5 aromatic side chain SA is inserted in the
β-CD cavity. The stoichiometry and binding constant of 1:1 inclusion
complex was calculated using the Benesi–Hildebrand plot derived
UV and fluorescence studies and the thermodynamic parameters
(ΔG, ΔH and ΔS) of inclusion process were also determined. The
results indicated that the inclusion process was an exergonic and
spontaneous process. The complex was investigated by FTIR, XRD,
Semi empirical and molecular docking methods. The SA:β-CD
inclusion complex obtained by molecular docking studies was in good
correlation with the results obtained through experimental methods.
Keywords: β-Cyclodextrin; Salicylanilide; Inclusion Complex;
Salicylanilides (SA) is the amide of salicylic acid and aniline.
It is classified as both a salicylamide and an anilide. SA is a
group of anthelmintics which exert their action by uncoupling
mitochondrial reactions which are critical to electron transport
and associated phosphorylation in the metabolic system of the
parasite. They are effective against cestodes and trematodes but
not nematodes. Some are active against Haemonchus contortus.
Derivatives of salicylanilide have a variety of pharmacological
uses. Chlorinated derivatives including niclosamide, oxyclozanide
and rafoxanide are used asanthelmintics, especially as
flukicides. Brominated derivatives including dibromsalan,
metabromsalan and tribromsalan are used as disinfectants with
antibacterial and antifungal activities.
CycloDextrins (CDs) are cyclic oligosaccharides obtained
from enzymatic hydrolysis of starch. The β-CD is the most
abundant natural oligomers and corresponds to the association
of seven glucose units with cavity, which exhibits a hydrophobic
character whereas the exterior is strongly hydrophilic. Their
ability to form host-guest complexes has led to the use of CDs in a number of fields [1, 2]. CDs have been used in the pharmaceutical
industry, as solubilizers, diluents and as tablet ingredients which
improve the chemical stability, solubility, bioavailability and
pharmacokinetic properties of drugs. In this paper, we report the
photo physical and computational studies on the complexation
SA and β-CD at different conditions. In addition to the UV
and Fluorescence studies, we have utilized the complexation
behavior of SA for the stoichiometry and binding constant of
SA:β-CD inclusion complex. Further it was also supported by
XRD, semiemprical method and molecular docking studies.
Materials and Methods
Salicylanilide and β-CD were obtained from Aldrich, Hi
Media Laboratories and used without further purification. Triply
distilled water was used to prepare all solutions and spectro
grade solvents were used. Solutions in the pH 7 were prepared
by adding the appropriate amount of NaOH and H3PO4. The
concentration of β-CD was varied from zero to 1.2×10−3 mol dm−3.
From the stock solution 2, 4, 6, 8, 10 and 12 ×10−3 mol dm−3 of
β-CD were prepared using pH~ 7 buffers. The concentrations of
the solutions were of the order 10−4 mol dm−3. All experiments
were carried out at 30oC. The solid inclusion complex was also
prepared by coprecipitation method.
The pH values were measured using Elico pH meter LI-
120. The UV spectra were recorded with Specord 200+
spectrophotometer, Germany. The Fluorescence spectra were
recorded using Spectro fluorometer, Perkin Elmer, USA. The
IR spectra of all samples were recorded using Alpha-T FTIR
Spectrometer (Bruker optics) equipped KBr by using a clean glass
pestle and mortar. Powder X-ray diffraction spectra were taken
by XPert PRO PANalytical diffractometer. The most probable
structure of the SA:β-CD inclusion complex was determined by
molecular docking studies using PatchDock server .
Results and Discussion
Host–Guest Interaction of SA with β-CD
The absorption spectral data of SA in different concentrations
of β-CD recorded in pH~7 are compiled in Table 1. In SA upon
increasing the concentration of β-CD a slight red shift is observed
in the absorption maxima in the LW and SW absorption bands.
No clear isosbestic point is observed in absorption spectrum. The
absorption spectra show only very slight change in absorption
maxima even in the presence of highest concentration of β-CD
used (12x10-3 M) in pH~7. This behavior has been attributed
to the enhanced dissolution of SA molecules through the
hydrophobic interaction between guest molecule (SA) and nonpolar
cavity of β-CD [4-6] as reported by others also [7, 8]. Since,
this indicates the formation of 1:1, host–guest inclusion complex
of SA: β-CD.
The binding constant for the formation of SA:β-CD complex
has been determined by analyzing the changes in the intensity of
absorption maxima with the β-CD concentration. In the case of
inclusion complex formed between SA and β-CD, the equilibrium
can be written as,
The binding constant 'K' and stoichiometric ratios of the
inclusion complex of SA can be determined according to the
Benesi–Hildebrand  relation assuming the formation of a 1:1
Where, A and A0 is the difference between the absorbance
of SA in the presence and absence of β-CD, Δε is the difference
between the molar absorption coefficient of SA and the inclusion
complex, [SA]0 and [β-CD]0 are the initial concentration of SA and
β-CD respectively. The plot of 1/A-A0 verses 1/ [β-CD] for SA in
pH~7. For pH~7 solutions, a good linear correlation was obtained,
confirming the formation of a 1:1 inclusion complex. From the
intercept and slope values of this plot, the binding constant 'K'
was evaluated. The 'K' value for SA in neutral condition (258.5
M-1 at pH~7) at 303K.
The effect of β-CD on the fluorescence spectra of SA (Table
1) is different from absorption spectra and more pronounced
than the relative effect on the absorption spectra. In SA, there
is no significant change is observed in emission maxima (~528
nm) at pH~7. The emission intensity of SA in pH~7 is increases
when the β-CD concentration is increased (Figure 1), whereas the
intensity is increased. Figure 1 shows the Benesi–Hildebrand plot
of observed changes in the fluorescence intensity with increasing
concentration of β-CD. It is seen from this plot that the emission
intensity of SA initially increases with β-CD concentration
and then saturates to a limiting value at 0.012M β-CD, indicating
the maximum inclusion of SA molecule in the β-CD cavity. The
binding constant for the formation of complex has been
Figure 1: The Fluorescence spectra of Salicylanilide (pH~7) in different
β-CD concentrations (mol dm−3): (1) 0.0 M, (2) 0.002 M, (3) 0.004 M, (4)
0.006 M, (5) 0.008 M, (6) 0.010M and (7) 0.012 M. (Inside- Benesi–Hildebrand
plot of 1/F−F0 vs. 1/[β-CD] for Salicylanilide in pH~7 solution).
determined by analyzing the changes in the intensity of
emission maxima with the β-CD concentration using the Benesi-
Hildebrand relation assuming the formation of a 1:1 host –
Where, [β-CD]0 represents the initial concentration of β-CD,
"I0" and "I" are the fluorescence intensities in the absence and
presence β-CD respectively, and I' is the limiting intensity of
florescence. The 'K' value was estimated from the slope and
intercept of the Benesi–Hildebrand plot which shows a good
linear correlation supporting the assumption of 1:1, SA: β-CD
inclusion complex. The binding constant 'K' value is SA evaluated
as 375.14 M−1.
The thermodynamics of inclusion process
The thermodynamic parameters ΔG for the binding of guest
molecule to β-CD cavity can be calculated from the binding
constant 'K' by using the following equation
The thermodynamic parameters ΔG for the binding of guest
molecules (SA) to β-CD cavity are given in Table 1. The negative
value of ΔG suggests that the inclusion process proceeded
spontaneously at 303K. Considering the above discussions,
the possible inclusion mechanism is proposed. Naturally, The
inclusion complex formation of SA with β-CD, both guest
molecules with the –NH-CO-C6H5 aromatic side chain and benzene
ring of SA inserted in the β-CD cavity as shown in Scheme 1.
Characterization of SA: β-CD complex
The infrared (FTIR) spectra of wave number from 4000 to
400 cm-1 of salicylanilide, β-CD and the solid inclusion complex of
Salicylanilide with β-CD were registered by FTIR spectrometer.
The host molecule (β-CD) reacts with guest molecule (SA) to
form host–guest solid complex (SA: β-CD). The solid complex
formation can be confirmed by FTIR spectroscopy because, the
bands resulting from the included part of the guest molecule are
Table 1: Absorption and fluorescence maxima (nm) of SA (0.001 M) at different concentrations of β-CD in pH~7 solutions.
l max (nm)
Binding constant (M-1)
DG (kJ mol-1)
generally shifted or their intensities altered . If β-CD and SA
forms a solid inclusion complex, the non-covalent interactions
between them such as hydrophobic interactions, vander Waal's
interactions and hydrogen bonding lowers the energy of the
included part of SA and reduces the absorption intensities of
the corresponding bands. We can see that there are apparent
differences between the FTIR spectra of β-CD, SA and SA:β-CD
solid inclusion complex. The FTIR spectra of SA:β-CD and that
of β-CD are alike due to (i) the presence of large number of polar
groups such as O–H, C–O, etc., that are responsible for intense
absorption bands; (ii) the excess of free (unreacted) β-CD in
the SA:β-CD inclusion complex sample. However, the inclusion
of SA into the β-CD cavity is evidently confirmed by the bands
at 3299, 1501 cm-1, 1334 and 828 cm-1. The IR spectrum of SA
examined in KBr pellet, showed one absorption band at 3299
cm-1 for N-H stretching with benzene. 1501 cm-1 was noted for
bending vibration of N-H group attached with benzene ring.
The characteristic peak of C–N stretching vibration in aromatic
ring appeared at 1334 cm-1, For N–H wagging vibration the peak
appeared at 828 cm-1. However, in the IR spectrum of SA:β-CD
Inclusion complex, (formed by stirring method) absorption band
due to the stretching (at 3299 cm-1) and bending (at 1501 cm-
1) vibrations of N-H disappeared, infers that the N-H of SA was
entrapped into the β-CD cavity in the inclusion complex. The C-N
stretching vibration peak at 1334 cm-1, N–H wagging vibration
peak at 828 cm-1 shifted in the inclusion complex, indicating the
restriction in the vibration of N–H group due to the complete
entrapment of N-H containing aromatic ring into β-CD. The
aromatic C-H bending vibration at 790 cm-1 and C-H out of plane
at 895 cm-1 disappeared in the inclusion complex. Stretching
vibration of C-C in aromatic ring at 1558 cm-1 of SA shifted to
1560 cm-1 in the SA:β-CD complex. The inclusion complex FTIR
spectra peaks are 30-40% weaker than the free SA molecule.
The SA:β-CD inclusion complex did not show any new peaks, indicating that no chemical bonds were created between SA
and β-CD in the formed complex which was also confirmed by
molecular modeling studies.
Powder X-ray diffraction spectra
The formation of inclusion complex can be confirmed by
X-ray diffractometry [11,12]. The Figure2 shows the powder
X-ray diffraction spectra of β-CD, , SA and SA:β-CD solid complex.
The X-ray spectrum of the inclusion complex shown in Figure 2
b and c was evidently different from that of β-CD Figure 2 a. The
difference between the spectra of β-CD and inclusion complex is
due to the interaction of β-CD with SA.
Semi empirical quantum mechanical calculations
The internal diameter of the β-CD is approximately 6.5 Å
and its height is 7.8 Å (Scheme 1). Considering the shape and
dimensions of β-CD, SA cannot be completely embedded in the
Figure 2: XRD pattern of (a) β-CD, (b) Salicylanilide and (c)
Salicylanilide:β-CD solid complex.
Figure 3: Ball and stick representation of (a) β-CD, (f) SA, and (g) SA:β-
CD 1:1 inclusion complex. oxygen atoms are shown as red balls, carbon
atoms as golden balls and sticks, nitrogen atoms as blue balls and hydrogen
atoms are shown in grey balls and sticks.
β-CD cavity. The ground state of SA molecule was optimized using
AM1 method. SA the vertical distance between H17-H25 is 11.3
and this is higher than the height of β-CD. The horizontal distance
between H23-H27 is 5 Å and is less than the internal diameter of
β-CD. Since, the height of SA is higher than that of upper-lower
rim of β-CD, the –NH-CO- C6H5 aromatic side chain and benzene
ring of SA insertion in the β-CD cavity is possible as shown in
Molecular docking study of inclusion process
The 3D structure of β-CD and SA obtained from
crystallographic databases are shown in Figure 3. The guest
molecule, SA was docked into the cavity of β-CD using PatchDock
server. The PatchDock server gave several possible docked
models for the most probable structure based on the energetic
parameters; geometric shape complementarity score ,
approximate interface area size and atomic contact energy 
of the SA:β-CD inclusion complex. The docked SA:β-CD model
(Figure 3c) with the highest geometric shape complementarity
score 3212 approximate interface area size of the complex
381.90 Å2 and atomic contact energy -248.24 kcal/mol was the
highly probable and energetically favourable model.
In summary, the inclusion complex with 1:1 molar ratio was
formed between β-CD and SA. The –NH-CO-C6H5 aromatic side
chain and part of benzene ring of SA was inserted in the β-CD. The
inclusion complex formation of SA with β-CD, the guest molecules
with the –NH-CO-C6H5 aromatic side chain and benzene ring of
SA inserted in the β-CD. Thermodynamic parameter values
show the inclusion processes are spontaneous. UV, Fluorescence,
XRD, semi empirical and molecular docking results confirms the
formation of SA:β-CD inclusion complex. The inclusion complex formation which was also confirmed by molecular docking
- Scott Loethen, Jong-Mok Kim, David H. Thompson. Biomedical Applications of Cyclodextrin Based Polyrotaxanes. Polymer Rev. 2007;47(3):383-418.
- AR Hedges. Industrial Applications of Cyclodextrins. Chem Rev. 1998;98(5):2035-2044. DOI: 10.1021/cr970014w
- Dina Schneidman-Duhovny, Yuval Inbar, Ruth Nussinov, Haim J Wolfson. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucl Acids Res. 2005;33(suppl 2); 363-367.
- YH Kim, DW Cho, M Yoon. Observation of Hydrogen-Bonding Effects on Twisted Intramolecular Charge Transfer of p-(N, N-Diethylamino)benzoic Acid in Aqueous Cyclodextrin Solutions. J Phys Chem. 1996;100(39):15670-15676. DOI: 10.1021/jp9613652
- YB Jiang J. Photochem. Photobiol. A Chem. 1995;88:109-116.
- YV Ilichev, W Kuhnle, KA Zachariasse. Intramolecular Charge Transfer in Dual Fluorescent 4-(Dialkylamino) benzonitriles. Reaction Efficiency Enhancement by Increasing the Size of the Amino and Benzonitrile Subunits by Alkyl Substituents. J Phys Chem. 1998;102(28):5670-5680.
- S Santra, SK Dogra, J Photochem. Photobiol. A Chem. 1996;101:221-227.
- P Bortolos, S Monti. cis .dblharw. trans Photoisomerization of azobenzene-cyclodextrin inclusion complexes. J Phys Chem. 1987;91:(19):5046-5050.
- HA Benesi, JH Hildebrand. A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons. J Am Chem Soc. 1949;71(8):2703-2707.
- L Szente. Analytical Methods for Cyclodextrins, Cyclodextrin derivatives and Cyclodextrin Complexes, in J. Szejtli, and T Osa, Cyclodextrins, in J Lehn et al., (Editor), Comprehensive Supramolecular Chemistry. Pergamon Press, Oxford. 1996;3:253-278.
- S Scalia, A Molinari, A Casolari, A Maldotti. Euro J Pharm Sci. 2004;22:241-249.
- T Pralhad, K Rajendrakumar. Study of freeze-dried quercetin–cyclodextrin binary systems by DSC, FT-IR, X-ray diffraction and SEM analysis. J Pharm Biomed Anal. 2004;34(2):333-339
- D Duhovny, R Nussinov, HJ Wolfson. Efficient Unbound Docking of Rigid Molecules. Algorithms in Bioinformatics. Lecture Notes in Computer Science. Springer Verlag. 2002;2452:185-200.
- C. Zhang, G. Vasmatzis, J.L.Cornette, C. DeLisi. Determination of atomic desolvation energies from the structures of crystallized proteins. J Mol Biol. 1997;267(3):707-726.