Preparation and characterization of host–guest system between inosine and β-cyclodextrin through inclusion mode

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 147 (2015) 151–157

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Preparation and characterization of host–guest system between inosine and b-cyclodextrin through inclusion mode Samikannu Prabu a,c, Krishnamurty Sivakumar e, Meenakshisundaram Swaminathan d, Rajaram Rajamohan a,b,⇑ a

Research and Development Centre, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India Department of Chemistry, SKP. Institute of Technology, Tiruvannamalai 606 611, Tamil Nadu, India Department of Chemistry, SKP. Engineering College, Tiruvannamalai 606 611, Tamil Nadu, India d Department of Chemistry, Annamalai University, Annamalainagar 608002, Tamil Nadu, India e Department of Chemistry, SCSVMV University, Kanchipuram 631 561, Tamil Nadu, India b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Enhancement of absorbance and

fluorescence intensity occurs together with complex formation of INS with b-CDx.  BH plot confirms the 1:1 stoichiometric ratio.  Lifetime analysis confirms the complex between INS and b-CD in liquid state.  FT-IR, SEM, DSC and XRD analysis confirms the complex formation between INS and b-CDx in solid state.  The orientation of the complex is confirmed by molecular docking study.

a r t i c l e

i n f o

Article history: Received 1 November 2014 Received in revised form 6 February 2015 Accepted 2 March 2015 Available online 9 March 2015 Keywords: Inosine b-cyclodextrin Inclusion complex XRD DSC Patch – Dock server

a b s t r a c t Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a b-N9-glycosidic bond. Inosine is commonly found in tRNAs. Inosine (INS) has been used widely as an antiviral drug. The inclusion complex of INS with b-CDx in solution phase is studied by ground and excited state with UV–visible and fluorescence spectroscopy, respectively. A binding constant and stoichiometric ratio between INS and b-CDx are calculated by BH equation. The lifetime and relative amplitude of INS is increases with increasing the concentrations of b-CDx, confirms the formation of inclusion complex in liquid state. The solid complexes are prepared by kneading method (KM) and co-precipitation method (CP). The solid complex is characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (XRD) and differential scanning colorimetry (DSC). CP method gives the solid product with good yield than that of physical mixture and KM method. The structure of complex is proposed based on the study of Patch – Dock server. Ó 2015 Published by Elsevier B.V.

Introduction ⇑ Corresponding author at: Department of Chemistry, SKP. Institute of Technology, Tiruvannamalai 606 611, Tamil Nadu, India. Tel.: +91 9865233802. E-mail address: [email protected] (R. Rajamohan). http://dx.doi.org/10.1016/j.saa.2015.03.056 1386-1425/Ó 2015 Published by Elsevier B.V.

Inosine is a naturally occurring purine nucleoside, stimulates the growth of projections from the undamaged hemisphere into denervated areas of the spinal cord [1]. It is a non-toxic to human

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body. INS is widely available as a nutritional supplement in health food stores [2]. The theoretical study of the conformational preferences and intra molecular interactions in INS has been carried out with full relaxation of all geometric parameters, if the different geometric in INS is alternative forms of hydrogen bonding make significant contributions to the conformational behaviors of INS [3]. It is effective, selective, and non-toxic antiviral agents, a variety of strategies has been devised to design nucleoside analogs [4,5]. Inosine have been used to be pharmaceutically important as antiviral and antitumor drug. The supramolecular chemistry gives a broad idea of intermolecular interactions has been performed by host–guest system. The cyclodextrin is mostly hopeful to form inclusion complexes, especially with various guest molecules with proper structure [6]. In supramolecular chemistry, host–guest chemistry describes complexes that are composed of two or more molecules or ions that are held together in unique structural relationships by forces other than those of full covalent bonds. Host–guest chemistry encompasses the idea of molecular recognition and interactions through non covalent bonding. There are four commonly mentioned types of non-covalent interactions: hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. Cyclodextrins are the neutral macromolecule and oligosaccharides with consists of six, seven and eight a-D-glucopyranose units are represented as a, b and c-cyclodextrins, respectively. a-cyclodextrin is not interacting with many drugs because of their small size than that of others and c-cyclodextrin is more expensive. b-Cyclodextrin is very chief and widely used because it is readily available and it’s cavity size is suitable for many more guest molecules [7]. The b-cyclodextrin is well known core structure with the hydrophilic outer surface and a hydrophobic inter cavity [8]. The b-CDx with inclusion complex properties had been widely used in different areas, such as the medicine [9–12], chemistry [13,14], agriculture [15] and so on. The possible driving forces leads to the formation of inclusion complex between guest and b-CDx which includes electrostatic interaction, van der Waals interaction, hydrophobic interaction, hydrogen bonding, relief of conformational strain, exclusion of cavity – bound high – energy water and Charge – transfer interaction [16]. The length (6.6 Å), Heat capacity (1342 J mol1 K1) and Solubility water (0.0163 mol L1) of b-CDx is reported [17]. Two type of hydration takes place in b-CDx such as undecahydrate (b-CDx11H2O), dodecahydrate (b-CDx12H2O) [18,19]. Due to more flexibility, water molecules are displaced by more hydrophobic guest molecules to yield a polar – a polar association and decrease of the cyclodextrin ring strain, b-CDx possess more stable lower energy state [20]. In our earlier results are focused the study of complexation of some organic compounds with b-CDx based on proton shift effect [21–23]. Our aim of the present work is to evaluate the effect of inclusion complexation of inosine with b-cyclodextrin in aqueous solution. Here, we report the complex formation between INS and b-CDx in liquid as well as solid state too with the accordance of characterization of the solid complex. Materials and methods Materials Inosine (INS) and b-cyclodextrin (b-CDx) are purchased from Alfa Acer and used as received. Distilled water is used throughout the study. The concentration of the experimental solution is 1.04  104 mol dm3. Solutions for absorptiometric and fluorimetric titrations are prepared just before taking measurements.

Preparation of solid inclusion complexes The solid complexes between inosine and beta-cyclodextrin are prepared at 1:1 M ratio as per the following methods. Preparation of physical mixture of the INS and b-CDx In the physical mixture process, the accurate weight of INS and b-CDx (molar ratio, 1:1) are allowed to continuous agitation using a mortar and pestle for 10 min. Finally we obtained the homogeneous mixture of INS and b-CDx. Preparation of the INS and b-CDx by kneading method The INS and b-CDx are accurately weighed in a molar ratio of 1:1. Further sufficient quantity of water added to make a pasty form. The half an hour grinding are carefully done in a mortar itself. The kneading mass is obtained after it is dried for 48 h in an oven at 303 K. Preparation of the INS and b-CDx by co-precipitation method The inclusion complex of INS and b-CDx at 1:1 M ratio is prepared using the co-precipitation method. The accurate weight (0.75 g) of b-CDx is dissolved in distilled water to become a saturated solution. Other hand accurate weight of (0.75 g) of INS is dissolved in distilled water to get a saturated solution. The INS solution is added slowly to the b-CDx solution up to suspension is formed. The suspension is stirred continuously for 48 h at 303 K. The solution is kept in a refrigerator for 24 h. After 24 h, the solution become as white precipitate, then it is slowly changed to clear solution. In order to get the solid product we allowed the whole solution into Freeze-drying, also known as lyophilisation (is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase) Process. Then it gives the white product called as solid complex between INS and b-CDx. Instruments Absorption spectra are recorded between wavelength range of 200–400 nm with SHIMADZU model UV – 2450PC spectrophotometer while fluorescence spectra of each solution are recorded between the wavelength of 270–800 nm with Perkin Elmer LS-45 fluorescence spectrophotometer. Single photon counting picosecond spectrofluorimeter is used for the measurements of fluorescence lifetime of INS. The excitation fixed at 280 nm and emission measured at 350 nm. The fluorescence decay measurements were recorded using an IBH time-correlated-singlephoton-counting spectrometer (TSUNAMI, SPECTRA PHYSICS, USA), with a micro-channel plate photomultiplier tube (MCPPMT) (Hamamatsu, R3809U) as detector and LED as an excitation source. The emission slit width was fixed at 4 nm for all measurements. The total photon counts were 10,000 and the time calibration was 24.5 ps per channel. FT-IR spectra are obtained with iD1 Thermo nicolate iS5 FT-IR spectrophotometer. The pellet is made by using KBr. The wave number range of FT-IR spectra is from 500 to 4000 cm1. Microscopic morphological structure measurements are performed with FEI Quanta FEG 200 scanning electron microscope (SEM). DSC analysis is carried out from 20 °C to 800 °C with NETZSCH STA 449 differential scanning calorimeter

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Results and discussion Analysis of complex in liquid state If the concentration of b-CDx is increased in INS aqueous solution, the absorbance of INS is also increased at 248 nm. This b-CDx concentration dependent, hyper chromic shift in spectral maximum of INS to higher absorbance is resulted due to host–guest interaction between b-CDx and INS shown in (Fig. 1). INS exists as a neutral molecule at pH 6.5. The absorption spectra of INS with non-polar solvents are observed at 248.0 nm. Thus the neutral form exists at pH 6.5. The significant changes in absorption spectral maximum and its absorbance values observed for INS molecule while the addition of b-CDx at pH 6.5. Upon increasing the concentration of b-CDx, the spectral maximum (kmax) is slightly blue shifted with a gradual increase in absorbance (Table 1). This may be due to the transfer of the INS molecule from a more protic environment (bulk aqueous phase) to a less protic environment (the cavity of b-CDx). For 1:1 and 1:2 inclusion complexes between b-CDx and INS, the following equilibrium can be written as, K 1:1

INS þ b-CDx ¢ INS : b-CDx

The binding constant ‘K’ and stoichiometric ratio of the inclusion complex of INS can be determined according to the Benesi– Hildebrand Eqs. (3) and (4) for the formation of 1:1 and 1:2 host–guest complexes

1 1 1 ¼ þ A  A0 De½INS0 K½INS0 De½b-CDx0

ð3Þ

1 1 1 ¼ þ A  A0 De½INS0 K½INS0 De½b-CDx02

ð4Þ

where, A is the absorbance of INS in the presence of b-CDx and A0 is absorbance of INS in the absence of b-CDx, De is the difference between the molar absorption co-efficient of free INS and the inclusion complex, [INS]0 and [b-CDx]0 are the initial concentrations of INS and b-CDx, respectively. The (inset Fig. 1) depicts a plot of 1/A  A0 as a function of 1/[b-CDx]. Good linear correlation is obtained (regression coefficient, r = 0.99) which confirms the formation of a 1:1 stoichiometric complex. The binding constant ‘K’ calculated from the slope of the straight line is found to be 33.59 M1 at 303 K using the equation.

K¼

1 slopeðA  A0 Þ

ð5Þ

Fig 2 shows the fluorescence spectra of INS with different concentrations of b-CDx in aqueous solution. The fluorescence maximum (kflu) is observed for INS in aqueous solution at 380.5 nm. When increase the concentration of b-CDx, the blue shifted maximum is appeared. It is observed that the fluorescence intensity increases (Table 1) with increasing concentrations of b-CDx. The data confirms that the stable complex is formed between INS and b-CDx. As the earlier reports [28,29] confirm that the formation of an inclusion complex between guest and host due to the increases in fluorescence intensity of a guest molecule when increasing the concentration of b-CDx. The stoichiometry and binding constants of the INS – b-CDx inclusion complex are determined using the Benesi–Hildebrand Eq. (6) for the 1:1 stoichiometric complex

1 1 1 ¼ þ I  I0 I0  I0 K½I0  I0 ½b-CDx

1.1

7

1.0

40 35

0.9

ð1Þ

30

INS þ 2½b-CDx ¢ INS : 2½b-CDx

ð2Þ

Table 1 Absorption and fluorescence spectral maxima of INS with different concentrations of b-CDx at pH = 6.5. S. No.

1 2 3 4 5 6 7

Conc. of b-CDx (M)

0 0.002 0.004 0.006 0.008 0.010 0.012

Absorption spectrum

Fluorescence spectrum

kmax (nm)

Absorbance

kflu (nm)

Intensity

250.5 250.0 249.3 249.0 247.0 246.0 245.0

0.276 0.302 0.346 0.416 0.505 0.606 0.640

380.5 380.0 370.0 360.5 349.5 340.0 332.5

3.53 6.20 8.50 10.45 12.25 14.80 16.48

Absorbance

0.8 K 1:2

ð6Þ

1 / A - Ao

(DSC) (NETZSCH Corporation, Germany), Powder X-ray diffraction spectra are taken by D8 Advance X-ray instrument (BRUKER, Germany) with 2.2 KW Cu anode, ceramic X-ray tube as the source, Lynx Eye (Silicon strip detector technology) as the detector, Ni filter as the Beta filter and zero back ground sample holder, PMMA sample holder. The most probable structure of the INS: b-CD inclusion complex is determined also by molecular docking studies using the Patch Dock server [24]. The 3D structural data of b-CD and INS is obtained from crystallographic databases. The guest molecule (INS) is docked into the host molecule (b-CD) cavity using Patch Dock server by submitting the 3D coordinate data of INS and b-CD molecules. Docking is performed with complex type configuration settings. Patch Dock server follows a geometry-based molecular docking algorithm to find the docking transformations with good molecular shape complementarity. Patch Dock algorithm separates the Connolly dot surface representation [25,26], of the molecules into concave, convex and flat patches. These divided complementary patches are matched in order to generate candidate transformations and evaluated by geometric fit and atomic desolvation energy scoring [27], function. RMSD (root mean square deviation) clustering is applied to the docked solutions to select the non-redundant results and to discard redundant docking structures.

0.7

25 20 15 10

1

0.6

5 0 0

0.5

100

200

300

400

500

1 / [β - CDx]

0.4 0.3 0.2 0.1 0.0 200

250

300

350

400

Wavelength (nm) Fig. 1. Absorption spectra of INS with increasing concentrations of b-CDx at pH = 6.5 (1, 0; 2, 0.002; 3, 0.004; 4, 0.006; 5, 0.008, 6, 0.010; and 7, 0.012 M b-CDx). Inset figure: Benesi–Hildebrand absorption plot for 1:1 complexation of INS with bCDx.

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28

Fluorescence intensity

24 20 16 12 8

1

4 0 270

310

350

390

430

470

Wavelength (nm) Fig. 2. Fluorescence spectra of INS with increasing concentrations of b-CDx at pH = 6.5 (1, 0; 2, 0.002; 3, 0.004; 4, 0.006; 5, 0.008, 6, 0.010; and 7, 0.012 M b-CDx). Inset figure: Benesi–Hildebrand fluorescence plot for 1:1 complexation of INS with b-CDx.

where K is the binding constant, I0 is the intensity of fluorescence of INS in the absence of b-CDx, I is the fluorescence intensity with a certain concentration of b-CDx and I0 is the intensity with the highest concentration of b-CDx. The binding constant K is calculated from the slope of BH plot using the equation.

K¼

1 slopeðI  I0 Þ

ð7Þ

(Inset Fig. 2) shows the BH plot of 1/(I  I0) vs 1/[b-CDx]. The linearity of the plot (regression coefficient, r2 = 0.99) indicates the formation of 1:1 complex between INS and b-CDx. From the slope of the straight line, the binding constant K is found to be 104.53 M1 at 303 K. Time resolved fluorescence spectral changes in INS by the addition of b-CDx have been analyzed (Fig. 3). The fluorescence decay of INS at different concentrations of b-CDx is recorded. The

Fig. 3. Fluorescence decay curves of INS with increasing concentrations of b-CDx at pH = 6.5 (1, 0; 2, 0.002; 3, 0.004; 4, 0.006; 5, 0.008, 6, 0.010; and 7, 0.012 M b-CDx).

excitation wavelength is fixed at 280 nm and the emission wavelength is fixed at 350 nm. Before the addition of b-CDx, the decay curve of INS gave a best fit for the three exponential decay with good v2 values (1.26). If INS has no isomer or it is pure, it gives only single exponential decay in aqueous medium and it confirm the presence of single species with relative amplitude is 100. Hence, we observed tri-exponential decay with 3 split up of relative amplitude values, and then it is due to the presence of three species, namely pure INS, their isomer and impurity. Upon the gradual increasing the concentration of b-CDx, the lifetime and relative amplitude of the complexed form are increased (Table 2). Here the isomer also forms an inclusion complex with b-CDx in liquid state. But it is not in a gradual mode. Pure INS and their isomer have better lifetime and relative amplitude than that of its impure form. All the analysis is carried out not beyond the v2 value is 1.28. Thus we concluded that, the lifetime analysis gives the support for the formation of complex between INS and b-CDx. In order to find the reaction of complex formation whether thermodynamically favors or not, the free energy change (DG) is calculated from the binding constant K using the following equation.

DG ¼ RT ln K

ð8Þ

We have known that the reaction thermally favorable if the DG value is negative. The thermodynamic parameter DG values have been calculated using the binding constants of the INS: b-CDx obtained from absorption and fluorescence data are found to be 8.88 and 11.71 kJ mol1, respectively. The negative values of DG indicates that the formation of inclusion complex between INS and b-CDx in exergonic and spontaneous at 303 K. Effect of aqueous D(+) glucose with INS instead of b-CDx, the absorption and fluorescence spectra of INS are remain unaltered, which confirms that all the spectral changes between INS and bCDx due to only the formation of inclusion complex and not due to any non-inclusion complex formation. Analysis of inclusion complex in solid state FT-IR Study Inclusion complex formation may be confirmed by FT-IR spectral study through comparison of guest molecule with solid Table 2 Fluorescence lifetime and amplitudes of INS with increasing concentrations of b-CDx. Concentrations of bCDx (M)

Lifetime (s)

Relative amplitude

v2

Standard deviation

0

5.16  1011 9.74  1010 5.62  109

34.99 27.56 37.45

1.26

2.28  1012 1.45  1011 3.26  1011

0.002

6.81  1010 3.22  109 7.92  109

36.68 39.54 23.79

1.09

3.59  1011 1.37  1010 1.64  1010

0.004

6.04  1011 1.72  109 6.37  109

26.71 27.52 45.77

1.28

1.33  1012 3.54  1011 4.44  1011

0.006

5.65  1011 1.74  109 5.78  109

28.28 26.97 44.75

1.09

1.53  1012 3.69  1011 3.55  1011

0.008

5.65  1011 2.00  109 6.42  109

27.66 29.79 42.54

1.16

6.48  1013 4.07  1011 5.09  1011

0.010

4.05  1010 2.52  109 7.73  109

22.89 40.32 36.79

0.99

2.81  1011 5.83  1011 8.94  1011

0.012

3.13  1010 2.27  109 6.96  109

28.40 35.08 36.53

1.11

1.69  1011 5.98  1011 7.46  1011

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Exothermic (mW/mg)

E D C

B A

0

50

100

150

200

250

300

350

400

Temperature

Fig. 4. The Fourier transform-infrared (FT-IR) spectra of (1) b-cyclodextrin; (2) inosine; (3) inclusion complex of INS-b-CDx; (4) physical mixture of INS and b-CDx; and (5) kneading method of INS and b-CDx.

complex. Because of bands resulting from the included ‘‘guest’’ molecule are generally shifted or their intensities are altered. Based on the changes, we can give some idea about the complex formation in solid state. IR spectrum of b-CDx, INS, Physical mixture of INS and b-CDx, solid prepared by Kneading method (KM) and co-precipitation(CP) are given in (Fig. 4). The FT-IR spectrum of b-CDx observed at 3385.23 cm1 due to symmetric stretching of m [OH]. Also in the IR spectrum of b-CDx, the absorption band with maximum at 2924.70 cm1 is observed. It belongs to the valence vibration of the CAH bonds in the CH & CH2 groups. There are absorption peaks at 1157.30, 1079.98 and 1028 cm1 which corresponds to the symmetry of m[CAC], m[CAOAC] and bending vibration of m[OH] respectively. The absorption bands in the region 950–700 cm1 belong to the deformation vibration of the CAH bonds and the pulsation vibration in glucopyranose cycle. In the IR spectrum of INS a strong absorption peak at 1698.51 cm1 for [C@O] stretching band due to six membered condensed with five membered rings and the presence of N atom. The frequencies for INS observed at 3432.67, 3296.66 and 2932.04 cm1 which corresponds to the m[OH], m[–NH] and m[–CH2] respectively. The Aromatic m[C@C], m[CAH] and m[CAOAC] is observed IR spectrum of the frequency 1534.70, 1197.34 and 1055.53 cm1 respectively. The IR spectrum of the inclusion complex ‘‘b-CDx – INS’’ differs from the IR spectrum of b-CDx and INS. The band of the valance vibration of the m[OH] functional group of INS & b-CDx is shifted

Fig. 6. The differential scanning calorimetry (DSC) spectra of (A) b-cyclodextrin; (B) inosine; (C) inclusion complex of INS-b-CDx; (D) physical mixture of INS and b-CDx; (E) kneading method of INS and b-CDx.

to lower wavenumber in spectral pattern of the inclusion complex and registered at 3357.89 cm1. At the same time, the band of the vibration and m[CAOAC] bonds shifted to higher wavenumber and observed at 1084.72 cm1 with 24% of transmittance. There is a very strong absorption band at 1705.01 cm1 almost appeared in the b-CDx/INS inclusion complex, indicating that the group [C@O] are not entrapped into the host cavities. The decrease in the m[OH] frequency between the inclusion complex between INS and b-CDx, its constituent INS is due to the changes in the microenvironment which lead to the formation of hydrogen bonding and the presence of the van der waals force during their interaction to form the inclusion complex. On the other hand, the FT-IR spectrum of physical mixture and kneading method imitated the characteristic peaks of b-CDx and INS, which can be regarded as a simple superimposition of those host and guest molecules. Thus, the FT-IR spectrum significantly proves the formation of the INS/b-CDx inclusion complex. SEM image analysis One more secondary and fruitful evidence for the complexation of INS with b-CDx is also observed from morphological studies (Fig. 5). SEM images of INS and b-CDx show that the clear rock structure. The complexed form of INS shows that the colloidal image. It is completely different from the morphology of INS and b-CDx. Further SEM picture showed that the shape and size of the inclusion complex are completely different from the areas of free INS and b-CDx. From the comparison of SEM images, we

Fig. 5. Scanning electron microscopy (SEM) images of (A) beta-cyclodextrin, (B) Inosine, (C) INS and b-CD inclusion complex (1:1 M ratio).

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E

Intensity (cps)

D C

B

A 5

10

15

20

25

30

35

40

45

50

2 Theta (Degrees) Fig. 7. The X-ray diffraction patterns (XRD) spectra of (A) b-cyclodextrin; (B) inosine; (C) inclusion complex of INS-b-CDx; (D) physical mixture of INS and b-CDx; (E) kneading method of INS and b-CDx.

Fig. 8. Ball and stick representation of (a) b-CD (b) INS (c) 1:1 inclusion complex; the oxygen atoms are shown as red, nitrogen as blue, carbon as golden and hydrogen atoms are not shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

strongly believe the complex formation between INS and b-CDx in solid state. DSC Study The DSC of INS, b-CDx, INS: b-CDx (PM), INS: b-CDx (KM) and INS: b-CDx (CP), complexes are shown in (Fig. 6). Thermal behavior of INS: b-CDx is studied by DSC in order to confirm the formation of solid complex of INS with b-CDx. When guest molecules are incorporated in b-CDx cavity or in crystal lattice, their melting, boiling and sublimation points usually shifted to a different temperature

or disappear within the temperature range, where b-CDx lattice is decomposed. DSC thermogram of INS showed an endothermic peak at 229 °C corresponding to its melting point. Thermogram of b-CDx showed a very broad peak, which attained a maximum around 100 °C due to release of water molecules and peak at 330 °C corresponding to its melting point. Thermogram of INS and b-CDx prepared by physical and kneading method showed endothermic peak at 288 °C. This may be due to shift to characteristic peak of INS, which is observed at 229 °C, indicates weak interaction of INS with b-CDx. Thermal curve of

S. Prabu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 147 (2015) 151–157 Table 3 Scores of the top 10 docked models of INS: b-CD inclusion complex computed using Patch Dock server. Model

Geometric shape complementarity score

Approximate interface area size of the complex (Å2)

Atomic contact energy (kcal/mol)

1 2 3 4 5 6 7 8 9 10

3250 3236 3234 3124 3104 3096 3070 3038 3034 2976

381.2 388.5 373.5 383 377.5 364.3 364.8 354.3 363.3 360.4

273.4 282.8 266.8 276.6 275.4 262.6 276.1 258.9 280.93 271.2

INS: b-CDx solid complex showed no endothermic peak at 229 °C. Hence, it is disappeared due to formation of complex between INS and b-CDx. XRD Study (Fig. 7) showed the XRD patterns of pure INS, b-CDx, inclusion complex by CP method, physical mixture and KM method. The diffraction peaks are observed to INS within 5–50 °C 2h range with characteristic peaks at 11.59, 16.15, 18.24, 25.87 and 32.86 (Fig 7B). Some sharp peaks at the diffraction angle of 2h 10.75, 12.67, 15.43, 19.62 and 22.82 are present in the b-CDx powder (Fig 7A). The diffraction pattern of solid inclusion complex of INS by CP method contains the peaks at 10.62, 15.17, 17.94, 24.38 and 30.75 (Fig 7C). The intensity peaks at 11.59, 16.15, 18.24, 25.87 and 32.86, 2h of INS are reduced significantly in INS:b-CDx inclusion complex diffraction patterns which suggests the reduction in crystallinity of INS. The change in diffraction patterns due to the formation of inclusion of INS molecule into cavity of b-CDx. Further new intense diffraction peaks are also observed for INS–b-CDx inclusion complex which indicates change in INS–b-CDx environment after inclusion complex. Only a slight decrease in peak intensity is noted for PM and KM. Hence no inclusion complex is obtained by physical mixtures and kneading method. Molecular docking study of inclusion process The 3D structure of b-CD and INS obtained from crystallographic databases are shown in (Fig. 8a and b). The guest molecule, INS is docked into the cavity of b-CD using Patch Dock server. The Patch Dock server program gave several possible docked models for the most probable structure based on the energetic parameters; geometric shape complementarity score [30], approximate interface area size and atomic contact energy [27], of the INS: b-CD inclusion complex Table 3. The docked INS:b-CD 1:1 model (Fig. 8c) with the highest geometric shape complementarity score 3250, approximate interface area size of the complex 381.2 Å2 and atomic contact energy (273.4 kcal/mol) is the highly probable and energetically favorable model and it is in good correlation with results obtained through experimental methods.

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