Energy efficient, facile and cost effective methodology for formation of an inclusion complex of resveratrol with hp-β-CD

Article abstract of DOI:10.1039/c5nj00924c

A distinctive, effortless, proficient and innovative process for preparation of an inclusion complex (IC) of resveratrol (Res) with hp-β-CD was developed. The methodology overcame the hindrance of low solubility of Res which is a potential anticancer agent and makes its use more practical. The strategy involved the utilization of a microwave meant for conventional use and the process of sonication in the laboratory. It considerably saved time and labour besides cutting down on energy consumption. The prepared inclusion complexes were characterised by FTIR, UV-visible absorption spectroscopy, NMR, TGA, DSC, XRD and CHNS analysis. Phase solubility studies along with Gaussian studies proved the formation of a 1:1 complex whose physical nature and stability were studied using XRD and UV-visible absorption spectroscopy, respectively. This effortless and economical process could pave the way for the formation of inclusion complexes of other nutraceuticals and pharmaceuticals.

Full text of DOI:10.1039/c5nj00924c

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Energy efficient, facile and cost effective
methodology for formation of an inclusion
Cite this:DOI: 10.1039/c5nj00924c
complex of resveratrol with hp-b-CD
Khushwinder Kaur,* Shivani Uppal, Ravneet Kaur, Jyoti Agarwal and
Surinder Kumar Mehta
A distinctive, effortless, proficient and innovative process for preparation of an inclusion complex (IC) of
resveratrol (Res) with hp-b-CD was developed. The methodology overcame the hindrance of low
solubility of Res which is a potential anticancer agent and makes its use more practical. The strategy
involved the utilization of a microwave meant for conventional use and the process of sonication in the
laboratory. It considerably saved time and labour besides cutting down on energy consumption. The
prepared inclusion complexes were characterised by FTIR, UV-visible absorption spectroscopy, NMR,
TGA, DSC, XRD and CHNS analysis. Phase solubility studies along with Gaussian studies proved the
formation of a 1 : 1 complex whose physical nature and stability were studied using XRD and UV-visible
absorption spectroscopy, respectively. This effortless and economical process could pave the way for
the formation of inclusion complexes of other nutraceuticals and pharmaceuticals.
Received (in Montpellier, France)
14th April 2015,
Accepted 31st August 2015
DOI: 10.1039/c5nj00924c
www.rsc.org/njc
and inhibits platelet activation and their aggregation. It also
protects lipoproteins from oxidative and free radical damage
1. Introduction
Nutraceutical, as defined in the literature, are a portmanteau of besides inhibiting tumour initiation and their promotion and
the words ‘‘nutrition’’ and ‘‘pharmaceuticals’’.1,2 These molecules progression. However, the problems concerning the physico-
possess a variety of physiological benefits and provide protection chemical properties, such as poor solubility and photosensitivity
against diseases and ailments like cardiovascular problems, have meant that no novel food has actually been fortified with this
obesity, diabetes, carcinoma, chronic inflammatory disorders antioxidant.8
and degenerative diseases.3 In broader terms, they are defined
Concerted efforts are now being made to overcome physico-
as food or a part of food that provides medical and health chemical challenges and instability of natural bio-actives in
benefits. In conformity with their conjectured safety, prospective order to improve their commercial viability and efficacy vis-a-vis
nutritional values and therapeutic effects, nutraceuticals have thus their conventional counterparts. Nutraceutical formulation
been of great interest to scientists.
strategies reported in the literature include: liposomal carrier
These naturally occurring bioactive compounds are now systems, electrospun fiber mats, microsponges and nanosponges,
being considered as the single most successful discovery of cyclodextrin complexation, biodegradable hydrogels, nano-
modern medicine.4–6 The polyphenolic nature of compounds emulsions, nanostructured lipid carriers, nanomicelles, nano-
like quercetin, curcumin, resveratrol, etc. explain their antioxidant particles, nanocapsules and nano-encapsulation, solid dispersions,
activities and enable them to quench the free radicals produced self-emulsifying drug delivery systems (SEDDS) and microparticulate
under diseased conditions. However, high hydrophobicity, low systems like microparticles, microspheres and microcapsules.9
water solubility and sensitivity to external agents such as air, light Cyclodextrin and modified cyclodextrin based nutraceutical
and oxidative enzymes pose a serious threat to the stability and the carriers have an exceptional ability to enhance the therapeutic
bioavailability of these formulations.7 These characteristics limit index of the bioactive agents by improving their inherent
their use as food additives and therapeutics.
solubility, stability, permeation and bioavailabililty.10–13 Pinho
Resveratrol i.e. (trans-3,5,40-trihydroxystilbene) (Res) is a et al. reported cyclodextrins as cyclic oligomers capable of
potential anticancer agent found free and in conjugated form in enhancing the biological, physical and chemical properties of
grapes, peanuts and other plants. It modulates lipid metabolism bioactive molecules like stilbenes.14 The advantages of using
these ‘‘cage-like’’ molecules include their high biocompatibility
and versatility to form complexes with a wide range of hydro-
phobic naturally occurring drugs. A number of complexation
Department of Chemistry and Centre of Advanced Studies in Chemistry,
Panjab University, Chandigarh – 160 014, India. E-mail: makkarkhushi@gmail.com
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studies with cyclodextrins (CDs) are available in the literature15–18 IC is formed only in 100 s using the microwave methodology and
demonstrating the formation of the 1 : 1 b CD complex with Res. within 15 min with sonication. This simple and quick approach
Zou et al.16 proved that hp-b CD was a better complexing agent for not only produces instant results similar to the traditional stirring
Res than b CD. Nunez-Delicado17 and his co-workers investigated methods but also avoids the use of toxic solvents.
complexation of Res with b CD and G2-b CD. They confirmed that
in addition to increasing Res hydrosolubility, entrapment in
internal CD cavity delayed Res oxidation by lipoxygenase. The
authors concluded that CDs could be used as Res carrier systems
2. Experimental
Materials and methods
since they acted as substrate reservoirs in a dosage controlled
manner. In another report, Venuti et al.18 prepared the Res/
Resveratrol (purity 499%), hp-b-CD, (purity 499%), ethanol,
(purity 499%) were purchased from Sigma-Aldrich. Triple
sulfobutyl ether–cyclodextrin inclusion complex (IC) by 30 min
sonication followed by stirring the mixture for 3 days. The
prepared complex was found to be a potential drug for treatment
of breast cancer. Apart from this, ICs of various other bioactive
distilled water with conductance less than 3 mS cmꢀ1 was used
for the preparations. The 2-D structures of the materials used
are reported in Scheme 1.
molecules were also prepared. These studies clearly illustrated
the merits of forming ICs with cyclodextrin derivatives for
Method of preparation
enhancing the solubility and stability of these hydrophobic wonder
molecules and lay their pathway to being capable, productive and prepared by the standard procedure (Med I) reported in the
an economical remedy for numerous infirmities.
literature.30 The second method (Med II) involved mixing of
Preparation of inclusion complex (IC). Firstly, the IC was
For efficient incorporation in food or to be administered as 4.38 ꢁ 10ꢀ3 mol hp-b-CD and 4.38 ꢁ 10ꢀ3 mol Res in a glass
effective pharmacological drugs various other ICs have also container with the minimum amount of the solvent mixture
been reported in the literature (Table 1)18–29 (only a few of them have (ethanol : water = 1 : 9). The mixture was microwave (IFB 20 PG
been listed) but all the reported methods are highly time engrossing. 2S) processed for 100 s at 800 W to get the product. Water was
In the present day contemporary world, time minimization and evaporated under vacuum. The data obtained after 25, 50 s at
energy efficiency have become fundamental conditions for any 200, 400, 600 W revealed incomplete complexation. The third
new discovery. New methods of providing energy are being method (Med III) involved the same compositions of reactants
explored for the quick formation of the products without adversely as in Med II. However, the sample was sonicated for 15 min at
affecting their desired healing properties.
In the present report, an inexpensive, quick and facile microwave. The end product was obtained by lyophilization.
methodology has been developed for the formation of the IC Preparation of physical mixture (PM). The physical mixture
180 W using a Hielsher UP200St ultrasonic device instead of a
of Res which can find potential applications if loaded in daily of Res and hp-b-CD in a 1 : 1 molar ratio was prepared by
diet. It does not require laborious and endless stirring and the grinding in a pestle mortar.
Table 1 List of ICs and their synthesis procedures as reported in the literature
Targeting drug/nutraceutical
Time taken
Ref.
A characterization study of resveratrol/sulfobutyl ether-b-
cyclodextrin inclusion complex and in vitro anticancer activity
Synthesis and spectroscopy studies of the inclusion complex
of 3-amino-5-methyl pyrazole with beta-cyclodextrin
Preparation, characterisation and bioactivity evaluation of the
inclusion complex formed between picoplatin and g-cyclodextrin
Inclusion complexes of poly(4-vinylpyridine)–dodecylbenzene-
sulfonic acid complex and cyclodextrins
30 min sonication followed
by stirring for 3 days
72 h of stirring
Colloids Surf., B, 2014, 115, 22–28
Spectrochim. Acta, Part A, 2015, 134,
276–282
Carbohydr. Res., 2014, 396, 54–61
5 days stirring
2 days of stirring
Macromolecules, 2002, 35, 3997–4002
J. Agric. Food Chem., 2002, 50, 1355–1361
Carbohydr. Polym., 2014, 113, 577–587
Fluorometric determination of curcumin in yogurt and mustard Agitation of mixture at 70 1C
for 4 h
48 h of stirring
2,6-Dinitroaniline and b-cyclodextrin inclusion complex
properties studied by different analytical methods
N-Phenyl-1-naphthylamine/b-cyclodextrin inclusion complex as a Stirred for 48 h at room
Spectrochim. Acta, Part A, 2014, 133,
73–79
Food Res. Int., 2014, 62, 424–431
new fluorescent probe for rapid and visual detection of Pd2+
Functional electrospun polymeric nanofibers incorporating
geraniol–cyclodextrin inclusion complexes: high thermal
stability and enhanced durability of geraniol
temperature
Stirring for 2 h at 80 1C +
additional 5 h at room
temperature
Inclusion complexes of b-cyclodextrin–dinitrocompounds as
UV absorber for ballpoint pen ink
48 h at room temperature
Spectrochim. Acta, Part A, 2014, 129,
551–564
Carbohydr. Polym., 2014, 114, 558–566
Spectral investigations of host–guest IC of 4,40-methylene-bis(2- Stirring at room temperature for
chloroaniline) with beta-cyclodextrin
24 h and then freezed for 24 h
48 h stirring at room temperature Carbohydr. Polym., 2014, 107, 72–84
Preparation and characterizations of solid/aqueous phases
inclusion complex of 2,4-dinitroaniline with b-cyclodextrin
Transdermal delivery of the in situ hydrogels of curcumin and
its inclusion complexes of hydroxypropyl-b-cyclodextrin for
melanoma treatment
0.5 h stirring using grinding
method
Int. J. Pharm., 2014, 469, 31–39
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with AgCl windows. The samples were scanned at a resolution
of 4 cmꢀ1 over the range.
UV-visible absorption spectra. UV-visible absorption spectra
were obtained in the spectral range 200–380 nm using a JASCO
V 530 (4-21, Sennin-cho 2-chome, Hachioji, Tokyo 193-0835,
Japan) model spectrophotometer with a precision of ꢂ0.2 nm
using quartz cells with a path length of 1 cm.
NMR. 1H NMR spectra were recorded at 400 MHz on a
Brucker FT-NMR spectrometer (Bruker, Switzerland) at 400 MHz
in D2O using TMS as an internal standard. Chemical shifts of
1H NMR spectra were measured in parts per million with respect
to TMS.
Stability studies. Comparative studies of the stability of free
Res and the IC were carried out in solution form (ethanol : water
= 1 : 9) at room temperature under high energy UV exposure.
The solutions were prepared dissolving 2 millimoles of Res and
IC in 20 ml of solvent. At specified time intervals, the samples
were taken and the concentration of trans-Res was determined
by UV-visible absorption spectroscopy. The results were
expressed as the percentage of the remaining trans-Res and
the percentage of Res converted to its non-functional cis form
in the IC by UV-visible absorption spectra. The results were
obtained by a curve-fitting algorithm using a Gaussian–Lorentzian
function.32,33 The best curve fitting procedure was performed by
iterative fits toward a minimum standard error. The relative
composition of the component was computed to be the fractional
area of the corresponding peak, divided by the sum of the area for
all the peaks.
Powder X-ray diffraction. The information on the crystal-
linity of the prepared complex was obtained through X-ray
diffraction (XRD; PANalycital X’pert Pro.) with Cu-K radiation
(=1.5418 Å) in the range 15–70 1C at a 8 1C minꢀ1 scanning speed.
CHNS analysis. The elemental analysis was carried out using
a CHNS-O ANALYZER thermo scientific flash 2000 organic
elemental analyzer.
Scheme 1 Representative structures of (a) trans Res (b) cis Res (c) hp-b-CD.
Phase solubility studies. Solubility studies were carried out
by using the method reported by Higuchi and Connors31 with
some modifications. Excess Res was added to aqueous solution
with increasing concentrations of hp-b-CD (0.5–4 mmol lꢀ1) at
25 1C, 30 1C and 37 1C. The system was left under stirring for
24 h at 850 rpm. The samples were centrifuged for 15 min and
the supernatants were filtered through 0.45 mm nylon filters in
order to separate excess Res. Further, the concentration of Res in
each aqueous solution or ‘‘apparent solubility’’ was determined
spectrophotometrically. The stability constant, Kc, was calculated
from the straight line portion of the phase solubility diagram
according to Higuchi–Connors eqn (1).
Slope
Kc ¼
(1)
S0 ꢁ ð1 ꢀ slopeÞ
SEM. The morphological estimations were done using scanning
electron microscopy (SEM; JEOL-JSM-7600F).
Where S0 is the Res solubility in the absence of b-CD.
The experimental data obtained from phase solubility were
used to calculate thermodynamic properties of the complex
formation using the experimentally determined Kc values and
the integrated form of the Van’t Hoff equation
TGA. The thermal behaviour was investigated using an SDT-
Q-600 (TA instruments New Castle, DE). Sample measurements
were carried out using in alumina pans under nitrogen atmosphere.
The flow rate of nitrogen was 100.0 ml minꢀ1. Alumina crucibles
were used for recording the curves. The samples were heated
(B10 mg) to the given temperature at a fixed heating rate (b)
DH DS
ln Kc ¼ ꢀ
þ
(2)
RT
R
which was 10 1C minꢀ1
.
where DH is the enthalpy change for the reaction (kJ molꢀ1), R is the
universal gas constant (J molꢀ1 Kꢀ1), T is the absolute temperature
(K), and DS is the entropy change for the reaction (J molꢀ1 Kꢀ1).
The Gibbs free energy (DG) was calculated using the above-
mentioned parameters and eqn (3) under standard conditions
DSC. DSC measurements were performed using a DSC TA
Q20 instrument equipped with a refrigerated cooling system
(TA Instruments, New Castle, DE). An indium standard (melt-
ing point = 156.66 C, DH melting = 28.41 J gꢀ1) was used for
calibration. The solid samples (6–11 mg) were pipetted out to
40 ml DSC aluminium pans, which were hermetically sealed
with the lid quickly to prevent evaporation. DSC tests for samples
were run the under conditions (i) equilibrating at 25 1C for 1 min,
(ii) cooling the sample at a ramp rate of 10 1C minꢀ1 to ꢀ50 1C,
DG = DH ꢀ TDS
(3)
Characterization of inclusion complex
FTIR spectra. FTIR spectra were recorded in the spectral isothermal for 0.5 min, (iii) heating the sample at a ramp rate of
region 4000–500 cmꢀ1 using a Perkin-Elmer (RX1) spectro- 10 1C minꢀ1 to 300 1C. An empty hermetically sealed pan was
photometer (Perkin-Elmer, Beaconsfield, Buck, U.K.) equipped used as a reference.
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Gaussian studies. All the computational calculations for the peaks at 1654 cmꢀ1, 1156 cmꢀ1, and 1032 cmꢀ1 were assigned to
synthesized complexes were performed using Gaussian 03 the H–O–H bending, C–O and C–O–C stretching. In the spectra
package. The structure of complexes was built using Gauss of the PM, all characteristic bonds of Res and hp-b-CD were
view 3.0 implemented in the Gaussian 03 package.
evident, indicating weaker or no interaction between Res and
hp-b-CD when physically mixed.34 However, in the spectra of IC,
some characteristic bands of Res were found to be shifted
or absent. Fig. 1(c) depicts the FTIR spectra of the IC prepared
by different methods at different times in a microwave. The
results showed that in the IC formed by either of the methods, the
band intensities decreased at 1384 and 965 cmꢀ1 and disappeared
at 1606 and 1587 cmꢀ1. This clearly evidenced the formation
of the IC.35
Clear spectral overlap indicated the compatibility between
the ICs formed by the three methods. Also, the shapes and
intensities of the bands changed dramatically for the formation
of ICs as compared to pure Res or hp-b-CD. It further supported
the formation of ICs.36
3. Results and discussion
The results obtained by FTIR, UV-visible absorption spectro-
scopy, TGA, XRD, 1H NMR and CHNS for the three methods
were found to be comparable and are discussed below.
The FTIR spectra of Res, hp-b-CD, and the physical mixture are
shown in Fig. 1(a). Res showed peaks in the range 1750–450 cmꢀ1
with characteristic peaks at 1606, 1587, 1384, and 965 cmꢀ1
corresponding to the C–C aromatic double bond stretching, C–C
olefinic, C–O stretching and trans olefinic bond, respectively.30 The
spectrum of hp-b-CD was characterized by the intense band at
3407 cmꢀ1, corresponding to the vibration of the hydrogen-
bonded OH groups, the band at 2926 cmꢀ1 correspond to the
absorption of CH and CH2 groups, and some other prominent
Time and power optimization remained major challenges
while carrying out reactions using the microwave methodology.
Fig. 1 FTIR spectra (a) comparison of Res, the PM and hp-b-CD (b) the IC synthesized using Med II at different times and (c) comparison of ICs
synthesized using Med (I, II, and III).
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For this, the reaction was carried out at different time intervals show photoisomerisation when exposed to daylight; they undergo
of 25, 50 and 100 s. The FTIR results indicated the complete changes in the trans stereoisomer ratio as the amount of the cis
absence of characteristic peaks when the reaction was carried isomer increases affecting the trans forms amount. The same
out for 25 s. The band intensities at 1384 and 965 cmꢀ1 reaction takes place when the solution is exposed to UV light.
decreased whereas they disappeared at 1606 and 1587 cmꢀ1 The longer the irradiation the higher the cis-Res concentration,
indicating the formation of the IC after 50 and 100 s. Similar until about 2 h, where the ratio of the cis isomer reaches its
experiments were also carried out at different power modes of maximum.42 Bertacche et al.30 reported the stability of Res and the
150, 300, and 600 W, however, no product was obtained.
corresponding IC both in the solid state and in ethanol solution at
UV-visible absorption spectroscopy has proved to be a room temperature in the dark, at 4 1C and under light exposure
very convenient method to explore the structural change and which proved the stability of Res and the IC in solid state. They
formation of the complex.37,38 The absorption spectra of Res, concluded that the isolation rate of isomerization of trans to cis
hp-b-CD, the PM and the IC were recorded according to the decreased with inclusion. The present study also revealed the
standard procedure. The UV-visible absorption spectra of Res decrease in conversion with inclusion when the system was nearly
showed two bands, one centred at around 215 nm and the other aqueous. The obtained %ages of cis and trans products with time
one at about 300 nm.39 After conversion from trans- to cis Res, are tabulated in Table 2.
the band at 300 nm disappeared and a band at 285 nm was
From the TG analysis of various moieties viz., Res, hp-b-CD and
observed. The distinct and deformed peaks observed in the PM the IC it was observed that the IC formed by the three methods
indicates that Res and hp-b-CD are in their natives states and followed similar thermal behaviours. The results have been
very weak interactions are occurring which lead to overlapping depicted in the TG curve (Fig. 3(a)). Comparing the decomposition
of peaks and thereby broadening of peaks. The studies on the behaviour it was observed that while Res decomposed in a single
IC further revealed not only simple superposition but also a step, hp-b-CD, Res and the IC decomposed following a two-step
slightly shifted peak at 310 nm (Fig. 2(c)). This may be taken as
indirect proof of complex formation.40 The results further
indicated that the value of absorption (DA) at the peak wavelength
Table 2 Stability of the IC under high energy UV exposure
of the IC was almost 20 times that of free Res. This increase in the
Time (min)
%age cis
%age trans
peak absorption was attributed to enhanced solubility of the IC as
compared to free Res and hp-b-CD. Yet again the peaks obtained
by the three methods completely overlapped indicating the
formation of the IC by the three methods.
0
20
40
100
120
160
200
220
0
7.57
8.04
12.50
27.36
31.91
38.00
45.85
100
90.26
89.74
85.70
66.00
63.45
52.54
48.73
It has been reported in the literature41 that Res exists in
trans and cis isomeric forms of which the trans form is more
photo and thermo stable. Standard trans-Res solutions also
Fig. 2 UV-visible absorption spectra of (a) Res (H2O + C2H5OH) and hp-b-CD (H2O) (b) PM (H2O) (c) comparison of ICs synthesized using Med (I, II, and
II) (H2O).
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proposed method, if eqn (4) given below is satisfied by the
relative parameters then the stoichiometry of Res : hp-b-CD can
be defined as 1 : n.
À
Á
aWpure CD ꢀ kbWpure CD MCD
nMguest ꢀ 2M H2O ꢃ
ꢃ nMguess
Wpure CD
(4)
where Mguest, MH O and MCD are the molar weights of the guest
2
0
(Res), water and hp-b-CD, respectively. Wpure CD, and Wpure CD
represent the mass of the total hp-b-CD with and without any
guest, respectively. The parameters k, b and a were calculated
using equations as already reported in the literature. Substituting
the values obtained from TG curves in the above equation with
k = 0.917, b = 0.1279, a = 0.3442, the ratio 1 : n was satisfied by the
value 1: 1, which confirmed the formation of the complex in a
1: 1 stoichiometric ratio.
Fig. 3 TG curves for (a) the ICs formed by the three methods, (b) Res,
hp-b-CD and ICs; (c) the Coats–Redfern plot.
Perusal of the literature revealed that very few researchers
have commented on the stoichiometry of the formed IC.46,47
Bru et al.47 reported that the 1 : 1 IC had the highest correlation
coefficient, thus, specifying the complex formation in a 1 : 1
ratio. The present study also evidenced the formation of a 1 : 1
stoichiometric ratio for the IC.
Molecular modelling calculations were further carried out to
compare the experimental TG data with theoretical computa-
tions. Minimum energy optimized structures for the complexes
were obtained using Gaussian software as given in Fig. 4. The
minimum energy of the optimized structure and activation
energy calculated using TG were in sync and followed a similar
trend suggesting that the IC formed was more stable than
hp-b-CD (Table 3).
XRD is a useful technique for the detection of CD encapsulation
and is a useful method to assess the degree of crystallinity of the
given sample. Supporting evidence for complex formation was
obtained from this. Perusal of the literature reveals that the
formation of an IC between hp-b-CD and a crystalline guest meant
that the latter would no longer exist in the crystalline state and,
consequently, the diffraction pattern of the complex would not
be a simple superposition of those of the two compounds. When
a true complex is formed, the overall number of crystalline
structures reduce and that of amorphous structures increase.34
Hence the IC exhibits less intense peaks. It was observed that
pure Res was highly crystallized and exhibited intense crystal-
line peaks between 51 and 501.48 The XRD pattern of hp-b-CD
showed no crystalline peak indicating an amorphous nature.49,50
The obtained XRD of the PM confirmed the loss of identity of the
individual compounds (Fig. 5) showing characteristic peaks of the
mixture of Res or hp-b-CD but as it was not a true complex and
the peaks retained their intensity and sharpness. This type of
behaviour was found to be consistent with other studies in the
literature reported for the formation of ICs.51–53 Meanwhile, the
XRD pattern of the three ICs showed no characteristic peak of
Res, which was distinct from the PM and hp-b-CD. The diffraction
peaks relating to crystalline behaviour were no longer detectable in
the complex system and the reduction of the crystalline degree were
further considered as an indirect proof of complex formation52
mechanism (Fig. 3(b)); the first step being the removal of water in
both hp-b-CD and the IC.
TG plots exhibited high thermal stability along with well-
defined decomposition steps. It was observed that hp-b-CD was
thermally the most stable of all the given moieties and decomposed
at 360 1C, whereas Res was the least stable. The higher decom-
position temperature of the IC (360 1C) vis-a-vis Res (340 1C) is
an indication of superior thermal stability. The IC showed
decomposition between 340 and 360 1C. This could be attributed
to the fact that pure Res showed decomposition at 340 1C.
Therefore, Res present in the cavity of hp-b-CD also started
decomposing at a similar temperature. A similar decomposition
behaviour was also observed by Macedo et al.43 The activation
energy for thermal decomposition was calculated using the Coats–
Redfern method.44 The calculated activation energy came out to be
nearly the same for all the three methods, i.e., EMedI = 74.94 kJ molꢀ1
,
EMedII = 74.90 kJ molꢀ1, and EMedIII = 73.39 kJ molꢀ1. From this it
could be inferred that the product obtained by any of the
methods followed the same decomposition behaviour. Also,
the apparent activation energy and behaviour for thermal
decomposition varied as: ECD 4 EIC 4 ERes (Table 3) indicating
that the IC formed was more stable compared to hp-b-CD and
hence required less energy for decomposition.
Evidence for the formation of the 1 : 1 IC was carried out by
determining the stoichiometric ratio from TG curves. Bai et al.45
had successfully devised a scheme to calculate the stoichiometric
ratio of the a-CD-based ICs in a solid-state form with four guests,
4-cresol, benzyl alcohol, ferrocene and decanoic acid, to verify the
possibility and accuracy of a new method. According to the
Table 3 Comparison of the activation energy using TG analysis and the
minimum energy optimized structure using Gaussian package
Eact
Moieties
TGA (kJ molꢀ1
)
Energy optimization (a.u.)
Res
hp-b-CD
IC
63.30
90.67
74.90
ꢀ761.00
ꢀ5592.52
ꢀ6353.96
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Fig. 4 Minimum energy optimized structure of the IC (a) top and (b) side views.
The exact overlapping XRD pattern indicated the similarity of the chemical shifts of pure Res and Med I were found to be in
ICs formed by any of the methods.
agreement with the literature55 and confirmed the formation of
Nuclear Magnetic Resonance spectroscopy (NMR) is one of ICs. Data in Table 4 showed that the chemical shift (Dd) for Res
the most useful techniques to study the interactions of host–guest protons in ICs were in range 0.006–0.163 ppm. As expected, the
complexes. It provides the information about the right orientation NMR signals of protons H-2 and H-6 showed the maximum
of the guest molecule inside the cavity of host molecules.54 So, if the downfield chemical shift Dd, i.e., 0.163 ppm and confirmed the
IC of hp-b-CD with Res is synthesized, then the protons inside the insertion of an aromatic ring bearing two hydroxyl groups inside
hp-b-CD cavity shall show the shift in the hp-b-CD value due to their the hp-b-CD bucket. The H-4 proton showed little chemical shift
interaction with the Res molecule. Similarly, Res protons shall also 0.027 ppm as it is positioned at the bottom of the hollow hp-b-CD
be sensitive to this changed environment and as a result, they will cavity and is not affected much by the –OH group of hp-b-CD.
also show the chemical shift in the 1H NMR spectrum. The proton Furthermore, vinylic protons H-7 and H-8 in ICs shifted towards
NMR spectra of Res, hp-b-CD and inclusion compounds (Med I, downfield and appeared at d 7.018 and 6.826 ppm, with Dd values
Med II and Med III), obtained from three different methods were of 0.120 ppm and 0.130 ppm, respectively. H-11 and H-13 showed
recorded in D2O solvent.
the slightest chemical shift at around 0.006 ppm, as they are away
The proton NMR spectra of pure Res, physical mixture (PM) from the hp-b-CD cavity. It proved that the second aromatic ring
and ICs are shown in Fig. 6 and their d values in Table 4. The 1H with one –OH group should not be capped with the second
Fig. 5 XRD pattern of (a) Res and hp-b-CD (b) the PM (c) comparison of ICs.
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Fig. 6 1H NMR (400 MHz, D2O) spectra (expanded between d 7.4 and 6.0 ppm) of (a) Res; (b) PM; (c) ICs synthesized using Med I; (d) Med II and (e) Med III.
The IC of Res was obtained in the solid and solution phases.
The stoichiometric ratio and stability constants were derived
from the changes in the solubility of Res in the presence of
increasing concentrations of hp-b-CD. The obtained phase
solubility plots at 25,30 and 37 1C are shown in Fig. 7. The
plots indicated that the solubility increased with the increase in
temperature with a linear dependence. Consequently, according
to Higuchi and Connors53 the plots could be considered to be of
the AL type suggesting the formation of the 1 : 1 IC. Further, for
all the plots the slope of the straight line was less than 1, thus it
was inferred that the increase in solubility was due to the
formation of the 1 : 1 IC.30 The results were in correlation with
the results obtained from TGA studies and Gaussian calculations
which also indicated the formation of the 1: 1 IC.
Table 4 Comparison of the d value for various protons of Res and ICs
synthesized using Med I, Med II and Med III
Entry
H-2 H-4 H-6 H-7 H-8 H-10 H-11 H-13 H-14
Res
PM
6.643 6.306 6.643 7.139 6.954 7.490 6.897 6.897 7.490
6.465 6.259 6.465 7.027 6.815 7.430 6.888 6.888 7.430
Med I (t) 6.491 6.286 6.491 7.018 6.826 7.439 6.903 6.903 7.439
Med II (M) 6.480 6.281 6.480 7.017 6.825 7.439 6.901 6.901 7.439
Med III (S) 6.484 6.281 6.484 7.019 6.828 7.439 6.902 6.902 7.439
molecule of hp-b-CD and confirmed the formation of 1 : 1 ICs.
The other ICs synthesized using Med II and Med III also showed
similar types of downfield chemical shifts as in Med I and
validated the formation of ICs using microwave and sonication
methods too. However, in Med II and Med III protons showed
the same Dd value with ꢂ0.01. This might be due to the different
morphological structures obtained from using three different
methodologies (discussed in the next section).
Furthermore, when the proton NMR (d 5.8 and 0.5 ppm
region) of hp-b-CD and Med I, Med II and Med III ICs were
compared, hp-b-CD protons in the IC synthesized by Med I
showed the chemical shift which matched with the literature
value. ICs synthesized by Med II and Med III also showed similar
types of shifts which proved the formation of similar ICs.
The PM is formed by simply mixing both the reactants Res
and hp-b-CD without using any external energy, therefore the
Res molecule should not be inserted inside the hp-b-CD cavity.
But, protons still showed a downfield chemical shift because
there was a little interaction between these two molecules due
to the presence of –OH groups on both the compounds.
Fig. 7 Straight line portion of the phase solubility diagrams for Res at
different concentrations of hp-b-CD.
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The stability constants (Kc) for the IC were calculated from
Paper
the straight line plots of Fig. 7 considering a 1 : 1 stoichiometry.
The values are listed in Table 5. The Kc values obtained at
different temperatures were plotted in a van’t Hoff plot. The IC
showed a linear function between Kc and the inverse of absolute
temperature (1/T). The stability constant decreased with the
increase in temperature indicating an exothermic process. The
results were comparable to the other reports in the literature on
the IC formation of Res.56,57 Also the obtained high Kc values
indicated that the equilibrium was displaced towards the
formation of the IC.
The phase solubility data were further used to calculate the
thermodynamic parameters using eqn (2) and (3). The calculated
thermodynamic parameters are tabulated in Table 4. The negative
value of the enthalpy change (ꢀDH) indicated that the interaction
process of Res with hp-b-CD was exothermic in nature. The
negative value of DG further indicated that the interaction
process was spontaneous in nature. The obtained negative value
for entropy changes could be explained considering the fact that
inclusion moderately hinders the free rotation of the included
molecule around the symmetry axis57
Elemental analysis is an important tool for characterization
of newly synthesized complexes. CHNS analysis of pure Res,
hp-b-CD and ICs are recorded in Table 6. The C and H %
observed were found to be well in agreement with the calculated
values and considered a good measure to assess the purity of the
complexes. It was observed that complexes formed were the
same irrespective of the employed methodology. It also sup-
ported the evidence given by Gaussian studies that a 1 : 1 (Res :
hp-b-CD) IC formation took place.
The DSC data also provide evidence for the formation of the
IC. It has been reported by Marques et al.58 that the inclusion of
a guest into the host molecules such as CDs causes changes in
the crystal lattice, and melting, boiling, and sublimation points
shift at different temperatures or disappear. The thermograms
of Res, hp-b-CD, the PM and IC are shown in Fig. 8. The obtained
DSC of Res was typical of a crystalline substance showing a sharp
endothermic peak at 269.9 1C corresponding to the melting point
Fig. 8 DSC curves of Res, ICs, the PM, and hp-b-CD.
of pure Res. The thermogram of hp-b-CD showed a wide endo-
thermic peak at about 110 1C, which referred to the release of
water. In the physical mixture the characteristic thermal profile of
Res was shifted to a lower temperature in the region 152.8 1C.
Furthermore, the thermogram of the PM indicated a super-
imposition of thermograms of Res and hp-b-CD indicating no or
very weak interaction in the PM. The thermogram of the IC
showed a shifted peak at 105 1C, the shift of peak to the lower
temperature could be attributed to the change in the properties
followed by complete complexation indicating an interaction
between Res and hp-b-CD. This also indicated an increase in the
amorphous character in the complex which was also supported
by the XRD data. All these results showed that Res was wrapped
into the cavity of hp-b-CD.59
Table 5 Aqueous solubility of Res in the absence of hp-b-CD (S0)
solubility, stability constants (Kc) and thermodynamic parameters
Temperature S0
(1C)
Kc
ꢀDG
ꢀDH
ꢀDS
(mmolꢀ1
)
(Mꢀ1
)
(kJ molꢀ1
)
(kJ molꢀ1
)
(J Kꢀ1 molꢀ1
)
25
30
37
0.00090
0.00125
0.00139
719.42 15.74
652.55 16.32
575.00 16.84
5.33
3.33
1.59
17.33
Table 6 Analytical data depicting obtained %ages of C, H, and N in Res,
hp-b-CD and ICs synthesized using Med (I), (II) and (III)
Compounds
C
H
Res
72.95 (73.61)
42.76 (42.96)
46.76 (46.48)
46.33 (46.48)
48.85 (46.48)
5.32 (5.25)
6.87 (7.72)
6.61 (7.44)
6.84 (7.44)
hp-b-CDꢄ12H2O
Med (I)
Fig. 9 SEM images of the ICs prepared by the three methods (a) Med I (b)
Med (II)
Med (III)
6.43 (7.44) Med II (c) Med III and (d) the PM.
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SEM (Fig. 9) is a reliable technique for studying the structural
aspects of materials and it was employed to investigate the
microstructures of the IC in solid state.60–62 The obtained shapes
were very different from pure Res which had been reported to be
bar shaped by Le et al.60 and pure hp-b-CD which were reported as
spherical particles with cavity structures by Chen et al.36
The PM indicated the presence of both the bars and open
spherical structures indicating the presence of both the com-
pounds and retention of their individual identity. SEM images
of the IC prepared by all the three methods clearly indicated the
formation of the irregular complex in which the original morphology
of hp-b-CD and Res disappeared. Tiny aggregates of amorphous and
irregular pieces were seen. The observed shape was in accordance
with the literature60–62 Liu et al.51 reported for the formation of ICs
under long stirring conditions.
NJC
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4. Conclusion
In light of our findings it can be concluded, that the complex
was prepared under solvent free conditions using a new innovative
simple microwave methodology involving only 100 s of reaction
time and the powerful sonication laboratory method. The obtained
23 K. Srinivasan, K. Sivakumar and T. Stalin, Carbohydr.
Polym., 2014, 113, 577–587.
1
product was characterized using FTIR, H NMR, TGA, DSC, XRD
24 M. Maniyazagan, S. Mohandoss, K. Sivakumar and T. Stalin,
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and CHNS analysis. The product was found to be exactly similar to
the one reported in the literature synthesized under 120 h long
refluxing conditions. This inexpensive and quick methodology will
find potential applications in food industry and may also provide a
gateway for cancer treatment for patients using quick microwave
methodology at home. The sonication method can also be applied
for the synthesis of ICs of other nutraceuticals. Further work in
this direction is in progress in our laboratory.
27 R. Periasamy, S. Kothainayaki and R. Rajamohan, Carbohydr.
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