Betulonic acid—cyclodextrins inclusion complexes

Article abstract of DOI:10.1007/s10973-019-08359-6

Betulonic acid (BA) is a pentacyclic lupane-type triterpenoid possessing valuable pharmacological activities, exhibiting very low water solubility. Inclusion complexes between the substance and cyclodextrins (CDs) were obtained in order to improve its solubility and consequently its bioavailability. The aim of this study was to investigate the guest–host interaction of BA with γ-cyclodextrin (γ-CD) and its derivative, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), in solution and in solid state in order to prove the formation of inclusion complexes between the components. The kneading method was used for the inclusion complexes preparation, and different analytical techniques such as thermal analysis, powder X-ray diffractometry, universal attenuated total reflectance Fourier transform IR spectroscopy (UATR-FTIR) and UV spectroscopy were employed to investigate the interaction between substances. The stoichiometry of the inclusion complex BA/HP-γ-CD was found to be 1:1 by employing continuous variation method, and the apparent stability constant was calculated as 1855.55?M^?1 using Benesi–Hildebrand equation. Structural studies of the inclusion complexes were carried out using molecular modeling techniques in order to explain the complexation mechanism. The results of this study confirm the formation of inclusion complexes between BA and cyclodextrins both in solution and in solid state.

Full text of DOI:10.1007/s10973-019-08359-6

Journal of Thermal Analysis and Calorimetry
https://doi.org/10.1007/s10973-019-08359-6
Betulonic acid—cyclodextrins inclusion complexes
1
2
1
4
5
Laura Sbârcea · Adriana Ledeţi · Lucreţia Udrescu · Renata-Maria Văruţ · Paul Barvinschi ·
6
3
Gabriela Vlase · Ionuţ Ledeţi
Received: 27 October 2018 / Accepted: 1 May 2019
Akadémiai Kiadó, Budapest, Hungary 2019
©
Abstract
Betulonic acid (BA) is a pentacyclic lupane-type triterpenoid possessing valuable pharmacological activities, exhibiting
very low water solubility. Inclusion complexes between the substance and cyclodextrins (CDs) were obtained in order to
improve its solubility and consequently its bioavailability. The aim of this study was to investigate the guest–host
interaction of BA with γ-cyclodextrin (γ-CD) and its derivative, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), in solution
and in solid state in order to prove the formation of inclusion complexes between the components. The kneading method
was used for the inclusion complexes preparation, and different analytical techniques such as thermal analysis, powder
X-ray diffractometry, universal attenuated total reflectance Fourier transform IR spectroscopy (UATR-FTIR) and UV
spectroscopy were employed to investigate the interaction between substances. The stoichiometry of the inclusion complex
BA/HP-γ-CD was found to be 1:1 by employing continuous variation method, and the apparent stability constant was
−1
calculated as 1855.55 M using Benesi–Hildebrand equation. Structural studies of the inclusion complexes were carried
out using molecular modeling techniques in order to explain the complexation mechanism. The results of this study confirm
the formation of inclusion complexes between BA and cyclodextrins both in solution and in solid state.
Keywords Betulonic acid · Triterpenoid · γ-Cyclodextrin · 2-Hydroxypropyl-γ-cyclodextrin · Inclusion complex ·
Thermoanalysis
Introduction
&
Adriana Lede ¸t i
afulias@umft.ro
Natural products have always been an important source of
starting compounds for drug discovery and development
[
1–3]. They have played a substantial role in the antitumor
1
Department of Drug Analysis, Faculty of Pharmacy, “Victor
Babe s¸ ” University of Medicine and Pharmacy, 2 Eftimie
Murgu Square, 300041 Timisoara, Romania
drug development [4, 5], a great majority of antitumor
compounds originate in natural products [4, 6–8]. The
biological active compounds contained in natural products
have special selectivity to cellular targets [3, 9].
2
3
4
Department of Analytical Chemistry, Faculty of Pharmacy,
“Victor Babe s¸ ” University of Medicine and Pharmacy, 2
Eftimie Murgu Square, 300041 Timisoara, Romania
Pentacyclic triterpenoids are one of the most abundant
natural compounds in the plant kingdom [4, 8] and, in the
last decade, have received particular attention due to their
important medicinal properties [10–12]. Betulonic acid
(BA, 3-oxolup-20(29)-en-28-oic acid, Fig. 1) is a triter-
penoid with lupane skeleton, presenting poor water solu-
bility (in silico predicted aqueous solubility − 8.0\log
Department of Physical Chemistry, Faculty of Pharmacy,
“Victor Babe s¸ ” University of Medicine and Pharmacy, 2
Eftimie Murgu Square, 300041 Timisoara, Romania
Department of Physical Chemistry, Faculty of Pharmacy,
University of Medicine and Pharmacy Craiova, 2-4 Petru
Rares Str., 200349 Craiova, Romania
5
6
Faculty of Physics, West University of Timisoara, 4 Vasile
P aˆ rvan Blvd, 300223 Timisoara, Romania
(Sw)\−6.0) [13]. BA and its derivatives have proved
biological activities including antiviral [14–16], anti-in-
Research Center for Thermal Analysis in Environmental
Problems, Faculty of Chemistry-Biology-Geography, West
University of Timisoara, 16 Pestalozzi Street, Timisoara,
Romania
flamatory [17, 18], antitumor [4, 19], hepatoprotective
[
18, 20], anti-HCMV activity [21], antimicrobial [22],
immunostimulant [23] and antioxidant effects [24].
123

L. Sbârcea et al.
(
a)
(b)
(c)
OH
O -R
6
6
5
O
1
5
O
1
O
O
OH
O
O
O-R
O
4
4
HO
R-O
2
2
H
O
3
O
3
HO
OH
O
HO
OH
HO
HO
OH
OH
OH
HO
O
OH
O
HO
O
OH
O
HO
HO
OH
OH
OH
HO
HO
O
OH
OH
O-R
O
γ-CD
O
O
γ-CD
HO
O
R-O
H
O
OH
HO
O
O
HO
O
OH
HO
O
O
OH
O
HO
O
HO
OH
OH
O
O
O
HO
OH
OH
O
O -R
HO
O
O
R-O
O
HO
O -R
Fig. 1 Chemical structure of betulonic acid (BA) (a) and of γ-cyclodextrin, with the location attachment of the hydroxypropyl group (b) or
R-hydroxypropyl (c)
Cyclodextrins (CDs) belong to the family of the cyclic
oligosaccharides and possess six, seven and eight glu-
copyranose units linked by α-(1,4) bonds (α, β and γ-cy-
clodextrin, respectively). CDs molecules have torus macro-
ring shape, like a truncated cone, having a hydrophobic
internal cavity and a hydrophilic outer surface, responsible
for their water solubility [25–27]. This particular structure
of CDs offers them the ability to form host-guest inclusion
complexes with a wide variety of drug substances of
appropriate shape and size. The inclusion complex for-
mation is of biomedical and pharmaceutical interest
because of the major changes in drug candidate physico-
chemical and biopharmaceutical properties, such as solu-
bility, chemical reactivity, stability, volatility, melting
point, unpleasant taste or odors, which lead to significant
amelioration of formulation design and improvement of
drugs bioavailability [27–33]. Additionally, CDs can also
reduce ocular and gastrointestinal irritation and prevent
drug-excipient and drug-drug interactions [25, 29, 34].
Thus, the CD complexation results in significant
improvement of drugs formulation design and enhance-
ment of solubility, dissolution and bioavailability of drugs
based on thermal analyses, PXRD, FTIR and UV spec-
troscopy have been used to characterize the inclusion
complexes both in solid state and in solution.
Experimental
Materials
Betulin, as starting material, was obtained in our laboratory
by Soxhet extraction from the white outer birch bark (Be-
tula pendula) [45]. The functionalizated method for the BA
obtaining was previously described by our group [45]:
’
Betulin was oxidized with freshly prepared Jones reagent
(consisting in dissolved CrO in a mixture of concentrated
3
H SO and water) in acetone. After purification procedure,
2
4
BA heaving purity [96% was obtained. γ-CD and HP-γ-
CD were purchased from Cyclolab R&L Ltd (Budapest,
Hungary). The substances were used as received. All other
chemicals and reagents were of analytical grade.
Characterization of inclusion complexes
in solution
[
25, 35–37].
Some studies have reported the inclusion complexes
formation between pentacyclic triterpenoids such as betu-
linic, oleanolic and ursolic acid, betulin and cyclodextrins,
including β, γ-cyclodextrin and its derivatives [38–44].
In order to overcome the very low water solubility of
betulonic acid, Saxena B et al. [19] have synthesized a
hydrophilic lysinated derivative of drug with anti-prostate
cancer activity. Up to the present, there has been no sci-
entific report on BA inclusion complex with γ-CD and its
derivatives. Following this, the aim of this paper has been
to investigate the molecular encapsulation of BA in γ-cy-
clodextrin and 2-hydroxypropyl-γ-cyclodextrin cavity. The
binary products have been prepared using the kneading
method in molar ratio 1:1. Physicochemical determinations
All spectrophotometric measurements were recorded using
SPECTRONIC UNICAM—UV 300 UV-VISIBLE double
beam spectrophotometer with 1 cm matched quartz cells.
Stoichiometry and stability constant
determination
The stoichiometry of the BA/γ-CD and BA/HP-γ-CD
inclusion complexes was investigated by Job’s method
[29, 46]. Job plot was obtained from UV spectrophoto-
metric measurements. For this purpose, equimolar 2.29
10− M solution of BA and CDs (γ-CD and HP-γ-CD,
respectively) prepared in 0.1 M phosphate buffer (pH 8.5)
4
1
23

Betulonic acid—cyclodextrins inclusion complexes
was mixed to a standard volume, varying the molar ratio,
but keeping the total concentration of the species constant.
An analogous dilution set of the BA stock solution was
prepared using the same solvent. After stirring, the absor-
bance was measured at 205 nm for all solution, and the
Characterization of solid-state inclusion
complexes
Thermal analysis
difference in absorbance ΔA (ΔA=A –A) of BA in the
presence (A) and in the absence (A ) of CDs was plotted
0
TG/DTG/HF measurements were performed using a Perkin
Elmer DIAMOND TG/DTA instrument. In these experi-
ments, the samples of about 3–4 mg were placed in the
aluminum crucibles. The thermal behavior of BA, γ-CD,
HP-γ-CD, the binary systems BA/γ-CD and BA/HP-γ-CD
has been studied under air atmosphere at a flow rate of
0
against BA mole fraction R; R=[BA]/{[BA]?[CD]}.
The apparent stability constant of the BA/HP-γ-CD
inclusion complex was determined employing Benesi–
Hildebrand model. The spectrophotometric data were col-
lected for BA in the presence of increasing concentration of
−1
100 mL min , in the temperature range of 40–500 °C, with
−1
CD and also under free complexation conditions. The BA
concentration was maintained constant, at 1.759910−
heating rate of 10 °C min .
4
M
in 0.1 M phosphate buffer (pH 8.5), and the CD concen-
tration was varied from 0 to 1.759910− M, in the same
solvent. The absorption spectra were recorded in the range
of 200–230 nm, using 1.0 cm quartz cells.
Powder X-ray diffractometry
3
X-ray diffraction patterns of BA, γ-CD, HP-γ-CD and of
the binary systems BA/γ-CD and BA/HP-γ-CD were
obtained using a Bruker D8 Advance powder X-ray
diffractometer, at ambient temperature, in the range of 10°–
45° angular domain (2θ). The samples were irradiated with
Ni-filtered CuKα radiation (40 kV, 40 mA).
The binding constant determination is achieved by using
the changes in absorbance at maximum wavelength of 205
for BA in the presence of increasing CD concentration. The
stability constant value for the 1:1 inclusion complex can
be obtained by means of the Benesi–Hildebrand equation
[
29, 46, 47]
Fourier transform infrared spectroscopy
1
1
1
¼
þ
ð1Þ
The FTIR spectra were recorded using a Perkin Elmer
SPECTRUM 100 device. The data were collected directly
on solid samples, in the spectral domain 4000–600 cm− on
an UATR device. Spectra were built up after a number of 16
DA De Á ½BAŠ Á K Á ½CDŠ De Á ½BAŠ
1
where Δε is change in molar attenuation coefficient, ΔA is
change in absorbance and K is stability constant.
−
1
coadded acquisitions, with a spectral resolution of 4 cm .
Preparation of solid inclusion complexes BA/γ-CD
and BA/HP-γ-CD
Molecular modeling
The kneading method in 1:1 molar ratio was used to pre-
pare the inclusion complexes. This method was applied
because of its simplicity, accessibility, rapidity and eco-
nomical advantages in comparison with other methods
The Gaussian program suite at DFT/B3LYP/6-311G for BA
optimization was used. The γ-CD X-ray crystal structure
˚
with 5E70 code and X-ray diffraction at 2.33 A resolution)
(
was taken from the Protein Data Bank [49] and then opti-
mized with the Gaussian software Hartree–Fock/6-311G. The
HP-γ-CD molecular structure was modeled by adding eight
hydroxypropyl moieties to the γ-CD structure, attached to the
primary hydroxyl groups from carbon six D-glycopyranosyl
units, then optimized in the same manner (DFT/B3LYP/6-
[
48]. The amounts of 0.0389 g BA and 0.1111 g γ-CD were
weighed, and the obtained mixture was pulverized in a
mortar and triturated with 0.15 g ethanol-water solution
(
1:1, m/m). In the case of BA/HP-γ-CD inclusion complex
preparation, 0.0378 g BA and 0.1119 g HP-γ-CD were
weighed and the obtained mixture was pulverized and
triturated with 0.15 g ethanol-water solution (1:1, m/m) in
order to get a homogeneous paste. The paste thus obtained
was kneaded for 45 min, and during this process, a few
drops of solvent were added in order to ensure a suit-
able consistency. The product was dried at room temper-
ature and then in the oven, at 40 °C for 24 h. The dried
kneaded products named BA/γ-CD and BA/HP-γ-CD were
pulverized and passed through a 75 µm size sieve.
311G). The molecular docking analysis was performed using
the AutoDock 4.2.6 software together with the Auto-
DockTools, (a molecular viewer and graphical support for
setup and analysis of docking runs). The preparation of γ-CD
host molecule involves adding all the hydrogens, computing
the Gasteiger charge; the grid box was created using Autogrid
˚ ˚
with 50940940 A in x, y and z directions with 0.375 A
4
spacing. All the calculations were performed in vacuum.
For the docking process, we chose the Lamarckian
genetic algorithm (Genetic Algorithm combined with a
123

L. Sbârcea et al.
local search), with a population size of 150 and a number
of 30 runs. We exported all AutoDock results in the
PyMOL molecular visualization system [50].
pure BA, supporting the hypothesis of an inclusion complex
formation. Also, from Fig. 3a it can be seen an increase of
BA absorption intensity as a consequence of increasing HP-
γ-CD concentrations. The variation of the absorbance ΔA=A
−
A0 was calculated as the difference between BA absor-
Results and discussion
bance in the presence of different HP-γ-CD concentrations
and the pure BA absorbance.
Characterization of inclusion complexes
in solution
For the aim of verifying the inclusion complex stoi-
chiometry, a double reciprocal plot was represented, 1/
ΔA versus 1/[HP-γ-CD], as it is shown in Fig. 3b. A good
linear relationship was achieved when 1/ΔA was plotted
Stoichiometry and stability constant determination
2
against 1/[HP-γ-CD], (R =0.9884), confirming the 1:1
According to the continuous variation method [51], the
stoichiometry of the inclusion complex is given by the
value of the molar ratio R that corresponds to the maximum
concentration of the complex. The molar ratio is identified
when the absorbance (a physical parameter which is
directly related to the complex concentration) was mea-
sured for a series of samples having continuously varied
molar fraction of its components [29, 46, 52]. The maxi-
mum absorbance variation (ΔA) for BA, as shown in
Fig. 2a, is reached, when BA mole fraction is 0.667 in the
presence of γ-CD, which may indicate that the main stoi-
chiometry of BA/γ-CD inclusion complex is 2:1. In the
case of BA/HP-γ-CD inclusion complex formation, the
maximum variation in absorption is observed for R=0.5,
thus indicating that the stoichiometry is 1:1 (Fig. 2b).
The Benesi–Hildebrand method is one of the common
strategies for estimation of the stoichiometry and binding
constant of the inclusion complexes [29, 46, 47]. For the
purpose of confirming the 1:1 stoichiometry of the BA/HP-
γ-CD inclusion complex and calculating its stability con-
stant, changes in the absorption intensity of BA at 205 nm
have been evaluated as a function of HP-γ-CD concentra-
tion. A hypochromic shift is observed in the absorption
maxima of BA in the presence of HP-γ-CD compared with
stoichiometry of the BA/HP-γ-CD inclusion complex.
The value of the apparent stability constant was obtained
using Benesi–Hildebrand Eq. (1), as a ratio of the intercept
to slope of the straight line in the Benesi–Hildebrand
double reciprocal plot. The stability constant value was
−
1
determined as K1:1=1855.55 M .
Characterization of solid-state inclusion
complexes
Thermal analysis
In order to evaluate the interaction between BA and CDs,
the thermal behavior of the pure substances and their
kneaded products was investigated using TG, DTG and HF.
TG, DTG and HF thermoanalytical curves of BA, γ-CD,
HP-γ-CD and their inclusion complexes are shown in
Fig. 4a–e.
The TG/DTG curves of BA reveal a multistage
decomposition process in air atmosphere (Fig. 4a). A good
thermal stability of the substance is noticed, the fist mass
loss starting up at 222 °C with a DTGmax=273 °C (Δm=
12%), followed by another process which begins at 286.7 °
C, DTGmax=307.6 °C (Δm=41%). These two mass loss
(
a) 0.007
(b) 0.012
0
0
0
0
0
0
0
.006
.005
.004
.003
.002
.001
.000
0
0
0
0
0
.010
.008
.006
.004
.002
BA/HP-γ-CD
BA/γ-CD
0.000
0
.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
R
R
Fig. 2 Job’s plot for BA/γ-CD (a) and BA/HP-γ-CD (b) inclusion complexes from absorbance measurements
1
23

Betulonic acid—cyclodextrins inclusion complexes
(
a)
(b) 0
0
0
0
0
0
0
0
0
0
.40
.35
.30
.25
.20
.15
.10
.05
.00
BA 0.1759mM/HP-γ -CD 0mM
BA 0.1759mM/HP-γ -CD 0.880 mM
BA 0.1759mM/HP-γ -CD 1.100 mM
BA 0.1759mM/HP-γ -CD 1.320 mM
BA 0.1759mM/HP-γ -CD 1.540 mM
BA 0.1759mM/HP-γ -CD 1,759 mM
–
5
y= 14.5376x – 26.9759
2
R = 0.9884
– 10
Α
–
–
–
15
20
25
2
00
205
210
215
220
225
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Wavelength/nm
–1
1
/[HP-γ-CD]/mM
Fig. 3 UV spectra of pure BA and BA/HP-γ-CD inclusion complex in presence of increasing HP-γ-CD concentration (a); Benesi–Hildebrand
linear plot for 1/ΔA versus 1/[HP-γ-CD] (b)
steps are accompanied by a strong exothermic effect in HF
curve (Tpeak=310 °C), which probably corresponds to the
thermo-oxidations of the triterpenic moiety. The BA
heating at 525 °C induces a total mass loss of 100% [53].
The thermoanalytical curves of γ-CD present a small
mass loss between 40 and 130 °C (Δm=9.4%) corre-
sponding to the loss of crystal water (Fig. 4b); a stability
stage of dehydrated γ-CD is noticed between 130–260 °C,
but after 260 °C the mass loss process continues. The γ-CD
melting occurs at 318.6 °C, as the endothermic peak of the
HF curve shows.
also, the exothermic peak due to the thermo-oxidations of
the BA triterpenic moiety is shifted to higher temperature
(i.e., 331.6 °C).
The thermal analysis represents a valuable technique
regarding the evaluation of the interactions between CDs
and drug substances. The melting point of the guest
molecules which are embedded in CDs cavity generally
shifts to a different temperature and decreases its intensity
or disappears [54, 55]. The area reduction and the shift
toward a higher temperature of the thermo-oxidations of
BA or shift toward a lower temperature of the melting
peaks of CDs in the kneaded products indicate a molecular
interaction between the guest and host molecules.
The results obtained by using the thermal analysis
indicate a molecular interaction between the BA and CDs,
through the inclusion complexes formation when the
kneading method was employed.
The TG/DTG curves of HP-γ-CD indicate a mass loss
up to 100 °C (Δm=8.4%) due to the cyclodextrin dehy-
dration (Fig. 4d), followed by a thermal stability stage in
the temperature range of 100–265 °C. The HF curve
reveals the HP-γ-CD melting at 334.5 °C.
The thermal profiles of the binary systems reveal dif-
ferences in comparison with those of the pure substances
(
Fig. 4c, e). The thermoanalytical curves of BA/γ-CD
Powder X-ray diffractometry
kneaded product present a mass loss process between 40
and 95 °C (Δm=8.2%) corresponding to γ-CD dehydration;
in the temperature range of 95–225 °C a stability stage is
observed, but after 225 °C the mass loss process continues.
It is also noticed a marked reduction in intensity of the
DTG peak corresponding to the BA first mass loss step in
the range 200–400 °C and a shift toward higher tempera-
ture (DTGmax=285 °C). The HF curve of the binary system
BA/γ-CD indicates the significant reduction and the dis-
placement of both melting endothermic peak of γ-CD (i.e.
The powder X-Ray diffractograms of BA, γ-CD, HP-γ-CD
and BA/γ-CD, respectively, BA/HP-γ-CD inclusion com-
plexes are depicted in Fig. 5.
The XRD pattern of BA had one broad peak and many
undefined, diffused peaks with low intensities, reflecting
the amorphous nature of substance, while the diffraction
pattern of γ-CD contains a number of sharp and intense
peaks at 2θ 12.03; 13.64; 15.13; 16.23; 18.42, reflecting its
crystalline nature. The binary system BA/γ-CD diffracto-
metric profile presents different degrees of changes in
comparison with those of the pure substances. Thus, the
characteristic diffraction peaks of γ-CD have disappeared
in the XRD pattern of kneaded product, while there can be
noticed the appearance of some broad peaks indicating that
a new amorphous solid phase is formed.
3
08 °C) and the BA thermo-oxidations exothermic peak (i.
e., 328.4 °C), as compared with the parent substances.
Furthermore, the endothermic peak corresponding to the
HP-γ-CD melting exhibits a marked reduction in its
intensity and shifts to lower temperature (i.e., 322.6 °C) in
the HF curve of the BA/HP-γ-CD kneaded product and
123

L. Sbârcea et al.
(a)
(b)100
90
0
.025
35
20
1
00
TG
0.2
.0
TG
BA
1
8
3
2
2
1
0
5
0
5
γ-CD
9
0
0
0
0
0
0
0
0
0
0.000
DTG
16
14
12
0
DTG
8
7
6
5
4
3
2
1
80
–
0.025
0.050
0.075
0.100
0.125
–
–
0.2
0.4
70
60
1
8
6
4
2
0
–
0
–
–
–
–
–
50
–0.6
–0.8
–1.0
10
5
40
30
20
0
HF
10
2
HF
0.150 – 5
0
–
1.2
– 4
0
0
100
200
300
400
500
0
100
200
300
400
500
Temperature/°C
Temperature/°C
(c)1
(d) 100
1
1
1
8
6
4
2
0
4
2
0
18
TG
0.2
00
TG
0.03
.00
–0.03
BA/γ -CD
16
14
90
80
HP-γ-CD
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
DTG
0
.0
DTG
0
1
1
8
6
4
2
0
–
–
2
–0.2
–0.4
–0.6
–0.8
–1.0
–1.2
7
6
5
0
0
0
0
–
–
–
0.06
0.09
0.12
40
30
20
10
0
–0.15
0.18
HF
HF
2
–
2
–
4
– 4
0
100
200
300
400
500
0
100
200
300
400
500
Temperature/°C
Temperature/°C
7
0
(
e)100
TG
0.2
BA/HP-γ -CD
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
DTG
60
0
.0
5
4
0
0
–
0.2
–0.4
–
–
0.6 30
0.8 20
–1.0 10
1.2
HF
–
0
0
0
100
200
300
400
500
Temperature/°C
Fig. 4 The thermal profile of: BA (a); γ-CD (b); BA/γ-CD kneaded product (c); HP-γ-CD (d) and BA/HP-γ-CD (e) binary system in air
atmosphere (100 mL min− ), temperature range of 40–500 °C and heating rate of 10 °C min
1
−1
The diffraction pattern of HP-γ-CD indicates its amor-
phous structure. The binary system BA/HP-γ-CD diffrac-
togram emphasizes its amorphous nature, different from
that of BA, by the two broad peaks and many undefined,
diffused peaks with low intensities, similar to that of pure
cyclodextrin.
UATR-FTIR analysis
The UATR-FTIR spectra of pure BA, γ-CD, HP-γ-CD and
the BA binary products with CDs are presented in Fig. 6.
The IR spectra of the kneaded products have been evalu-
ated as compared to those of the pure substances in order to
assess the inclusion complexes formation.
The XRD analysis suggests that new compounds with an
amorphous state have probably been formed.
The UATR-FTIR spectral pattern of BA shows a strong
band at 1698 cm− associated to the C=O stretching
1
1
23

Betulonic acid—cyclodextrins inclusion complexes
(
a)
(b)
BA/γ-CD
γ-CD
BA/HP-γ-CD
HP-γ -CD
BA
BA
1
0
15
20
25
30
35
40
45
10
15
20
25
30
35
40
45
2θ
/°
2θ/°
Fig. 5 XRD patterns of BA, γ-CD, BA/γ-CD kneaded product (a) and BA, HP-γ-CD, BA/HP-γ-CD binary system (b)
(
a)
(b)
BA
BA
1
698
1698
2947
2947
HP-γ -CD
γ-CD
1014
BA/γ-CD
BA/HP-γ -CD
1027
1021
4
000
3500
3000
2500
2000
1500
1000
500
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber/cm–
1
Wavenumber/cm–
1
Fig. 6 UATR-FTIR spectra of BA, γ-CD and their inclusion complex (a); BA, HP-γ-CD and their kneaded product (b)
vibration of ketone and carboxyl group. It also presents
other characteristic bands for the carboxyl group (the C–O
stretching vibration and characteristic peaks that corre-
spond to the C-H stretching vibration from CH (at
2
−1
−1
2927 cm−1 for γ-CD; 2926 cm for HP-γ-CD), to C–H
stretching at 1180 cm , the hydrogen bonded O-H stretch
−1
−1
−1
vibration at 3442 cm ), for the vinylidene group (the C=C
bending from CH (1365 cm for γ-CD; 1366 cm for
2
−
1
stretching vibration at 1642 cm [45], the C–H stretch
HP-γ-CD), to the C–C–O stretching vibration from C–OH
−
1
3
−1
−1
vibration at 3081 cm ) and for the lupan skeleton (the sp
C-H stretching vibration from CH and cycle CH at 2947
(1021 cm for γ-CD; 1014 cm for HP-γ-CD) and to the
−1
skeletal vibration involving α 1-4 linkage (at 935 cm for
both γ-CD and HP-γ-CD). These data are in agreement
with previously reported results [58].
3
2
−1
−1
and 2867 cm , the CH scissoring at 1459 cm and the
2
methyl C-H antisymmetric/symmetric bending at
1
−1
1
459 cm− and 1376 cm , respectively) [45, 56]. The band
The IR spectra of the kneaded products reveal major
differences in comparison with the corresponding parent
substances. As can be seen, almost all BA characteristic
peaks have either reduced their intensity and shift to other
wavenumbers or have disappeared in the IR spectrum of
the binary compounds. Spectral data indicate that the BA
located at 888 cm− can be assigned to either the C-H out-
of-plane bending from vinylidene group or the antisym-
metric cyclohexane ring stretching vibration [56, 57].
The FTIR spectra of the CDs indicate a broad band
between 3600 and 3000 cm−1 associated to the O-H
1
123

L. Sbârcea et al.
characteristic bands corresponding to the carboxyl group,
of experimentally determined binding constants. The first
term is a 6/12 potential for dispersion/repulsion interac-
tions. The parameters are based on the Amber force field.
The second term is a directional H-bond term based on a
10/12 potential. The parameters C and D are assigned to
vinylidene group and the BA bands from 1376 and
1
8
88 cm− are present neither in BA/γ-CD kneaded product
nor in BA/HP-γ-CD binary system. Furthermore, the BA
CH scissoring band is still present in the BA/HP-γ-CD
2
−1
give a maximal well depth of 5 kcal mol−1 at 1.9 A for
˚
kneaded product spectrum and shifted to 1452 cm , but is
no more present in the spectrum of the BA/γ-CD binary
product and the BA band assigned to C-H stretching
hydrogen bonds with oxygen and nitrogen, and a well
depth of 1 kcal mol− at 2.5 A for hydrogen bonds with
1
˚
−
1
vibration from CH shifted from 2947 cm in the pure
sulfur. The function E(t) provides directionality based on
the angle t from ideal H-bonding geometry. The third term
is a screened Coulomb potential for electrostatics. The final
term is a desolvation potential based on the volume of
atoms (V) that surround a given atom and shelter it from
solve nt, weighted by a solvation parameter (S) and an
3
compound to 2940 cm− in both inclusion complexes
spectra, as a consequence of BA-CDs interaction when the
kneading method was employed. Also, the bands assigned
1
to C-H stretching vibration in CDs spectra shifted to
1
2
940 cm− in spectra of inclusion complexes.
˚
The results of the spectral analysis reveal changes in the
exponential term with distance-weighting factor ∫ = 3.5 A
[59].
characteristic bands of the pure compounds, thus indicating
an interaction between the BA and CDs through the
inclusion complexes formation.
The free energy of binding that we will attempt to
estimate is calculated as ΔbindG=ΔH−TΔS, where the
ΔH represents the enthalpic, and ΔS the entropic contri-
bution (only a negative ΔG value is energetically favorable
and the process is considered spontaneous).
The binding free energy values are calculated as fol-
Molecular modeling
Molecular modeling is a powerful tool that theoretical
chemistry employs for quantitative predictions on host-
guest interaction [52].
−
1
lows: −8.90 kcal mol for BA/γ-CD inclusion complex, −
5.29 kcal mol for the 2 BA/γ-CD inclusion complex and
−1
−1
AutoDock(1) is an automated procedure for predicting
the interaction of ligands with their macromolecular tar-
gets. An ideal procedure should be able to find the global
minimum in the interaction energy between the substrate
and the target protein, exploring all available degrees of
freedom for the system. AutoDock thus combines two
methods to achieve these goals: rapid grid-based energy
evaluation and efficient search of torsional space. The
conformations are then evaluated using a semi-empirical
free energy force field. The force field includes six pair-
wise evaluations (V) and an estimate of the conformational
entropy lost upon binding ðꢀSconfÞ:
−5.95 kcal mol for the BA/HP-γ-CD. The negative
binding free energy values hint that the inclusion com-
plexation process is favorably driven by enthalpy, thus
confirming the interaction between the host molecules,
namely γ-CD and HP-γ-CD, and the guest BA molecules.
According to our data, the BA/HP-γ-CD inclusion complex
is more stable than the 2 BA/γ-CD, because it has a lower
value of binding free energy. Figures 7 and 8 present the
theoretical BA/γ-CD and BA/HP-γ-CD inclusion com-
plexes, as rendered in the PyMOL molecular visualization
system. Figure 7 shows the molecular modeled inclusion
complex BA/γ-CD, simulated in 2:1 molar ratio.
Figure 8 presents the molecular modeling images of the
ꢀ
G ¼ ðV
À V
Þ
boundLÀL
boundPÀP
unboundLÀL
unboundPÀP
BA/HP-γ-CD inclusion complex.
þ ðV
À V
Þ
Our molecular docking results bring out a structural
insight about BA interactions with both γ-CD and HP-γ-
CD, which supports the experimental results that indicate
the formation of inclusion complexes between the BA and
the two host cyclodextrins. However, in our future studies,
we will investigate also the influence of complexation over
decomposition mechanism of inclusion complexes, by
already established protocols in our research group
þ ðVboundPÀL À VunboundPÀL þ ꢀSconfÞ
where L refers to the “ligand” and P refers to the “protein”
in a ligand-protein docking calculation. Each of the pair-
wise energetic terms includes evaluations for dispersion/
repulsion, hydrogen bonding, electrostatics and
desolvation:
!
!
X
X
Aij
Bij
16
Cij
12
Dij
10
[
60–64].
V ¼ Wvdw
À
qiqj
þ Whbond
À
1
ij
2
r
r
r
r
i;j
ij
i;j
ij
ij
X
XÀ
Á
2
2
ðÀr =2r Þ
ij
þ Welec
þ Wsol
SiVj À SjVi e
ꢀðr Þr
ij ij
i;j
i;j
The weighting constants W have been optimized in
order to calibrate the empirical free energy based on a set
1
23

Betulonic acid—cyclodextrins inclusion complexes
clearly demonstrate that the binary products possess dif-
ferent physicochemical properties in relation to the pure
compounds. The above results indicate that the kneading
method leads to formation of solid-state complexes
between BA and the two cyclodextrins.
Acknowledgements This work was supported by a grant from the
“Victor Babe s¸ ” University of Medicine and Pharmacy Timisoara
(
Grant PIII-C1-CFI-2014/2015-03 to L.S., A.L., L.U. and I.L.).
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