Comparison and correlation of in vitro, in vivo and in silico evaluations of alpha, beta and gamma cyclodextrin complexes of curcumin

Article abstract of DOI:10.1007/s10847-013-0322-1

In the present study investigated the effect of curcumin (CUR) alpha (α), beta (β) and gamma (γ) cyclodextrin (CD) complexes on its solubility and bioavailability. CUR the active principle of turmeric is a natural antioxidant agent with potent anti-inflammatory activity along with chemotherapeutic and chemopreventive properties. Poor solubility and poor oral bioavailability are the main reasons which preclude CUR use in therapy. Extent of complexation was β-CD complex (82 %) > γ-CD (71 %) > α-CD (65 %). Pulverization method resulted in significant enhancement of CUR (0.002 mg/ml) solubility with CUR α-CD complex (0.364 mg/ml) > CUR β-CD complex (0.186 mg/ml) > CUR γ-CD complex (0.068 mg/ml). Gibbs-free energy and in silico molecular docking studies favour formation of α-CD complex > β-CD complex > γ-CD complex. With reference to CUR, relative bioavailability of CUR α-CD, CUR β-CD and CUR γ-CD complexes were 460, 365 and 99 % respectively. CUR-CD complexes exhibited increased bioavailability with an increase in t_/, tmax, Cmax, AUC, Ka, and MRT; and a decrease in Ke, clearance and Vd values. AUC increase was CUR α-CD complex > CUR β-CD complex > CUR γ-CD complex. Significant difference (p < 0.05) was observed between CUR α-CD complex and CUR γ-CD complex by one-way ANOVA and Dunnett's post hoc test for multiple comparison analysis. Correlation observed between in vitro, in vivo and in silico methods indicates potential of in silico and in vitro methods in CD selection.

Full text of DOI:10.1007/s10847-013-0322-1

J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-013-0322-1
ORIGINAL ARTICLE
Comparison and correlation of in vitro, in vivo and in silico
evaluations of alpha, beta and gamma cyclodextrin complexes
of curcumin
•
Nagaraju M. Patro Azmi Sultana Keiji Terao Daisuke Nakata
•
•
•
•
•
•
•
Ayako Jo Akihito Urano Yoshiyuki Ishida Raghu N. Gorantla
•
Vinay Pandit Kshama Devi Shishir Rohit Baljinder K. Grewal
•
•
•
•
Elizabeth M. Sophia Anand Suresh Vineeth K. Ekbote Sarasija Suresh
•
•
Received: 7 February 2013 / Accepted: 10 April 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract In the present study investigated the effect of
curcumin (CUR) alpha (a), beta (b) and gamma (c) cyclo-
dextrin (CD) complexes on its solubility and bioavailability.
CUR the active principle of turmeric is a natural antioxidant
agent with potent anti-inflammatory activity along with che-
motherapeutic and chemopreventive properties. Poor solu-
bility and poor oral bioavailability are the main reasons which
preclude CUR use in therapy. Extent of complexation was b-
CD complex (82 %) [c-CD (71 %) [a-CD (65 %). Pul-
verization method resulted in significant enhancement of
CUR (0.002 mg/ml) solubility with CUR a-CD complex
(0.364 mg/ml)[ CUR b-CDcomplex(0.186 mg/ml)[ CUR
c-CD complex (0.068 mg/ml). Gibbs-free energy and in silico
molecular docking studies favour formation of a-CD com-
plex[b-CD complex [ c-CD complex. With reference to
CUR, relative bioavailability of CUR a-CD, CUR b-CD and
CUR c-CD complexes were 460, 365 and 99 % respectively.
CUR–CD complexes exhibited increased bioavailability with
one-way ANOVA and Dunnett’s post hoc test for multiple
comparison analysis. Correlation observed between in vitro,
in vivo and in silicomethodsindicatespotentialofin silicoand
in vitro methods in CD selection.
Keywords Curcumin ꢀ Alpha cyclodextrin ꢀ Beta
cyclodextrin ꢀ Gamma cyclodextrin ꢀ Solubility ꢀ
Bioavailability
Introduction
Curcumin (CUR) or diferuloyl methane is a yellow poly-
phenol isolated from the rhizome of turmeric (Curcuma
longa). From ancient times in Ayurveda (Indian system of
medicine) turmeric has been used in treating sprains,
inflammation, infections and hepatic disorders [1]. The
active principle of turmeric, CUR, has shown therapeutic
benefits in cardiovascular diseases [1], diabetes [2], Alz-
heimer’s disease [3], multiple sclerosis [4], HIV [5], cata-
ract [6] and inflammatory bowel disease [7]. Potent
antioxidant and anti-inflammatory activity of CUR leads to
an array of metabolic, cellular and molecular activities that
individually or in combination exhibit diverse therapeutic
activities including chemopreventive and chemotherapeu-
tic activities [8–17]. CUR exhibits poor oral bioavailability
and poor photostability which precludes its use in therapy
despite its therapeutic potential. Poor water solubility and
extensive pre-systemic and systemic metabolism of cur-
cumin results are responsible for its poor oral bioavail-
ability (1 % or lower) [18, 19].
an increase in t , tmax, Cmax, AUC, Ka, and MRT; and a
‘
decrease in Ke, clearance and Vd values. AUC increase was
CUR a-CD complex [ CUR b-CD complex[CUR c-CD
complex. Significant difference (p\ 0.05) was observed
between CUR a-CD complex and CUR c-CD complex by
N. M. Patro ꢀ A. Sultana ꢀ R. N. Gorantla ꢀ V. Pandit ꢀ
K. Devi ꢀ A. Suresh ꢀ S. Suresh
Al-Ameen College of Pharmacy, Hosur Road,
Bangalore 560027, India
K. Terao ꢀ D. Nakata ꢀ A. Jo ꢀ A. Urano ꢀ Y. Ishida
Cyclochem Co., Ltd, Tokyo, Japan
S. Rohit ꢀ B. K. Grewal ꢀ E. M. Sophia ꢀ
V. K. Ekbote ꢀ S. Suresh (&)
Cyclodextrin (CD) inclusion complexation of drugs is
an important method for increasing solubility of poorly
water-soluble drugs [20]. CDs are cyclic (a-1,4)-linked
oligosaccharides composed of a-D-glucopyranose residues.
National Institute of Pharmaceutical Education and Research
(NIPER), Mohali, Punjab, India
e-mail: ssuresh@niper.ac.in; sarasija_s@hotmail.com
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J Incl Phenom Macrocycl Chem
Most common natural CDs are alpha CD (a-CD), beta CD
(b-CD) and c-CD consisting of six, seven and eight
D-glucopyranose residues. CDs contain a relatively hydro-
phobic central cavity because of methane hydrogen atoms
directed towards inside of the ring which is capable of
entrapping hydrophobic molecules to form inclusion
complexes; OH groups on the outside imparts hydrophi-
licity to CD molecule [21].
Estimation of CUR by HPLC
Developed a HPLC method of the estimation of CUR in
Shimadzu HPLC system (Shimadzu, Japan) equipped with
Shimadzu LC-10A pump and UV–Vis SPD-10 AVP
detector. Selected Merck C18 (250 9 4.6 mm) column
packed with 5 lm particles. Mobile phase consisted of a
mixture of acetonitrile: tetrahydrofuran: 2 % acetic acid
(50:30:20). Optimized flow rate at 1 ml/min to obtain
3.2 min retention time at ambient conditions at 423 nm. A
linear calibration curve was obtained in 0.05–5 lg/ml
concentration range. Plotted the area under curve values
versus concentration to obtain the following regression
equation:
The research activity of CUR is intensively studied. A
recent Medline search indicated over thousands of publica-
tions describing its various activities. However, studies on
increasing oral bioavailability of CUR are not extensive. For
instance, CUR-hydroxypropyl CD complex exhibited potent
anti-inflammatory activity of CUR-hydroxypropyl CD
complex in synthetic dextran sulphate induced rat colitis
model [22]. We investigated the effect of CUR–CD com-
plexes on CUR solubility and oral bioavailability. We
hypothesized increasing solubility of CUR could decrease
the extent of its pre-systemic and systemic metabolism and
enhance bioavailability. We studied the effect of a-CD, b-
CD and c-CD complexes on CUR bioavailability. Pulveri-
zation technique, also called co-grinding, resulted in CD
complexes with maximum CUR content and solubility with
potential of scale-up for large scale manufacture. CUR–CD
complexes were characterized by FTIR, X-ray diffractom-
etry, DSC and H-1NMR studies. The in vitro dissolution
behaviour of CUR and CD complexes was studied in simu-
lated gastric fluid (SGF) and fasted state simulated intestinal
fluid (FaSSIF) medium. The in vivo bioavailability of CUR
and the CUR–CD complexes was evaluated in New Zealand
rabbits. Further, computed IVIVC Level A correlation for
CUR and CUR–CD complexes by linear regression analysis
of a plot of percentage drug dissolved in vitro and percentage
drug absorbed. Determined by in silico molecular modelling
studies the most stable CUR–CD complex by determining
the ease of docking CUR in CD molecule. A comparison of in
silico data, Gibbs-free energy and in vivo results indicated
the potential of in silico modelling studies in predicting the
most favourable CD for formulation development.
À
Á
Y ¼ 0:189X ꢁ 5:295 R2 ¼ 0:999
ð1Þ
Phase-solubility studies
Conducted phase-solubility studies by Higuchi and Con-
nors method to determine the stoichiometry of CUR–CD
binary systems [23, 24]. Added an excess amount of CUR
to different molar concentrations (2–8 mM) of a-CD, b-
CD and c-CD in 5 ml of distilled water (pH 5.5–6) to form
saturated solutions at RT and agitated in Water Bath Sha-
ker (Model RSB-12, REMI Laboratory Instruments, India)
at 120–130 stokes/min for 2 h at 37 °C. Estimated CUR
dissolved by HPLC at 423 nm after filtration of samples
and suitable dilution with methanol (40 % v/v). Figure 1
presents the phase-solubility diagram obtained by plotting
molar concentration of CD versus solubility of CUR.
Calculated the apparent stability constant (Ks) from the
slope of the phase-solubility diagram by Eq. 2.
Slope
Ks þ
ð2Þ
S0ð1 ꢁ SlopeÞ
where S0 is the solubility of curcumin in the absence of
CD.
Materials and methods
Curcumin (C3 complex) was a gift sample from Sami Labs
Limited, Bangalore, India. Samples of a-CD (CAVAMAX
W6), b-CD (CAVAMAX W7), c-CD (CAVAMAX W8)
were kindly provided by Cyclo Chem Ltd, Japan. Enzymes
b-glucuronidase, type B-1 from bovine liver and sulfatase,
type H-1 from Helix pomatia were procured from Sigma–
Aldrich (Germany). Acetonitrile and methanol (HPLC
grade) were procured from Merck, India. All the other
solvents and reagents were of A.R. grade. All solutions
were prepared using ultra pure water (MILLI Q, Millipore).
Fig. 1 Phase solubility profiles of curcumin in cyclodextrins in
water. Mean SD, n = 3; filled diamond alpha CD, filled square
beta CD, filled triangle gamma CD
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J Incl Phenom Macrocycl Chem
Calculated the Gibbs-free energies of transfer, DGo, of
CUR from an aqueous solution to cavity of CD using Eq. 3
Computational studies for optimization of stability
determination
ꢀ
ꢁ
S0
St
G0 ¼ ꢁ2:303RT log
ð3Þ
Further optimized stability of each of the CUR–CD complex
obtained from in silico studies. From PDB, downloaded
complex crystal structures of a-, b- and c CDs (25YE, 2Y4S
and 2ZyK) and extracted a-, b- and c cyclodextrin structures
[28, 29]. Sketched and minimized the CUR molecule by
Powell’s conjugate gradient method and Tripos force field.
Minimum energy difference of 0.001 kcal/mol was set as the
convergence criteria. Applied Gasteiger-Hu¨ckel partial
atomic charges for minimization of CUR and performed over
1,000 iterations to ascertain energy minimized conforma-
tions of each structure [30]. Subjected CUR–CD complexes
to an optimization process by using GAUSSIAN 03 software
package [34]. Performed complete optimizations by HF
(Hartree–Fock) method with 3-21?G* and 6-31?G* basis
set where Mulliken charges were used in the calculations.
Computed frequencies of optimized species analytically to
characterize each stationary point as a minimum or transition
state and estimate the zero point vibrational energies (ZPE).
The calculated ZPE values (at 298.15 K) were scaled by a
factor of 0.9153 in the HF method. Calculated the energy of
complex formation with respect to molecular form of CDs
and CUR by the following equation:
where S0 and St are the solubility of CUR in the absence
and presence of CD, respectively.
Determination of Job plot
Confirmation of stoichiometry of inclusion complexes was
by Job plot. Job plot also known as the continuous variation
method or Job’s method involves determination of binding
stoichiometry of CUR and cyclodextrin [25–27]. Briefly,
added equimolar (10 9 10-6 M) solutions of CUR to CD
(with a-CD, b-CD and c-CD) solutions individually in
varying ratios (1:9, 2:8, 3:7 and so on) of drug and CD to
make 10 ml, keeping the total concentration of solutes
constant. In addition, prepared an analogous set of CUR
solutions without CDs in distilled water. Finally, absor-
bance of all the solutions was measured at 423 nm in
Shimadzu UV-1700 PharmaSpec UV–Vis spectrophotom-
eter (Shimadzu, Japan). The difference in absorbance of
CUR in the presence and in absence of CD was plotted
against R where R is obtained by the following equation:
Ecomplex ¼ Edockedꢁcomplex ꢁ ðEcurcumin þ Ecyclodextrin
Here,
Þ
ð5Þ
½CURꢂ
R ¼
ð4Þ
½CURꢂ þ ½CDꢂ
Ecomplex = Energy of complex formation.
where [CUR] and [CD] are concentrations of curcumin and
cyclodextrin, respectively.
Edocked-complex = Energy of optimized structure of docked-
complex.
All the determinations were conducted in triplicate.
Ecurcumin = Energy of optimized structure of curcumin.
Ecyclodextrin = Energy of optimized structure of cyclodextrin.
In silico molecular modelling studies
Investigated in silicomolecular modellingand docking study
to study the ease with which CUR complexes with a-CD, b-
CD and c-CD. Calculated the transition energies for docking
CUR into each CD molecule. Firstly obtained crystal struc-
tures of a-, b- and c CD (25YE, 2Y4S and 2ZyK) from
protein data bank (PDB) and structures of a-, b- and c CD
was extracted [28, 29]. Performed molecular modeling
studies in SYBYL7.1 installed on a silicon graphics fuel
station. Manually docked CUR into a-, b- and c CD by
visualization and 3D tools of SYBYL7.1 and further mini-
mized the structures by calculation of Powell’s conjugate
gradient. For all the four structures calculated Gasteiger-
Hu¨ckel partial atomic charges by using over 1,000 iterations
in order to ascertain the energy minimized conformations for
each structure [30, 31]. After setting minimum energy dif-
ference of 0.005 kcal/mol as convergence criteria obtained
CUR–CD complexes using tools in SYBYL7.1. [32, 33].
Preparation of CUR–CD complexes by pulverization
method
For preparation of CUR–CD complexes equimolar ratios
(1:1) of CUR and the CDs (5 of CUR, 13.2 of a-CD, 15.25
of b-CD and 17.6 gm of c-CD) were weighed. Triturated
individually CUR and CD and mixed thoroughly (30 min)
to form CUR–CD complex. Characterized and evaluated
the CUR–CD complexes.
Evaluation studies of CUR–CD complex
Curcumin content
The CUR content was estimated in CUR–CD complexes by
HPLC at 423 nm. Briefly, dissolved in methanol (40 %
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J Incl Phenom Macrocycl Chem
v/v), sample containing 10 mg of CUR/equivalent amount
of CUR–CD complexes (36.4 of CUR a-CD, 40.5 of CUR
b-CD and 55.2 mg of CUR c-CD) for estimation. Esti-
mation was carried out in triplicate.
X’pert PRO diffractometer (The Netherlands) equipped
with a rotating target X-ray tube and wide-angle goniom-
etry. The X-ray source was Ka radiation from a copper
target (k = 1.5418). The X-ray tube was operated at a
potential of 40 kV and a current of 30 mA. The range (2h)
of the scans was from 0° to 50° and the scan speed was
maintained at a rate of 2° per minute and increased at
increments of 0.02°.
Saturation solubility studies
Determined CUR and CUR–CD complexes solubility in
three media: distilled water, SGF and FaSSIF. Dissolved
excess amount of CUR/CUR–CD complexes in water
(5 ml) in Schott Duran bottles subjected to constant shak-
ing in Water Bath Shaker (Model RSB-12, REMI Labo-
ratory Instruments, India) for 2 h at 37 °C. Estimated CUR
dissolved by HPLC at 423 nm after filtration of samples
and suitable dilution with methanol (40 % v/v). The solu-
bility study was conducted in triplicate.
Nuclear magnetic resonance (NMR) studies
The 1H-NMR spectra was recorded on a BRUKER
AVANCE 11 400 MHz spectrometer at 298 K. CUR was
dissolved in DMSO while pure CDs and CUR–CD com-
plexes were dissolved in deuterated water (D2O). 1H-NMR
chemical shift measured in d parts per million (d ppm)
caused upon complexation were measured to determine
inclusion of CUR in the CDs. Processing and analysis of
the data was performed with MestReNova-Software
(Mestrelab Research S.L., Version 6.1.1).
Dissolution studies
The in vitro dissolution study of CUR (100 mg) and CUR–
CD complexes (*100 mg) were in USP Dissolution Test
apparatus type II (Electrolab TDT-06P, India). Dissolution
study was for 2 h in SGF (900 ml) and 10 h in FaSSIF
(900 ml) at 75 rpm stirring speed at 37° 0.5 °C. Sam-
pled (5 ml) at time intervals of 0.25, 0.5, 0.75, 1, 1.5, 2,
2.5, 4, 8, and 12 h. Estimated CUR dissolved by HPLC at
423 nm after filtration and suitable dilution of samples. The
in vitro dissolution studies were conducted in triplicate.
In vivo bioavailability study
The in vivo bioavailability studies of CUR and CUR–CD
complexes in Male New Zealand rabbits (1.5–2.5 kg from
Biogen Laboratory, Bangalore). Animals were maintained
under standard laboratory conditions. (20–25 °C,
50 5 % RH; 12 h light/12 h dark cycle) and allowed
access food and water ad libitum. All experiments were
performed in accordance to the guidelines of committee for
control and supervision of experiments on animal (CPC-
SEA), Ministry of Culture, and Government of India with
approval from the Institutional Animal Ethics Committee.
Formed four groups containing three rabbits randomly
assigned in each group for the bioavailability study. The
animals were fasted overnight before the experiment with
free access to water. Orally administered CUR (1 gm/kg)
and CUR–CD complexes (*1 gm/kg of CUR) in water to
different groups of animals. Group I received pure CUR
while Group II received CUR a-CD, Group III received
CUR b-CD and Group IV received CUR c-CD. Blood
samples (1 ml) were collected from the marginal ear vein
of animals into heparinized tubes at time intervals of 0, 5,
10, 15, 20, 25, 30 and 60 min after administration. Pre-
liminary studies indicated plasma concentration of pure
CUR was below the minimum detectable level after 60 min
while it was detectable for longer duration in CUR–CD
groups. Blood samples were therefore collected from
CUR–CD administered groups at 0, 0.25, 0.5, 0.75, 1, 1.5,
2, 4, 8, 12 and 24 h after administration. Blood samples
were centrifuged at 12,000 rpm for 10 min to obtain
plasma. Mixed the plasma aliquots (0.5 ml) with 2,000
units of b-glucuronidase in 0.1 M sodium phosphate buffer
Fourier-transform infrared spectra (FTIR)
Fourier-transform infrared spectra (FTIR) of CUR, CDs
and CUR–CD complexes were recorded in Perkin Elmer
Spectrum BX infrared spectrophotometer. Samples were
thoroughly mixed with dry KBr and compressed under high
pressure to obtain transparent discs. Placed the disc in
sample holder of IR spectrophotometer and scanned the
spectrum range from 4,000–400 cm-1
.
Differential scanning calorimetry (DSC)
The thermal behaviour of CUR, CDs and CUR–CD complexes
were examined by DSC using Mettler–Toledo DSC module
controlled by Star software (Mettler–Toledo GmbH, Swit-
zerland). Samples were weighed(5.00–8.00 mg)and placed in
sealed aluminium pans. DSC analysis was carried out at a
heating rate of 10 °C/min under flow of nitrogen. The obser-
vations were recorded over a temperature range of 40–400 °C.
Powder X-ray diffraction studies (PXRD)
Powder X-ray diffraction (PXRD) of CUR, CDs and CUR–
CD complexes were determined using a PAN Analytical
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J Incl Phenom Macrocycl Chem
(pH 6.8) and b-sulfatase solution in 0.1 M sodium acetate
buffer (pH 6.5) and incubated at 37 °C for 1 h. Acidified
the obtained solution with 50 ll of 1 M glacial acetic acid
and thoroughly mixed in cyclomixer (REMI Laboratory
instruments, India). Added 50 ll of quercetin (internal
standard) and 1 ml of acetonitrile to the solution and cen-
trifuged at 1,000 rpm for 10 min. Analyzed by HPLC at
423 nm and quantified the amount of CUR present in the
supernatant. Selected Hibar C18 column (150 9 4.6 mm)
packed with 5 lm particles while the mobile phase con-
sisted of acetonitrile, tetrahydrofuran and 2 % acetic acid
mixture in 50:5:45 ratios.
peak plasma concentration (Cmax) and time to reach the
maximum plasma concentration (Tmax) elimination rate
constant Ke, and mean retention time MRT.
All the results were expressed as mean standard
deviation (SD). Statistical analysis of AUC0-? was per-
formed with GraphPad Prism (Demo version) by one-way
ANOVA and Dunnett’s post hoc test for multiple com-
parisons. p \ 0.05 was considered as statistically signifi-
cant difference.
In vitro–in vivo correlation
Computed IVIVC Level A correlation for CUR and CUR–
CD complexes by linear regression analysis of a plot of
percentage drug dissolved in vitro and percentage drug
absorbed. Determined by Wagner–Nelson method the
percentage of drug absorbed from the CUR plasma con-
centration versus time data [35].
Development of bio-analytical method for estimation
of curcumin
Prepared individually stock standard solutions of CUR
(200 lg/ml) and quercetin (internal standard, IS, 6 mg/ml)
in methanol. Prepared five concentrations of standard CUR
solutions (0.1–5 lg/ml) from the CUR stock solution for
standard calibration curve. For the bio-analytical method,
acidified rabbit plasma (900 ll) with 1 M glacial acetic
acid (50 ll), added CUR (50 ll) and thoroughly mixed.
Spiked the resultant solution (500 ll) with 50 ll of IS and
mixed thoroughly. Added Acetonitrile (1 ml) to precipitate
proteins mixed thoroughly and centrifuged for 10 min at
1,000 rpm. Analyzed by HPLC at 423 nm and quantified
the amount of CUR present in the supernatant. In
0.1–5 lg/ml concentration range the calibration curve was
linear for CUR was linear. Determined by Kinetica soft-
ware (Thermo Scientific) the following pharmacokinetic
parameters: area under the curve AUC0-t, total area under
the curve (AUC)0-? (lg/ml h), terminal phase half-life
(t1/2) (h), peak plasma concentration (Cmax) (lg/ml), time
to reach the maximum plasma concentration (tmax), elimi-
nation rate constant Ke (h-1), and mean retention time
MRT (h). Relative bioavailability (F) of CUR–CD com-
plexes were calculated from the following equation:
Results and discussion
Phase-solubility studies
Figure 1 presents the phase-solubility diagrams for CUR
with a-CD/b-CD/c-CD binary systems. A linear increase
was observed in the aqueous solubility of CUR with an
increasing concentration of CD. This is indicative of AL type
solubility diagram where stoichiometry of CUR–CD is 1:1
which was further confirmed by slope \1 in the solubility
diagrams [26]. Calculated apparent stability constant, Ks, of
CUR–CD complexes from Eq. 2 and observed Ks of a-CD
complex [ c-CD complex [ b-CD complex (Table 1).
Considerable high Ks observed in a-CD complex indicates
its increased stability in comparison to c-CD complex and b-
CD complex. Further, confirmed stoichiometry of CUR–CD
complexes by Job’s plot (Fig. 2) and double-reciprocal plots
(Fig. 3). In Job’s plot obtained R value of 0.5 and observed a
linear relationship when 1/(A-A0) was plotted against 1/CD
(R2 = 0.9898, 0.9966 and 0.9936 for CUR a-CD, CUR b-
CD and CUR c-CD respectively).
½AUCA ꢃ DBꢂ
F ¼ 100 ꢃ
ð6Þ
½AUCB ꢃ DAꢂ
where F is relative bioavailability; AUCA is area under
curve of curcumin; AUCB is area under curve of cyclo-
dextrin; DA is dose of curcumin and DB is dose of
curcumin.
The driving force for inclusion complexation between
CD and CUR includes van der Waals interaction, hydrogen
bonding, and hydrophobic interaction which results in the
release of high-energy water molecules from CD cavity
and release of strain energy from the CD ring. The opti-
mum apparent stability constant Ks for enhancement in
solubility and stability of poorly soluble drug molecules
lies between 200 and 5,000 M-1 [36]. The Ks values of
CUR–CD complexes were all in the range of
559–1,125 M-1 indicating the capability of complexes in
improving the solubility and stability of CUR with a-
CD [ c-CD [ b-CD.
Statistical analysis
Non-compartment model analysis of pharmacokinetic
plasma concentration–time data was by Kinetica software
(Thermo Scientific). Pharmacokinetic parameters deter-
mined include area under AUC0-t, the curve total area
under the curve (AUC)0-?, terminal phase half-life (t1/2),
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J Incl Phenom Macrocycl Chem
Table 1 Stability constant of complexes of curcumin
Table 2 Gibbs free energy data of Curcumin-CD complex in water
Type of cyclodextrin
Stability constant (Ks; M-1
)
Concentration
of CD (mM)
Gibb’s free energy (-DtransG°/cal mol-1
)
a CD
b CD
c CD
1,124.606 (r2 = 0.9859)
558.909 (r2 = 0.9845)
746.714 (r2 = 0.9710)
a CD
b CD
c CD
2.5
5
421.863
556.697
610.095
683.037
296.749
356.143
461.893
511.535
357.691
416.051
492.632
590.683
7.5
10
of CUR a-CD complex (130.5) \ CUR b-CD complex
(145.6)\CUR c-CD complex (171.2 kcal/mol). Based on
the energy values most favoured complex is CUR a-CD
complex. Table 3 presents the data of optimization studies.
Based on the energy of complex formation, CUR a-CD and
CUR b-CD complexes are more stable than CUR c-CD
complex by hf/6-31G* level of calculations. However,
when hf/3-21G* is adopted, CUR b-CD is less stable than
CUR a-CD and CUR c-CD complexes. Considering that
hf/6-31G* level of calculations are more accurate than hf/
3-21G*, stability of CUR a-CD and CUR b-CD complexes
may be more than CUR c-CD complex.
Fig. 2 Job’s plot of curcumin cyclodextrin complexes filled diamond
alpha CD, filled square beta CD, filled triangle gamma CD
Evaluation of curcumin cyclodextrin complexes
Evaluated spray drying, pH modified method (procedure
not provided in the text) in the preparation of CUR–CD
complexes. However, CUR degraded in the presence of
water. Hence, pulverization or co-grinding technique was
selected for the preparation of CUR–CD complexes. Pul-
verization ensured stability of CUR and can be easily
scaled-up.
Figure 5 presents aqueous solubility of CUR–CD com-
plexes expressed in mg/ml. Observed considerable
improvement in solubility of CUR in all the CD complexes
with a-CD complex [ b-CD complex [ c-CD complex. In
comparison to pure CUR (0.002 mg/ml) a-CD complex
(0.364 mg/ml) had almost 135-fold increase in solubility
while b-CD complexes (0.186 mg/ml) exhibited 69-fold
increase and c-CD (0.068 mg/ml) exhibited 25-fold
increase in solubility. In our earlier studies observed
enhanced in solubility of CUR in hydroxy propyl b-CD
(HPbCD) and methyl b-CD (MbCD) [22]. Increased sol-
ubility of CUR is due to decrease in crystallinity of CUR in
CUR–CD complexes during pulverization and increased
wetting [37].
Fig. 3 Dependence of curcumin cyclodextrin complex absorbance on
cyclodextrin concentrations filled diamond alpha CD, filled square
beta CD, filled triangle gamma CD
Gibbs-free energy is a thermodynamic function. Change
in Gibbs-free energy (DG°) is the net energy available to do
useful work and is a measure of ‘‘free energy’’. Negative
DG° indicates a spontaneous process [36]. Table 2 presents
DG° obtained by substituting solubility of CUR in presence
and absence of CD in Eq. 3. DG° values were negative
which increased with an increase in CD concentration.
Hence CD solution is a more favourable environment than
water for CUR molecule and order of spontaneity of a-CD
complex [ c-CD complex [ b-CD complex.
Dissolution study
In silico molecular modelling studies
In Fig. 6 presented CUR and CUR–CD complexes disso-
lution profiles in SGF and FaSSIF. Observed an enhanced
dissolution in all the CUR–CD complexes as shown by the
Figure 4 presents structures of CUR–CD complexes from
molecular modelling studies. The energy values obtained
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J Incl Phenom Macrocycl Chem
Fig. 4 In silico molecular modelling structures of A CUR a-CD complex B CUR b-CD complex C CUR c-CD complex
time taken for 50 % CUR release. At the end of 2 h, the
extent of dissolution in SGF was 13.24, 77.18, 69.4 and
45.33 % from CUR, CUR a-CD complex, b-CD complex
and c-CD complex, respectively. The extent of dissolution
at the end of 12 h in FaSSIF were 14.53, 91.84, 83.89 and
60.28 % from CUR, CUR a-CD, CUR b-CD and CUR c-
CD complexes, respectively. This observed increase in the
dissolution of CUR may be due to formation of readily
soluble complexes because of increased wettability of
complexes. Enhancement in dissolution was maximum in
CUR a-CD followed by CUR b-CD and CUR c-CD.
Compounds with aliphatic chains form inclusion com-
plexes with a-CD because of its small cavity size while
compounds with aromatic rings, exemplified by paclitaxel,
form complexes with b-CD [38]. Consequently, CUR may
form inclusion complexes to a greater extent in a-CD than
in b-CD or c-CD because of its low molecular weight
(354.49).
Table 3 Calculation of energy of CUR–CD complex formation by
Hartree–Fock Method
Particulars
Ehf
ZPC
Ecorr
Ecomplex
hf/3-21G*
CUR
Alp
–
–
–
–
–
–
–
0.4
-783,274.30
1.21
1.31
1.49
1.65
1.71
1.89
-2,273,254.46
-2,605,484.87
-2,984,364.74
-3,056,801.98
-3,389,041.29
-3,767,914.03
-21.51
-30.41
-23.28
Beta
Gam
Acd
Bcd
Gcd
hf/6-31G*
CUR
Alp
–
–
–
–
–
–
–
0.40
1.12
1.31
1.49
1.53
1.71
1.90
-787,590.30
-2,284,924.62
-2,618,784.47
-2,999,608.71
-3,072,538.43
-3,406,397.29
-3,787,206.00
-23.51
-22.52
-06.99
Beta
Gam
Acd
Bcd
Gcd
Characterization of CUR–CD complexes
CUR curcumin, Alp a-CD, Beta b-CD, Gam c-CD, Acd CUR a-CD
complex, Bcd CUR b-CD complex, Gcd CUR c CD complex, Ehf is
absolute energy values calculated in Hartree, ZPC is the zero point cor-
rection energy, Ecorr is corrected energy in kcal/mol after zero point
energy correction process Ecomplex is the energy of complex formation in
kcal/mole
CUR–CD complexes were characterized by various spec-
troscopy techniques. FTIR spectrum was recorded to ana-
lyse the structure of CUR–CD complex and determine
occurrence of any chemical change in the complex (Fig. 7).
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J Incl Phenom Macrocycl Chem
Fig. 5 Solubility of curcumin and CUR–CD complexes in distilled
water, simulated gastric fluid (SGF) and fasted state simulated
intestinal fluid (FaSSIF) Mean SD, n = 3; red filled square
Distilled water, green filled square SGF pH1.2, violet filled square
FaSSIF
In the FTIR spectra, the characteristic peaks of CUR
appear at 3,664.97 corresponding to phenolic (OH) vibra-
tions; 3,014.33 corresponding to aromatic C–H stretching
vibrations; 1,602 cm-1 corresponding to the stretching
vibration of the benzene ring skeleton, 1,510.68 to the
mixed (C=O) and (C=C) vibration; 1,272.85 cm-1 to enol
C–O peak; and 1,023 cm-1 to C–O–C peak [39]. A sharp
peak of hydroxyl (-OH) group at 3,510.20 cm-1 indicates
the presence of free hydroxyl (-OH) group. Observed no
change in IR spectra of CUR–CD complexes except for
Fig. 6 Dissolution profiles of curcumin and CUR–CD complex.
Mean SD, n = 3; filled diamond Pure drug, filled square CUR- a
CD complex, filled triangle CUR- b CD complex, filled circle CUR- c
CD complex
Similarly, observed a slight shift in the endothermic peak at
300 °C in c-CD complex. This phenomenon is indicative
of interaction between CUR and CDs. From the area of
endothermic peak obtained the extent of complexation of
CUR in CD complexes with b-CD complex (82 %)[c-CD
complex (71 %) [ a-CD complex (65 %).
broadening of the hydroxyl group peak at 3,379.05 cm-1
,
In P-XRD studies, presented in Fig. 9, CUR has two
intense peaks at 9.0485 and 17.4957 degrees apart from
other characteristic peaks which indicate its crystalline
nature. Pure a-CD has intense peaks at 11.9508, 12.2235,
13.5856, 14.3873 and 21.7351 degrees. Similarly, pure b-
CD has intense peaks at 9.1595 and 12.7292; however pure
c-CD is amorphous, with decreased intensity of peaks in
comparison to a-CD and b-CD. All the CUR–CD com-
plexes exhibited a decrease in intensity of peaks when
compared to pure CUR and pure CDs indicating a decrease
in the crystallinity of CUR in CD complexes. This decrease
in CUR crystallinity is responsible for increased solubility
of CUR in all the CD complexes.
which may suggest occurrence of interaction of the
hydroxyl (-OH) group. The bands within the range of
3,079–3,000 cm-1 were observed, which can be attributed
to aromatic C–H stretching vibration, indicating possible
intercalation of CUR in CD complex.
DSC has been extensively adopted to evaluate thermal
properties of cyclodextrin complexes. It has the potential to
provide qualitative and quantitative information regarding
the physicochemical state of CUR inside the CD com-
plexes. In general, complexation results in the absence of
endothermic peak or shift of peak to different temperature
because of change in the initial crystal lattice, melting,
boiling or sublimation points. In the present study, as
observed in Fig. 8, DSC thermogram of CUR had a sharp
endothermic characteristic peak at 150 °C. Thermograms
of pure a-CD and b-CD had two peaks, one peak at about
60 °C and another peak at around 300 °C while the ther-
mogram of pure c-CD had a peak at 300 °C. DSC ther-
mograms of CUR–CD complexes exhibited a pattern of
reduction in the sharp endothermic peak of CUR with
slight broadening without any change in temperature.
Further, observed in a-CD complex and b-CD complex a
similar broadening of peak at around 60 °C with a slight
shift in temperature of the second endotherm at 300 °C.
NMR spectroscopy may provide a direct evidence of
inclusion complex formation. An overview of 1H-NMR
chemical shifts of hydrogen atoms of cyclodextrin in D2O
the complexation induced chemical shifts in their respective
complexes is presented in Table 4. In a-CD complexes,
maximum involvement appears to be H1 hydrogen followed
by H5 hydrogen. In CUR b-CD complex maximum
involvement is of H1 and H3 hydrogen. In c-CD complex
there is involvement of H1, H2, H4 and H5 hydrogen atoms.
Among the CD complexes, in a-CD complexes the
involvement of hydrogen atoms of CD appears to be the
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J Incl Phenom Macrocycl Chem
Fig. 7 FTIR Spectra of A CUR, B Pure a-CD, C Pure b-CD, D Pure
c-CD, E CUR a-CD complex, F CUR b-CD complex and G CUR c-
CD complex
Fig. 8 DSC thermograms of pure drug and complexes of A CUR,
B Pure a-CD, C Pure b-CD, D Pure c-CD, E CUR a-CD complex,
F CUR b-CD complex, and G CUR c-CD complex
least. All the CD complexes exhibited involvement of H3 or
H5 hydrogen atoms which is normally expected in cyclo-
dextrin complexation. In addition they exhibited involve-
ment of H1, H2, H4 hydrogen atoms. This phenomenon may
be explained by the pulverization technique adopted in the
preparation of CUR–CD complex Table 5.
of CUR a-CD, CUR b-CD and CUR c-CD complexes were
460, 365 and 99 % with reference to pure CUR.
Increase in bioavailability exhibited by CUR a-CD com-
plex can be attributed to the observed increase in inclusion of
CUR in a-CD. Increase in CUR solubility in CUR–CD
complexes may decrease the extent of its pre-systemic and
systemic metabolism because of enzyme saturation at the site
of metabolism. This phenomenon can increase the amount of
CUR available for absorption and systemic circulation
resulting in enhanced bioavailability of CUR in the CD
complexes. Reports of enhanced CUR solubility and bio-
availability from curcumin-phospholipid complex are avail-
able in the literature. One study reported 5-fold increase CUR
oral bioavailability from CUR formulation with phosphatidyl
choline (Meriva) [19]. Another study reported 3-fold increase
in CUR aqueous solubility with increased hepatic protective
action [40]. Reports of enhancement in solubility and bio-
availability of drugs with poor solubility undergoing pre-
systemic and systemic metabolism are available in the liter-
ature. For instance, raloxifene exhibits poor absolute oral
bioavailability (*2 %) attributed to its extensive first-pass
metabolism by glucuronidation and poor aqueous solubility
[38]. Wempe et al. reported a two-fold increase in Cmax and a
3-fold increase in raloxifene AUC from raloxifene-hydro-
xybutenyl-beta cyclodextrin complex in male Wistar-Han-
nover rats [41, 42].
In vivo bioavailability studies
Figure 10 illustrates CUR plasma concentration profiles
from pure CUR and CUR–CD complexes. Table 5 sum-
marizes the pharmacokinetic parameters obtained from one
compartmental analysis of experimental data by Kinetica
2000 software (Thermo Scientific). CUR–CD complexes
exhibited an increase in t , tmax, Cmax, AUC, Ka, and MRT;
‘
and decrease in Ke, clearance and Vd. Statistically analyzed
AUC0-? values by one-way analysis of variance
(ANOVA) and Dunnett’s post hoc test for multiple com-
parisons. CUR–CD complexes exhibited significant
(p \ 0.05) enhanced AUC in comparison with CUR.
Increase in AUC of CUR a-CD complex (3.88 lg/ml h)[
CUR b-CD complex (3.149 lg/ml h) [ CUR c-CD com-
plex (0.801 lg/ml h).
There is a significant difference (p \ 0.05) in the AUC
of CUR a-CD complex and CUR c-CD complex. However,
there is no significant difference between CUR a-CD and
CUR b-CD complexes. To summarize, observed an
increase in the AUC of CUR a-CD complex [ CUR b-CD
complex [ CUR c-CD complex. Relative bioavailability
123

J Incl Phenom Macrocycl Chem
Fig. 9 X-ray diffraction
patterns of A CUR, B pure a-
CD, C pure b-CD, D pure c-CD,
E CUR a-CD complex, F CUR
b-CD complex and G CUR c-
CD complex
Table 4 1H-NMR chemical shifts of cyclodextrin hydrogen atoms in D2O and their complexation induced chemical shifts in their respective
complexes
Hydrogen
a- CD
D
CUR a-CD
d (D d)
b-CD
d
CUR b-CD
d (D d)
c- CD
d
CUR c-CD
d (D d)
1
2
3
4
5
4.96
4.72
3.86
3.76
3.51
4.92 (0.04)
4.69 (0.03)
3.84 (0.02)
3.74 (0.02)
3.48 (0.03)
4.91
4.64
3.78
3.71
3.46
5.00 (0.09)
4.69 (0.05)
3.87 (0.09)
3.72 (0.01)
3.44 (0.02)
5.07
4.80
3.89
3.84
3.60
4.96 (1.01)
4.69 (1.11)
3.84 (0.05)
3.75 (0.09)
3.50 (1.00)
Table 5 Pharmacokinetic data of CUR and CUR–CD Complex
Parameters
CUR
CUR-aCD Complex
CUR-bCD Complex
CUR-cCD Complex
AUC0–n (lg/ml h)
AUCtot (lg/ml h)
0.072 0.01
3.438 0.24
3.879 0.33
0.289 0.02
5.174 0.25
2.396 0.16
4.255 0.14
259 23.07
896 22.87
2.525 0.097
3.149 0.35
0.146 0.036
2.64 0.26
0.684 0.045
0.801 0.13
0.089 0.01
1.88 0.12
Ke (h-1
Ka (h-1
T1/2 (h)
)
)
0.290 0.11
2.303 0.02
1.289 0.14
0.369 0.02
4.940 1.314
7.52 1.56
2.631 1.03
MRT (h)
Clearance (ml h-1
Vd (ml)
0.561 0.03
4.320 1.32
)
11,301.89 1,076
6,023.03 64.13
320 36.37
2,241 356.44
1,271 219.37
4,646.67 1,046.39
There is a correlation between pharmacokinetic and
computation data. Energy values from molecular docking
indicate CUR a-CD complex \ CUR b-CD com-
plex \ CUR c-CD complex. Similarly, computational
studies and from Gibbs-free energy values indicate CUR a-
CD complex to be most stable CD complex. The stability
of CUR b-CD complex [ CUR c-CD complex from the
computational studies; while stability of CUR c-CD
complex [ CUR b-CD from apparent (Ks) and Gibbs-free
energy (DG°) values.
In vitro–in vivo correlation (IVIVC)
In the present study attempted prediction of Level A cor-
relation. Plotted percentage of CUR absorbed versus per-
centage of CUR dissolved from CUR–CD complexes
123

J Incl Phenom Macrocycl Chem
Fig. 10 Plasma concentration–
time profile of CUR and CUR
cyclodextrin complexes.
Mean SD, n = 6 Pure drug,
filled pink square CUR- a CD
complex, filled brown diamond
CUR- b CD complex, filled
violet circle CUR- c CD
Complex; Inset Fig.
drug, CUR- a CD complex,
CUR- b CD complex,
Pure
CUR- c CD complex. Values of
***p \ 0.001; *p \ 0.1
Fig. 11 In vitro-in vivo correlation of percentage drug absorbed and percentage drug released of A CUR a-CD complex, B CUR b-CD complex
and C CUR c-CD complex
(Fig. 11). From the correlation plots obtained regression
coefficient (R2) of 0.805, 0.578 and 0.844 for CUR a-CD,
CUR b-CD and CUR c-CD complexes, respectively. On
the contrary, obtained no correlation plot from pure CUR
which may be explained by its inherent poor solubility and
permeability considering it is a BCS Class IV drug.
123

J Incl Phenom Macrocycl Chem
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IVIVC has gained considerable significance because of
it can be successfully applied to predict in vivo perfor-
mance from in vitro behavior [43]. Among the four levels
of IVIVC, Level A is considered to be the highest level of
correlation which aims to predict the entire in vivo con-
centration time course from the in vitro dissolution profile.
Prediction of Level A correlation essentially involves
evaluation of point-to-point correlation between in vitro
and in vivo profiles [44].
In conclusion, observed altered IVIVC behaviour by
inclusion complexation of CUR with CUR a-CD complex
and CUR c-CD exhibiting greater correlation than CUR b-
CD complex. Altered IVIVC behaviour has been observed
in pioglitazone, a BCS Class 2 drug, where increase in drug
solubility leads to better correlation between in vitro and
in vivo profiles [45].
Conclusions
From ancient times in Ayurveda turmeric has been used in
treating a number of ailments. Curcumin the active princi-
ple of turmeric has poor aqueous solubility and oral bio-
availability which precludes its use in therapy. In this study
investigated the effect of curcumin-CD complexation on its
solubility and bioavailability. Pulverization method resulted
in significant enhancement of CUR solubility with CUR a-
CD complex [ CUR b-CD complex [ CUR c-CD com-
plex. Gibbs-free energy and in silico molecular docking
studies favour formation of a-CD complex [ b-CD com-
plex [ c-CD complex. Maximum enhancement in disso-
lution and bioavailability of curcumin was observed from
CUR a-CD complex. Enhanced relative bioavailability was
observed in all CUR–CD complexes with CUR a-CD
complex [ CUR b-CD complex [ CUR c-CD complex.
16. Joe, B., Vijaykumar, M., Lokesh, B.R.: Biological properties of
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Cyclodextrin-complexed curcumin exhibits anti-inflammatory
and antiproliferative activities superior to those of curcumin
through higher cellular uptake. Biochem. Pharmacol. 80,
1021–1032 (2010)
Acknowledgments Authors would like to thank Prof. B.G. Shiva-
nanda, Principal, Al-Ameen College of Pharmacy for his kind support
and encouragement to carry out this project.
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