Host-guest inclusion system of glycyrrhetic acid with polyamine-β-cyclodextrin: Preparation, characterization, and anticancer activity

Article abstract of DOI:10.1016/j.molstruc.2017.07.104

The inclusion complexation behaviors of glycyrrhetic acid (CTA) with four polyamine-modified β-cyclodextrins (CDs) have been investigated by ¹H and 2D NMR, thermal gravimetric analysis, X-ray power diffraction and scanning electron microscopy. The results showed that Glycyrrhetic acid was encapsulated into the cavity of cyclodextrin to form the complexes with 1:1 stoichiometry. The water solubility of GTA was significantly enhanced by inclusion complexation with polyamine-modified β-cyclodextrins. The calculated IC₅₀ values indicated that the antitumor activities of inclusion complexes were better than that of GTA. Satisfactory aqueous solubility, along with high thermal stability of inclusion complexes will be potentially useful for their application on the formulation design of natural medicine.

Full text of DOI:10.1016/j.molstruc.2017.07.104

Journal of Molecular Structure 1149 (2017) 155e161
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Host-guest inclusion system of glycyrrhetic acid with polyamine-b-
cyclodextrin: Preparation, characterization, and anticancer activity
*
Zhi Shen, Qi Qin, Xiali Liao, Bo Yang
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2017
Received in revised form
The inclusion complexation behaviors of glycyrrhetic acid (CTA) with four polyamine-modified b-cy-
clodextrins (CDs) have been investigated by 1H and 2D NMR, thermal gravimetric analysis, X-ray power
diffraction and scanning electron microscopy. The results showed that Glycyrrhetic acid was encapsu-
lated into the cavity of cyclodextrin to form the complexes with 1:1 stoichiometry. The water solubility of
28 July 2017
Accepted 29 July 2017
Available online 29 July 2017
GTA was significantly enhanced by inclusion complexation with polyamine-modified b-cyclodextrins.
The calculated IC50 values indicated that the antitumor activities of inclusion complexes were better than
that of GTA. Satisfactory aqueous solubility, along with high thermal stability of inclusion complexes will
be potentially useful for their application on the formulation design of natural medicine.
Keywords:
Glycyrrhetic acid
Polyamine-modified
Inclusion complex
Solubilization
©
2017 Published by Elsevier B.V.
b-cyclodextrins
Anticancer activity
1
. Introduction
reduce the adverse effect of GTA such as structural modification,
micelle [21], or liposome formation [22,23]. Under the current
situations, it will be of great significance to design a novel formu-
lation to improve the biopharmaceutical properties and reduce
manufacture for the mass production of GTA.
Glycyrrhetic acid (GTA, Fig. 1), a 3
b
-hydroxy-1-oxo-18b, 20b-
oleanane-12-acrylic, is a pentacyclic triterpenoid from the extracts
of the root of plant Glycyrrhiza glabra L. It was widely used in
Chinese medicinal preparations with various pharmacological and
therapeutic properties, including anti-allergic [1], antiulcerative
It is well known that cyclodextrins (CDs) are truncated cone
oligosaccharides mainly composed of 6e8
D-glucose monomers
[2], antioxidant [3,4], anti-inflammatory [5] and antibacterial ac-
linked by -1, 4-glycosidic bonds, referred to as
a
a
-, - and -CD,
b
g
tivities [6,7]. In addition, it has been clinically used as a remedy for
the treatment of chronic hepatitis [8] and immunodeficiency virus
infection [9]. Moreover, it is recently reported to inhibit the repli-
cation of the SARS-associated viruses [10] and induce cell apothe-
osis in the liver cancer cell line [11], and used as a ligand for liver
targeting [12e18] because of the abundant receptors for GTA on
hepatocyte membranes. Even though GTA has been used clinically
for a long time, it is a lipophilic drug with a very low solubility in
water, poor bioavailability. Furthermore, overconsumption of CTA
results in mineralocorticoid-like side effects such as salt retention,
hypokalaemic hypertension and aldosteronism [19,20]. It has pre-
sented a challenge in the development of formulations for oral
administration and reducing the adverse effect. In previous study,
great efforts have been made to improve its water solubility and
respectively. They have a hydrophobic central cavity and a hydro-
philic outer surface, which enable them to form host-guest com-
plexes with different kinds of guest molecules. Intensive studies
revealed that it can enhance drug solubility in aqueous solutions,
reduce the adverse effect of drug, and affect the chemical charac-
teristics of encapsulated drug [24,25]. Those features of cavity
render them a new function, successfully being utilized as drug
carriers [26e29], enzyme mimics [30]. The applications of un-
modified
solubility. Fortunately, chemically modified
as a promising solution to this problem. Compared with native
CD, chemically modified -CD with specific functional groups can
b-CD, however, are restricted by its relatively low aqueous
b
-CD has been applied
b
-
b
significantly improve molecular-binding ability as well as water
solubility [31,32]. Since GTA is an acidic triterpenoid, it is believed
that the binding ability of polyamine-modified CDs to GTA could be
higher than native b-CD. According to the previous work [33e36],
we wish to report the preparation and characterization of the in-
clusion complexes formed by GTA and polyamine-modified CDs
*
Corresponding author.
E-mail address: yangbo6910@sina.com (B. Yang).
http://dx.doi.org/10.1016/j.molstruc.2017.07.104
022-2860/© 2017 Published by Elsevier B.V.
0

156
Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
O
of CD), 4.98 (s, 7H, H-1 of CD). DETA-
b
CD, TETA-
b
CD and TEPA-
b
CD
were prepared by the same approach.
29
30
OH
2.3. Preparation inclusion complexes
20
The GTA/EN-
bCD, GTA/DETA-bCD, GTA/TETA-bCD and GTA/
19
21
TEPA- CD inclusion complex were prepared using a reported
b
H
method [37]. The CDs (0.01 mmol) and GTA (0.03 mmol, 14.1 mg)
were added to ultrapure water (10 mL), and the mixture was stirred
for 72 h at room temperature. Then, the precipitate was removed by
1
2
1
8
2
2
O
1
1
13
14
17
2
5
filtration, and the filtrate was lyophilized to dryness to obtain the
26
2
8
1
GTA/CDs complexes. ( H NMR; 500 MHz, D
2
O):
d
0.76 (s, 3H, H-24
1
4
9
of GTA), 0.98 (s, 3H, H-25 of GTA), 1.25 (s, 2H, H-29 of GTA), 1.4 (s,
3H, H-25 of GTA), 2.7e3.0 (m, 4H, CH CH ), 3.3e3.6 (m, 14H, H-2, 4
of CD), 3.7e3.9 (m, 26H, H-3, 5, 6 of CD), 4.94 (s, 7H, H-1 of CD).
16
2
3
2
2
1
0
8
15
27
5
7
2
.4. Preparation of physical mixture
HO
6
The GTA and EN- CD, DETA-bCD, TETA-bCD and TEPA-bCD
b
physical mixtures were prepared [38]. By mixing GTA and CD de-
rivatives thoroughly in an agate mortar, giving the 1:1 physical
mixture.
2
3
24
Fig. 1. Structure of glycyrrhetic acid (GTA).
2
.5. Phase-solubility diagram
with different tethered chain lengths, including ethylenediamine-
-cyclodextrin (EN- CD), diethylenetriamine- -cyclodextrin
DETA- CD), triethylenetetramine- -cyclodextrin (TETA- CD), and
Tetraethylenepentamine- -cyclodextrin (TEPA- CD).
b
(
b
b
The phase-solubility diagram was investigated by using the
b
b
b
Higuchi and Connors method [39]. An excess amount of GTA was
suspended in ultrapure water containing increasing amounts of
b
b
In this paper, the evaluation of the inclusion behaviors between
GTA and polyamine-modified CDs have been studies by phase-
solubility diagram. Additionally, the solid inclusion complexes
b
CD, EN-
bCD, DETA-bCD, TETA-bCD and TEPA-bCD (from 0.001 to
0.2 M). The mixtures were placed in an ultrasonic bath for 2 h at
25 C. After that, all suspensions were centrifuged and the super-
natants were collected, and filtered over 0.45 m millipore mem-
ꢀ
1
have been characterized by means of H and 2D NMR spectra,
m
powder X-ray diffraction, differential scanning calorimetry and
scanning electron microscopy. In vitro cytotoxicity of these solid
inclusion complexes was also measured with three cancer cell lines,
HT26, HCT116, and SW480, as well as normal human lung fibroblast
WI-38, to evaluate the potential of GTA in the form of inclusion
complexes with polyamine-modified CDs, which would provide a
useful approach to developing novel GTA products with high water
solubility and bioavailability.
branes. These experiments were repeated three times. The phase-
solubility profiles were obtained by plotting the solubility of GTA
versus the concentration of
TEPA- CD. The apparent stability constant, Ks of GTA and
CD, DETA- CD, TETA- CD and TEPA- CD complexes can be
b
CD, EN-
b
CD, DETA-bCD, TETA-bCD and
b
bCD, EN-
b
b
b
b
calculated from the slope and the intercept of the linear segment of
the phase solubility line using the following equation:
slope
S0ð1 ꢁ slopeÞ
K
s
¼
2
. Materials and methods
The value slope is found in the linear regression and S
0
is the
2
.1. Reagents and materials
aqueous solubility of the drug at pH 7 (S ¼ 0.00632 mg/mL) in the
0
absence of CD, EN- CD, DETA- CD, TETA- CD and TEPA- CD.
b
b
b
b
b
Glycyrrhetinic acid (GA, MW ¼ 470.79, PC > 99%) was purchased
from Sigma (USA). b-CD was purchased from Mengzhou Huaxin
2
.6. X-ray powder diffraction
Biological Technology (Shanghai, China). Other chemicals and re-
agents were of analytical grade. All experiments were conducted by
ultrapure water.
The X-ray Powder diffraction (XRD) patterns were performed
with
a
D/Max-3B diffractometer using Cu-K
a
radiation
ꢀ
(
k ¼ 1.5460 Å, 40 kV, 100 mA), with a 5 /min scanning rate. Powder
samples were mounted on a vitreous sample holder and scanned
2.2. Synthesis of polyamine-b-cyclodextrin
ꢀ
ꢀ
with a step size of 2
q
¼ 0.02 between 2
q
¼ 5e70 .
The method to synthesis EN-
text as a typical example. Mono-6-O-(tolylsulfonyl)-
.32 mmol) was dissolved in 20 mL ethanediamine solution, stirred
b
CD was given in the succeeding
b
-CD (3.0 g,
2.7. Differential scanning calorimetry
2
ꢀ
at 85 C for 10 h under N
2
atmosphere with a condense pipe. After
Differential scanning calorimetry (DSC) measurements were
analyzed using a STA449 F1 Jupiter (Netzch Corp, Germany).
that, the solution was poured into 300 mL of acetone. A yellow
precipitate was formed and collected by filtration. Finally, the
precipitate was dissolved in small amount of water and dropped in
acetone again and a light yellow precipitate was formed and
2
Samples were placed under an N flow in the temperature range
ꢀ
ꢀ
ꢀ
ꢁ1
from 25 C to 450 C at a heating rate of 10 C$min
.
collected by filtration and dried under vacuum to yield a stable light
2.8. Scanning electron microscopy
1
yellow powder. ( H NMR; 500 MHz, D
2
O):
d
2.6e2.8 (m, 4H,
CH
2
CH
2
), 3.4e3.6 (m, 14H, H-2, 4 of CD), 3.7e3.9 (m, 26H, H-3, 5, 6
The morphologies of the samples were performed on a scanning

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
157
electron microscope (TESCAN, model VEGA3). Samples were
distributed on metal stubs with double-sided adhesive tapes.
Before examination, the samples were gold sputter-coated to
render them electrically conductive, and the micrographs were
obtained under reduced pressure.
2.9. Solubilization test
The water solubility of the GTA/
b
CD, GTA/EN-bCD, GTA/DETA-
b
CD, GTA/TETA-bCD and GTA/TEPA-bCD complex are assessed by
preparation of its saturated solution [40]. An excess amount of the
complex was placed in 2 mL water (pH 7.0), protected from light,
ꢀ
and the mixture was stirred for 1 h at 25 ± 2 C. The solution was
then filtered on a 0.45
mm Millipore membrane and the filtrate was
lyophilized to dryness, and the resulting solid weighed.
Fig. 2. Phase solubility diagram of Structure of glycyrrhetic acid (GTA) in water
depending on the cyclodextrin (CD) concentration.
2.10. In vitro cytotoxicity studies
The cytotoxicity tests for GTA and its inclusion complexes with
EN-bCD, DETA-bCD, TETA-bCD and TEPA-bCD were evaluated
than the native
decreasing, the Ks value also decreases. These may be because that
the native -CD can slip onto the guest molecular chain like a bead
41], and the amino groups at the rim of CDs can reduce the
effective cavity of the CDs and then hold the drugs more tightly
42].
b-CD. Besides, with the number of amino
in vitro for antitumor activity against human HT29, HCT116,
SW480 cell lines and normal human lung fibroblast WI-38 as
reference cell lines by the MTT cytotoxicity assay. The IC50 values
that represented the concentration of drug required for 50%
reduction of cellular growth have been calculated. Cells were
b
[
[
5
ꢁ1
cultured at 5 ꢂ 10 cells mL in RPMI 1640 supplemented with 10%
ꢀ
heat-inactivated fetal bovine serum at 37 C in a humidified at-
mosphere of 5% CO
5
ꢁ1
3.2. Inclusion mode
2
in air. Cells were seeded at 1 ꢂ 10 cells mL
and treated with the indicated amounts of the GTA and its inclusion
complexes. The cytotoxic activities of the GTA and its inclusion
complexes were evaluated as cell survival after treatment. Cell
viability was evaluated by a microculture tetrazolium reduction
assay using 3-(4,5-dimethyltriazol-2-yl) 2,5-diphenyltetrazolium
bromide (MTT).
To explore the possible inclusion mode of GTA/CDs inclusion
1
complex, the H NMR and 2D NMR spectra of CDs, GTA or their
inclusion complex were investigated. As shown in Fig. 3, protons of
1
GTA displayed chemical shifts at
spectra of GTA/EN-
from the CDs protons (
d
0.5e2.0 ppm in the H NMR
b
CD complex, which were obviously different
d
3.0e5.0 ppm), indicating the formation of
inclusion complex regarding the poor water solubility of GTA. After
inclusion complexation with GTA, the H-3 and H-5 protons of CDs
both shifted of 0.04 and 0.02 ppm, respectively. Given that both H-3
and H-5 protons are located in the interior of the CD cavity, which
may indicate that GTA should be included in the CDs cavity.
It is well know that spatial proximity between host and guest
molecules can be identified by the nuclear Overhauser effect (NOE)
cross-correlations between relevant protons through 2D NMR ex-
periments, such as NOESY and ROESY. To further confirm the
3
. Results and discussion
3.1. Phase-solubility
Judging from the phase solubility diagram results (Fig. 2), the
solubility of GTA increased as a linear function of the CD concen-
tration. The regression equations are as follows:
binding mode, a 2D ROESY spectrum of the GTA/EN-bCD inclusion
2
complex was obtained (Fig. 4). It is apparent that the H-12, 27, 29
protons of GTA are strongly correlated with the H-3 protons of EN-
[
[
[
[
[
GTA] ¼ 0.0162[
b
-CD] þ0.0295
R
R
R
R
R
¼ 0.9922
¼ 0.9918
¼ 0.9901
¼ 0.9921
¼ 0.9939
2
2
2
2
GTA] ¼ 0.0234[EN-
b
CD] þ0.0283
GTA] ¼ 0.0290[DETA-
b
CD] þ0.0287
b
CD, whereas, H-24, 25 protons of GTA is correlated with the H-5
protons of EN- CD. Besides, correlations between ethylene protons
of the amino side chain of EN- CD and H-3, 5 protons of EN- CD
showed that the amino side chain was inserted into the cavity of
EN- CD. Based on these observations, together with the 1:1 in-
GTA] ¼ 0.0416[TETA-
GTA] ¼ 0.0529[TEPA-
bCD] þ0.0191
b
bCD] þ0.0167
b
b
b
clusion stoichiometry, we deduced the possible inclusion modes
According to Higuchi and Connors's theory [38], these five re-
lationships are classified as a typical A -type, suggesting that the
stoichiometry of GTA in the -CD, EN- CD, DETA- CD, TETA- CD or
TEPA- CD inclusion complex was 1:1. To the best of our knowledge,
L
b
b
b
b
Table 1
b
Complex stability constant Ks, log Ks and Gibbs free energy change(-DG) for inclu-
several weak intermolecular forces, such as ion-dipole, van der
Waals, electrostatic, hydrogen bond, and hydrophobic interactions,
are cooperatively contribute to inclusion complexation. The
apparent stability constant (Ks) value is usually at the range from 50
sion complexation of GTA guest with host
b
-CD, EN-
b
CD, DETA-
b
CD, TETA-
b
CD and
ꢀ
TEPA-
Host
-CD
b
CD at 25 C.
ꢁ
ꢁ1
Guest
Ks(L$mol 1
)
log Ks
-△G(KJ$mol
)
ꢁ
1
b
210
2.32
2.78
3.04
3.08
3.13
13.2
15.8
17.39
17.45
17.93
to 2000 M . In our study (Table 1), the calculated Ks values of the
-CD, EN- CD, DETA- CD, TETA- CD or TEPA- CD complexes were
EN- CD
b
604
b
b
b
b
b
DETA- CD
b
Glycyrrhetic acid
1047
1191
1362
ꢁ
1
210, 604, 1047, 1191 and 1362 M , respectively, showing that the
TETA-
b
CD
CD
polyamine-modified
b
-cyclodextrins have stronger binding ability
TEPA-b

158
Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
H-3,6,5 of EN- CD
H-1 of EN- CD
H-2,4 of EN- CD
EN- CD
8
8
7
6
5
4
4
3
2
1
0
0
CH of GTA
3
GTA
H-12 of GTA
7
6
5
3
2
1
H-1 of EN- CD H-3,6,5 of EN- CD
H-2,4 of EN- CD CH of GTA
3
GTA/EN- CD
H-12 of GTA
8
7
6
5
4
3
2
1
0
Fig. 3. Proton nuclear magnetic resonance spectra of EN-
bCD (in D
2
O), GTA (in CDCl
3
), and GTA/EN-
bCD (in D
2
O) inclusion complex.
H-5 of EN- CD
H-3 of EN- CD
H-25 of GTA
H-29 of GTA
H-24 of GTA
Ethylenediamine
H-27 of GTA
H-12 of GTA
0.5
1.0
1.5
2.0
2.5
3.0
(
A)
(
B)
(C)
3.5
4.0
4.5
5.0
5.5
6.0
(A)
(B)
(C)
6.0
5.5
5.0
4.5
4.0
3.5
f2 (ppm)
3.0
2.5
2.0
1.5
1.0
0.5
ꢀ
Fig. 4. ROESY of GTA/EN-
bCD inclusion complex in D
2
O at 25 C.
between GTA and EN-
b
CD (Fig. 5).
ray powder diffraction. As Fig. 6 depicted, GTA displayed series of
characteristic peaks, which demonstrated its high degree of
crystallinity. In contrast, their inclusion complexes displayed a
very different amorphous halo pattern from the diffractogram, in
which the feature diffraction peaks of GTA were completely dis-
appeared, but different from their physical mixtures. These results
3.3. X-ray powder diffraction
The crystalline states of GTA, EN-
inclusion complexes and physical mixtures were examined by X-
bCD, DETA-bCD, and their

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
159
H-3
H-5
H-3
2
9
O
H-5
12
H C
3
H
29
O
O
H
H
12
H C
3
OH
NH2
25
O
H
CH3
OH
HO
2
5
HN
HO
CH3
H C
27
3
CH3
24
H C
27
H
3
H
CH3
24
H
N
H-5
H-3
H-5
H N
2
H-3
Fig. 5. Possible inclusion mode of the GTA/EN-
bCD complex.
5
000
500
0
1
2
3
4
2
0
0
0
0
20
20
20
20
40
40
40
40
60
60
60
60
5
000
500
0
2
5
000
500
0
2
3000
2000
1000
0
2
[degree]
Fig. 6. XRD patterns of (1) GTA, (2) EN-bCD, (3) GTA/EN-bCD physical mixture (1: 1), and (4) GTA/EN-bCD inclusion complex.
further proved that the formation of GTA and CDs inclusion
complexes.
3.5. Scanning electron microscopy
From scanning electron microscopy analysis (Fig. 8), using GTA,
EN- CD, their physical mixture and inclusion complex for example.
GTA was seen as claviform crystals that formed aggregates (a),
while EN- CD looked like irregularly 3D-shaped crystals (b). Their
b
3.4. Differential scanning calorimetry
b
physical mixture was just like a simple overlay of their morpho-
logical characteristics (c). In contrast, their inclusion complex
Thermal properties of GTA, GTA/EN-bCD inclusion complex
were investigated by DSC. As showed in Fig. 7, the DSC curves of
appeared similar to that of EN-
bCD, which was distinct from the
GTA and EN-
thermic peak at ca 310 C (a), and the free EN-
broad endothermic bands between ca 100 and 400 C (b). Their
physical mixtures DSC curve was nearly a combination of the two
b
CD showed that the free GTA had obvious endo-
ꢀ
morphology of their physical mixture (d). That proved that the
formation of GTA and EN-bCD inclusion complex once more.
b
CD displayed two
ꢀ
components (c). However, the DSC curve of GTA/EN-
b
CD inclusion
3.6. Water solubility
complex was much different from their physical mixtures (d),
indicating a different thermal stability from that of above. All these
The water solubility of GTA/EN-
TETA- CD and GTA/TEPA- CD inclusion complex was assessed by
the preparation of its saturated solution. An excess amount of the
bCD, GTA/DETA-bCD, and GTA/
results showed that a new formation of GTA and EN-
their inclusion complex.
bCD, namely
b
b

160
Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
3.0
1.5
0.0
3.0
(
a)
(b)
1.5
0.0
100
200
300
400
500
100
200
300
400
500
2
0
2
0
(
c)
(d)
100
200
300
400
500
100
200
300
400
500
o
T ( C)
Fig. 7. DSC curves of (a) GTA, (b) EN-
b
CD, (c) GTA/EN-bCD physical mixture (1: 1), and (d) GTA/EN-bCD complex.
complex was placed in 2 mL of water (ca pH 7.0), and the mixture
was stirred vigorously at room temperature for 2 h. Subsequently,
the insoluble substance was removed by filtration and then
lyophilized to dryness, and the residue was dosed by the weighing
method. The results show that the water solubility of this GTA
(Table 2), compared with that of native GTA (ca. 0.00632 mg/mL),
was markedly increased to approximately 2.34, 4.78, 7.15 and
8.33 mg/mL by the solubilizing effects of EN-
bCD, DETA-bCD, TETA-
b
CD, and TEPA- CD, respectively. This confirmed the reliability of
b
the obtained more satisfactory water solubility of GTA/CDs com-
plexes, which would be beneficial for the utilization of this com-
pound as medicine products.
3.7. In vitro cytotoxicity studies
The cytotoxicity of GTA and four GTA/CDs solid inclusion com-
plex on human cancer cell lines (HT29, HCT116, and SW480) and
normal human lung fibroblast WI-38 were individually evaluated
using the MTT assay, using cisplatin (DPP) as a reference compound.
The IC50 values that represented the concentration of a drug
required for 50% reduction of cellular growth had been calculated
and showed in Table 3. As seen, the IC50 values of GTA/CDs solid
inclusion complex were better than that of the free GTA, but also as
Fig. 8. Scanning electron microscopy images of (a) GTA, (b) EN-bCD, (c) physical
mixture of EN-bCD and GTA, and (d) GTA/EN-bCD complex.
Table 3
Table 2
IC50 (mM) of GTA and GTA/CDs complexes in various colon cancer cell lines.
ꢀ
The water solubility of Glycyrrhetic acid/CDs complex at 25 C.
Samples
EN- CD
HT-29
HCT116
SW480
WI-18
Compound
Water Solubility
b
>100
>100
>100
>100
10.8
71.8
13.4
17.5
11.5
16.4
>100
>100
>100
>100
8.9
>100
>100
>100
>100
9.3
>100
>100
>100
>100
30.5
Solubility (mg/mL)
Fold increase
DETA- CD
b
TETA-
TEPA-
DPP
b
b
CD
CD
GTA
GTA/EN-
GTA/DETA-
GTA/TETA-
0.00632
2.34
4.78
7.15
8.33
1.0
b
CD complex
370
756
1132
1318
bCD complex
GTA
GTA/EN-
GTA/DETA-
GTA/TETA-
53.9
12.8
17.1
9.7
97.8
13.7
21.5
10.8
19.7
99.3
b
b
CD complex
CD complex
b
CD complex
CD complex
>100
>100
>100
>100
GTA/TEPA-
b
b
CD complex
CD complex
GTA/TEPA-
b
14.1

Z. Shen et al. / Journal of Molecular Structure 1149 (2017) 155e161
Chemother. 57 (2013) 241.
161
well as the reference compound DPP, and GTA/TETA-bCD complex
[
[
[
7] K. Oyama, M. Kawada-Matsuo, Y. Oogai, T. Hayashi, N. Nakamura,
H. Komatsuzawa, PLoS One 11 (2016) 1.
8] S. Iino, T. Tango, T. Matsushima, G. Toda, K. Miyake, K. Hino, H. Kumada,
K. Yasuda, T. Kuroki, C. Hirayama, Hepatol. Res. 19 (2001) 31.
was the best one among them. Additionally, cytotoxicity of GTA/CDs
solid inclusion complexes against normal human lung fibroblast
bCD, DETA-bCD, TETA-bCD and
TEPA- CD had no significant toxicity. In vitro toxicity study is
essential to prove the safety of any drug carriers. These results
would provide much useful information for the study of GTA in the
future.
WI-38, which indicates that, the EN-
9] H. Sasaki, M. Takei, M. Kobayashi, R.B. Pollard, F. Suzuki, Pathobiology 70
b
(
2003) 229.
10] G. Hoever, L. Baltina, M. Michaelis, et al., J. Med. Chem. 48 (2005) 1256.
[11] H. Hibasami, H. Iwase, K. Yoshioka, H. Takahashi, Int. J. Mol. Med. 17 (2006)
15.
12] Y. Cong, B. Shi, Y. Lu, S. Wen, R. Chung, D. Jin, Sci. Rep. 6 (2016) 21891.
13] W. Huang, W. Wang, P. Wang, Q. Tian, et al., Acta Biomater. 6 (2010) 3927.
[
2
[
[
4
. Conclusions
[14] W.W. Qi, H.Y. Yu, H. Guo, J. Lou, et al., Mol. Pharm. 12 (2015) 675.
[15] Q. Tian, X.H. Wang, W. Wang, C.N. Zhang, P. Wang, Z. Yuan, Nanomed.
Nanotechnol. Bio. Med. 8 (2012) 870.
In the present work, solid inclusion complexes between GTA and
[16] Q. Tian, C.N. Zhang, X.H. Wang, et al., Biomaterials 31 (2010) 4748.
four polyamine-modified
the characterization, inclusion complexation behavior, and binding
ability of the inclusion complexes were investigated. The results
b
-CDs were successfully prepared, and
[17] F.Z. Wang, L. Xing, Z.H. Tang, et al., Mol. Pharm. 13 (2016) 1298.
[18] C. Zhang, W. Wang, T. Liu, Y. Wu, H. Guo, P. Wang, Q. Tian, Y. Wang, Z. Yuan,
Biomaterials 33 (2012) 2187.
[19] L.J. Ming, A.C. Yin, Nat. Prod. Commun. 8 (2013) 415.
showed that polyamine-modified
water solubility of GTA. The cytotoxicity assay showed that com-
plexes of GTA with polyamine-modified -CD exhibited higher
anticancer activity on human cancer cell lines than that of free GTA.
Considering the shortage of applications of GTA and the conve-
niently and environmentally friendly preparation of the GTA/CDs
complex, inclusion complexation should be regarded as a useful
approach to the design of a novel formulation of GTA for herbal
medicine. Furthermore, high anticancer activity of inclusion com-
plexes will be potentially applied to the cancer chemotherapy.
b
-CD dramatically enhanced the
[20] N. Ohtake, A. Kido, K. Kubota, N. Tsuchiya, T. Morita, Y. Kase, S. Takeda, Life Sci.
80 (2007) 1545.
[
21] S. Jin, S. Fu, J. Han, S. Jin, Q. Lv, Y. Lu, J. Qi, W. Wu, H. Yuan, J. Drug Target 20
2012) 615.
22] J. Li, Y. Hua, L. Shuai, G.J. Wang, J. Pharm. Biomed. 51 (2010) 1147.
b
(
[
[23] X. Zhao, Y. Fan, D. Wang, Y. Hu, L. Guo, S. Ruan, J. Zhang, J. Yuan, Vaccine 29
2011) 9611.
(
[
[
24] T. Loftsson, M.E. Brewster, J. Pharm. Pharmacol. 62 (2010) 1607.
25] T. Loftsson, M.E. Brewster, J. Pharm. Sci. 101 (2012) 3019.
[26] R. Arun, Sci. Pharm. 76 (2008) 567.
[
[
[
[
[
[
27] X. Chen, Y.K. Qiu, C. Owh, X.J. Loh, Y.L. Wu, Nanoscale 8 (2016) 18876.
28] L. Ding, W. Song, R. Jiang, L. Zhu, Polym. Chem. 7 (2016) 6992.
29] P.K. Shende, R.S. Gaud, R. Bakal, D. Patil, Colloids Surf. B 136 (2015) 105.
30] R.B. And, S.D. Dong, Chem. Rev. 98 (1998) 1997.
31] Y. Liu, G. Chen, L. Li, H. Zhang, D. Cao, Y.J. Yuan, J. Med. Chem. 46 (2003) 4634.
32] H. Wang, R. Cao, C. Ke, Y. Liu, A. Takehiko Wada, Yoshihisa Inoue, J. Org. Chem.
Acknowledgments
7
0 (2005) 8703.
This work was supported by National Natural Science Founda-
tion of China (NNSFC) (No. 21362016 and 21642001), which are
gratefully acknowledged.
[33] X. Ma, B. Yang, Y. Zhao, H. Xie, X. Gong, Aust. J. Chem. 68 (2015) 946.
34] Y. Ren, Y. Liu, Z. Yang, R. Niu, K. Gao, B. Yang, X. Liao, J. Zhang, Mater. Sci. Eng.
C 69 (2016) 68.
35] B. Yang, Y.L. Zhao, X. Yang, X.L. Liao, J. Yang, J.H. Zhang, C.Z. Gao, Carbohydr.
Polym. 92 (2013) 1308.
[
[
References
[36] X. Yang, Y. Zhao, Y. Chen, X. Liao, C. Gao, D. Xiao, Q. Qin, D. Yi, B. Yang, Mater.
Sci. Eng. C 33 (2013) 2386.
[
[
[
[
[
37] B. Shen, L. Tong, D. Jin, Synth. Commun. 21 (1991) 635.
[
1] H.Y. Park, S.H. Park, H.K. Yoon, M.J. Han, D.H. Kim, Arch. Pharm. Res. 27 (2004)
38] Y. Liu, B. Li, T. Wada, Y. Inoue, Supramol. Chem. 10 (1998) 279.
39] T.A. Higuchi, K.A. Connors, Adv. Anal. Chem. Instrum. 4 (1965) 117.
40] P. Montassier, D. Duch e^ ne, M.C. Poelman, Int. J. Pharm. 153 (1997) 199.
57.
[
[
[
[
2] S. Yano, M. Harada, K. Watanabe, et al., Chem. Pharm. Bull. 37 (1989) 2500.
3] H.E. Jian, Chin. J. Aesthet. Med. 10 (2001) 191.
4] K.H. Nwe, A. Hamid, P.B. Morat, B.A. Khalid, Steroids 65 (2000) 40.
5] D. Maitraie, C.F. Hung, H.Y. Tu, Y.T. Liu, et al., Bioorg. Med. Chem. 17 (2009)
41] K. Miyajima, H. Komatsu, K. Inoue, T. Handa, M. Nakagaki, Bull. Chem. Soc. Jpn.
6
3 (1990) 6.
42] J. Gu, S.M. Chi, Y. Zhao, P. Zheng, Q. Ruan, Y. Zhao, H.Y. Zhu, Helv. Chim. Acta
4 (2011) 1608.
[
2785.
9
[
6] D.R. Long, J. Mead, J.M. Hendricks, M.E. Hardy, J.M. Voyich, Antimicrob. Agents