Host-Guest Inclusion System of Luteolin with Polyamine-β-cyclodextrin: Preparation, Characterisation, Anti-oxidant and Anti-cancer Activity

Article abstract of DOI:10.1071/CH15194

The characterisation, inclusion complexation behaviours, and binding ability of inclusion complexes of luteolin (LU) with four polyamine-modified β-cyclodextrins (NH₂-βCD, EN-βCD, DETA-βCD, TETA-βCD; where EN=ethylenediamine; DETA=diethylenediamine; TETA=triethylenetetramine) were investigated in both the solid and solution forms by photoluminescence spectroscopy, ¹H and 2D NMR spectroscopy, differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy. The results showed that the water solubility, and the anti-oxidant activity and anti-cancer activity of LU were significantly increased in the inclusion complex with polyamine-β-cyclodextrin. The LU/CDs complex will be useful for its application as herbal medicine or healthcare product.

Full text of DOI:10.1071/CH15194

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH15194
Host–Guest Inclusion System of Luteolin
with Polyamine-b-cyclodextrin: Preparation,
Characterisation, Anti-oxidant and Anti-cancer Activity
A
A
B
A C
,
Manshuo Liu, Rongqiang Liao, Yulin Zhao, and Bo Yang
A
Faculty of Life Science and Technology, Kunming University of Science and
Technology, Kunming 650500, China.
B
Faculty of Chemical Engineering, Kunming University of Science and Technology,
Kunming 650500, China.
C
Corresponding author. Email: yangb_2015@163.com
The characterisation, inclusion complexation behaviours, and binding ability of inclusion complexes of luteolin (LU) with
four polyamine-modified b-cyclodextrins (NH2-bCD, EN-bCD, DETA-bCD, TETA-bCD; where EN ¼ ethylenediamine;
DETA¼ diethylenediamine; TETA¼ triethylenetetramine) were investigated in both the solid and solution forms by
photoluminescence spectroscopy, 1H and 2D NMR spectroscopy, differential scanning calorimetry, X-ray diffraction, and
scanning electron microscopy. The results showed that the water solubility, and the anti-oxidant activity and anti-cancer
activity of LU were significantly increased in the inclusion complex with polyamine-b-cyclodextrin. The LU/CDs complex
will be useful for its application as herbal medicine or healthcare product.
Manuscript received: 16 April 2015.
Manuscript accepted: 30 June 2015.
Published online: 7 August 2015.
cavity.[9] They form inclusion complexes with various hydro-
phobic molecules.[10,11] Recently, Ficarra et al. and Duncan
et al. reported the inclusion complex formation of CDs and
flavonoids.[12,13] However, the unmodified or substituted b-CD
has relatively poor water solubility and may be at risk of
nephrotoxicity.[14] To tackle these problems, modified b-CD
was applied.[15]
The chemically modified b-CDs can largely alter the original
molecular-binding ability by stereochemical complementarity of
the functional branch located in the CD cavity.[16–19] Encourag-
ingly, the complexation of LU with b-CD, HP-bCD, DM-bCD,
and SBE-bCD has been achieved (HP-bCD¼ hydroxypropy-b-
cyclodextrin; DM-bCD¼ carboxymethyl-b-cyclodextrin; SBE-
bCD¼ sulfobutyl ether-b-cycoldextrin).[20] Meanwhile, other
types of CDs as molecular hosts for LU have not been well
explored. LU is a weak acid flavonoid and polyamine-modified
CDs are alkaline. Thus, we anticipate that the binding affinity
between polyamine-modified CDs and LU will be slightly
stronger than that between native b-CD and LU.
Introduction
Luteolin (LU; Chart 1), a 30,40,5,7-tetrahydroxyflavone, is widely
found in a variety of plants, especially vegetables such as celery,
green pepper, and Perilla leaves.[1] LU exhibits a wide range
of pharmacological activities, including anti-inflammation, anti-
microbial, anti-cancer, anti-allergic, cardio-protective proper-
ties,[2–6] and other metabolic disorders.[7] It iswellknown that LU
possesses strong anti-oxidative activity, even stronger than vita-
min E and butylated hydroxytoluene.[8] Consequently, numerous
studies reported the inhibition effect against a wide range of
oxidative stress-associated pathologies such as cancer and heart
diseases. However, its poor solubility (1.93 ꢀ 10ꢁ5 mol kgꢁ1 at
208C) and stability in water severely restrict its application as a
drug. Therefore, it is necessary to find an efficient and non-toxic
carrier for LU to further its clinical application.
It is known that cyclodextrins (CDs) are truncated-cone
polysaccharides that are mainly made up of six-to-eight
D-glucose monomers linked by a-1,4-glycosidic bonds. Addi-
tionally, they have a hydrophilic exterior and a hydrophobic
We herein reported the preparation and characterisation of
the inclusion complexes of LU with polyamine-modified CDs
with different tethered chain lengths i.e. amino-b-cyclodextrin
(NH2-bCD), ethylenediamine-b-cyclodextrin (EN-bCD),
diethylenetriamine-b-cyclodextrin (DETA-bCD), and triethy-
lenetetramine-b-cyclodextrin (TETA-bCD) (Chart 2). The
binding behaviour of the water-soluble polyamine-modified
CDs with LU, and the anti-oxidant and anti-cancer activity of
LU after formation of the inclusion complexes were also
investigated. We believe that such LU polyamine-modified
CDs complexes could potentially be applied in healthcare
OH
OH
5ꢀ
2ꢀ
8
B
HO
O
A
C
6ꢀ
6
3
OH
O
Chart 1. The structure of luteolin.
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

B
M. Liu et al.
NH2
n
HN
O
O
HO
O
OH
HO
OH
O
H
OH
N
HO
HO
O
OH
NH2
n
O
O
H-5
H-3
OH
O
OH
H-5
H-3
HO
OH
O
OH
OH
OH
O
O
OH
O
HO
O
OH
HO
n ꢁ 0: NH2-βCD
1: EN-βCD
O
OH
2: DETA-βCD
3: TETA-βCD
Chart 2. The structure of the polyamine-b-cyclodextrins.
products upon further development. To our knowledge, this is
the first investigation of the inclusion behaviour of LU and
polyamine-modified CDs complexes.
12
10
8
Result and Discussion
Stoichiometry
The stoichiometry for the inclusion complex of LU with CDs
was determined by the Job’s method. Fig. 1 illustrates the Job
plot for the LU/EN-bCD system. The plot for EN-bCD showed
a maximum value at a molar fraction of 0.5, which indicated
the 1 : 1 inclusion complexation between LU and CDs. Similar
results were obtained for the complexes of LU with b-CD, NH2-
bCD, DETA-bCD, and TETA-bCD. The results were consis-
tent with the previous study by Liu who used the phase solubility
method.[21]
6
4
2
0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Spectral Titration
Fraction of EN-βCD
The inclusion complex binding behaviour between host b-CDs
and LU was studied in phosphate buffered solution using fluo-
rescence spectroscopy. The complex stability constants (KS)
were determined by changes in the absorbance intensity
upon addition of the host molecule. As the Job plot showed the
stoichiometry for the inclusion complexes of b-CDs with the
guest molecule LU is 1 : 1, the inclusion complex of host (H)
with guest (G) was expressed by Eqn 1.
Fig. 1. Job plot of the LU/EN-bCD system at lem 430 nm ([LU ]þ[EN-
bCD] ¼ 4.0 ꢀ 10ꢁ5 M) obtained in pH 7.4 buffer.
Parameter De is the proportionality coefficient, which may
be regarded as a sensitivity factor for the fluorescence intensity
change.
As illustrated in Fig. 2, the fluorescence intensity of LU
increased with the gradual addition of CDs. The fluorescence
quantum yield could be improved by formation of an inclusion
complex.[22] By using a non-linear least-squares curve-fitting
method, the complex constant could obtained.[23] It also showed
the perfect fit between the calculated and experimental data. For
all the hosts examined, a perfect fit was obtained for the plot of
DF as a function of [G]0 (total concentration of guest), thereby
verifying the validity of the 1 : 1 complex stoichiometry as
assumed above. Table 1 lists the KS values and Gibbs free
energy (ꢁDG) for the inclusion complexes of CDs with LU.
KS
Ð
H þ G
H ꢂ G
ð1Þ
Parameter KS was calculated according to Eqn 2.
½LU ꢃ CDꢄ
½LUꢄ½CDꢄ
DF=De
ꢀ
ꢁꢀ
ꢁ
KS ¼
¼
ð2Þ
½CDꢄ0 ꢁ DF=De ½LUꢄ0 ꢁ DF=De
Here [CD]0 and [LU]0 refer to the total concentration of CD
and LU, and DF can be expressed by Eqn 3.
ðDeÞð½CDꢄ0 þ ½LUꢄ0 þ 1=KSÞ
Binding Ability
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
2
2
Many studies have shown that the size and shape-fit concept
plays a vital role in the formation of inclusion complexes of host
CDs with guest molecules which have various structures.
Accordingly, some weak intermolecular forces, such as van der
ꢅ
ðDeÞ ð½CDꢄ0 þ ½LUꢄ0 þ 1=KSÞ ꢁ 4ðDeÞ ½CDꢄ0½LUꢄ0
DF ¼
2
ð3Þ

Host–Guest Inclusion System
C
100
H (free host) mole fraction
HG complex mole fraction
400
350
300
250
200
150
100
50
1.0
0.8
0.6
0.4
0.2
0
j
80
60
Calculated
a
Experimental
40
0
0
0.2
0.4
0.6
0.8
1.0
[G]0 [nM]
20
0
500
600
Wavelength [nm]
0
2
4
6
8
Fig. 2. Fluorescence emission spectra of LU (4.0 ꢀ 10ꢁ5 M) containing
Equivalent guest ([G]0/[H]0) added
various concentrations of DETA-bCD (from a to k: 0.0 ꢀ 10ꢁ4, 0.1 ꢀ 10ꢁ4
0.2 ꢀ 10ꢁ4, 0.3 ꢀ 10ꢁ4, 0.4 ꢀ 10ꢁ4, 0.5 ꢀ 10ꢁ4, 0.6 ꢀ 10ꢁ4, 0.7 ꢀ 10ꢁ4
,
,
Fig. 3. Speciation plots of the DETA-bCD (red curve) and LU/DETA-
bCD complex (black curve) at lUV 353 nm in pH 7.4 buffer.
0.8 ꢀ 10ꢁ4, and 1.0 ꢀ 10ꢁ4 M of DETA-bCD); lem 509 nm. The inset shows
the non-linear least-squares curve-fitting analysis for the inclusion
complexation.
calculated binding constants.[26,27] To understand the conditions
and molar proportions, speciation plots revealing the theoretical
proportions of CDs and LU/CDs complexes under increasing
concentrations of LU were prepared. Fig. 3 shows that when the
proportion of the guest/host gradually increased from 0 to 4, the
concentration of free DETA-bCDs gradually decreases. How-
ever, when the proportion of the guest/host was greater than 4,
the concentration of free DETA-bCDs was ,2 %.
Table 1. Stability constant (KS) and Gibbs free energy change (DG) for
inclusion complex of host CD with LU (pH 7.4) at 258C
Host
Guest
KS [L molꢁ1
]
Log KS
DG [kJ molꢁ1
]
NH2-bCD
b-CD
Luteolin
698 ꢅ 30
604 ꢅ 20
2.84
2.78
3.25
3.06
3.04
ꢁ16.24
ꢁ15.88
ꢁ18.56
ꢁ17.45
ꢁ17.39
EN-bCD
DETA-bCD
TETA-bCD
1789 ꢅ 80
1141 ꢅ 50
1117 ꢅ 60
1H NMR and 2D NMR analysis
1H NMR spectroscopy is a useful tool to investigate the for-
1
mation of the LU/CDs. The H NMR spectra of LU in the
Waals, ion–dipole, hydrogen bond, dipole–dipole, electrostatic,
as well as hydrophobic interactions, are accepted for coopera-
tively contributing to the formation of inclusion complexes. The
structural characteristics of b-CD are very important, whereby
b-CD has a cyclic truncated-cone cavity with a height of
0.79 nm, an inner diameter of 0.62–0.78 nm, and a cavity
volume of 0.262 nm3.[24] The host–guest size match may dom-
inate the stability of the complexes formed by CDs and LU.
From Table 1, the data indicated that the binding constants for
the complexes of LU with b-CD, NH2-bCD, EN-bCD, DETA-
bCD, and TETA-bCD decreased in the following order:
EN-bCD . TETA-bCD . DETA-bCD . NH2-bCD . b-CD.
By comparing the enhancement effect of some types of CDs for
LU, the modified derivatives gave a stronger KS value than the
native b-CD.[25] Furthermore, the number of amino groups may
influence the KS value. In other words, host EN-bCD is more
suitable for forming an inclusion complex with LU. The result is
understandable because the cavity of b-CD cannot encapsulate
LU tightly, and native b-CD can slip onto the guest molecular
chain like a bead. Meanwhile the other CDs (NH2-bCD, DETA-
bCD, TETA-bCD) display a lower binding ability than
EN-bCD, which has a moderate side chain length. CDs with a
longer side chain may increase the level of steric hindrance, thus
affecting the entrance of LU in the cavity of the CDs.
presence of host CDs were compared with the spectrum of LU in
order to know the possible inclusion mode of CDs/LU com-
plexes (Fig. 4). The 1H resonances of b-CD, NH2-bCD,
EN-bCD, DETA-bCD, and TETA-bCD were studied in
accordance with the reported method.[28,29] Because of its poor
water solubility, LU is transparent to 1H NMR under most
conditions when D2O is used as the solvent. Assessment of the
1
LU/CDs complexes by H NMR perfectly demonstrated the
presence of the framework protons of LU molecule and implied
a significant solubility increase for LU/CDs relative to that of
native LU (Fig. 4). The chemical shifts of LU or CDs in the 1H
NMR spectra can prove the formation of inclusion complexes.
Specifically, LU protons (4H) displayed chemical shifts at d
6.0–7.5 ppm, which were different from the CD protons (usually
observed at d 3.0–5.0 ppm). After formation of the inclusion
complex with LU, the H-3 proton of EN-bCD shifted to
0.030 ppm, and the H-5 proton of EN-bCD shifted to 0.049 ppm.
The detailed changes in the hydrogen chemical shift values of
LU and EN-bCD before and after formation of the inclusion
complexes are presented in Table 2. The shift in the proton
signals of LU upon addition of CDs shows that the B-ring and
C-ring are included in the cavity. Both H-3 and H-5 protons are
located in the interior of the CD cavity, and the H-3 protons
are located near the wide side of cavity, whereas H-5 protons are
located near the narrow side. This result could indicate that LU
should be included in the EN-bCD cavity from the narrow side.
It is known that 2D NMR spectroscopy is a powerful tool for
studying inter- and intramolecular interactions. Two protons,
Speciation Plot
The speciation plot that shows the molar fraction of CDs for-
mation inclusion complexes was constructed based on the

D
M. Liu et al.
(a)
Table 2. Chemical shifts of EN-bCD and LU/EN-bCD complex in D2O
and LU in DMSO at 258C
H-3,5,6 of CD
H-2,4 of CD
i
H-1 of CD
dd [ppm]
EN-bCD
8
6
4
2
LU
EN-bCD complex
H-3,5,6 of CD
H-2,4 of CD
ii
H-1 of CD
H-1 of EN-bCD
H-2 of EN-bCD
H-3 of EN-bCD
H-4 of EN-bCD
H-5 of EN-bCD
H-6 of EN-bCD
H-20 of LU
d
dd
dd
dd
m
dd
s
4.919
3.695
3.830
3.506
3.783
3.807
4.945
3.654
3.860
3.503
3.734
3.748
7.148
6.696
6.176
7.207
6
6
4
2
2
8
8
H-3,5,6 of CD
H-2,4 of CD
iii
H-1 of CD
4
7.395
6.894
6.442
7.406
H-3,5,6 of CD
H-2,4 of CD
H-3 of LU
H-50 of LU
H-60 of LU
s
H-1 of CD
iv
d
d
8
6
4
2
(b)
i
the superimposition of the patterns of LU and the LU/DETA-
bCD physical mixture. The results confirm that the formation of
an inclusion complex between CD and LU.
7.5
7.5
7.0
H-2ꢀ of LU
6.5
6.0
ii
H-3 of LU
H-6ꢀ of LU
H-5ꢀ of LU
Differential Scanning Calorimetry Analysis
7.0
6.5
6.0
iii
The DSC curves of LU, CDs, physical mixture, and inclusion
complex are illustrated in Fig. 8. As observed, LU displayed a
sharp endothermic peak at 3358C. DETA-bCD showed two
broad and shallow endothermic bands between 80 and 3158C.
The DSC curve of the physical mixture was mainly a combi-
nation of the curves of the two elements, whereby the LU peaks
were only weakly observed for the lower proportions. The curve
of the inclusion complex mainly showed features of the DETA-
bCD curve. In other words, the characteristic endothermic peaks
of LU disappeared, suggesting that an inclusion complex was
formed. Similarly, the characteristic DSC peaks of the guest
molecules were no longer observed.[31]
7.5
7.0
6.5
6.0
iv
H-2ꢀ of LU
H-3 of LU
H-6ꢀ of LU
H-5ꢀ of LU
7.5
7.0
6.5
6.0
δ [ppm]
Fig. 4. (a) Complete and (b) enlarged (d ,6.0–7.7 ppm) 1H NMR spectra
of LU in the absence and presence of NH2-bCD and DETA-bCD measured
in D2O at 258C: NH2-bCD (curve i), LU/NH2-bCD complex (curve ii),
DETA-bCD (curve iii), and LU/DETA-bCD complex (curve iv).
Scanning Electron Microscopy (SEM) Analysis
From the SEM analysis (Fig. 9), LU (Fig. 9a) aggregated as ball-
like crystals. DETA-bCD (Fig. 9b) is observed as irregularly
3D-shaped crystals. The physical mixture (Fig. 9c) showed
characteristics of LU and the 3D-shaped crystals of DETA-bCD
coincidently and separately. In contrast, a drastic change in
the morphology and shape of the particles was observed in
LU/DETA-bCD inclusion complex (Fig. 9d). These images
proved that when the powders of LU and CDs were simply
mixed together, and existed in their original individuals forms
(i.e. when the solutions of the two compounds were mixed), they
formed a close association, revealing an apparent interaction in
the solid state.
when closely located in space, can generate a NOE cross-
correlation between the relevant protons in the NOESY or
ROESY spectrum. The presence of NOE cross-peaks between
protons of two species indicates spatial contacts within
0.4 nm.[30] To obtain more conformation information, we per-
formed 2D ROESY analysis of the inclusion complexes of LU
with CDs. The ROESY spectrum of the LU/EN-bCD complex
(Fig. 5) showed obvious correlation between LU protons and
EN-bCD protons. The results indicated that not only the B-ring,
but also part of the C-ring of LU were included in the EN-bCD
cavity in accordance with the above results. Based on these
results together with the 1 : 1 inclusion stoichiometry deduced
by Job plot, the possible inclusion modes of LU with EN-bCD
are illustrated in Fig. 6.
Water Solubility
The inclusion complexes were clearly more soluble than native
LU (,0.93 mg mLꢁ1).[32] The solubility remarkably increased
to 0.363, 0.705, 0.990, and 1.192 mg mLꢁ1 upon the solubilising
effects of NH2-bCD, EN-bCD, DETA-bCD, and TETA-bCD,
respectively. In the control experiment, a clear solution was
obtained after dissolving LU/NH2-bCD (28.9 mg mLꢁ1),
EN-bCD (33.5 mg mLꢁ1), DETA-bCD (75.6 mg mLꢁ1),
TETA-bCD (94.3 mg mLꢁ1), equivalent to 0.363, 0.705, 0.990,
1.192 mg of LU, respectively, in 1 mL of H2O at room tem-
perature. The result confirmed the reliability of the obtained
satisfactory water solubility of the inclusion complexes. How-
ever, the solubility of b-CD and SBE-bCD only increased to
1.51- and 15.53-fold.[33] The satisfactory water solubility of LU
X-Ray Diffraction (XRD) Analysis
The XRD patterns of LU, DETA-bCD, their physical mixture,
and inclusion complex are illustrated in Fig. 7. LU (spectrum a)
was in a crystalline form, but DETA-bCD (spectrum b)
is amorphous. The XRD patterns of the physical mixture
(spectrum c) confirmed the presence of both species as isolated
as the diffractogram showed both LU peaks and amorphous
peak features of DETA-bCD. In contrast, the XRD pattern of the
LU/DETA-bCD complex (spectrum d) displayed amorphous
features and showed halo patterns, and was quite different from

Host–Guest Inclusion System
E
H-5 of EN−βCD
H-3 of EN−βCD
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
H-3 of LU
H-5ꢀ of LU
H-2ꢀ,6ꢀ of LU
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
f2 [ppm]
Fig. 5. ROESY spectrum of LU/EN-bCD complex in D2O.
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method. The IC50 (half-maximal inhibitory concentra-
tion) values have been calculated and presented in Table 3. These
complexes revealed a satisfactory anti-proliferative activity
when compared with native LU. The IC50 values of the four
complexes and LU were 10.36, 8.64, 4.21, 7.65, and 43.25 mM,
respectively in Hep G2 cell line. In the control tests, the IC50
values of NH2-bCD, EN-bCD, DETA-bCD, and TETA-bCD
were tested following addition (5 mM) to Hep G2 cell line. The
results indicated that the derivatives of b-CD were non-toxic.
The increase in cytotoxicity could be due to the formation of the
inclusion complexes. The inclusion of LU into CDs can increase
the transport of LU through the cellular membranes, thus
improving its cytotoxicity activity.[34]
H3
H5
N
NH2
H
HO
HO
H
OH
ꢀ
2
O
ꢀ
5
3
ꢀ
6
H
H
OH
H
O
H5
H3
Fig. 6. Possible inclusion mode of EN-bCD/LU complex.
with polyamine-b-cyclodextrin will be beneficial to the medical
application of this compound.
Conclusion
Anti-oxidant Activity
In this study, the inclusion complex of LU with polyamine-b-
cyclodextrin was characterised by its binding behaviour, bind-
ing ability, speciation plot, solubility, and anti-oxidant activity
and anti-cancer activity. The results indicated that polyamine-
modified b-CDs could improve the water solubility of LU sig-
nificantly. EN-bCD was the most promising host for inclusion
complex of LU. The anti-oxidant studies showed that the
inclusion complexes had better anti-oxidant activities than free
LU. In vitro cytotoxicity studies showed that the inclusion
complex had much higher anti-proliferative activities than
native LU. Thus, this type of inclusion complex is expected to
represent a significant step in the design of a novel formulation
of LU for application in herbal medicine and natural health
products.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a free radical, stable
at room temperature, which presents a violet solution in ethanol.
And it is reduced in the presence of an anti-oxidant material. The
use of DPPH provides an easy method to evaluate anti-oxidants.
We investigated the scavenging ability of LU with DPPH in the
presence and absence of CDs. When CD only is mixed with
DPPH, the absorbance did not change. In other words, CD had
no effect on DPPH. The results are expressed as remaining
DPPH (%) as a function of time (Fig. 10). By measuring the
activity of LU and its complexes by spectrophotometry, the
results clearly showed that the LU complexes displayed higher
anti-oxidant activity than the free form of LU. This enhance-
ment in the anti-oxidant activity could be due to stabilisation of
the radical in the cyclodextrin cavity. In other words, in the
presence of CDs, LU radical is more stable, probably due to the
hindered oxidation of the apolar cavity of CD.
Experimental
Materials
Luteolin (30,40,5,7,-tetrahydroxyflavone, .98 %) was pur-
chased from Nanjing Jingzhu Bio-technology Co., Ltd. Modi-
fied CDs, NH2-bCD, EN-CD, DETA-bCD, and TETA-bCD,
were synthesised in accordance with reported procedure.[35]
Cytotoxic Activity
The cytotoxicity of LU and the four LU/CD complexes (NH2-
bCD, EN-bCD, DETA-bCD, TETA-bCD) were studied using

F
M. Liu et al.
5000
2500
0
a
0
20
20
20
20
40
40
40
40
60
60
60
60
5000
2500
0
b
0
5000
2500
0
c
0
3000
1500
0
d
0
2θ [ ]
ꢂ
Fig. 7. XRD patterns of (a) LU, (b) DETA-bCD, (c) LU/DETA-bCD physical mixture (1 : 1), and (d) LU/DETA-
bCD inclusion complex.
2
1
0
a
ꢃ1
100
100
200
200
300
300
400
400
2
1
b
0
ꢃ1
2
1
c
0
ꢃ1
100
100
200
200
300
300
400
400
2
1
d
0
ꢃ1
Temperature [ꢂC]
Fig. 8. DSC patterns of (a) Lu, (b) DETA-bCD, (c) LU/TETA-bCD physical mixture (1 : 1), and (d) LU/TETA-bCD
inclusion complex.
b-CD was purchased from MengZhou Huaxin Biological
Technology Co., Ltd (Shanghai, China). DPPH was purchased
from Sigma. Other chemicals and reagents were of analytical
grade. Double-distilled and deionised water was used.
room temperature in the dark. The precipitate was removed by
filtration using a 0.45 mm Millipore membrane. Then, the filtrate
was evaporated under reduced pressure to remove the solvent
and dried under vacuum to obtain the LU/CD complexes.
Preparation of LU/NH2-bCD, LU/EN-bCD, LU/DETA-bCD,
LU/TETA-bCD Inclusion Complexes
Preparation of LU and NH2-bCD, EN-bCD, DETA-bCD,
TETA-bCD Physical Mixtures
LU (0.03 mM) and the required CD (0.01 mM) were added to
distilled water in a round flask, and then stirred for 4 days at
The physical mixture was made by grinding together a 1 : 1
molar mixture of LU and CD in an agate mortar for 15 min.

Host–Guest Inclusion System
G
(a)
(b)
WD: 8.14 mm
Det: SE
VEGA3 TESCAN
WD: 7.96 mm
Det: SE
VEGA3 TESCAN
SEM HV: 5.0 kV
BI: 10.00
SEM HV: 10.0 kV
BI: 10.00
100 μm
20 μm
Date(m/d/y) 11/19/14
Date(m/d/y) 11/19/14
Performance in nanospace
SEM MAG: 2.04 kx
Performance in nanospace
SEM MAG: 306 x
(c)
(d)
WD: 8.20 mm
Det: SE
VEGA3 TESCAN
SEM HV: 5.0 kV
BI: 10.00
WD: 8.11 mm
Det: SE
VEGA3 TESCAN
Performance in nanospace
SEM HV: 5.0 kV
BI: 10.00
20 μm
200 μm
Date(m/d/y) 11/19/14
Performance in nanospace
SEM MAG: 969 x
Date(m/d/y) 11/19/14
SEM MAG: 155 x
Fig. 9. EMS images of (a) LU, (b) TETA-bCD, (c) physical mixture of TETA-bCD and LU, and (d) LU/TETA-bCD
inclusion complex.
100
Table 3. IC50 of LU and LU/CD complexes in human hepatoma cell
line Hep G2
100
75
50
25
0
68.9
54.5
52.9
51.1
Sample
IC50 [mM]A
48.4
LU
43.25
10.36
8.64
NH2-CD/LU
EN-CD/LU
DETA-CD/LU
TETA-CD/LU
4.21
7.65
AThe concentrations of free LU, physical mixtures, and inclusion complexes
mentioned in this table are expressed as per mole of LU.
Fig. 10. Percentage content of DPPH remaining for samples blank, free
LU, and LU in the presence of NH2-bCD, EN-bCD, DETA-bCD, and
TETA-bCD.
3.5, 4.0, 4.5, 5.0 mL; DETA-bCD, or TETA-bCD: 0.0, 0.3,
0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7 mL) were added sequentially.
The mixed solution was diluted to the mark with buffer, and
then ultrasonically oscillated for 30 min at 308C, after which the
fluorescence spectra were measured at lex/lem ¼ 420 nm/510nm
(lex ¼ excitation wavelength; lem ¼ emission wavelength).
Stoichiometry
The stoichiometry of the inclusion complexes of LU with CDs
was measured by Job’s methods.[36] The Job plot was deter-
mined using fluorescence spectroscopy conducted in a pH 7.4
buffer solution. The total molar concentration (i.e. the combined
concentration of LU and CD was kept constant (4.0 ꢀ 10ꢁ5 M),
and the molar ratio of LU (i.e. [LU]/([LU] þ [CD]) was varied
from 0.1 to 0.9.
Speciation Plot
The speciation plots were obtained using CD (4.0 ꢀ 10ꢁ4 M)
and LU (4.0 ꢀ 10ꢁ4 M) solution in a KH2PO4–NaOH (pH ¼ 7.4)
buffer solution. In a 10 mL colourimetric tube, 0.5 mL CD and
varied amounts of LU (0.0, 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0,
2.5, 3.0, 4.0 mL) were added sequentially. The mixed solution
was diluted to the mark with buffer, then ultrasonically oscil-
lated for 30 min at 308C, after which the ultraviolet spectra were
measured at 353 nm. The speciation plot showing the molar
fraction of LU bound to the CDs at steady-state was calculated
according to the website www.supramolecular.org.
Spectral Titration
The spectral titration studies were conducted using CD
(2.0 ꢀ 10ꢁ3 M) and LU (4.0 ꢀ 10ꢁ4 M) solution in a KH2PO4–
NaOH (pH 7.4) buffer solution. In a 10 mL colorimetric tube,
1.0 mL of 4.0 ꢀ 10ꢁ4 M LU and varied amounts of 2.0 ꢀ 10ꢁ3 M
CD (b-CD, NH2-bCD, EN-bCD: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,

H
M. Liu et al.
1H NMR and 2D NMR
4 mg mLꢁ1 in PBS was added to 150 mL of the cell cultures in the
96-microwell flat-bottom plate for 24 h incubation at 378C.
Then, the solutions in the plates were centrifuged (1000 g for
5 min.), and the MTT-containing culture medium was removed.
The precipitated formazan was dissolved in 120 mL DMSO.
Results were read within 15 min using a spectrometer at 490 nm.
Finally, the means of the triplicates were calculated. Cell inhi-
bition rate was expressed as a percentage of the control samples.
1H NMR spectra of the LU/CD inclusion complexes and LU
monomer were obtained using a Bruker Avance DRX spec-
trometer at 500 MHz and 298 K using D2O and [D6]DMSO,
respectively. Data were collected without an external reference
in order to avoid potential interactions with the CDs. ROESY
data were obtained on a Bruker Avance DRX500 instrument and
were implemented in D2O.
XRD
Supplementary Material
Stoichiometry and speciation plots; absorption, 1H NMR
and ROESY spectra, and SEM images for various inclusion
complexes are available on the Journal’s website.
The XRD patterns were recorded on a PHI 5000 VersaProbe II
˚
temperature, and a scanning speed was 48 minꢁ1. Powder
samples were mounted on a vitreous sample holder and scanned
with a step size of 2y 0.028 in the 2y range of 5–658.
using Cu-Ka radiation (l 1.5460 A, 40 kV, 100 mA) at room
Acknowledgements
We gratefully acknowledge support from the National Natural Science
Foundation of China (NNSFC) (Nos 21362016, 21361014, and 21302074).
DSC
DSC measurements were performed on
a NETZSCH
STA449F3, using a heating rate of 108C minꢁ1 in the temper-
ature range of 30–4608C in a dynamic nitrogen atmosphere
(flow rate ¼ 100 mL minꢁ1).
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