Crassicauline A/β-cyclodextrin host-guest system: Preparation, characterization, inclusion mode, solubilization and stability

Article abstract of DOI:10.1016/j.carbpol.2011.01.031

The inclusion complexation behavior, characterization and binding ability of crassicauline A (CLA) with β-cyclodextrin (β-CD) has been investigated in both solution and the solid state by means of UV-vis spectroscopy, FT-IR, ¹H and 2D NMR, XRD,

Full text of DOI:10.1016/j.carbpol.2011.01.031

Carbohydrate Polymers 84 (2011) 1321–1328
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Crassicauline A/␤-cyclodextrin host–guest system: Preparation,
characterization, inclusion mode, solubilization and stability
Wen Chena, Li-Juan Yanga,b,∗, Shui-Xian Maa, Xiao-Dong Yangb, Bao-Min Fana, Jun Linb,∗∗
a Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry and Biotechnology,
Yunnan University of Nationalities, Kunming, 650031, PR China
b Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology,
Yunnan University, Kunming 650091, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The inclusion complexation behavior, characterization and binding ability of crassicauline A (CLA) with
␤-cyclodextrin (␤-CD) has been investigated in both solution and the solid state by means of UV–vis
spectroscopy, FT-IR, 1H and 2D NMR, XRD, SEM and DSC. The results showed that the water solubility and
thermal stability of CLA were obviously increased in the inclusion complex with ␤-CD. This satisfactory
water solubility and high stability of the CLA/␤-CD complex will be potentially useful for its application
as herbal medicine or healthcare products.
Received 3 December 2010
Received in revised form 5 January 2011
Accepted 18 January 2011
Available online 25 January 2011
Keywords:
© 2011 Elsevier Ltd. All rights reserved.
Crassicauline A
␤-Cyclodextrin
Inclusion complex
Solubilization
Stability
1. Introduction
was found to be 65 times as potent as morphine, without any phys-
ical dependence (Tang, Liu, Lu, Wang, & Li, 1986). To date, CLA has
The genus Aconitum, mostly growing on the mountainous parts
of the northern hemisphere, produce highly toxic norditerpenoid
alkaloids that have attracted considerable interest because of
their complex structures, interesting chemistry, and noteworthy
physiological effects (Pelletier, 1984). Among these norditerpenoid
alkaloids, crassicauline A (CLA, also named as bulleyaconitine A,
Scheme 1) is considered to be one of the most important active
ingredients due to its various effects. CLA was isolated from the
traditional Chinese medicine Aconitum crassicaule for the first time
(Wang & Fang, 1981). From then on, CLA was isolated widely from
the Aconitum species growing on the Yunnan-Tibet Plateau (Jing
et al., 2009; Yang, Chen, et al., 2008; Yang, Yang, Zhao, Zhang, & Li,
2007; Zheng, Gao, Hao, Wang, & Shen, 1997). CLA is an excellent
analgesic and antiinflammatory, and can be used for treating
osteoarthritis, periarthritis humeroscapularis, lumbar muscle
strain and sprain (Sha & Mao, 1993). The analgesic effect of CLA
been approved for the treatment of chronic pain and rheumatoid
arthritis in China (Wang, Gerner, Wang, & Wang, 2007). However,
the use of CLA as biopesticides or herbal medicines is greatly
limited by its low water solubility and bioavailability. Although
much effort has been made to improve its water solubility and
stability, such as microencapsule (Zhao, 2005), microemulsion
(Feng, Shang, & Xu, 2007) and multivesicular liposome (Weng
et al., 2003), CLA still cannot be sufficiently dissolved in water.
Therefore, the search for an efficient and nontoxic carrier for CLA
has become important in order to further its clinical application.
It is well known that ␤-cyclodextrin (␤-CD) is truncated-cone
polysaccharides mainly composed of seven D-glucose monomers
linked by ␣-1,4-glucose bonds (Scheme 1). It has a hydrophobic
central cavity and hydrophilic outer surface and can encapsulate
various inorganic/organic molecules to form host–guest complexes
or supramolecular species. This usually enhances drug solubility
in aqueous solution and affects the chemical characteristics of the
encapsulated this drug in pharmaceutical industry (Liu & Chen,
2006; Misiuk & Zalewska, 2009; Wu, Liang, Yuan, Wang, & Yan,
2010). This fascinating property enables them to be successfully
utilized as drug carriers (Bian et al., 2009; Uekama, Hirayama, &
Irie, 1998; Wang & Cai, 2008), separation reagents (Szejtli, 1998),
enzyme mimics (Breslow & Dong, 1998), photochemical sensors
(Ueno, 1996), etc.
∗
Corresponding author at: Key Laboratory of Ethnic Medicine Resource Chem-
istry, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry
and Biotechnology, Yunnan University of Nationalities, Kunming 650031, PR China.
Tel.: +86 871 503 3215; fax: +86 871 503 3215.
∗∗
Corresponding author. Tel.: +86 871 503 3215; fax: +86 871 503 3215.
E-mail addresses: ljyyang@gmail.com (L.-J. Yang), linjun@ynu.edu.cn (J. Lin).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.01.031

1322
W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
OH
OMe
2'
3'
13
OH
12
7'
O16C
OMe
4'
OMe
14
O
17
10
1'
1
H-5
H-5
9
O
15
2
5'
6'
11
21
HO
N 5
8
4
OH
3
O C Me
7
6
O
22
7
H-3
H-3
23
18OMe
19
O
MeO
β-CD
crassicauline A
Scheme 1. The structures of crassicauline A (CLA) and ␤-CD.
More recently, we reported that the inclusion complexation of
CDs with natural medicines such as nimbin (Yang et al., 2010),
artemether (Yang, Lin, Chen, & Liu, 2009), scutellarin (Yang, Yang,
Lin, Chen, & Liu, 2009) and azadirachtin B (Yang, Chen, Lin, & Liu,
2008; Yang & Lin, 2009), significantly enhanced the water solubil-
ity and bioavailability of the medicines. For example, CDs increased
the water solubility of nimbin from 50 ␮g/mL to 1.3–4.7 mg/mL,
and increased the bioavailability of artemether by 1.81-fold (Yang,
Lin, et al., 2009; Yang et al., 2010). As a continuation of our studies
on natural medicines/cyclodextrin inclusion complex, an inclusion
complex of CLA with ␤-CD was investigated. To the best of our
knowledge, no scientific study on this inclusion complex has hith-
erto been reported.
In this paper, we aim to report the preparation and characteriza-
tion of a water-soluble inclusion complex formed by CLA and ␤-CD.
We applied UV–vis spectral titration techniques to calculate the
inclusion stoichiometry and stability constant of CLA/␤-CD com-
plex, and characterized the complex by FT-IR, 1H NMR and 2D NMR,
powder X-ray diffraction (XRD), differential scanning calorimetry
(DSC) and scanning electron microscopy (SEM). We also focused
on the solubilization and stability effect of ␤-CD on CLA and the
binding ability of the resulting inclusion complexes, which would
provide a useful approach for obtaining novel CLA-based healthcare
products with high water solubility, stability and bioavailability.
sion, was prepared by grinding together a 1:1 molar mixture of CLA
and ␤-CD for 5 min with a small amount of water (the minimum
amount to form a slurry) in an agate mortar.
2.2.3. Determination by UV spectra
Absorption spectra measurements were carried out with a
Shimadzu UV 2401 (Japan) using
a conventional 1 cm path
(1 cm × 1 cm × 4 cm) quartz cell in a thermostated compartment,
which was kept at 25 ◦C through a Shimadzu TB-85 Thermo Bath
unit. Given the poor water solubility of CLA, a water/ethanol
(V:V = 4:1) solution was used in the spectral measurements. The
concentration of CLA was held constant at 0.021 mM. Then, an
appropriate amount of ␤-CD was added with the final concentra-
tions varied from 0 to 6.00 mM (0, 0.24, 0.50, 1.01, 1.44, 2.06, 2.94,
4.20, 6.00 mM). The absorption spectra measurement was taken
after 1 h. The measurements were done in the 200–400 nm spectral
range. All experiments were carried out in triplicate.
2.2.4. Standard curve of CLA
A series of CLA ethanol solutions with their concentrations
ranging from 2.96 to 13.50 × 10−3 mg/mL were configured. The
absorbance was recorded at 261 nm in UV at 37 ◦C in order to make
a standard curve using concentrations (C, mg/mL) as x-coordinate
and absorbance (A) as y-coordinate. We found the standard curve
of CLA could be depicted by the equation: A = 40.648C + 0.0017
(R = 0.9993).
2. Materials and methods
2.1. Materials
2.2.5. FT-IR spectra
FT-IR spectra were measured on a Nicolet Avatar 360 FTIR
spectrometer with 4 cm−1 resolution and 64 scans between wave
number of 4000 cm−1 and 400 cm−1. Samples were prepared as KBr
disks with 1 mg of complex in 100 mg of KBr.
CLA (FW = 643, PC > 95%) used in this work was obtained from
Kunming Pharmaceutical Corporation or isolated from the roots
of Aconitum macrorhynchum in Yunnan Province, PR China. ␤-CD
was purchased from ABCR GmbH & Co. KG and used without fur-
ther purification. Other reagents and chemicals were of analytical
reagent grade. All experiments were carried out using ultrapure
water.
2.2.6. 1H and 2D NMR
All NMR experiments were carried out in D2O. Tetramethylsi-
lane was used as a reference. Samples were dissolved in 99.98%
D2O and filtered before use. 1H NMR spectra were acquired on
a Bruker Avance DRX spectrometer at 500 MHz and 298 K. The
one-dimensional spectra of both solutions were run with FID res-
olution of 0.18 Hz/point. The residual HDO line had a line width at
half-height of 2.59 Hz. Two-dimensional (2D) ROESY spectra were
acquired at 298 K with presaturation of the residual water reso-
nance and a mixing (spin-lock) time of 350 ms at a field of ∼2 kHz,
using the TPPI method, using a 1024 K time domain in F2 (FID reso-
lution 5.87 Hz) and 460 experiments in F1. Processing was carried
out with zero-filling to 2K in both dimensions using sine (F2) and
qsine (F1) window functions, respectively.
2.2. Methods
2.2.1. Preparation of CLA/ˇ-CD complex
The inclusion complex was prepared by a solution-ultrasonic
method (Miecznik & Kaczmarek, 2007). CLA (0.03 mM, 19.3 mg)
and ␤-CD (0.01 mM, 12.6 mg) were completely dissolved in a mixed
solution of alcohol and water (ca. 10 ml, V:V = 1:4), and the mixture
was reacted in a ultrasonic wave equipment for 1 h at room tem-
perature in the dark. After evaporating the reaction mixture, the
surplus CLA was removed by a 0.45 ␮m millipore membrane. The
filtrate was evaporated under reduced pressure to isolate the sol-
vent and dried in a vacuum dryer at 60 ◦C for 12 h to give ␤-CD/CLA
inclusion complex (15.6 mg, yield 82%).
2.2.7. Powder X-ray diffraction (XRD)
XRD patterns were obtained using a D/Max-3B diffractometer
with Cu K␣ radiation (40 kV, 100 mA), at a scanning rate of 5◦/min.
Powder samples were mounted on a vitreous sample holder and
scanned with a step size of 2ꢀ = 0.02◦ between 2ꢀ = 3◦ and 50◦.
2.2.2. Preparation of CLA/ˇ-CD physical mixture
The physical mixture was prepared by our reported method
(Yang, Lin, et al., 2009). A physical mixture, to test for possible inclu-

W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
1323
100
80
60
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.06
0.05
0.04
0.03
0.02
0.01
0.00
i
40
20
0
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
Exp.
Calc.
a
a
0
1
2
3
4
5
6
[CD]/mM
b
c
200
250
300
350
400
λ/nm
Fig. 1. UV–vis spectral changes of CLA (0.021 mM) upon addition of ␤-CD (from 0
to 6.00 mM, a: 0 mM, b: 0.24 mM, c: 0.50 mM, d: 1.01 mM, e: 1.44 mM, f: 2.06 mM,
g: 2.94 mM, h: 4.20 mM, and i: 6.00 mM) in a water/alcohol (V:V = 4:1, ca. pH 10.5)
mixed solution, and the nonlinear least-squares analysis (inset) of the differential
intensity (ꢁA at 261 nm) to calculate the complex stability constant (Ks).
d
4000 3500 3000 2500 2000 1500 1000
Wavenumber/cm-1
500
Fig. 2. FT-IR spectra of (a) ␤-CD, (b) CLA, (c) CLA/␤-CD inclusion complex, (d) CLA
and ␤-CD physical mixture (1:1 molar ratio).
2.2.8. Differential scanning calorimetry (DSC)
DSC measurements were performed with a 2960 SDT V3.0F
instrument, at a heating rate of 10 ◦C/min from room tem-
perature to 400 ◦C in a dynamic nitrogen atmosphere (flow
rate = 70 mL/min).
expressed by Eq. (1), and the stability constant (Ks) can be calculated
from Eq. (2), where [CLA·CD], [CLA], [CD], [CLA]0 and [CD]0 refer to
the equilibrium concentration of the CLA/␤-CD inclusion complex,
the equilibrium concentration of CLA, the equilibrium concentra-
tion of ␤-CD, the original concentration of CLA, and the original
concentration of ␤-CD, respectively, and ꢁε is thedifferential molar
extinction coefficient of CLA in the absence and presence of ␤-CD.
According to Lambert–Beer law, we can obtain that the concentra-
tion of the CLA/␤-CD complex is equal to ꢁA/ꢁε (Eq. (2)). We can
then derive Eq. (3) from Eq. (2). Finally, Ks can be obtained from
the analysis of the sequential changes of absorption (ꢁA) at var-
ious ␤-CD concentrations, with a nonlinear least squares method
according to the curve-fitting Eq. (3).
2.2.9. Scanning electron microphotographs (SEM)
SEM photographs were determined on a FEI QUANTA 200. The
powders were previously fixed on a brass stub using double-sided
adhesive tape and then were made electrically conductive by coat-
˚
ing, in a vacuum with a thin layer of gold (approximately 300 A) for
30 s and at 30 W. The pictures were taken at an excitation voltage of
15, 20 or 30 kV and a magnification of 1080, 1200, 1400 or 2000×.
2.2.10. Solubilization test
An excess amount of complex was placed in 2 mL of water (ca.
pH 6.0), under nitrogen, sheltered from light and the mixture was
stirred for 1 h at 37 2 ◦C. The solution is then filtered on a 0.45 ␮m
cellulose acetate membrane. The absorbance of the filtrate was
recorded at 261 nm in UV at 37 ◦C and the residue is dosed by the
standard curve of CLA.
K
s
CLA + CDꢀCLA · CD
(1)
(2)
[CLA · CD]
(ꢁA/ꢁε)
Ks =
=
([CLA][CD])
{([CLA]0 − ꢁA/ꢁε)([CD]0 − ꢁA/ꢁε)}
ꢀ
ꢂ
ꢁ
ꢁε([CLA]0 + [CD]0 + 1/Ks)
ꢁε2([CLA]0 + [CD]0 + 1/Ks)2 − 4ꢁε2[CLA]0[CD]0
ꢁA =
(3)
2
Using a nonlinear least squares curve-fitting method (Liu, Li,
Wada, & Inoue, 1999), we obtained the complex stability constant
for each host–guest combination. Fig. 1 (inset) illustrates a typ-
ical curve-fitting plot for the titration of CLA with ␤-CD, which
shows the excellent fit between the experimental and calculated
data and the 1:1 stoichiometry of the CLA/␤-CD inclusion complex.
In repeated measurements, the Ks values were reproducible within
an error of 5%. The stability constant (Ks) and Gibbs free energy
change (−ꢁG◦) for the inclusion complexation of ␤-CD with CLA
are listed in Table 1.
3. Results and discussion
3.1. Spectral titration
Quantitative investigation of the inclusion complexation behav-
ior of ␤-CD with CLA was carried out in a water/ethanol (v:v = 4:1)
mixed solution using a spectrophotometric titration method owing
to the rather low water solubility of CLA. As illustrated in Fig. 1, the
absorbance intensity of CLA gradually increased with the stepwise
addition of ␤-CD. In all experiments, the pH of the solution did
not change appreciably during the experimental procedure. As the
size-fit, shape-fit, and charge-fit effects are the dominant control-
ling factors on the formation of inclusion complexes of ␤-CDs (Liu
& Chen, 2006), these results indicate that the binding behavior is
mainly dependent on the individual structural features of the host
and guest. Assuming 1:1 stoichiometry for the CLA/␤-CD inclusion
complex, the inclusion complexation of CLA with ␤-CD could be
Table 1
The stability constant (Ks and log Ks), molar extinction coefficient change (ꢁε) and
Gibbs free energy change (−ꢁG0) for the inclusion complexation of ␤-CD with CLA
guest in a water/alcohol (V:V = 4:1, ca. pH 10.5) mixed solution.
Ks/M−1
log Ks
ꢁε/M−1 cm−1
−ꢁG0/kJ mol−1
−14.25
313
2.49
4210

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W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
H-5
*
H-6
H-1
H-3
H-2
H-4
a
*
b
ppm
0.5
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
YUNNAN UNIVER. AV.DRX500
H-3 of
β-CD
yanglijuan 503-YLJ-CC roesy
H-3'/H-5' of
crassicauline A
H-2'/H-6' of
crassicauline A
ppm
0.5
1.0
1.5
2.0
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-23 of
crassicauline A
b
H-5 of β-CD
H-3 of β-CD
b
a
a
c
8.5
ppm
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Fig. 3. 1H NMR spectra of CLA in the absence and presence of ␤-CD in D2O at 25 ◦C, respectively: (a) ␤-CD, (b) CLA/␤-CD complex (asterisk highlights the water peak); and
ROESY spectrum in D2O at 25 ◦C: (c) CLA/␤-CD complex.
3.2. Binding ability
sess a cyclic truncated cone cavity with a height of 0.79 nm,
an inner diameter of 0.60–0.65 nm, and a cavity volume of
0.262 nm3 (Liu, Chen, Chen, & Lin, 2005; Szejtli, 1998). The
host–guest size match may dominate the stability of the com-
plex formed between ␤-CD and CLA. Additionally, considering
the structural features of the host and guest, we deduced that
the hydrogen bond between the hydrogen atoms of ␤-CD and
the oxygen atoms of CLA may strengthen the host–guest associ-
ation.
Extensive studies have revealed that the size/shape-fit con-
cept plays a crucial role in the formation of inclusion complex
of host ␤-CD with guest molecules of various structures. On
the basis of this concept, several weak intermolecular forces
such as ion–dipole, dipole–dipole, van der Waals, electrostatic,
hydrogen bond, and hydrophobic interactions are known to
cooperatively contribute to the inclusion complexation. ␤-CD pos-

W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
1325
Table 2
The chemical shifts (ı) of ␤-CD and CLA/␤-CD complex.
H-3
ı (ppm)
␤-CD
H-5
CLA/␤-CD
complex
H
2'
H
3'
OMe
OH
H-1
H-2
H-3
H-4
H-5
H-6
d
4.99
3.57
3.88
3.50
3.80
3.78
4.91
3.49
3.79
3.42
3.73
OMe
O C
O
OMe
dd
dd
dd
m
6'
H
5'
N
H
23
dd
3.69
O C Me
OMe
O
MeO
3.3. FT-IR analysis
H-5
H-3
The FT-IR spectra of ␤-CD, CLA, their inclusion complex, and
their physical mixture are shown in Fig. 2. The spectrum of
CLA shows strong absorption bands in the range of 1840–1650,
1340–1190 and 1140–1050 cm−1 (Fig. 2b). Due to the absorption
disturbance of ␤-CD, these characteristic bands can not all exist
in the spectra of CLA/␤-CD inclusion complex, but we can find
the obvious band with a negligible rightward shift in the range of
1840–1650 cm−1, which indicates the existence of CLA in the inclu-
sion complex (Fig. 2c). In addition, the IR spectra of ␤-CD can be
characterized by the intense band at 3780–3010 cm−1 correspond-
ing to vibration of the hydrogen-bonded –OH groups as well as the
band at 3000–2800 cm−1 assigned to absorption by the –CH and
–CH2 groups (Fig. 2a). After association with CLA, the spectra of the
inclusion complex manifests a reduced intensity and a certain shift
of the peak assigned to absorption by the hydrogen-bonded –OH
groups (from 3390 to 3392 cm−1). However, the spectra of the phys-
ical mixture correspond simply to the superposition of the spectra
of the individual component (Fig. 2d). All these phenomena jointly
indicate that some of the existing hydrogen bonds formed between
–OH groups of ␤-CD are broken after inclusion complexation.
Fig. 4. Possible inclusion mode and significant NOESY (↔) correlations of the CLA/␤-
CD complex.
as a solvent. Assessment of the CLA complex by 1H NMR clearly
demonstrated the presence of the framework protons of the CLA
molecule, consistent with the significant solubilization. As illus-
trated in Fig. 3, the majority of CLA protons displayed chemical
shifts at ı 1.0–3.4 and 6.5–8.5 ppm, which were distinct from the
␤-CD protons (ı 3.4–5.0 ppm). By comparing the integration area
of these protons with that of the ␤-CD’s H-1 protons, we calculated
the inclusion stoichiometry of the CLA/␤-CD complex, that is, 1:1
for the CLA/␤-CD.
To further explore the inclusion mode, the chemical shifts of
␤-CD protons in the absence and presence of CLA were listed in
Table 2. As can be seen from Table 2, after inclusion complexation
with CLA, a relatively weak effect was observed on the ı values of
H-5 protons of ␤-CD. In contrast, those values of H-1, H-2, H-3, H-4
and H-6 protons exhibited the significant changes (0.08–0.09 ppm).
It is fairly noteworthy that H-3 protons shifted ca. 0.09 ppm, but H-
5 protons showed relatively weak shifts (0.07 ppm) after resulting
inclusion complex. Because both H-3 and H-5 protons are located in
the interior of ␤-CD cavity, and H-3 protons are near the wide side
of cavity while H-5 protons near the narrow side, this phenomenon
may indicate that CLA should penetrate into the ␤-CD cavity from
the wide side.
3.4. 1H and 2D NMR analysis
In order to explore the possible inclusion mode of the CLA/␤-CD
complex, we compared the 1H NMR spectra of CLA in the pres-
ence of host ␤-CD (Fig. 3). Owing to its poor water solubility, CLA
is transparent to 1H NMR under most conditions when D2O is used
4000
A
B
a
3000
a
2000
1000
0
99OC
4000
b
b
c
3000
2000
1000
0
202OC
163OC
1000
c
750
500
250
0
97OC
3000
d
d
2000
1000
0
163OC
99OC
10
20
30
40
50
0
100
200
300
2θ [deg.]
Temperature / ºC
Fig. 5. (A) XRD patterns: (a) ␤-CD, (b) CLA, (c) CLA/␤-CD inclusion complex, (d) CLA and ␤-CD physical mixture (1:1 molar ratio). (B) DSC thermogram: (a) ␤-CD, (b) CLA, (c)
CLA/␤-CD inclusion complex, (d) CLA and ␤-CD physical mixture (1:1 molar ratio).

1326
W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
Fig. 6. Scanning electron microphotographs: (a) ␤-CD, (b) CLA, (c) CLA/␤-CD inclusion complex, (d) CLA and ␤-CD physical mixture (1:1 molar ratio).
Two-dimensional (2D) NMR spectroscopy provides important
from a superimposition of the XRD of ␤-CD and the CLA, indi-
cating the formation of the inclusion complex between ␤-CD and
CLA. In addition, most of the crystalline diffraction peaks of ␤-CD
disappeared after complexation with CLA, indicating that the com-
plexation of CLA reoriented the ␤-CD molecule to some extent.
information about the spatial proximity between host and guest
atoms by observation of intermolecular dipolar cross-correlations
(Yang, Lin, et al., 2009). Two protons closely located in space can
produce a nuclear Overhauser effect (NOE) cross-correlation in NOE
spectroscopy (NOESY) or ROESY. The presence of NOE cross-peaks
between protons from two species indicates spatial contacts within
0.4 nm (Correia et al., 2002). To gain more conformational infor-
mation, we obtained 2D ROESY of the inclusion complex of CLA
with ␤-CD. The ROESY spectrum of the CLA/␤-CD complex (Fig. 3c)
showed appreciable correlations of H-2ꢀ/H-6ꢀ and H-3ꢀ/H-5ꢀ pro-
tons of CLA with H-3 and H-5 protons of ␤-CD (peaks a), as well as
a key correlation of H-23 protons of CLA with H-3 protons of ␤-CD
(peak b). These results indicate that the aryl ring and acetyl group
of CLA are included in the ␤-CD cavity.
3.6. DSC analysis
The differential scanning calorimetry (DSC) diagram of ␤-CD,
CLA, their inclusion complex and their physical mixture are pre-
sented in Fig. 5B. The thermogram of ␤-CD shows a very board
endothermic band, between 50 and 120 ◦C, which gains a maxi-
mum at 99 ◦C (Fig. 5B(a)), indicating dehydration process. The trace
of CLA illustrates a sharp endothermic peak at 163 ◦C (Fig. 5B(b)),
corresponding to the melting point of CLA, followed by another
endothermic peak at 202 ◦C. However, in the DSC curves of the
CLA/␤-CD complex, we can find that two endothermic peaks at
about 163 and 202 ◦C (Fig. 5B(c)), corresponding to the free CLA
disappear, and the board endothermic peak, corresponding to the
free ␤-CD shifts from 99 ◦C to 97 ◦C. In contrast, the physical mix-
ture of CLA and ␤-CD contains the melting peak at 163 ◦C and the
board endothermic peak at 99 ◦C, without any shifts (Fig. 5B(d)).
These fascinating results indicate the usual thermal properties were
changed after inclusion complex formation between CLA and ␤-CD.
Based on these observations, together with the 1:1 stoichiom-
etry deduced by 1H NMR spectra and UV–vis spectrophotometric
titration, we deduced the possible inclusion modes for the CLA/␤-
CD complex as illustrated in Fig. 4.
3.5. XRD analysis
The powder X-ray diffraction (XRD) patterns of ␤-CD, CLA, their
inclusion complex and their physical mixture are illustrated in
Fig. 6. As indicated in Fig. 5A, both ␤-CD (Fig. 5A(a)) and CLA
(Fig. 5A(b)) are in crystalline form. The XRD of their physical mix-
ture (Fig. 5A(d)) just is a superimposition of the XRD of ␤-CD and
the CLA. In contrast, the XRD of their inclusion complex (Fig. 5A(c))
exhibited the amorphous halo pattern, which was quite different
3.7. SEM analysis
Scanning electron microscopy (SEM) is a qualitative method
used to study the structural aspects of raw materials, i.e., CDs and

W. Chen et al. / Carbohydrate Polymers 84 (2011) 1321–1328
1327
pH 1.5) or the simulated intestinal fluid (ca. pH 7.6) (Liu, Chen,
Chen, Ding, & Chen, 2005). The solid CLA or CLA/␤-CD complex
was quickly dissolved in the buffer solution, and the absorbance
was recorded at 261 nm in UV at 37 ◦C with an interval of 12 2 h.
Fig. 7 illustrates the relative absorbance A/A0 (A is the absorbance
at the recording time and A0 is the original absorbance) of CLA and
CLA/␤-CD complex at pH 1.5 and pH 7.6 with an interval of 12 2 h,
respectively. At pH 1.5, the relative absorbance of CLA and CLA/␤-
CD were similarly changed before 60 h, but the relative absorbance
of CLA/␤-CD declined with a slower rate than free CLA from 60 to
120 h. At pH 7.6, the relative absorbance of free CLA tapered off 7% at
the first 10 h, however, the relative absorbance of CLA/␤-CD dwin-
dled only 0.5%, and the relative absorbance of free CLA diminished
with a fast speed during the last 50 h. All these results indicate that
CLA/␤-CD is much more stable than free CLA at both pH 1.5 and 7.6.
100
95
90
85
80
a
b
c
d
0
20
40
60
80
100
120
Times / h
4. Conclusions
Fig. 7. The relative values (A/A0, A is the absorbance at the recording time and A0 is
the original absorbance) of (a) CLA at pH 1.5, (b) CLA/␤-CD complex at pH 1.5, (c)
CLA at pH 7.6, and (d) CLA/␤-CD complex at pH 7.6, with an interval of 12 2 h.
In summary, the inclusion complexation behavior, characteriza-
tion, binding ability, solubilization and stability of CLA with ␤-CD
was investigated. The results showed that ␤-CD could enhance not
only the water-solubility but also the stability of CLA. Given the
shortage of application of CLA and the easy and environmentally
friendly preparation of CLA/␤-CD complex, this inclusion complex-
ation should be regarded as an important step in the design of
a novel formulation of CLA for the herbal medicine or healthcare
products.
drugs or the products obtained by different methods of preparation
like physical mixture, solution complexation, coevaporation and
others (de Araujo et al., 2008; Duchêne, 1987). SEM photographs of
␤-CD, CLA, their inclusion complex and their physical mixture are
shown in Fig. 6. Typical crystal of ␤-CD and CLA are found in many
different sizes. ␤-CD crystallizes in polyhedral form (Fig. 6a) and
pure CLA appears as irregular-shaped crystal particles with large
dimensions (Fig. 6b). The physical mixture CLA/␤-CD reveals some
similarities with the crystal of the free molecules and shows both
crystalline components (Fig. 6d). However, the CLA/␤-CD inclusion
complex appears as compact and homogeneous plate-like structure
crystal particles and is quite different from the sizes and shapes
of ␤-CD and CLA (Fig. 6c), which confirms the formation of the
inclusion complex.
Acknowledgments
This work was supported by NSFC (30860342), Natural Sci-
ence Foundation of Yunnan Province (2010CD090), Natural Science
Foundation of Yunnan Education Department (2010Y429), the
Opening Foundation of Key Laboratory of Chemistry in Ethnic-
medicine Resources (Yunnan Nationalities University), Ministry of
Education (MJY090103), which are gratefully acknowledged.
3.8. Solubilization
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